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Vivarium: an interface and engine for integrative multiscale modeling in computational biology AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSMotivationC_ST_ABSThis paper introduces Vivarium - software born of the idea that it should be as easy as possible for computational biologists to define any imaginable mechanistic model, combine it with existing models, and execute them together as an integrated multiscale model. Integrative multiscale modeling confronts the complexity of biology by combining heterogeneous datasets and diverse modeling strategies into unified representations. These integrated models are then run to simulate how the hypothesized mechanisms operate as a whole. But building such models has been a labor-intensive process that requires many contributors, and they are still primarily developed on a case-by-case basis with each project starting anew. New software tools that streamline the integrative modeling effort and facilitate collaboration are therefore essential for future computational biologists.

ResultsVivarium is a Pythonic software for building integrative multiscale models. It provides an interface that makes individual models into modules that can be wired together in large composite models, parallelized across multiple CPUs, and run with Vivariums simulation engine. Vivariums utility is demonstrated by building composite models that combine several modeling frameworks: agent based models, ordinary differential equations, stochastic reaction systems, constraint-based models, solid-body physics, and spatial diffusion. This demonstrates just the beginning of what is possible - future efforts can integrate many more types of models and at many more biological scales.

AvailabilityThe models, simulation pipelines, and notebooks developed for this paper are available at the vivarium-notebooks repository: https://github.com/vivarium-collective/vivarium-notebooks. Vivarium-core is available at https://github.com/vivarium-collective/vivarium-core, and has been released on PyPI. The Vivarium Collective (https://vivarium-collective.github.io) is a repository of freely-available Vivarium processes and composites, including the processes used in Section 3. Supplementary materials provide with an extensive methodology section, with several code listings that demonstrate the basic interfaces.

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General object-based features account for letter perception better than specialized letter features After years of experience, humans become experts at perceiving letters. Is this visual capacity attained by learning specialized letter features, or by reusing general visual features previously learned in service of object categorization? To explore this question, we first measured the perceptual similarity of letters in two behavioral tasks, visual search and letter categorization. Then, we trained deep convolutional neural networks on either 26-way letter categorization or 1000-way object categorization, as a way to operationalize possible specialized letter features and general object-based features, respectively. We found that the general object-based features more robustly correlated with the perceptual similarity of letters. We then operationalized additional forms of experience-dependent letter specialization by altering object-trained networks with varied forms of letter training; however, none of these forms of letter specialization improved the match to human behavior. Thus, our findings reveal that it is not necessary to appeal to specialized letter representations to account for perceptual similarity of letters. Instead, we argue that it is more likely that the perception of letters depends on domain-general visual features.

AUTHOR SUMMARYIn literate societies, the ability to visually recognize letters is a highly important skill. For over a century, scientists have conducted behavioral experiments to investigate how the visual system recognizes letters, but it has proven difficult to propose a model of the feature space underlying this capacity. Here we leveraged recent advances in machine learning to model a wide variety of features ranging from specialized letter features to general object-based features. Across two large-scale behavioral experiments we find that general object-based features account well for letter perception, and that adding letter specialization did not improve the correspondence to human behavior. It is plausible that the ability to recognize letters largely relies on general visual features unaltered by letter learning.

sentences: CuAAC stabilization of an NMR mixed labeled dimer Homo dimers are the most abundant type of enzyme in cells and as such, they represent the archetypal system for studying the remarkable phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on the similar event at the second site, the most informative state is the asymmetric single bound (lig1) form, yet it tends to be elusive thermodynamically. Here we take significant steps towards obtaining milligram quantities of pure lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins, and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of pure, fully intact, and active mixed labeled dimer. These data not only will make possible NMR-based investigations of allostery envisioned by us, but should also facilitate other structural applications where specific linkage of proteins is helpful.

sentences: Cephalopod Retinal Development Shows Vertebrate-like Mechanisms of Neurogenesis Neurogenesis, the regulation of cellular proliferation and differentiation in the developing nervous system, is the process that underlies the diversity of size and cell type found in animal nervous systems. Our understanding of how this process has evolved is limited because of the lack of high resolution data and live-imaging methods across species. The retina is a classic model for the study of neurogenesis in vertebrates and live-imaging of the retina has shown that during development, progenitor cells are organized in a pseudostratified neuroepithelium and nuclei migrate in coordination with the cell cycle along the apicobasal axis of the cell, a process called interkinetic nuclear migration. Eventually cells delaminate and differentiate within the boundaries of the epithelium. This process has been considered unique to vertebrates and thought to be important in maintaining organization during the development of a complex nervous system. Coleoid cephalopods, including squid, cuttlefish and octopus, have the largest nervous system of any invertebrate and convergently-evolved camera-type eyes, making them a compelling comparative system to vertebrates. Here we have pioneered live-imaging techniques to show that the squid, Doryteuthis pealeii, displays cellular mechanisms during cephalopod retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo interkinetic nuclear migration until they exit the cell cycle, we identify retinal organization corresponding to progenitor, post-mitotic and differentiated cells, and we find that Notch signaling regulates this process. With cephalopods and vertebrates having diverged 550 million years ago, these results suggest that mechanisms thought to be unique to vertebrates may be common to highly proliferative neurogenic primordia contributing to a large nervous system.

sentences: Kinetic principles underlying pioneer function of GAGA transcription factor in live cells How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that the majority of GAF is chromatin-bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for over 2 minutes, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAFs DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAFs neighbors such as HSF. Evaluation of GAF kinetics together with its endogenous abundance indicates that despite on-off dynamics, GAF constitutively and fully occupies chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental, and developmental signals.

sentences: Subspace based Multiple Constrained Minimum Variance (SMCMV) Beamformers In MEG and EEG brain imaging research two popular approaches are often used for spatial localization of focal task- or stimuli-related brain activations. One is a so called MUSIC approach applied in the form of RAP or TRAP MUSIC algorithms. Another one is the beamformer approach, specifically multiple constrained minimum variance (MCMV) beamformer when dealing with significantly correlated activations. Either method is using its own source localizer functions. Considering simplicity, accuracy and computational efficiency both approaches have their advantages and disadvantages. In this study we introduce a novel set of so called Subspace MCMV (or SMCMV) beamformers whose localizer functions combine MUSIC and MCMV localizers. We show that in ideal situations where forward modeling, data recording and noise measurements are error-free, SMCMV localizers allow precise identification of n arbitrarily correlated sources irrespective to their strength in just n scans of the brain volume using RAP MUSIC type algorithm. We also demonstrate by extensive computer simulations that with respect to source localization errors and the total number of identified sources SMCMV outperforms both the TRAP MUSIC and MIA MCMV (which is the most accurate MCMV algorithm to our knowledge) in non-ideal practical situations, specifically when the noise covariance cannot be estimated precisely, signal to noise ratios are small, source correlations are significant and larger numbers of sources are involved.

sentences: Cerebellar lesions disrupt spatial and temporal visual attention. The current study represents the first comprehensive examination of spatial, temporal and sustained attention following cerebellar damage. Results indicated that, compared to controls, cerebellar damage resulted in a larger cueing effect at the longest SOA - possibly reflecting a slowed the onset of inhibition of return (IOR) during a reflexive covert attention task, and reduced the ability to detect successive targets during an attentional blink task. However, there was little evidence to support the notion that cerebellar damage disrupted voluntary covert attention or the sustained attention to response task (SART). Lesion overlay data and supplementary voxel-based lesion symptom mapping (VLSM) analyses indicated that impaired performance on the reflexive covert attention and attentional blink tasks were related to damage to Crus II of the left posterior cerebellum. In addition, subsequent analyses indicated our results are not due to either general motor impairments or to damage to the deep cerebellar nuclei. Collectively these data demonstrate, for the first time, that the same cerebellar regions may be involved in both spatial and temporal visual attention.

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Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis Succinate is a major focal point in mycobacterial metabolism and respiration, serving as both an intermediate of the TCA cycle and a direct electron donor for the respiratory chain. Mycobacterium tuberculosis encodes multiple enzymes predicted to be capable of catalyzing the oxidation of succinate to fumarate, including two different succinate dehydrogenases (Sdh1 and Sdh2) and a separate fumarate reductase (Frd) with possible bi-directional behavior. Previous attempts to investigate the essentiality of succinate oxidation in M. tuberculosis have relied on the use of single-gene deletion mutants, raising the possibility that the remaining enzymes could catalyze succinate oxidation in the absence of the other. To address this, we report on the use of mycobacterial CRISPR interference (CRISPRi) to construct single, double, and triple transcriptional knockdowns of sdhA1, sdhA2, and frdA in M. tuberculosis. We show that the simultaneous knockdown of sdhA1 + sdhA2 is required to prevent succinate oxidation and overcome the functional redundancy within these enzymes. Succinate oxidation was demonstrated to be essential for the optimal growth of M. tuberculosis, with the combined knockdown of sdhA1 + sdhA2 significantly impairing the activity of the respiratory chain and preventing growth on a range of carbon sources. Moreover, impaired succinate oxidation was shown to influence the activity of several antitubercular drugs against M. tuberculosis, including potentiating the activity of bioenergetic inhibitors and attenuating the activity of cell wall inhibitors. Together, these data provide fundamental insights into mycobacterial physiology, energy metabolism, and antimicrobial susceptibility.

ImportanceNew drugs are urgently required to combat the tuberculosis epidemic that claims 1.5 million lives annually. Inhibitors of mycobacterial energy metabolism have shown significant promise clinically; however, further advancing this nascent target space requires a more fundamental understanding of the respiratory enzymes and pathways used by Mycobacterium tuberculosis. Succinate is a major focal point in mycobacterial metabolism and respiration; yet the essentiality of succinate oxidation, and the consequences of inhibiting this process, are poorly defined. In this study, we demonstrate that impaired succinate oxidation prevents the optimal growth of M. tuberculosis on a range of carbon sources and significantly reduces the activity of the electron transport chain. Moreover, we show that impaired succinate oxidation both positively and negatively influences the activity of a variety of anti-tuberculosis drugs. Combined, these findings provide fundamental insights into mycobacterial physiology and drug susceptibility that will be useful in the continued development of bioenergetic inhibitors.

sentences: A chromosome-level, fully phased genome assembly of the oat crown rust fungus Puccinia coronata f. sp. avenae: a resource to enable comparative genomics in the cereal rusts Advances in sequencing technologies as well as development of algorithms and workflows have made it possible to generate fully phased genome references for organisms with non-haploid genomes such as dikaryotic rust fungi. To enable discovery of pathogen effectors and further our understanding of virulence evolution, we generated a chromosome-scale assembly for each of the two nuclear genomes of the oat crown rust pathogen, Puccinia coronata f. sp. avenae (Pca). This resource complements two previous released partially phased genome references of Pca, which display virulence traits absent in the isolate of historic race 203 (isolate Pca203) which was selected for this genome project. A fully phased, chromosome-level reference for Pca203 was generated using PacBio reads and Hi-C data and a recently developed pipeline named NuclearPhaser for phase assignment of contigs and phase switch correction. With 18 chromosomes in each haplotype and a total size of 208.10 Mbp, Pca203 has the same number of chromosomes as other cereal rust fungi such as Puccinia graminis f. sp. tritici and Puccinia triticina, the causal agents of wheat stem rust and wheat leaf rust, respectively. The Pca203 reference marks the third fully-phased chromosome-level assembly of a cereal rust to date. Here, we demonstrate that the chromosomes of these three Puccinia species are syntenous and that chromosomal size variations are primarily due to differences in repeat element content.

sentences: Neurodevelopmental Patterns of Early Postnatal White Matter Maturation Represent Distinct Underlying Microstructure and Histology During the early postnatal period, cerebral white matter undergoes rapid maturation through a complex series of interrelated cellular and histogenetic processes. Accurately quantifying these processes is important for improving understanding of early brain development, developmental abnormalities related to prematurity, and neurodevelopmental diseases. Past efforts have used magnetic resonance imaging (MRI) to track these developmental processes in vivo. However, most previous studies have relied on single imaging modality data and have often been limited by small samples and analytics that do not evaluate complex multivariate imaging patterns. Here, we applied an advanced unsupervised multivariate pattern analysis technique, non-negative matrix factorization (NMF), to T2w/T1w signal ratio maps from a large cohort of newborns (Developing Human Connectome Project [dHCP], n=342), revealing patterns of synchronous white matter maturation. These patterns showed divergent age-related maturational trajectories and differential susceptibility to premature birth, which were replicated in an independent large sample of newborns (Early Life Adversity, Biological Embedding, and Risk for Developmental Precursors of Mental Disorders [eLABE], n=239). Furthermore, we showed that T2w/T1w signal variations in white matter maturational patterns are explained by differential contributions of white matter microstructure indices (i.e., free water content and neurite density index) derived from neurite orientation dispersion and density imaging (NODDI) modeling of diffusion-weighted MRI. Finally, we demonstrated how white matter maturation patterns relate to distinct histological features by comparing our findings with postmortem late fetal/early postnatal brain tissue staining. Together, these results delineate a novel MRI representation of white matter microstructural and histological reorganization during the early postnatal development.

sentences: The ASCC2 CUE domain contacts adjacent ubiquitins to recognize K63-linked polyubiquitin Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the ALKBH3-ASCC repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains using its CUE domain, a type of ubiquitin-binding domain that typically binds monoubiquitin and does not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. Whereas the ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, the contacts with the proximal ubiquitin are unique to ASCC2. The N-terminal portion of the ASCC2 1 helix, including residues E467 and S470, contributes to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response.

sentences: Deep protein representations enable recombinant protein expression prediction A crucial process in the production of industrial enzymes is recombinant gene expression, which aims to induce enzyme overexpression of the genes in a host microbe. Current approaches for securing overexpression rely on molecular tools such as adjusting the recombinant expression vector, adjusting cultivation conditions, or performing codon optimizations. However, such strategies are time-consuming, and an alternative strategy would be to select genes for better compatibility with the recombinant host. Several methods for predicting soluble expression are available; however, they are all optimized for the expression host Escherichia coli and do not consider the possibility of an expressed protein not being soluble. We show that these tools are not suited for predicting expression potential in the industrially important host Bacillus subtilis. Instead, we build a B. subtilis-specific machine learning model for expressibility prediction. Given millions of unlabelled proteins and a small labeled dataset, we can successfully train such a predictive model. The unlabeled proteins provide a performance boost relative to using amino acid frequencies of the labeled proteins as input. On average, we obtain a modest performance of 0.64 area-under-the-curve (AUC) and 0.2 Matthews correlation coefficient (MCC). However, we find that this is sufficient for the prioritization of expression candidates for high-throughput studies. Moreover, the predicted class probabilities are correlated with expression levels. A number of features related to protein expression, including base frequencies and solubility, are captured by the model.

sentences: Drug repurposing based on a Quantum-Inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2 including vitamin B12 The COVID-19 pandemic has accelerated the need to identify new therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir (RDV), the only antiviral against SARS-CoV-2 currently approved for human use, using a quantum-inspired device. We modelled RDV and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. Lastly, we demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Our data reveal that BMS-986094 and different forms of vitamin B12 are effective at inhibiting replication of all these variants of SARS-CoV-2. While BMS-986094 can cause secondary effects in humans as established by phase II trials, these findings suggest that vitamin B12 deserves consideration as a SARS-CoV-2 antiviral, particularly given its extended use and lack of toxicity in humans, and its availability and affordability. Our screening method can be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

sentences: SARS-CoV-2 invades cognitive centers of the brain and induces Alzheimer's-like neuropathology Major cell entry factors of SARS-CoV-2 are present in neurons; however, the neurotropism of SARS-CoV-2 and the phenotypes of infected neurons are still unclear. Acute neurological disorders occur in many patients, and one-third of COVID-19 survivors suffer from "brain diseases". Here, we show that SARS-CoV-2 invades the brains of five patients with COVID-19 and Alzheimers, autism, frontotemporal dementia or no underlying condition by infecting neurons and other cells in the cortex. SARS-CoV-2 induces or enhances Alzheimers-like neuropathology with manifestations of {beta}-amyloid aggregation and plaque formation, tauopathy, neuroinflammation and cell death. SARS-CoV-2 infects mature but not immature neurons derived from inducible pluripotent stem cells from healthy and Alzheimers individuals through its receptor ACE2 and facilitator neuropilin-1. SARS-CoV-2 triggers Alzheimers-like gene programs in healthy neurons and exacerbates Alzheimers neuropathology. A gene signature defined as an Alzheimers infectious etiology is identified through SARS-CoV-2 infection, and silencing the top three downregulated genes in human primary neurons recapitulates the neurodegenerative phenotypes of SARS-CoV-2. Thus, SARS-CoV-2 invades the brain and activates an Alzheimers-like program.

sentences: The dynamics of decision making and action during active sampling Embodied Cognition Theories (ECTs) propose that the decision process continues to unfold during the execution of choice actions, and its outcome manifests itself in these actions. Scenarios where actions not only express choice but also help sample information can provide a valuable test of this framework. Remarkably almost no studies so far have addressed this scenario. Here, we present a study testing just this paradigmatic situation with humans. On each trial, subjects categorized a central object image, blurred to different extents (2AFC task) by moving a cursor toward the left or right of the display. Upward cursor movements, orthogonal with respect to choice options, reduced the image blur and could be freely used to actively sample information. Thus, actions for decision and actions for sampling were made orthogonal to each other. We analyzed response trajectories to test a central prediction of ECTs; whether information-sampling movements co-occurred with the ongoing decision process. Trajectory data revealed were bimodally distributed, with one kind being direct towards one response option (non-sampling trials), and the other kind containing an initial upward component before veering off towards an option (sampling trials). This implies that there was an initial decision at the early stage of a trial whether to sample information or not. Importantly, the trajectories in sampling trials were not purely upward, but rather had a significant horizontal deviation that was visible early on in the movement. This result suggests that movements to sample information exhibit an online interaction with the decision process. The finding that decision processes interact with actions to sample information supports the ECT under novel, ecologically relevant constrains.

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Discovering differential genome sequence activity with interpretable and efficient deep learning Discovering sequence features that differentially direct cells to alternate fates is key to understanding both cellular development and the consequences of disease related mutations. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two black-box methods that can interpret genome regulatory sequences for cell type-specific or condition specific patterns. We show that these methods identify relevant transcription factor motifs and spacings that are predictive of cell state-specific chromatin accessibility. Finally, we integrate these methods into framework that is readily accessible to non-experts and available for download as a binary or installed via PyPI or bioconda at https://cgs.csail.mit.edu/deepaccess-package/.

Author SummaryWithin the genome are the instructions to build all the cell types that make up the human body. However, understanding these instructions and how and when these instructions go wrong in cancer or genetically inherited disease is an open problem. Deep neural networks provide powerful models to learn the relationship between DNA sequence and functional consequence across many different cell types, such as whether a particular stretch of DNA is accessible and genes in that region can be expressed or is inaccessible and therefore genes are inactive. Despite these advances, a major setback in deep learning is that it is challenging to understand what patterns of DNA sequences a deep learning model has learned to associate with a particular genomic function, whether these patterns are significant, and how to determine whether these patterns are specific to a particular cell type or are general "housekeeping" patterns that function across many cell types. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two methods which allow us to evaluate the significance of particular patterns of DNA sequence features on models trained to predict function across multiple cell types, and apply this to problems of transcription factor binding and DNA accessibility across multiple cell types.

sentences: Run-on sequencing reveals nascent transcriptomics of the human microbiome Precise regulation of transcription initiation and elongation enables bacteria to control cellular responses to environmental stimuli. RNAseq is the most common tool for measuring the transcriptional output of bacteria, comprising predominantly mature transcripts. To gain further insight into transcriptional dynamics, it is necessary to discriminate actively transcribed loci from those represented in the total RNA pool. One solution is to capture RNA polymerase (RNAP) in the act of transcription, but current methods are restricted to culturable and genetically tractable organisms. Here, we apply precision run-on sequencing (PRO-seq) to profile nascent transcription, a method amenable to diverse species. We find that PRO-seq is well-suited to profile small, structured, or post-transcriptionally modified RNAs, which are often excluded from RNAseq libraries. When PRO-seq is applied to the human microbiome, we identify taxon-specific RNAP pause motifs. We also uncover concurrent transcription and cleavage of guide RNAs and tRNA fragments at active CRISPR and tRNA loci. We demonstrate the specific utility of PRO-seq as a tool for exploring transcriptional dynamics in diverse microbial communities.

sentences: AliSim: A Fast and Versatile Phylogenetic Sequence Simulator For the Genomic Era Sequence simulators play an important role in phylogenetics. Simulated data has many applications, such as evaluating the performance of different methods, hypothesis testing with parametric bootstraps, and, more recently, generating data for training machine-learning applications. Many sequence simulation programs exist, but the most feature-rich programs tend to be rather slow, and the fastest programs tend to be feature-poor. Here, we introduce AliSim, a new tool that can efficiently simulate biologically realistic alignments under a large range of complex evolutionary models. To achieve high performance across a wide range of simulation conditions, AliSim implements an adaptive approach that combines the commonly-used rate matrix and probability matrix approach. AliSim takes 1.3 hours and 1.3 GB RAM to simulate alignments with one million sequences or sites, while popular software Seq-Gen, Dawg, and INDELible require two to five hours and 50 to 500 GB of RAM. We provide AliSim as an extension of the IQ-TREE software version 2.2, freely available at www.iqtree.org, and a comprehensive user tutorial at http://www.iqtree.org/doc/AliSim.

sentences: The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. Here we identified the long noncoding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to ABA. MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel noncoding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

sentences: Development of an open source program to identify the size of objects using bright-field microscopy - an automated calibration tool In light microscopy, eyepiece graticules are commonly used to gauge the size of objects at the micron scale. While this is a relatively simple tool to use, not all microscopes possess this feature. Furthermore, calibrating an eyepiece graticule with a stage micrometer can be time-consuming, particularly for inexperienced microscopists. Similarly, calculating the size of individual objects may also take some time. We present an open-source program to determine the size of objects under a microscope using Python and OpenCV. Taking photos of a stage micrometer under a microscope, we identify gradations on the micrometer and calculate the distance between lines on the micrometer in pixels. From this, we can infer the size of objects from bright-field microscopy images. We believe this will improve access to quantitative microscopy techniques and increase the speed at which samples may be analyzed by light microscopy. Future studies may aim to integrate this with machine learning for object identification.

sentences: Effective drug release from safe ultrasound-triggered nanocarriers Selective delivery of medication into specified tissue targets would realize the promise of personalized medicine with minimal side effects. Focused ultrasound provides noninvasive and practical means to release drugs from nanocarriers selectively at its target. However, which nanocarrier formulations provide safe and effective release and under which ultrasound parameters has been unclear. To expedite regulatory approval, we tested release effectiveness from nanocarriers containing perfluorocarbon cores of relatively high boiling points (up to 142{degrees}C) which show excellent stability compared with those used previously. We confirmed the safety of these nanocarriers and characterized their clearance kinetics and organ biodegradation in non-human primates. Crucially, we found that these safe, high-boiling-point nanocarriers can be used for effective release so long as they are activated by low-frequency ultrasound (300 kHz). This study informs the formulation and release parameters for safe and effective drug delivery in specific parts of the body or brain regions.

sentences: Molecular determinants of phase separation for Drosophila DNA replication licensing factors Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificity a priori.

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Wnt1-Cre mediated deletion of BMP7 suggests a role for neural crest-derived BMP7 in retina development and function Neural crest (NC) contributes to various structures of the eye including cornea, ciliary body and retina. The association of NC-derived cells with hyaloid vessels in the form of pericytes is established. Similarly, persistence of NC-derived cells in the inner retina layer of the mature retina has been suggested. To date, no specific function has been attributed to them. NC-derived Bone morphogenetic protein 7 (BMP7) controls neurogenic properties in the brain and regulates glia differentiation. Here, we assessed the role of NC-derived BMP7 in the adult retina.

BMP7 expression was determined using Bmp7LacZ reporter mice. BMP7 was expressed in GCL, IPL, OPL, and photoreceptors in P0, P14 and P30 retinas. Lineage tracing confirmed the presence of NC-derived cells in the GCL, INL, and ONL. Some but not all cells associated with vasculature. To test the function of NC-derived Bmp7, Bmp7fl/flWnt1cre (Bmp7ncko) mice were assessed by histological and functional methods. Loss of NC-derived cells in the GCL and INL and mild structural abnormalities were observed in the Bmp7ncko retina. Electroretinography revealed reduced a wave under photopic conditions and b wave under both scotopic and photopic conditions. The neuronal circuitry in the inner retina appeared affected, evidenced by decreased Calbindin in the GCL, IPL and INL. In the outer retina, S-opsin was increased. BMP7 expression in the mutant retina was strongly decreased at birth, but increased expression from cells other than NC was observed in the adult retina. This was associated with an increase in IBA1, suggestive that loss of NC-derived BMP7 predisposes to development of gliosis-like changes in the adult retina. Overall, our data reveal an important contribution of NC-derived BMP7 for the development and function of the inner and outer retina.

sentences: Viability of AMURA biomarkers from single-shell diffusion MRI in Migraine Clinical Studies Diffusion Tensor Imaging (DTI) is the most employed method to assess white matter properties using quantitative parameters derived from diffusion MRI, but it presents known limitations that restricts the evaluation of complex structures. The objective of this study was the assessment of complementary diffusion measures extracted with a novel approach, Apparent Measures Using Reduced Acquisitions (AMURA), in comparison with DTI in clinical studies. Fifty healthy controls, 51 episodic migraine and 56 chronic migraine patients underwent single-shell diffusion MRI. Three DTI-based and eight AMURA-based parameters were compared between groups with tract-based spatial statistics. On the other hand, following a region-based analysis, the measures were assessed for multiple subsamples with diverse reduced sample sizes and their stability was evaluated with the coefficient of quartile variation. Compared to DTI, statistically significant differences in additional regions and group comparisons were found with AMURA-based measures. Additionally, some AMURA-based measures showed a remarkable discriminative power for quite reduced sample size and, simultaneously, lower stability (higher coefficient of quartile variation) than DTI-based measures. These findings suggest that AMURA presents favorable characteristics to identify differences of specific microstructural properties between clinical groups in regions with complex fiber architecture and lower dependency on the sample size or assessing technique than DTI.

sentences: Systemic infection with highly pathogenic H5N8 of avian origin produces encephalitis and mortality in wild mammals at a UK rehabilitation centre Europe has experienced extensive outbreaks of highly pathogenic avian influenza (HPAI) during the autumn/winter 2020/21 season. These avian influenza A viruses are highly transmissible and have infected over 1000 commercial and backyard poultry premises in Europe in this period causing high mortality. The impact on wild bird populations has also been significant, with over 400 detections in at least 47 different species reported across Europe as being positive with the H5N8 virus. Although different H5Nx combinations within the H5 clade 2.3.4.4b have been detected, the H5N8 subtype has predominated both in wild birds and domestic poultry outbreaks. In the UK there have been 22 outbreaks of H5N8 in domestic poultry and captive birds and more than 300 wild bird detections involving H5N8 over the autumn/winter 2020/21 period to April 2021. Here we detail the series of events surrounding the detection of an H5N8 influenza A virus of avian origin in five swans, a fox and three seals in a wildlife rehabilitation centre.

sentences: Analysis of migration of pathogenic drug-resistant bacteria to soils and groundwater after fertilization with sewage sludge The paper discusses the analysis of the effect of using sewage sludge for fertilization on the level of soil and groundwater contamination with drug-resistant bacteria. Other sanitary contaminants in these environments were also analysed. Composted sewage sludge was introduced into the sandy soil over a period of 6 months. The examinations were conducted under conditions of a lysimetric experiment with the possibility of collecting soil leachates (in natural conditions). The following doses of sewage sludge were used: 0, 10, 20, 30 and 40 t/ha calculated per experimental object containing 10 kg of sandy soil. The research were carried out within the time frame of one year. Dactylis glomerata grass was grown on the fertilized soils. In soils and leachates from soils (which may have polluted groundwater) collected from fertilized experimental objects, the sanitary condition and quantity of drug-resistant bacteria (mainly from the families Enterobacteriaceae and Enterococcus) were analysed one year after fertilization. Their drug resistance to selected antibiotics was also analysed based on current recommendations.

sentences: The Resting-State Causal Human Connectome is Characterized by Hub Connectivity of Executive and Attentional Networks We demonstrate a data-driven approach for calculating a "causal connectome" of directed connectivity from resting-state fMRI data using a greedy adjacency search and pairwise non-Gaussian edge orientations. We used this approach to construct n=442 causal connectomes. These connectomes were very sparse in comparison to typical Pearson correlation-based graphs (roughly 2.25% edge density) yet were fully connected in nearly all cases. Prominent highly connected hubs of the causal connectome were situated in attentional (dorsal attention) and executive (frontoparietal and cingulo-opercular) networks. These hub networks had distinctly different connectivity profiles: attentional networks shared incoming connections with sensory regions and outgoing connections with higher cognitive networks, while executive networks primarily connected to other higher cognitive networks and had a high degree of bidirected connectivity. Virtual lesion analyses accentuated these findings, demonstrating that attentional and executive hub networks are points of critical vulnerability in the human causal connectome. These data highlight the central role of attention and executive control networks in the human cortical connectome and set the stage for future applications of data-driven causal connectivity analysis in psychiatry.

sentences: Protein Structure Prediction Using a Maximum Likelihood Formulation of a Recurrent Geometric Network Only [~]40% of the human proteome has structural coordinates available from experiment (i.e., X-ray crystallography, NMR spectroscopy, or cryo-EM) or homology modeling with quality templates (i.e., 30% sequence identity or greater), leaving most of the proteome structurally unsolved. Deep learning (DL) methods for predicting protein structure can help close knowledge gaps where experimental and homology models are difficult to obtain. Recent advances in these DL methods have shown promising results in expanding structural coverage to the scale of the entire human proteome, providing researchers with more complete protein structural information. Here, we improve upon an existing DL algorithm for protein structure prediction, the Recurrent Geometric Network (RGN). We first expand the training dataset to include experimental uncertainty data in the form of atomic displacement parameters, then derive a maximum likelihood loss function that incorporates this uncertainty data into model training. Compared to the original RGN, our novel maximum likelihood model improves the rate of convergence of initial model training and ultimately results in more accurate structure prediction according to the root mean square deviation (RMSD) of backbone atoms, the Global Distance Test (GDT), the Global Distance Test High Accuracy (GDT-HA), and the Template-Modeling Score (TM-Score). Our model also predicts structures with more favorable backbone torsions, which provide more accurate starting coordinates for downstream physics-based simulations. Based on these results, our maximum likelihood reformulation provides a framework for improving existing or future machine learning algorithms for protein structure prediction. The augmented dataset, data collection scripts, reformulated RGN source code, and a series of trained models are publicly available at https://github.com/SchniedersLab/likelihood-rgn.

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APOE4 genotype increases neuronal calcium signals and decreases pial arteriole responsivity and vasomotion in visual cortex of awake mice Vascular dysfunction is an early feature of late onset Alzheimers disease (AD), preceding classic AD pathology such as beta amyloid accumulation and formation of hyperphosphorylated tau. Such vascular dysfunction may promote classic AD pathology by decreasing blood flow, impairing brain oxygenation and clearance of molecules such as beta amyloid. The main genetic risk factor for AD is the {varepsilon}4 allele of APOE, which has been found to increase blood brain barrier permeability and decrease vascular density, as well as decrease blood flow and functional hyperaemia in anaesthetised mice undergoing acute surgery. These results suggest that APOE4 may confer AD risk via its effects on the vasculature. However, the responses of neurons and individual vessels have not been studied, so neurovascular relationships are unknown, and no previous studies have looked at awake mice.

We therefore measured neurovascular responses at rest and in response to visual stimulation using 2 photon imaging of awake APOE3 and APOE4 targeted-replacement (APOE TR) mice that expressed the calcium indicator GCaMP6f in excitatory neurons, while labelling the vascular lumen with Texas Red dextran. In parallel, we measured cerebral blood flow, blood oxygenation and cerebral blood volume using combined laser Doppler flowmetry and haemoglobin spectrometry. Measurements were performed in mice aged between 3-4 months to 12-13 months.

We found a milder vascular deficit in awake mice than previous studies that used an acute surgical preparation: capillary responses to visual stimulation were the same in APOE3 and APOE4 TR mice, leading to unimpaired functional hyperaemia. However, neuronal calcium signals during visual stimulation were significantly enhanced in APOE4 mice, while there was a marked decrease in pial arteriole responsiveness and vasomotion. This pattern of results was unaffected by age, suggesting that APOE4 expression creates a stable, but mildly altered neurovascular state that does not itself cause degeneration. However, these changes likely make the system more sensitive to subsequent insults; for example, weaker vasomotion could impair clearance of beta amyloid as it starts to accumulate, and therefore may help explain how APOE4 expression increases risk of developing AD.

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METAbolomics data Balancing with Over-sampling Al-gorithms (META-BOA): an online resource for addressing class imbalance MotivationClass imbalance, or unequal sample sizes between classes, is an increasing concern in machine learning for metabolomic and lipidomic data mining, which can result in overfitting for the over-represented class. Numerous methods have been developed for handling class imbalance, but they are not readily accessible to users with limited computational experience. Moreover, there is no resource that enables users to easily evaluate the effect of different over-sampling algorithms.

ResultsMETAbolomics data Balancing with Over-sampling Algorithms (META-BOA) is a web-based application that enables users to select between four different methods for class balancing, followed by data visualization and classification of the sample to observe the augmentation effects. META-BOA outputs a newly balanced dataset, generating additional samples in the minority class, according to the users choice of Synthetic Minority Over-sampling Technique (SMOTE), Borderline-SMOTE (BSMOTE), Adaptive Synthetic (ADASYN), or Random Over-Sampling Examples (ROSE). META-BOA further displays both principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) visualization of data pre- and post-over-sampling. Random forest classification is utilized to compare sample classification in both the original and balanced datasets, enabling users to select the most appropriate method for their analyses.

Availability and implementationMETA-BOA is available at https://complimet.ca/meta-boa.

Supplementary InformationSupplementary material is available at Bioinformatics online.

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Microsecond simulation unravel the structural dynamics of SARS-CoV-2 Spike-C-terminal cytoplasmic tail (residues 1242-1273) All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions. Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region. This curiosity has led us to investigate the C terminal cytosolic region of the spike glycoprotein of SARS-CoV-2 in isolation. The cytosolic regions is supposed to be from 1235-1273 residues or 1242-1273 residues depending on what prediction tool we use. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Also, in temperature dependent replica-exchange molecular dynamics simulations it has shown its conformational dynamics. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm which is also adding the analysis as its intrinsically disordered nature. We believe that our findings will surely help us understand the structure-function relationship of the spike proteins cytosolic region.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Detection of allele-specific expression in spatial transcriptomics with spASE Allele-specific expression (ASE), or the preferential expression of one allele, can be observed in transcriptomics data from early development throughout the lifespan. However, the prevalence of spatial and cell type-specific ASE variation remains unclear. Spatial transcriptomics technologies permit the study of spatial ASE patterns genome-wide at near-single-cell resolution. However, the data are highly sparse, and confounding between cell type and spatial location present further statistical challenges. Here, we introduce spASE (https://github.com/lulizou/spase), a computational framework for detecting spatial patterns in ASE within and across cell types from spatial transcriptomics data. To tackle the challenge presented by the low signal to noise ratio due to the sparsity of the data, we implement a spatial smoothing approach that greatly improves statistical power. We generated Slide-seqV2 data from the mouse hippocampus and detected ASE in X-chromosome genes, both within and across cell type, validating our ability to recover known ASE patterns. We demonstrate that our method can also identify cell type-specific effects, which we find can explain the majority of the spatial signal for autosomal genes. The findings facilitated by our method provide new insight into the uncharacterized landscape of spatial and cell type-specific ASE in the mouse hippocampus.

sentences: Recognition and deubiquitination of free 40S for translational reset by Otu2 In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

sentences: Molecular Response to PARP1 Inhibition in Ovarian Cancer Cells as Determined by Mass Spectrometry Based Proteomics Poly (ADP)-ribose polymerase (PARP) inhibitors have entered routine clinical practice for the treatment of high-grade serous ovarian cancer (HGSOC), yet the molecular mechanisms underlying treatment response to PARP1 inhibition (PARP1i) are not fully understood. Here, we used unbiased mass spectrometry based proteomics with data-driven protein network analysis to systematically characterize how HGSOC cells respond to PARP1i treatment. We found that PARP1i leads to pronounced proteomic changes in a diverse set of cellular processes in HGSOC cancer cells, consistent with transcript changes in an independent perturbation dataset. We interpret decreases in the levels of the pro-proliferative transcription factors SP1 and {beta}-catenin and in growth factor signaling as reflecting the anti-proliferative effect of PARP1i; and the strong activation of pro-survival processes NF-{varkappa}B signaling and lipid metabolism as PARPi-induced adaptive resistance mechanisms. Based on these observations, we nominate several protein targets for therapeutic inhibition in combination with PARP1i. When tested experimentally, the combination of PARPi with an inhibitor of fatty acid synthase (TVB-2640) has a 3-fold synergistic effect and is therefore of particular pre-clinical interest. Our study improves the current understanding of PARP1 function, highlights the potential that the anti-tumor efficacy of PARP1i may not only rely on DNA damage repair mechanisms and informs on the rational design of PARP1i combination therapies in ovarian cancer.

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Acetylcholine boosts dendritic NMDA spikes in a CA3 pyramidal neuron model Acetylcholine has been proposed to facilitate the formation of memory ensembles within the hippocampal CA3 network, by enhancing plasticity at CA3-CA3 recurrent synapses. Regenerative NMDA receptor (NMDAR) activation in CA3 neuron dendrites (NMDA spikes) increase synaptic Ca2+ influx and can trigger this synaptic plasticity. Acetylcholine inhibits potassium channels which enhances dendritic excitability and therefore could facilitate NMDA spike generation. Here, we investigate NMDAR-mediated nonlinear synaptic integration in stratum radiatum (SR) and stratum lacunosum moleculare (SLM) dendrites in a reconstructed CA3 neuron computational model and study the effect of acetylcholine on this nonlinearity. We found that distal SLM dendrites, with a higher input resistance, had a lower threshold for NMDA spike generation compared to SR dendrites. Simulating acetylcholine by blocking potassium channels (M-type, A-type, Ca2+-activated, and inwardly-rectifying) increased dendritic excitability and reduced the number of synapses required to generate NMDA spikes, particularly in the SR dendrites. The magnitude of this effect was heterogeneous across different dendritic branches within the same neuron. These results predict that acetylcholine facilitates dendritic integration and NMDA spike generation in selected CA3 dendrites which could strengthen connections between specific CA3 neurons to form memory ensembles.

Highlights- Using biophysical computational models of CA3 pyramidal neurons we estimated the quantitative effects of acetylcholine on nonlinear synaptic integration.
- Nonlinear NMDA spikes can be triggered by fewer synapses in distal dendrites due to increased local input resistance.
- Acetylcholine broadly reduces the number of synapses needed to trigger NMDA spikes, but the magnitude of the effect varies across dendrite branches within a single neuron.
- No single potassium channel type is the dominant mediator of the excitability effects of acetylcholine.

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The smooth transition from many-legged to bipedal locomotion: Gradual ground force reduction and its impact on total ground reaction forces, body dynamics and gait transitions Most terrestrial animals move with a specific number of propulsive legs, which differs between clades. The reasons for these differences are often unknown and rarely queried, despite the underlying mechanisms being indispensable for understanding the evolution of multilegged locomotor systems in the animal kingdom and the development of swiftly moving robots. Moreover, when speeding up, a range of species change their number of propulsive legs. The reasons for this behaviour have proven equally elusive. In animals and robots, the number of propulsive legs also has a decisive impact on the movement dynamics of the centre of mass. Here, I use the leg force interference model to elucidate these issues by introducing gradually declining ground reaction forces in locomotor apparatuses with varying numbers of leg pairs in a first numeric approach dealing with these measures impact on locomotion dynamics. The effects caused by the examined changes in ground reaction forces and timing thereof follow a continuum. However, the transition from quadrupedal to a bipedal locomotor system deviates from those between multilegged systems with different numbers of leg pairs. Only in quadrupeds do reduced ground reaction forces beneath one leg pair result in increased reliability of vertical body oscillations and therefore increased energy efficiency and dynamic stability of locomotion.

Significance statementThe model grants access to the effects of gradual ground force reduction on total ground reaction forces, body dynamics and gait transitions.

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Cerebrospinal fluid amyloid beta and glial fibrillary acidic protein concentrations in Huntingtons disease IntroductionHuntingtons disease (HD) is a genetic incurable lethal disease. Biomarkers are needed for objective assessment of disease progression. Evidence supports both complex protein aggregation and astrocyte activation in HD. This study assesses the 42 amino acid long amyloid beta (A{beta}42) and glial fibrillary acidic protein (GFAP) as potential biomarkers in the cerebrospinal fluid (CSF) of HD mutation carriers.

MethodsCSF was obtained from manifest HD patients (ManHD), premanifest HD-gene-expansion carriers (PreHD) and gene-negative controls (controls). Disease Burden Score (DBS) and Total Functional Capacity (TFC) were calculated. Protein concentrations were measured by enzyme-linked immunosorbent assays (ELISA) and intergroup differences were analysed using Mann-Whitney U test. Spearman correlations were calculated to assess disease stage association. Age-adjustment was included in the statistical tests.

ResultsThe study enrolled 27 ManHD and 13 PreHD subjects. The number of controls differed in the analysis of A{beta}42 and GFAP (n = 19, and 8 respectively). A{beta}42 levels were higher in ManHD (mean 741 ng/l, SD 361) compared with PreHD (mean 468 ng/l, SD 184) (p = 0.025). Likewise GFAP concentration was higher in ManHD (mean 435 ng/l, SD 255) compared with both PreHD (mean 266 ng/l, SD 92.4)(p = 0.040) and controls (mean 208 ng/l, SD 83.7)(p = 0.011). GFAP correlated with DBS (r = 0.361, p = 0.028), TFC (r = - 0.463, p = 0.005), and 5-year risk of onset in PreHD (r = 0.694, p = 0.008). In contrast, there was no correlation between A{beta}42 concentration and DBS, TFC or 5-year risk of onset.

ConclusionCSF A{beta}42 levels did not correlate with disease stage suggesting no A{beta}aggregation in HD. GFAP is a potential biomarker in HD with association to disease stage. Validation in larger HD cohorts and potential correlations with clinical phenotype would be of interest.

sentences: Probing behavior of the corn leafhopper Dalbulus maidis on susceptible and resistant maize hybrids The corn leafhopper Dalbulus maidis is the main vector of the pathogens that cause corn stunt, a major disease of maize in the Americas. As host resistance is an efficient tool to control diseases, the findings of a previous report showed that some corn hybrids are resistant to D. maidis. In this work, we assessed the probing behavior of D. maidis on susceptible and resistant corn hybrids using EPG (Electrical Penetration Graph) technology. Fifteen-day-old females were monitored for 20 hours, with access to hybrids DK390, DK670, DK79-10, and DK72-10. Hybrids DK390 and DK72-10 showed resistance to D. maidis in phloem, since insects feeding on these hybrids presented more salivation events in phloem without subsequent ingestion, which are seen as failed attempts to ingest. A reduction of the total duration of phloem ingestion was observed, and accordingly of the time spent by insects with access to these hybrids on xylem ingestion. The hybrid DK390 also had mesophyll resistance, seen as less probing time and a higher number of probes of short duration. These findings support and are consistent with previous research, providing useful information to characterize maize hybrids resistant to D. maidis, and so to corn stunt.

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An analytical pipeline for DNA Methylation Array Biomarker Studies DNA methylation is one of the most commonly studied epigenetic biomarkers, due to its role in disease and development. The Illumina Infinium methylation arrays still remains the most common method to interrogate methylation across the human genome, due to its capabilities of screening over 480, 000 loci simultaneously. As such, initiatives such as The Cancer Genome Atlas (TCGA) have utilized this technology to examine the methylation profile of over 20,000 cancer samples. There is a growing body of methods for pre-processing, normalisation and analysis of array-based DNA methylation data. However, the shape and sampling distribution of probe-wise methylation that could influence the way data should be examined was rarely discussed. Therefore, this article introduces a pipeline that predicts the shape and distribution of normalised methylation patterns prior to selection of the most optimal inferential statistics screen for differential methylation. Additionally, we put forward an alternative pipeline, which employed feature selection, and demonstrate its ability to select for biomarkers with outstanding differences in methylation, which does not require the predetermination of the shape or distribution of the data of interest.

AvailabilityThe Distribution test and the feature selection pipelines are available for download at: https://github.com/uqjlu8/DistributionTest

sentences: SMCHD1 variants may induce variegated expression in Facio Scapulo Humeral Dystophy and Bosma Arhinia and microphtalmia syndrome An expanding number of genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Recently, distinct genetic syndromes have been linked to mutations in the SMCHD1 gene. However, the function of this non-canonical SMC protein remains partly defined in Human tissues. To address this question, we determined its epi-signature in type 2 Facio Scapulo Humeral Dystrophy (FSHD2) and Bosma Arhinia and Microphtalmia Syndrome (BAMS) linked to heterozygous mutations in this gene. By combining RNA-Seq, DNA methylation profiling and ChIP-Seq, we showed that SMCHD1 regulates repressed chromatin but also cis-regulatory elements and enhancers. Our results emphasize dual functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable outcomes and targets depending on tissues. We propose that altered DNA methylation and long-range chromatin organization at a number of loci required for development and tissue differentiation, trigger variegated gene expression in rare genetic diseases linked to heterozygous SMCHD1 mutations.

sentences: Subcellular proteomics of dopamine neurons in the mouse brain reveals axonal enrichment of proteins encoded by Parkinson's disease-linked genes Dopaminergic neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find a significant enrichment of proteins encoded by Parkinsons disease-linked genes in striatal dopaminergic axons, including proteins with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.

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Status of susceptibility of the visceral leishmaniasis vector, Phlebotomus argentipes (Diptera: Psychodidae: Phlebotominae), to insecticides used for vector control in Nepal BackgroundVisceral leishmaniasis (VL) is targeted for elimination as a public health problem in Nepal by 2023. For nearly three decades, the core vector control intervention in Nepal has been indoor residual spraying (IRS) with pyrethroids. Considering the long-term use of pyrethroids and possible development of resistance of the vector Phlebotomus argentipes sand flies, we monitored susceptibility status of their field populations to the insecticides of different classes, in villages with and without IRS activities in recent years.

Methodology/Principal findingsSand flies were collected from villages with and without IRS in five VL endemic districts from August 2019 to November 2020. The WHO susceptibility test procedure was adopted using filter papers impregnated at the discriminating concentrations of insecticides of the following classes: pyrethroids (alpha-cypermethrin 0.05%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%), carbamates (bendiocarb 0.1%) and organophosphates (malathion 5%). Pyrethroid resistance intensity bioassays with papers impregnated with 5x of the discriminating concentrations, piperonyl butoxide (PBO) synergist-pyrethroid bioassays and DDT cross resistance bioassays were also performed. In the IRS villages, the vector sand flies were resistant (mortality rate <90%) to alpha-cypermethrin and possibly resistant (mortality rate 90-97%) to deltamethrin and lambda-cyhalothrin, while susceptibility to these insecticides was variable in the non-IRS villages. The vector was fully susceptible to bendiocarb and malathion in all villages. A delayed knockdown time (KDT50) with pyrethroids was observed in all villages. The pyrethroid resistance intensity was low, and the susceptibility improved at 5x of the discriminating concentrations. Enhanced pyrethroid susceptibility after pre-exposure to PBO and the DDT-pyrethroid cross-resistance were evident.

Conclusions/SignificanceOur investigation showed that P. argentipes sand flies have emerged with pyrethroid resistance, suggesting the need to switch to alternative classes of insecticides such as organophosphates for IRS. We strongly recommend for the regular and systematic monitoring of insecticide resistance in sand flies to optimize the efficiency of vector control interventions to sustain VL elimination efforts in Nepal.

Author summaryVisceral leishmaniasis (VL), transmitted by P. argentipes sand flies, is endemic in South-East Asian countries such as Bangladesh, India and Nepal, and is on the verge of elimination as a public health problem in Nepal by 2023. As part of the WHO Global Vector Control Response, entomological surveillance including insecticide resistance monitoring is one of the four main pillars of this strategy. In the early 1990s, the historical use of DDT for sand fly vector control was replaced with deltamethrin or alpha-cypermethrin, which have now been in use for almost three decades in Nepal. Suspecting that this long-term use of pyrethroids might have selected resistance in sand fly populations which would jeopardize control efforts, we conducted the first comprehensive survey to generate contemporary evidence of insecticide resistance in Nepal. For this, we performed WHO susceptibility tests in five VL endemic districts and found strong evidence of pyrethroid resistance in vector populations from the areas receiving IRS. Resistance mechanisms involved would probably be kdr mutations and monooxygenase. This study also endorses regular insecticide resistance monitoring to inform evidence-based decisions on insecticide use for vector control and to maintain the effectiveness of vector control measures as a core intervention in the fight against VL.

sentences: Recut: a Concurrent Framework for Sparse Reconstruction of Neuronal Morphology Advancement in modern neuroscience is bottlenecked by neural reconstruction, a process that extracts 3D neuron morphology (typically in tree structures) from image volumes at the scale of hundreds of GBs. We introduce Recut, an automated and accelerated neural reconstruction pipeline, which provides a unified, and domain specific sparse data representation with 79x reduction in the memory footprint. Recuts reconstruction can process 111 Kneurons/day or 79 TB/day on a 24-core workstation, placing the throughput bottleneck back on microscopic imaging time. Recut allows the full brain of a mouse to be processed in memory on a single server, at 89.5x higher throughput over existing I/O-bounded methods. Recut is also the first fully parallelized end-to-end automated reconstruction pipeline for light microscopy, yielding tree morphologies closer to ground truth than the state-of-the-art while removing involved manual steps and disk I/O overheads. We also optimized pipeline stages to linear algorithmic complexity for scalability in dense settings and allow the most timing-critical stages to optionally run on accelerated hardware.

sentences: Single-molecule imaging reveals Tau trapping at nanometer-sized dynamic hot spots near the plasma membrane Accumulation of Tau aggregates is a pathological hallmark of Alzheimers disease, but little is known about where these aggregates assemble and how stable these assemblies are. Using quantitative, single-molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane, resulting in the formation of nanometer-sized hot spots smaller than the diffraction limit. The hot spots lasted tens of seconds, much longer than the short dwell time (~40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not affect hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.

sentences: Pathway dynamics can delineate the sources of transcriptional noise in gene expression Single-cell expression profiling has opened up new vistas on cellular processes. Among other important results, one stand-out observation has been the confirmation of extensive cell-to-cell variability at the transcriptomic and proteomic level. Because most experimental analyses are destructive we only have access to snapshot data of cellular states. This loss of temporal information presents significant challenges in inferring dynamics, as well as causes of cell-to-cell variability. In particular, we are typically unable to separate dynamic variability from within individual systems ("intrinsic noise") from variability across the population ("extrinsic noise"). Here we mathematically formalise this non-identifiability; but we also use this to identify how new experimental set-ups coupled to statistical noise decomposition can resolve this non-identifiability. For single-cell transcriptomic data we find that systems subject to population variation invariably inflate the apparent degree of burstiness of the underlying process. Such identifiability problems can, in principle, be remedied by dual-reporter assays, which separates total gene expression noise into intrinsic and extrinsic contributions; unfortunately, however, this requires pairs of strictly independent and identical gene reporters to be integrated into the same cell, which is difficult to implement experimentally in most systems. Here we demonstrate mathematically that, in some cases decomposition of transcriptional noise is possible with non-identical and not-necessarily independent reporters. We use our result to show that generic reporters lying in the same biochemical pathways (e.g. mRNA and protein) can replace dual reporters, enabling the noise decomposition to be obtained from only a single gene. Stochastic simulations are used to support our theory, and show that our "pathway-reporter" method compares favourably to the dual-reporter method.

sentences: Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

sentences: Chronic kidney disease alters Pin1 phosphorylation and parathyroid hormone mRNA binding proteins leading to secondary hyperparathyroidism Parathyroid hormone (PTH) regulates calcium metabolism and bone strength. Chronic kidney disease leads to secondary hyperparathyroidism (SHP) which increases morbidity and mortality. In experimental SHP, the increased PTH gene expression is due to enhanced PTH mRNA stability, mediated by changes in its interaction with stabilizing AUF1 and destabilizing KSRP. Pin1 isomerizes target proteins and leads to KSRP dephosphorylation. In SHP, Pin1 isomerase activity is decreased and phosphorylated KSRP fails to bind PTH mRNA, resulting in high PTH mRNA stability and levels. However, the up- and down-stream mechanisms by which kidney failure stimulates the parathyroid remain elusive. We now reveal a pathway where kidney failure induces parathyroid Pin1 phosphorylation, linking Pin1, KSRP and PTH mRNA stability as key players for the high PTH expression in SHP. We identified by mass-spectrometry, changes in rat parathyroid proteome and phosphoproteome profiles induced by impaired renal function, including KSRP phosphorylation at Pin1 target sites. Furthermore, both acute and chronic kidney failure led to parathyroid-specific Pin1 Ser16 and Ser71 phosphorylation, which disrupts Pin1 activity. Accordingly, pharmacologic Pin1 inhibition, that mimics the decreased Pin1 activity in SHP, increased PTH expression ex-vivo in parathyroid organ cultures and in transfected cells, through the PTH mRNA protein-interacting element and KSRP phosphorylation at potential Pin1-binding motifs. Therefore, kidney failure leads to loss of parathyroid Pin1 activity by inducing Pin1 phosphorylation. This predisposes parathyroids to increase PTH production through impaired PTH mRNA decay that is dependent on KSRP phosphorylation. Pin1 and KSRP phosphorylation and the Pin1-KSRP-PTH mRNA axis thus drive SHP.

sentences: Sensory plasticity in a socially plastic bee The social Hymenoptera have contributed much to our understanding of the evolution of sensory systems. Attention has focussed chiefly on how sociality and sensory systems have evolved together. In the Hymenoptera, the antennal sensilla are important for optimising the perception of olfactory social information. Social species have denser antennal sensilla than solitary species, which is thought to enhance social cohesion through nest-mate recognition. In the current study, we test whether sensilla numbers vary between regional populations of the socially plastic sweat bee Halictus rubicundus. We found region level differences in both olfactory and hygro/thermoreceptive sensilla numbers. We also found evidence that olfactory sensilla density is developmentally plastic: when we transplanted bees from Scotland to the south-east of England, their offspring (which developed in the south) had more olfactory hairs than the transplanted individuals themselves (which developed in Scotland). The transplanted bees displayed a mix of social (a queen plus workers) and solitary nesting, but neither individual nor nest phenotype was related to sensilla density. We suggest that this general, rather than caste-specific sensory plasticity provides a flexible means to optimise sensory perception according to the most pressing demands of the environment, and may support social plasticity in H. rubicundus.

sentences: Spleen voltammetry assesses in real time norepinephrine release elicited by autonomic stimulation Bioelectronic medicine uses neurostimulation devices to modulate the activity of autonomic nerves and restore function in visceral organs. The autonomic, noradrenergic, innervation of the spleen is implicated in the neural control of inflammation; for that reason, the spleen is the target of autonomic stimulation therapies tested in rheumatoid arthritis and other inflammatory diseases. However, there are no real time markers for successful engagement of the spleen by autonomic stimulation. Here, we use fast-scan cyclic voltammetry (FSCV) in the spleen to assess, in real time, the release of norepinephrine (NE) in response to autonomic stimulation in mice. We show that spleen voltammograms demonstrate oxidative current peaks at a voltage of ~0.8 V that is proportional up to a NE blood concentration of 30 to 300nM. The oxidative current increases within seconds in response to electrical splenic nerve stimulation and is blocked by interventions that deplete NE or inhibit NE release. Oxidative current increases in response to electrical vagus nerve stimulation (VNS) and is elicited by efferent, but not afferent, fiber-selective optogenetic VNS. Spleen cyclic voltammetry can potentially provide a real-time physiological marker for dose calibration and monitoring of the anti-inflammatory effectiveness of autonomic stimulation therapies targeting the spleen.

sentences: Lightsheet Optical Tweezer (LOT) for Optical Manipulation of Microscopic Particles and Live Cells A light sheet based optical tweezer (LOT) is developed to trap microscopic dielectric particles and live HeLa cells. The technique requires the generation of a tightly focussed diffraction-limited light sheet which is realized by a combination of cylindrical lens and high NA objective lens. The field pattern generated at the geometrical focus is a tightly focussed line (along x-axis) perpendicular to the beam propagation direction (z-axis). Spherical beads undergoing Brownian motion in the solution are trapped by the gradient potential, and the time (to reach trap-center under the influence of gradient potential) is estimated from the fast CMOS camera (operating at 230frames/sec). High-speed imaging of beads at varying laser power shows a steady increase in the stiffness of LOT with a maximum of 0.00118 pN/nm at 52.5 mW. This is an order less than traditional optical point-traps. The trapped beads displayed free movement along the light-sheet axis (x-axis), exhibiting a single degree of freedom. Subsequently, LOT is used to optically trap and pattern dielectric beads and HeLa cells in a line. We could successfully pattern 8 dielectric beads and 3 HeLa cells in a straight line. We anticipate that LOT can be used to study the 1D-physics of microscopic particles and help understand the patterned growth of live cells.

sentences: The Population Genetics of Gene Regulatory Networks The evolution of diverse phenotypes both involves and is constrained by molecular interaction networks. When these networks influence patterns of expression, we refer to them as gene regulatory networks (GRNs). Here, we develop a quasi-species model of GRN evolution. With this model, we prove that-across a broad spectrum of viability and mutation functions-the dynamics converge to a stationary distribution over GRNs. Next, we show from first principles how the frequency of GRNs at equilibrium will be proportional to each GRNs eigenvector centrality in the genotype network. Finally, we determine the structural characteristics of GRNs that are favored in response to a range of selective environments and mutational constraints. Our work connects GRN evolution to quasi-species models, and thus can provide a mechanistic explanation for the topology of GRNs experiencing various evolutionary forces.

sentences: Integrative phenotypic and genomic analyses reveal strain-dependent responses to acute ozone exposure and their associations with airway macrophage transcriptional activity Acute ozone (O3) exposure is associated with multiple adverse cardiorespiratory outcomes, the severity of which varies across human populations and rodent models from diverse genetic backgrounds. However, molecular determinants of response, including biomarkers that distinguish which individuals will develop more severe injury and inflammation (i.e., high responders), are poorly characterized. Here, we exposed adult, female and male mice from 6 strains, including 5 Collaborative Cross (CC) strains, to filtered air (FA) or 2 ppm O3 for 3 hours, and measured several inflammatory and injury parameters 21 hours later. Additionally, we collected airway macrophages and performed RNA-seq analysis to investigate influences of strain, treatment, and strain-by-treatment interactions on gene expression as well as transcriptional correlates of lung phenotypes. Animals exposed to O3 developed airway neutrophilia and lung injury, with varying degrees of severity. We identified many genes that were altered by O3 exposure across all strains, and examination of genes whose expression was influenced by strain-by-treatment interactions revealed prominent differences in response between the CC017/Unc and CC003/Unc strains, which were low- and high-responders, respectively (as measured by cellular inflammation and injury). Further investigation of this contrast indicated that baseline gene expression differences likely contribute to their divergent post-O3 exposure transcriptional responses. We also observed alterations in chromatin accessibility that differed by strain and with strain-by-treatment interactions, lending further plausibility that baseline differences can modulate post-exposure responses. Together, these results suggest that aspects of the respiratory response to O3 exposure may be mediated through altered airway macrophage transcriptional signatures, and further confirms the importance of gene-by-environment interactions in mediating differential responsiveness to environmental agents.

sentences: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in 	C. elegans Innate immunity in animals is orchestrated by multiple cell signaling pathways, including the TGF-{beta} superfamily pathway. While the role of TGF-{beta} signaling in innate immunity has been clearly identified, the requirement for this pathway in generating specific, robust responses to different bacterial challenges has not been characterized. Here, we address the role of DBL-1/TGF-{beta} in regulating signature host defense responses to a wide range of bacteria in C. elegans. This work reveals a role of DBL-1/TGF-{beta} in animal survival, organismal behaviors, and molecular responses in different environments. Additionally, we identify a novel role for SMA-4/Smad that suggests both DBL-1/TGF-{beta}-dependent and -independent functions in host avoidance responses. RNA-seq analyses and immunity reporter studies indicate DBL-1/TGF-{beta} differentially regulates target gene expression upon exposure to different bacteria. Furthermore, the DBL-1/TGF-{beta} pathway is itself differentially affected by the bacteria exposure. Collectively, these findings demonstrate bacteria-specific host immune responses regulated by the DBL-1/TGF-{beta} signaling pathway.

sentences: New tools for automated cryo-EM single-particle analysis in RELION-4.0 1We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a Variable-metric gradient Descent algorithm with Adaptive Moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on twelve publicly available cryo-EM data sets.

sentences: Validation of DeepLabCut as a tool for markerless 3D pose estimation Deep learning-based approaches to markerless 3D pose estimation have taken neuroscience by storm. Yet many of these tools remain unvalidated. Here, we report on the validation of one increasingly popular tool (DeepLabCut) against simultaneous measurements obtained from a reference measurement system (Fastrak) with well-known performance characteristics. Our results confirm close (mm range) agreement between the two, indicating that deep learning-based approaches can be used by the research community with confidence.

sentences: Maximize crop production and environmental sustainability: insights from an ecophysiological model of plant-pest interactions and multi-criteria decision analysis Satisfying the demand for agricultural products while also protecting the environment from negative impacts of agriculture is a major challenge for crop management. We used an ecophysiological model of plant-pest interaction and multi-criteria decision analysis to optimize crop management when considering two contrasting objectives: (1) maximizing crop production and (2) minimizing environmental impact related to fertilization, irrigation and pesticide deployment. The model provides an indicator of crop production for 27 management scenarios, obtained combining three levels of fertilization, irrigation and pesticide use, respectively. We computed the environmental impact relevant to each management scenario by means of a weighted sum of costs assigned to fertilization, irrigation and pesticide use. We identified the optimal scenarios with respect to the considered objectives analysing the Pareto front. These scenarios were mostly characterized by high fertilization and no pesticide use. We evaluated the multi-functionality of the optimal scenarios by mean of the Gini coefficient: the scenario better assuring the equality between the two objectives was characterized by high fertilization, intermediate irrigation and no pesticide. Although our results remain qualitative and not immediately transferable to agronomic practices, our analytical framework provides a useful tool to evidence trade-offs among two contrasting objectives and provide solutions to act in an efficient way by leaving a certain degree of freedom to the political decision maker.

sentences: Analyzing the relationship between distracted driving and eye movement using multimodal data collected during car driving Human error is the leading cause of traffic accidents and originates from the distraction caused by various factors, such as the drivers physical condition and mental state. One of the significant factors causing driver distraction is the presence of stress. In a previous study, multiple stressors were used to examine distraction while driving. Multiple stressors were given to the driver and the corresponding driver biometric data were obtained, and a multimodal dataset was published thereafter. In this study, we reiterate the results of existing studies and investigated the relationship between gaze variability while driving and stressor intervention, which has not yet been examined. We also examined whether biometric and vehicle information can estimate the presence or absence of secondary tasks during driving.

sentences: Phylogenomics of a new fungal phylum reveals multiple waves of reductive evolution across Holomycota Compared to multicellular fungi and unicellular yeasts, unicellular fungi with free-living flagellated stages (zoospores) remain poorly known and their phylogenetic position is often unresolved. Recently, rRNA gene phylogenetic analyses of two atypical parasitic fungi with amoeboid zoospores and long kinetosomes, the sanchytrids Amoeboradix gromovi and Sanchytrium tribonematis, showed that they formed a monophyletic group without close affinity with known fungal clades. Here, we sequence single-cell genomes for both species to assess their phylogenetic position and evolution. Phylogenomic analyses using different protein datasets and a comprehensive taxon sampling result in an almost fully-resolved fungal tree, with Chytridiomycota as sister to all other fungi, and sanchytrids forming a well-supported, fast-evolving clade sister to Blastocladiomycota. Comparative genomic analyses across fungi and their allies (Holomycota) reveal an atypically reduced metabolic repertoire for sanchytrids. We infer three main independent flagellum losses from the distribution of over 60 flagellum-specific proteins across Holomycota. Based on sanchytrids phylogenetic position and unique traits, we propose the designation of a novel phylum, Sanchytriomycota. In addition, our results indicate that most of the hyphal morphogenesis gene repertoire of multicellular fungi had already evolved in early holomycotan lineages.

sentences: Delayed auditory encoding and variable representation of stimulus regularity in cerebellar lesion patients The dynamic and fleeting nature of sound necessitates the rapid encoding and use of information distributed over time. Here we investigated cerebellar contributions to these abilities. We measured EEG from cerebellar patients and healthy controls while they listened to "oddball" sound sequences consisting of infrequent pitch-deviant and frequent standard tones. Inter-stimulus-intervals were temporally regular (600 ms) or irregular (200-1000 ms). This allowed probing early event-related potentials (ERP; P50, N100) that reflect repetitive and changing stimulus characteristics in temporally regular or less (irregular) predictable sequences. Further, time-frequency data provided an index of temporal processing variability at the stimulation frequencies. We expected that cerebellar lesions lead to aberrant encoding and use of auditory information, reflected in the ERP morphology of peak amplitudes, latencies and typical suppression effects linked to stimulus predictability. Results confirm longer P50 peak latencies in patients and variable processing at stimulation frequencies covarying with the location of cerebellar damage. These findings further support the idea that the cerebellum might play a generalizable role in the encoding of auditory stimulation over time.

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Zhx2 is a candidate gene underlying oxymorphone metabolite brain concentration associated with state-dependent oxycodone reward Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared to the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared to BALB/cByJ mice. Oxymorphone is a highly potent full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of CYP enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains.

Significance StatementOur findings show genetic variation can result in sex-specific alterations in whole brain concentrations of bioactive opioid metabolites following oxycodone administration, and reinforces the need for sex as a biological factor in pharmacogenomic studies. The co-occurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward which has implications for cue-induced relapse of drug-seeking behavior.

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Biceps femoris long head sarcomere and fascicle length adaptations after three weeks of eccentric exercise training PurposeEccentric exercise is widely used to increase muscle fascicle lengths and thus decrease the risk of muscle strain injuries. However, the mechanisms behind this protection are still unknown. The aim of this study was to determine whether Biceps femoris long head (BFlh) fascicle length increases in response to three weeks of eccentric exercise training are the result of addition of in-series sarcomeres within muscle fibres.

MethodsTen recreationally active participants (age: 27 {+/-} 3 years, mass: 70 {+/-} 14 kg, height: 174 {+/-} 9 cm) completed three weeks of Nordic hamstring exercise (NHE) training. We collected in vivo sarcomere and muscle fascicle images of the BFlh in two regions (central and distal), utilising microendoscopy and 3D ultrasonography. These images allowed us to estimate sarcomere length, sarcomere number and fascicle lengths before and after the training intervention.

ResultsEccentric knee flexion strength increased after the training (15%, P < 0.001, {eta}p2= 0.75). Further, we found a significant increase in fascicle (21%, P < 0.001, {eta}p2 = 0.81) and sarcomere (17%, P < 0.001, {eta}p2 = 0.9) lengths in the distal but not in the central portion of the muscle. The estimated number of in series sarcomeres did not change in either region.

ConclusionFascicle length adaptations appear to be heterogeneous in the BFlf in response to three weeks of NHE training. An increase in sarcomere length, rather than the addition of sarcomeres in series, appears to be underlying this adaptation. The mechanism driving regional increases in fascicle and sarcomere length remain unknown, but we speculate it may be driven by regional changes in the passive tension of muscle or connective tissue adaptations.

sentences: Joint inference of repeated evolutionary trajectories and patterns of clonal exclusivity or co-occurrence from tumor mutation trees Cancer progression is an evolutionary process shaped by both deterministic and stochastic forces. Multi-region and single-cell sequencing of tumors empower high-resolution reconstruction of the mutational history of each tumor. At the same time, it also highlights the extensive diversity across tumors and patients. Resolving the interactions among mutations and recovering the recurrent evolutionary processes may offer greater opportunities for successful therapeutic strategies. To this end, we present a novel probabilistic framework, called TreeMHN, for joint inference of repeated evolutionary trajectories and patterns of clonal exclusivity or co-occurrence from a cohort of intra-tumor phylogenetic trees. Through simulation studies, we show that TreeMHN outperforms existing alternatives that can only focus on one aspect of the task. By applying our method to an acute myeloid leukemia dataset, we find the most likely evolutionary trajectories and mutational patterns, consistent with and enriching known findings.

sentences: Statistical Transfer Learning with Generative Encoding for Spatial Transcriptomics AO_SCPLOWBSTRACTC_SCPLOWA flexible model is introduced which shares ideas with the Autoencoder, Canonical Correlation Analysis, Singular Value Decomposition, and Procrustes Analysis. It is proposed to find relevant maps to transform multiple datasets of various types from one modality to another. Here, the Generative Encoder is used to transform spatial gene expression from breast tissue, to the images of histology tissue measured with Spatial Transcriptomics. The model is directly interpretable given all parameters are linked to the data space. It is scalable on Big Data, training reasonably on several thousand RGB images of 100 by 100 pixels in under an hour, which equates to 30,000 pixel features per sample image.

sentences: Plant-associate interactions and diversification across trophic levels Interactions between species are widely understood to have promoted the diversification of life on earth, but how interactions spur the the formation of new species remains unclear. Interacting species often become locally adapted to each other, but they may also be subject to shared dispersal limitations and environmental conditions. Moreover, theory predicts that different kinds of interactions have different effects on diversification. To better understand how species interactions promote diversification, we compiled published genetic data for host plants and intimately associated herbivores, parasites, and mutualists. We first tested whether host and associate population structures were correlated -- an indication of associates locally adapting to hosts -- and tested for confounding correlations with geographic distance or climate variation. We used Bayesian multiple regression to estimate the effect of host plant genetic differentiation on associate genetic differentiation over and above the confounding effects of geography and climate. We found that plant and associate genetic structures are indeed often congruent, but isolation by distance and by climate are also common. Multiple regressions established that the effect of host plants on associates is robust to accounting for geographic distance and climate. Finally, associate genetic structure was significantly explained by plant genetic structure more often in antagonistic interactions than in mutualistic ones. This supports a key prediction of coevolutionary theory, that antagonistic interactions promote diversity through local adaptation of antagonists to hosts, while mutualistic interactions promote diversity via the effect of hosts geographic distribution on mutualists dispersal.

sentences: Analysis and Forecasting of Global RT-PCR Primers for SARS-CoV-2 Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

sentences: Sex-specific body mass ageing trajectories in adult Asian elephants In species with marked sexual dimorphism and where one sex undergoes stronger intrasexual competition, that sex is expected to age earlier or quicker. Here, we utilise a unique, longitudinal dataset of a semi-captive population of Asian elephants (Elephas maximus), a species with marked male-biased intrasexual competition, with males being larger and living shorter, and test the hypothesis that males show earlier and/or faster body mass ageing than females. We show sex-specific body mass ageing trajectories: adult males gained weight up to the age of 48 years old, followed by a decrease in body mass until natural death. In contrast, adult females gained body mass with age until a body mass decline in the last year of life. Our study shows that sex-specific life-histories shape ageing patterns, consistent with the predictions of the classical theory of ageing.

sentences: Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin-column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal-GSH interactions, in accordance with the order of the Irving-Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II) and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.

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Fast and accurate genome-wide predictions and structural modeling of protein-protein interactions using Galaxy Protein-protein interactions play a crucial role in almost all cellular processes. Identifying interacting proteins reveals insight into living organisms and yields novel drug targets for disease treatment. Here, we present a publicly available, automated pipeline to predict genome-wide protein-protein interactions and produce high-quality multimeric structural models.

Application of our method to the Human and Yeast genomes yield protein-protein interaction networks similar in quality to common experimental methods. We identified and modeled Human proteins likely to interact with the papain-like protease of SARS-CoV2s non-structural protein 3 (Nsp3). We also produced models of SARS-CoV2s spike protein (S) interacting with myelin-oligodendrocyte glycoprotein receptor (MOG) and dipeptidyl peptidase-4 (DPP4). The presented method is capable of confidently identifying interactions while providing high-quality multimeric structural models for experimental validation.

The interactome modeling pipeline is available at usegalaxy.org and usegalaxy.eu.

sentences: ArtSeg: Rapid Artifact Segmentation and Removal in Brightfield Cell Microscopy Images Brightfield cell microscopy is a foundational tool in life sciences. The acquired images are prone to contain visual artifacts that hinder downstream analysis, and automatically removing them is therefore of great practical interest. Deep convolutional neural networks are state-of-the-art for image segmentation, but require pixel-level annotations, which are time-consuming to produce. Here, we propose ScoreCAM-U-Net, a pipeline to segment artifactual regions in brightfield images with limited user input. The model is trained using only image-level labels, so the process is faster by orders of magnitude compared to pixel-level annotation, but without substantially sacrificing the segmentation performance. We confirm that artifacts indeed exist with different shapes and sizes in three different brightfield microscopy image datasets, and distort downstream analyses such as nuclei segmentation, morphometry and fluorescence intensity quantification. We then demonstrate that our automated artifact removal ameliorates this problem. Such rapid cleaning of acquired images using the power of deep learning models is likely to become a standard step for all large scale microscopy experiments.

sentences: A single amino acid in RTEL1 determines telomere length in mice Maintaining optimal telomere length throughout life limits cancer and enables healthy aging. Yet, it is unclear how the telomere length set point is determined. Telomeres in the house mouse, Mus musculus, are 2-4 times longer than human telomeres, limiting the use of the mouse as a model for telomere research. By introducing a single amino acid variation found in M. spretus into the helicase RTEL1 of M. musculus, we shortened its telomere length set point 2-3 times, implicating this variation in the dramatic difference in telomere length between the two mouse species. While the engineered mice are fertile and healthy, hepatocytes with short telomeres displayed reduced proliferation capacity. This  telomouse is a unique model for studying the implications of short telomeres in aging and cancer.

sentences: High salinity activates CEF and attenuates state transitions in both psychrophilic and mesophilic Chlamydomonas species In the last decade, studies have revealed the importance of PSI-driven cyclic electron flow (CEF) in stress acclimation in model organisms like C. reinhardtii; however, these studies focused on transient, short-term stress. In addition, PSI-supercomplexes are associated with CEF during state transition response to short-term stress. On the other hand, the role of CEF during long-term stress acclimation is still largely unknown. In this study, we elucidate the involvement of CEF in acclimation response to long-term high salinity in three different Chlamydomonas species displaying varying salinity tolerance. We compared CEF rates, capacity for state transitions, and formation of supercomplexes after salinity acclimation in the model mesophile C. reinhardtii and two psychrophilic green algae C. priscuii (UWO241) and C. sp. ICE-MDV. CEF was activated under high salt in all three species, with the psychrophilic Chlamydomonas spp. exhibiting the highest CEF rates. High salt acclimation was also correlated with reduced state transition capacity and a PSI-supercomplex was associated with high CEF. We propose that under long-term stress, CEF is constitutively activated through assembly of a stable PSI-supercomplex. The proteomic composition of the long-term PSI-supercomplex is distinct from the supercomplex formed during state transitions, and its presence attenuates the state transition response.

sentences: The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

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The proSAAS chaperone provides neuroprotection and attenuates transsynaptic α-synuclein spread in rodent models of Parkinson's disease Parkinsons disease is a devastating motor disorder involving the aberrant aggregation of the synaptic protein synuclein (aSyn) and degeneration of the nigrostriatal dopaminergic tract. We previously showed that proSAAS, a small secreted chaperone protein widely expressed in neurons within the brain, is able to block aSyn-induced dopaminergic cytotoxicity in primary nigral neuron cultures. We show here that coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This positive functional outcome was accompanied by significant amelioration of the human aSyn-induced loss of both nigral tyrosine hydroxylase-positive cells and striatal tyrosine hydroxylase-positive terminals, demonstrating clear proSAAS-mediated protection of the nigro-striatal tract. ProSAAS overexpression also reduced the content of human aSyn protein in both the nigra and striatum and reduced the loss of tyrosine hydroxylase protein in both regions. Since proSAAS is a secreted protein, we tested the possibility that proSAAS is able to block the transsynaptic spread of aSyn from the periphery to the central nervous system, increasingly recognized as a potentially significant pathological mechanism. The number of human aSyn-positive neurites in the pons and caudal midbrain of mice following administration of human aSyn-encoding AAV into the vagus nerve was considerably reduced in mice coinjected with proSAAS-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. We suggest that proSAAS may represent a promising target for therapeutic development in Parkinsons disease.

SignificanceThis paper describes two independent avenues of research that both provide support for the in vivo neuroprotective function of this small chaperone protein. In the first approach, we show that proSAAS overexpression provides remarkably effective protection against dopaminergic neurotoxicity in a rat model of Parkinsons disease. This conclusion is supported both by three independent assays of motor function as well as by quantitative analysis of surviving dopaminergic neurons in brain areas involved in the control of motor function. In the second line of research, we show that in mice, the spread of human synuclein across synapses can be blunted by proSAAS overexpression.

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MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors Aberrant epithelial differentiation and regeneration contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium (DSS)-induced colitis. Transcriptomic analyses implicated increased E box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that disrupts MTG16:E protein complex formation (Mtg16P209T), we established that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein-mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared to unaffected controls. Finally, uncoupling MTG16:E protein interactions only partially phenocopied the enhanced tumorigenicity of Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer.

Graphical Abstract

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sentences: The genetic origin of Daunians and the Pan-Mediterranean southern Italian Iron Age context The geographical location and shape of Apulia, a narrow land stretching out in the sea at the South of Italy, made this region a Mediterranean crossroads connecting Western Europe and the Balkans. Such movements culminated at the beginning of the Iron Age with the Iapygian civilization which consisted of three cultures: Peucetians, Messapians and Daunians. Among them, the Daunians left a peculiar cultural heritage, with one-of-a-kind stelae and pottery, but, despite the extensive archaeological literature, their origin has been lost to time. In order to shed light on this and to provide a genetic picture of Iron Age Southern Italy, we collected and sequenced human remains from three archaeological sites geographically located in Northern Apulia (the area historically inhabited by Daunians) and radiocarbon dated between 1157 and 275 calBCE. We find that Iron Age Apulian samples are still distant from the genetic variability of modern-day Apulians, they show a remarkable genetic heterogeneity, even though a few kilometers and centuries separate them, and they are well inserted into the Iron Age Pan-Mediterranean genetic landscape. Our study provides for the first time a window on the genetic make-up of pre-imperial Southern Italy, whose increasing connectivity within the Mediterranean landscape, would have contributed to laying the foundation for modern genetic variability. In this light, the genetic profile of Daunians may be compatible with an autochthonous origin, with plausible contributions from the Balkan peninsula.

sentences: Chromosome 1q amplification perturbs a ceRNA network to promote melanoma metastasis Somatic copy number alterations (CNAs) promote cancer, but the underlying driver genes are often not obvious when only the functions of the encoded proteins are considered. mRNAs can act as competitive endogenous miRNA sponges (ceRNAs) to post-transcriptionally regulate gene expression in a protein coding-independent manner. However, whether ceRNAs contribute to the oncogenic effects of CNAs is unknown. We report that chromosome 1q gains promote melanoma progression and metastasis at least in part through overexpression of three mRNAs with ceRNA activity: CEP170, NUCKS1, and ZC3H11A. Genetic studies reveal that these ceRNAs enhance melanoma metastasis by sequestering tumor suppressor miRNAs, thereby alleviating the repression of several pro-metastatic target genes. This regulatory RNA network is evident in other cancer types, suggesting that chromosome 1q ceRNA deregulation is a common driver of cancer progression. Taken together, our work demonstrates that ceRNAs mediate the oncogenicity of somatic CNAs.

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BATL: Bayesian annotations for targeted lipidomics MotivationBioinformatic tools capable of annotating, rapidly and reproducibly, large, targeted lipidomic datasets are limited. Specifically, few programs enable high-throughput peak assessment of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data acquired in either selected or multiple reaction monitoring (SRM and MRM) modes.

ResultsWe present here Bayesian Annotations for Targeted Lipidomics (BATL), a Gaussian naive Bayes classifier for targeted lipidomics that annotates peak identities according to eight features related to retention time, intensity, and peak shape. Lipid identification is achieved by modelling distributions of these eight input features across biological conditions and maximizing the joint posterior probabilities of all peak identities at a given transition. When applied to sphingolipid and glycerophosphocholine SRM datasets, we demonstrate over 95% of all peaks are rapidly and correctly identified.

Availability and implementationBATL software is freely accessible online at https://complimet.ca/batl/ and is compatible with Safari, Firefox, Chrome and Edge.

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Epitope classification and RBD binding properties of neutralizing antibodies against SARS-CoV-2 variants of concern Severe acute respiratory syndrome coronavirus-2 (SAR-CoV-2) causes coronavirus disease 2019 (COVID19) that is responsible for short and long-term disease, as well as death, in susceptible hosts. The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein binds to cell surface angiotensin converting enzyme type-II (ACE2) to initiate viral attachment and ultimately viral pathogenesis. The SARS-CoV-2 S RBD is a major target of neutralizing antibodies (NAbs) that block RBD - ACE2 interactions. In this report, NAb-RBD binding epitopes in the protein databank were classified as C1, C1D, C2, C3, or C4, using a RBD binding profile (BP), based on NAb-specific RBD buried surface area and used to predict the binding epitopes of a series of uncharacterized NAbs. Naturally occurring SARS-CoV-2 RBD sequence variation was also quantified to predict NAb binding sensitivities to the RBD-variants. NAb and ACE2 binding studies confirmed the NAb classifications and determined whether the RBD variants enhanced ACE2 binding to promote viral infectivity, and/or disrupted NAb binding to evade the host immune response. Of 9 single RBD mutants evaluated, K417T, E484K, and N501Y disrupted binding of 65% of the NAbs evaluated, consistent with the assignment of the SARS-CoV-2 P.1 Japan/Brazil strain as a variant of concern (VoC). RBD variants E484K and N501Y exhibited ACE2 binding equivalent to a Wuhan-1 reference SARS-CoV-2 RBD. While slightly less disruptive to NAb binding, L452R enhanced ACE2 binding affinity. Thus, the L452R mutant, associated with the SARS-CoV-2 California VoC (B.1.427/B.1.429-California), has evolved to enhance ACE2 binding, while simultaneously disrupting C1 and C2 NAb classes. The analysis also identified a non-overlapping antibody pair (1213H7 and 1215D1) that bound to all SARS-CoV-2 RBD variants evaluated, representing an excellent therapeutic option for treatment of SARS-CoV-2 WT and VoC strains.

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ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication RationaleMitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types1-3. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection1, 4. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure1. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models1, 4, 5. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue6, 7. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling.

ObjectiveThis study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication.

Methods and ResultsUsing high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound {beta}-ACTIN only exists in MAM and not in the purified mitochondria.

Additionally, co-immunoprecipitation pulls down both DRP1 and {beta}-ACTIN together. Inhibition of {beta}-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and {beta}-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts.

ConclusionsThe results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with {beta}-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through {beta}-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

sentences: Mitonuclear co-introgression opposes genetic differentiation between phenotypically divergent songbirds Comparisons of genomic variation among closely related species often show more differentiation in mitochondrial DNA (mtDNA) and sex chromosomes than in autosomes, a pattern expected due to the differing effective population sizes and evolutionary dynamics of these genomic components. Yet, introgression can cause species pairs to deviate dramatically from general differentiation trends. The yellowhammer (Emberiza citrinella) and the pine bunting (E. leucocephalos) are hybridizing avian sister species that differ greatly in appearance and moderately in nuclear DNA, but that show no mtDNA differentiation. This mitonuclear discordance is best explained by adaptive mtDNA introgression--a process that can select for co-introgression at nuclear genes with mitochondrial functions (mitonuclear genes). To better understand the extent of mitonuclear discordance and characterize nuclear differentiation patterns in this system, we investigated genome-wide differentiation between allopatric yellowhammers and pine buntings and compared it to what was seen previously in mtDNA. We found significant nuclear differentiation that was highly heterogeneous across the genome, with a particularly wide differentiation peak on the sex chromosome Z. We further tested for preferential introgression of mitonuclear genes and found statistical support for this process in yellowhammers. A role for mitonuclear coevolution in this system is supported by a stronger signal of co-introgression in genes coding for subunits of the mitoribosome and electron transport chain complexes. Altogether, our study emphasizes the extreme variation seen in differentiation across genomic components and study systems as well as highlights the ramifications of this variation in species evolution.

sentences: Latent functional connectivity underlying multiple brain states Functional connectivity (FC) studies have predominantly focused on resting state, where ongoing dynamics are thought to reflect the brains intrinsic network architecture, which is thought to be broadly relevant because it persists across brain states (i.e., is state-general). However, it is unknown whether resting state is the optimal state for measuring intrinsic FC. We propose that latent FC, reflecting shared connectivity patterns across many brain states, better captures state-general intrinsic FC relative to measures derived from resting state alone. We estimated latent FC independently for each connection using leave-one-task-out factor analysis in 7 highly distinct task states (24 conditions) and resting state using fMRI data from the Human Connectome Project. Compared to resting-state connectivity, latent FC improves generalization to held-out brain states, better explaining patterns of connectivity and task-evoked activation. We also found that latent connectivity improved prediction of behavior outside the scanner, indexed by the general intelligence factor (g). Our results suggest that FC patterns shared across many brain states, rather than just resting state, better reflects state-general connectivity. This affirms the notion of "intrinsic" brain network architecture as a set of connectivity properties persistent across brain states, providing an updated conceptual and mathematical framework of intrinsic connectivity as a latent factor.

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Context-aware information selection and model recommendation with ACCORDION Querying new information from knowledge sources, in general, and published literature, in particular, aims to provide precise and quick answers to questions raised about a system under study. In this paper, we present ACCORDION (ACCelerating and Optimizing model RecommenDatIONs), a novel methodology and a tool to enable efficient addressing of biological questions, by automatically recommending models that recapitulate desired dynamic behavior. Our approach integrates information extraction from literature, clustering, simulation and formal analysis to allow for automated, consistent, and robust assembling, testing and selection of context-specific models. We used ACCORDION in nine benchmark case studies and compared its performance with other previously published tools. ACCORDION is comprehensive as it can capture all relevant knowledge from literature, obtained by automated literature search and machine reading. At the same time, as our results show, ACCORDION is selective, recommending only the most relevant and useful subset (15-20%) of candidate model extensions found in literature, while guided by baseline model context and goal properties. ACCORDION is very effective, also demonstrated by our results, as it can reduce the error of the initial baseline model by more than 80%, recommending models that closely recapitulate desired behavior, and outperforming previously published tools. In this process, ACCORDION can also suggest more than one highly scored model, thus providing alternative solutions to user questions and novel insights for treatment directions.

Contact{yaa38@pitt.edu, nmzivanov@pitt.edu}

Supplementary informationSupplementary data are available.

Availabilityhttp://www.biodesignlab.pitt.edu/accordion

sentences: Single cell response landscape of graded Nodal signaling in zebrafish explants Nodal, as a morphogen, plays important roles in cell fate decision, pattern formation and organizer function. But because of the complex context in vivo and technology limitations, systematic studying of genes, cell types and patterns induced by Nodal alone is still missing. Here, by using a relatively simplified model, the zebrafish blastula animal pole explant avoiding additional instructive signals and prepatterns, we constructed a single cell response landscape of graded Nodal signaling, identified 105 Nodal immediate targets and depicted their expression patterns. Our results show that Nodal signaling is sufficient to induce anterior-posterior patterned axial mesoderm and head structure. Surprisingly, the endoderm induced by Nodal alone is mainly the anterior endoderm which gives rise to the pharyngeal pouch only, but not internal organs. Among the 105 Nodal targets, we identified 14 genes carrying varying levels of axis induction capability. Overall, our work provides new insights for understanding of the Nodal function and a valuable resource for future studies of patterning and morphogenesis induced by it.

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The Functional Logic of Odor Information Processing in the Drosophila Antennal Lobe The early olfactory system of the fruit fly, while sensing a complex odorant landscape, encodes the odorant object identity (semantic information) and the odorant concentration waveform (syntactic information) into a combinatorial neural code. Single-channel physiology recordings at the output of the Antenna Lobe (AL) exhibit concentration-invariance and contrast-boosting properties, indicating a decoupling of the odorant object identity from the concentration waveform in steady-state while responding strongly to odorant concentration onset and offset in transient states.

Through exhaustive computational explorations of the AL circuits, we show that the steady-state and transient response features of the AL are, respectively, due to presynaptic and postsynaptic Local Neurons (LNs). Theoretical analysis reveals that the LN pathways can be modeled as parallel differential Divisive Normalization Processors (DNPs). Differential DNPs robustly extract odorant identity (semantic information) and ON/OFF odorant event-timing (syntactic information), thereby providing for the AL the functional logic of ON-OFF odorant identity recovery.

sentences: A new monoclonal antibody enables BAR analysis of subcellular importin β1 interactomes Importin {beta}1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in detection of importin complex cargos, and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable Biotinylation by Antibody Recognition (BAR) analysis of importin {beta}1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1, and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little or no reaction with KPNB1 in the nucleus. BAR with this novel antibody revealed numerous new interactors of importin {beta}1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.

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The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins.

SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

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hei-tag: a highly efficient tag to boost targeted genome editing Precise, targeted genome editing by CRISPR/Cas9 is key for basic research and translational approaches in model and non-model systems1. While active in all species tested so far, editing efficiencies still leave room for improvement. To reach its target, the bacterial Cas9 needs to be efficiently shuttled into the nucleus as attempted by fusion of nuclear localization signals (NLSs) to the Cas9 protein2. Additional domains such as FLAG- or myc-tags are added for immediate detection or straight-forward purification3. To avoid steric hinderance impacting on activity, amino acid linkers are employed connecting Cas9 and additional domains.

We present the  hei-tag (high efficiency-tag), boosting the activity of the wide variety of CRISPR/Cas genome editing tools. The addition of the hei-tag to Cas9 or a C-to-T base editor dramatically enhances the respective targeting efficiency in model systems ranging from fish to mammals, including tissue culture applications. This allows to instantly upgrade existing and potentially highly adapted systems as well as establish novel highly efficient tools.

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Functional Near Infrared Spectroscopy (fNIRS) based Odor Detection and Classification using Functional Data Analysis An objective approach for odor detection is to analyze the brain activity using imaging techniques during the odor stimulation. In this study, Functional Near Infrared Spectroscopy (fNIRS) is used to record hemodynamic response from the frontal region of the brain by using a 4-channel fNIRS system. The fNIRs data is collected during the odor detection task in which the subjects were asked to press a button when they detect the given odor. Functional Data Analysis (FDA) was applied on fNIRs data to convert discrete measured samples of data to continuous smooth curves. The FDA method enables us to use the bases coefficients of fNIRS smoothed curves for features that represent the shape of the raw fNIRS signal. With the learning algorithm that we proposed, these features were used to train the support vector machine classifier. We evaluated the odor detection problem, in two binary classification cases: odorant vs. non-odorant and odorant vs. fingertapping. The model achieved a classification accuracy of 94.12% and 97.06% over the stimulus condition in the two cases, respectively. Moreover to find the actual predictors we used the extracted defined features (slope, standard deviation, and delta) to train our classifier. We achieved an average accuracy of 91.18 % on classifying odorant vs. non-odorant and an accuracy of 94.12% for odorant vs. fingertapping on the stimulus condition. The results determined that fNIRs signals of odorant and non-odorant are distinguishable without being affected by the motor activity during the experiment.

These findings suggest that fNIRs measurement on the forehead could be potentially used for objective and comparably inexpensive assessment of odor detection in cases that the subjective report is unreliable.

sentences: Dopamine reports reward prediction errors, but does not update policy, during inference-guided choice Dopamine is thought to carry reward prediction errors (RPEs), which update values and hence modify future behaviour. However, updating values is not always the most efficient way of adapting to change. If previously encountered situations will be revisited in future, inferring that the state of the world has changed allows prior experience to be reused when situations are reencountered. To probe dopamines involvement in such inference-based behavioural flexibility, we measured and manipulated dopamine while mice solved a sequential decision task using state inference. Dopamine was strongly influenced by the value of states and actions, consistent with RPE signalling, using value information that respected task structure. However, though dopamine responded strongly to rewards, stimulating dopamine at the time of trial outcome had no effect on subsequent choice. Therefore, when inference guides choice, rewards have a dopamine-independent influence on policy through the information they carry about the worlds state.

sentences: NMNAT1 facilitates energy metabolism and gene regulation in developing retinal neurons Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, large-scale transcriptomics reveals dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

sentences: Comparative assessment of computed tomography and magnetic resonance imaging of the spider morph of Python regius and wild type Python regius to evaluate the morphological correlate of the wobble syndrome. There is general awareness of artificial selection and its potential implications on health and welfare of animals. Despite growing popularity and increasing numbers of breeds of atypical colour and pattern variants in reptiles, only few studies have investigated the appearance and cause of various diseases associated with colour morphs. Ball pythons (Python regius) are among the most frequently bred reptiles and breeders selected for a multitude of different colour and pattern morphs. Among those colour variants, the spider morph of the ball python is frequently associated with the wobble syndrome. The aim of this study was to determine, whether a morphological variant can be found and brought in association with the clinical occurrence of the wobble syndrome in spider ball pythons, using MRI and CT-imaging as intra-vitam diagnostic methods. Data from eight ball pythons including five spider ball pythons and three wild type ball pythons was assessed and evaluated comparatively. We were able to identify distinctive structural differences in inner ear morphology in spider ball pythons highly probable to relate to the wobble syndrome. To our knowledge, these anomalies are described for the first time and represent a basis for further anatomical and genetic studies and discussions regarding animal welfare in reptile breeding.

sentences: Efficient prediction of a spatial transcriptomics profile better characterizes breast cancer tissue sections without costly experimentation Spatial transcriptomics is an emerging technology requiring costly reagents and considerable skills, limiting the identification of transcriptional markers related to histology. Here, we show that predicted spatial gene-expressions in unmeasured regions and tissues can enhance biologists histological interpretations. We developed the Deep learning model for Spatial gene Clusters and Expression, DeepSpaCE and confirmed its performance using the spatial-transcriptome profiles and immunohistochemistry images of consecutive human breast cancer tissue sections. For example, the predicted expression patterns of SPARC, an invasion marker, highlighted a small tumor-invasion region that is difficult to identify using raw data of spatial transcriptome alone because of a lack of measurements. We further developed semi-supervised DeepSpaCE using unlabeled histology images and increased the imputation accuracy of consecutive sections, enhancing applicability for a small sample size. Our method enables users to derive hidden histological characters via spatial transcriptome and gene annotations, leading to accelerated biological discoveries without additional experiments.

sentences: IntegronFinder 2.0: identification and analysis of integrons across Bacteria, with a focus on antibiotic resistance in Klebsiella Integrons are mobile genetic elements that contain multiple cassettes encoding accessory genes whose order is shuffled by a specific integrase. Integrons within mobile genetic elements often contain multiple antibiotic resistance genes that they spread among nosocomial pathogens and contribute to the current antibiotic resistance crisis. However, most integrons are presumably sedentary and encode a much broader diversity of functions. IntegronFinder is a widely used software to identify novel integrons in bacterial genomes, but has aged and lacks some useful functionalities to handle very large datasets of draft genomes or metagenomes. Here, we present IntegronFinder version 2. We have updated the code, improved its efficiency and usability, adapted the output to incomplete genome data, and added a few novel functions. We describe these changes and illustrate the relevance of the program by analyzing the distribution of integrons across more than 20,000 fully sequenced genomes. We also take full advantage of its novel capabilities to analyze close to 4 thousand Klebsiella pneumoniae genomes for the presence of integrons and antibiotic resistance genes within them. Our data shows that K. pneumoniae has a large diversity of integrons and the largest mobile integron in our database of plasmids. The pangenome of these integrons contains a total of 165 different gene families with most of the largest families being related with resistance to numerous types of antibiotics. IntegronFinder is a free and open-source software available at https://github.com/gem-pasteur/Integron_Finder.

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Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multidrug-resistant Aeromonas hydrophila Multidrug-resistant (MDR) bacteria has rapidly increased in aquaculture, which highlights the risk of production losses due to diseases and potential public health concerns. Previously, we reported that ozone nanobubbles (NB-O3) were effective at reducing concentrations of pathogenic bacteria in water and modulating fish immunity against pathogens; however, multiple treatments with direct NB-O3 exposures caused alterations to the gills of exposed-fish. Here, we set up a modified recirculation system (MRS) assembled with an NB-O3 device (MRS-NB-O3) to investigate whether MRS-NB-O3 were 1) safe for tilapia (Oreochromis niloticus), 2) effective at reducing bacterial load in rearing water, and 3) improved survivability of Nile tilapia following an immersion challenge with a lethal dose of MDR Aeromonas hydrophila. The results indicated no behavioral abnormalities or mortality of Nile tilapia during the 14 day study using the MRS-NB-O3 system. In the immersion challenge, although high bacterial concentration (~2 x 107 CFU/mL) was used, multiple NB-O3 treatments in the first two days reduced the bacteria between 15.9% to 35.6% of bacterial load in water while bacterial concentration increased 13.1% to 27.9% in the untreated control. There was slight up-regulation of non-specific immune-related genes in the gills of the fish receiving NB-O3 treatments. Most importantly, this treatment significantly improved survivability of Nile tilapia with relative percent survival (RPS) of 64.7 - 66.7% in treated fish and surviving fish developed specific antibody against MDR A. hydrophila. In summary, the result suggests that NB-O3 is a promising alternative to antibiotics to control bacterial diseases, including MDR bacteria, and has high potential for application in recirculation aquaculture system (RAS).

HighlightsO_LIMultiple treatments of NB-O3 in a modified recirculation system (MRS) were relatively safe for juvenile Nile tilapia
C_LIO_LINB-O3 treatments in MRS significantly improved survivability of Nile tilapia challenged with multidrug-resistant (MDR) A. hydrophila with RPS of 64.7 - 66.7%
C_LIO_LIConcentration of MDR A. hydrophila in MRS was reduced by 15.9 to 35.6% following each NB-O3 treatment, and increased by 13.1 to 27.9 % in untreated control
C_LIO_LISurviving fish developed specific antibody IgM against MDR A. hydrophila
C_LIO_LINB-O3 is a promising non-antibiotic approach to control diseases caused by MDR A. hydrophila
C_LI

sentences: Free fatty acid receptor 4 (FFAR4) regulates cardiac oxylipin balance to promote inflammation resolution in a model of heart failure preserved ejection fraction secondary to metabolic syndrome Free fatty acid receptor 4 (Ffar4) is a G-protein coupled receptor for long-chain fatty acids that improves metabolism and attenuates inflammation. Heart failure preserved ejection fraction (HFpEF) is a complex clinical syndrome, but a predominant subset of patients has meta-bolic syndrome (MetS). Mechanistically, systemic, non-resolving inflammation associated with MetS might promote HFpEF. Interestingly, we recently demonstrated that Ffar4 is cardioprotective in pressure overload. The beneficial effects of Ffar4 on metabolism/inflammation, the high incidence of MetS in HFpEF patients, and the cardioprotective effects of Ffar4 led us to hypothesize that loss of Ffar4 would worsen remodeling in HFpEF secondary to MetS (HFpEF-MetS). To test this, mice with systemic deletion of Ffar4 (Ffar4KO) were fed a high-fat/high-sucrose diet with L-NAME in their water (HFpEF-MetS diet) to induce HFpEF-MetS. In male Ffar4KO mice, the HFpEF-MetS diet induced similar metabolic deficits, but worsened diastolic function and microvascular rarefaction compared to wild-type mice. Conversely, in female Ffar4KO mice, the diet produced greater obesity but no worsening of HFpEF. Loss of Ffar4 in males altered the balance of inflammatory oxylipins in the heart, decreasing the eicosapentaenoic acid derived, pro-resolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE), while increasing the arachadonic acid derived, proinflammatory oxylipin 12-hydroxyeicosatetraenoic acid (12-HETE). This increased 12-HETE/18-HEPE ratio, reflecting a more proinflammatory state, was associated with increased macrophage numbers, which in turn correlated with worsened ventricular remodeling in male Ffar4KO hearts. In summary, our data suggest that Ffar4 controls the pro/anti-inflammatory oxylipin balance in the heart to modulate macrophage function and attenuate HFpEF remodeling.

sentences: Real-time object locator for cryo-EM data collection--- You only navigate EM once --- In cryo-electron microscopy (cryo-EM) data collection, locating a target object is the most error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation showed its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and for locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection.

sentences: Transmission of SARS-CoV-2 from humans to animals and potential host adaptation SARS-CoV-2, the agent of the COVID-19 pandemic, can infect a wide range of mammals. Since its spread in humans, secondary host jumps of SARS-CoV-2 from humans to a variety of domestic and wild populations of mammals have been documented. The evolution of SARS-CoV-2 in different host species is of fundamental interest while also providing indication of how SARS-CoV-2 may have adapted to human hosts soon after the initial host jump, a time window for which there are no genome sequences available. Moreover, the study of SARS-CoV-2 circulating in animals is critical to assess the risk that the transmission of animal-adapted viral lineages back into humans (i.e., spillback) may pose. Here, we compared the genomic landscapes of SARS-CoV-2 isolated from animal species relative to that in humans, profiling the mutational biases indicative of potentially different selective pressures in animals. We focused on viral genomes collected in infected mink (Neovison vison) and white-tailed deer (Odocoileus virginianus) for which reports of multiple independent spillover events and subsequent animal-to-animal transmission are available. We identified six candidate mutations for animal-specific adaptation in mink (NSP9_G37E, Spike_F486L, Spike_N501T, Spike_Y453F, ORF3a_T229I, ORF3a_L219V), and one in deer (NSP3a_L1035F), though these mutations appear to confer minimal advantage for circulation in humans. Additionally, circulation of SARS-CoV-2 in mink and deer has not caused considerable changes to the evolutionary trajectory of SARS-CoV-2 thus far. Finally, our results suggest that minimal adaptation was required for human-to-animal spillover and subsequent onward transmission in mink and deer, highlighting the  generalist nature of SARS-CoV-2 as a pathogen of mammalian hosts.

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Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity Pathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

sentences: Unique structure and positive selection promote the rapid divergence of Drosophila Y chromosomes Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposable elements, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. We generated de novo long read assemblies complemented with cytological validation to reveal Y chromosome organization in three closely related species of the Drosophila simulans complex, which diverged only 250,000 years ago and share >98% sequence identity. We find these Y chromosomes are divergent in their organization and repetitive DNA composition and discover new Y-linked gene families whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. We propose that this repair mechanism generally contributes to the convergent evolution of Y chromosome organization.

sentences: Cohort Profile: Genetic data in the German Socio-Economic Panel Innovation Sample (Gene-SOEP) The German Socio-Economic Panel (SOEP) serves a global research community by providing representative annual longitudinal data of private households in Germany. The sample provides a detailed life course perspective based on a rich collection of information about living conditions, socio-economic status, family relationships, personality, values, preferences, and health. We collected genetic data from 2,598 individuals in the SOEP Innovation Sample, yielding the first genotyped sample that is representative of the entire German population (Gene-SOEP). The Gene-SOEP sample is a longitudinal study that includes 107 full-sibling pairs, 501 parent-offspring pairs, and 152 parent-offspring trios that are overlapping with the parent-offspring pairs. We constructed a repository of 66 polygenic indices in the Gene-SOEP sample based on results from well-powered genome-wide association studies. The Gene-SOEP data provides a valuable resource to study individual differences, inequalities, life-course development, health, and interactions between genetic predispositions and environment.

sentences: uniPort: a unified computational framework for single-cell data integration with optimal transport Single-cell data integration can provide a comprehensive molecular view of cells. Here we introduce uniPort, a unified single-cell data integration framework which combines a coupled Variational Autoencoder (coupled-VAE) and Minibatch Unbalanced Optimal Transport (Minibatch-UOT). It leverages both highly variable common and dataset-specific genes for integration and is scalable to large-scale and partially overlapping datasets. uniPort jointly embeds heterogeneous single-cell multi-omics datasets into a shared latent space. It can further construct a reference atlas for online prediction across datasets. Meanwhile, uniPort provides a flexible label transfer framework to deconvolute spatial heterogeneous data using optimal transport space, instead of embedding latent space. We demonstrate the capability of uniPort by integrating a variety of datasets, including single-cell transcriptomics, chromatin accessibility and spatially resolved transcriptome data. uniPort software is available at https://github.com/caokai1073/uniPort.

sentences: Kinetic analysis of ASIC1a delineates conformational signaling from proton sensing domains to the channel gate Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. Molecular dynamics simulations were used to further evaluate VCF-predicted movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

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Coccolith Sr/Ca is a Robust Temperature and Growth Rate Indicator that Withstands Dynamic Microbial Interactions Coccolithophores are a diverse group of calcifying microalgae that have left a prominent fossil record on Earth. Various coccolithophore relics, both organic and inorganic, serve as proxies for reconstruction of past oceanic conditions.

Emiliania huxleyi is the most widely distributed representative of the coccolithophores in modern oceans, and is known to engage in dynamic interactions with bacteria. Algal-bacterial interactions influence various aspects of algal physiology and alter algal alkenone unsaturation (UK37), a frequently used organic coccolithophore-derived paleotemperature proxy. Whether algal-bacterial interactions influence inorganic coccolithophore-derived paleo-proxies, is yet unknown.

A commonly used inorganic proxy for past productivity and sea surface temperature is the Sr/Ca ratio of the coccolith calcite. Interestingly, during interactions between bacteria and a population of calcifying algae, bacteria were shown to physically attach only to non-calcified algal cells, suggesting an influence on algal calcification.

In this study we explore the effects of algal-bacterial interactions on calcification and coccolith Sr/Ca ratios. We find that while bacteria attach only to non-calcified algal cells, coccolith cell coverage and overall calcite production in algal populations with and without bacteria, is similar. Furthermore, we find that Sr/Ca values are impacted only by water temperature and algal growth rate, regardless of bacterial influences on algal physiology. Our observations reinforce the robustness of coccolith Sr/Ca ratios as a paleo-proxy independent of microbial interactions, and highlight a fundamental difference between organic and inorganic paleo-proxies.

Summary StatementThe current research investigates the effect of microbial interactions on coccolith Sr/Ca ratio and overall calcification in the coccolithophore Emiliania huxleyi. We co-cultured E. huxleyi with the marine bacterium Phaeobacter inhibens and compared coccolith Sr/Ca between different growth stages in a range of temperatures. Our results indicate that coccolith Sr/Ca depends on temperature and algal growth rate, and remains robust despite significant bacterial influences on algal physiology.

sentences: Glycolytic inhibitor 2-Deoxy-D-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions The COVID-19 pandemic is an ongoing public health emergency of international concern. While a lot of efforts are being invested in vaccinating the population, there is also an emergent requirement to find potential therapeutics to effectively counter this fast mutating SARS-CoV-2 virus-induced pathogenicity. Virus-infected host cells switch their metabolism to a more glycolytic phenotype. This switch induced by the virus is needed for faster production of ATP and higher levels of anabolic intermediates, required for new virion synthesis and packaging. In this study, we used 2-Deoxy-D-glucose (2-DG) to target and inhibit the metabolic reprogramming induced by SARS-CoV-2 infection. Our results showed that virus infection induces glucose influx and glycolysis resulting in selective high accumulation of the fluorescent glucose/2-DG analogue, 2-NBDG in these cells. Subsequently, 2-DG inhibits glycolysis in infected cells thereby reducing the virus multiplication and alleviates the cells from virus induced cytopathic effect (CPE) and cell death. Herein, we demonstrate that the crucial Nglycosites (N331 and N343) of RBD in spike protein of progeny virions produced from 2-DG treated cells were found unglycosylated and defective with compromised infectivity potential. In line with earlier reported observations, our study also showed that 2-DG mediated metabolic inhibiton can attenuate SARS-COV-2 multiplication. In addition, mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced un-glycosylation of spike protein. Our findings strengthen the notion that 2-DG effectively inhibits SARS-CoV-2 multiplication. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.

sentences: An invariant Trypanosoma vivax vaccine antigen inducing protective immunity Trypanosomes are protozoan parasites that cause infectious diseases including human African trypanosomiasis (sleeping sickness), and nagana in economically-important livestock animals1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms including antigenic variation3 that present an apparently insurmountable barrier to vaccination. Here we show using a systematic genome-led vaccinology approach4 and a murine model of Trypanosoma vivax infection5 that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell surface protein localised to the flagellum membrane termed "invariant flagellum antigen from T. vivax" (IFX) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to IFX could induce sterile protection and revealed multiple mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained the socioeconomic development of sub-Saharan African countries6 and challenges long-held views that vaccinating against trypanosome infections cannot be achieved.

sentences: A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development. Defects in embryonic patterning resulting in craniofacial abnormalities account for approximately 1/3 of birth defects. The regulatory programs that build and shape the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Large regions with coactivation of enhancer elements and co-regulation of multiple genes, referred to as superenhancers, are important in determining cell identity and perturbation could result in developmental defects. Building upon a previously published epigenomic atlas of human embryonic craniofacial tissue in which we identified over 75,000 putative embryonic craniofacial enhancer regions, we have identified 531 superenhancer regions unique to embryonic craniofacial tissue, including 37 which fall in completely noncoding regions. To demonstrate the utility of this data for the understanding of craniofacial development and the etiology of craniofacial abnormalities, we focused on a craniofacial-specific superenhancer in a [~]600kb noncoding region located between NPVF and NFE2L3. This region harbors over 100 individual putative craniofacial enhancer segments and 7 in vivo validated craniofacial enhancers from primary craniofacial tissue as well as strong enhancer activation signatures in a culture model of cranial neural crest cell (CNCC) development. However, none of the directly adjacent genes have been implicated in neural crest specification, craniofacial development, or abnormalities. To identify potential regulatory targets of this superenhancer region, we characterized three-dimensional chromatin structure of this region in CNCCs and mouse embryonic craniofacial tissues using multiple techniques (4C-Seq, HiC). We identified long range interactions that exclude most intervening genes and specifically target the anterior portion of the HOXA gene cluster located 1.2 to 1.8 Mb away. We demonstrate the specificity of the enhancer region for regulation of anterior HOXA genes through CRISPR/Cas9 editing of human embryonic stem cells. Mice homozygous for deletion of the superenhancer confirm the specificity of the enhancer region and demonstrate that the region is essential for viability. At fetal stages homozygotes develop at the same rate as heterozygous and wild type littermates but die at P0-P1 and have high penetrance of orofacial clefts that phenocopy previously described Hoxa2-/- mice. Moreover, we identified a de novo deletion partially overlapping the superenhancer in a human fetus with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and whose deletion is likely pathogenic in human patients.

sentences: Mechanosensitive Osteogenesis on Native Cellulose Scaffolds for Bone Tissue Engineering. In recent years, plant-derived cellulosic biomaterials have become a popular way to create scaffolds for a variety of tissue engineering applications. Moreover, such scaffolds possess similar physical properties (porosity, stiffness) that resemble bone tissues and have been explored as potential biomaterials for tissue engineering applications. Here, plant-derived cellulose scaffolds were seeded with MC3T3-E1 pre-osteoblast cells. Moreover, to assess the potential of these biomaterials, we also applied cyclic hydrostatic pressure (HP) to the cells and scaffolds over time to mimic a bone-like environment more closely. After one week of proliferation, cell-seeded scaffolds were exposed to HP up to 270 KPa at a frequency of 1Hz, once per day, for up to two weeks. Scaffolds were incubated in osteogenic inducing media (OM) or regular culture media (CM). The effect of cyclic HP combined with OM on cell-seeded scaffolds resulted in an increase of differentiated cells. This corresponded to an upregulation of alkaline phosphatase activity and scaffold mineralization. Importantly, the results reveal that well known mechanosensitive pathways cells which regulate osteogenesis appear to remain functional even on novel plant-derived cellulosic biomaterials.

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An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications.

To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmers barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmers fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmers fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

sentences: Necroptosis increases with age in the brain and contributes to age-related neuroinflammation Chronic inflammation of the central nervous system (CNS), termed neuroinflammation, is a hallmark of aging and a proposed mediator of cognitive decline associated with aging. Neuroinflammation is characterized by the persistent activation of microglia, the innate immune cells of the CNS, with damage-associated molecular patterns (DAMPs) being one of the well-known activators of microglia. Because necroptosis is a cell death pathway that induce inflammation through the release of DAMPs, we hypothesized that an age-associated increase in necroptosis contributes to increased neuroinflammation with age. The marker of necroptosis, phosphorylated form of MLKL (P-MLKL), and kinases in the necroptosis pathway (RIPK1, RIPK3, and MLKL) showed a region-specific increase in the brain with age, specifically in the cortex layer V and the CA3 region of the hippocampus of mice. Similarly, MLKL-oligomers, which causes membrane binding and permeabilization were significantly increased in the cortex and hippocampus of old mice relative to young mice. Nearly 70 to 80% of P-MLKL immunoreactivity was localized to neurons and less than 10% was localized to microglia, whereas no P-MLKL was detected in astrocytes. P-MLKL expression in neurons was detected in the soma, not in the processes. Blocking necroptosis using Mlkl-/- mice reduced markers (Iba-1 and GFAP) of neuroinflammation in the brains of old mice and short-term treatment with the necroptosis inhibitor, necrostatin-1s, reduced expression of proinflammatory cytokines, IL-6 and IL-1{beta}, in the hippocampus of old mice. Thus, our data demonstrate for the first time that brain necroptosis increases with age and contributes to age-related neuroinflammation in mice.

sentences: Histidine-rich protein 2: a new pathogenic factor of Plasmodium falciparum malaria Plasmodium falciparum causes serious malaria symptoms; when this protozoan parasite infects human erythrocytes, it produces and secretes large amounts of histidine-rich protein 2 (PfHRP2) into human blood. Thus, PfHRP2 is a well-known diagnostic marker for malaria infection. Here, however, we also identified PfHRP2 as a pathogenic factor produced by P. falciparum. PfHRP2 showed cell penetration and cytotoxicity against various human cells. In particular, PfHRP2 showed significant cytotoxicity over 5 days at the same concentration as in P. falciparum-infected patients blood (90-100 nM). This result is consistent with the mortality rate of P. falciparum malaria, which increases rapidly in untreated cases for 3-7 days. In addition, the cell penetration and cytotoxicity of PfHRP2 increased 2.5- and 2.6-fold, respectively, in the absence of serum, which suggests that low serum protein concentrations (occurring during malnutrition, for example) increase the risk of adverse effects from PfHRP2 (consistent with malnutrition increasing the lethality of malaria infection). We also showed that PfHRP2 bound to Ca2+ ions, localized to intracellular lysosomes, increased lysosomal Ca2+ levels, and inhibited the basal level of autophagy by inhibiting autolysosome formation. Furthermore, the Ca2+-dependent cytotoxicity of PfHRP2 was suppressed by the metal ion chelator ethylenediaminetetraacetic acid (EDTA). In summary, our findings suggest that PfHRP2 acts as a pathogenic factor in P. falciparum-infected patients and is associated with the exacerbation of malaria. Furthermore, EDTA is a promising candidate as a therapeutic agent for the suppression of PfHRP2 pathogenicity. Overall, this study provides new insights into P. falciparum malaria pathogenesis and treatment.

sentences: Global Importance Analysis: An Interpretability Method to Quantify Importance of Genomic Features in Deep Neural Networks Deep neural networks have demonstrated improved performance at predicting the sequence specificities of DNA- and RNA-binding proteins compared to previous methods that rely on k-mers and position weight matrices. To gain insights into why a DNN makes a given prediction, model interpretability methods, such as attribution methods, can be employed to identify motif-like representations along a given sequence. Because explanations are given on an individual sequence basis and can vary substantially across sequences, deducing generalizable trends across the dataset and quantifying their effect size remains a challenge. Here we introduce global importance analysis (GIA), a model interpretability method that quantifies the population-level effect size that putative patterns have on model predictions. GIA provides an avenue to quantitatively test hypotheses of putative patterns and their interactions with other patterns, as well as map out specific functions the network has learned. As a case study, we demonstrate the utility of GIA on the computational task of predicting RNA-protein interactions from sequence. We first introduce a convolutional network, we call ResidualBind, and benchmark its performance against previous methods on RNAcompete data. Using GIA, we then demonstrate that in addition to sequence motifs, ResidualBind learns a model that considers the number of motifs, their spacing, and sequence context, such as RNA secondary structure and GC-bias.

sentences: Ataxin-2 is essential for neurodevelopment in Drosophila and is a major regulator of the cytoskeleton. Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to Amyotrophic Lateral Sclerosis (ALS) or Spinocerebellar Ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of 3rd instar larvae. These include reduced brain size, impaired optic lobe innervation and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

sentences: Moment-Based Estimation of State-Switching Rates in Cell Populations In isogenic cell populations, cells can switch back and forth between different gene expression states. These expression states can be biologically relevant. For example, a certain expression state may cause a tumor cell to be resistant to treatment, while another state may leave it vulnerable to treatment. However, estimating the rates of state-switching can be difficult, because experimentally measuring a cells transcriptome often involves destroying the cell, so it can only be measured once. In this paper, we propose a computational method to estimate the rate of switching between expression states, given data from a Luria-Delbruck style fluctuation test that is experimentally simple and feasible. We then benchmark this method using simulated data to test its efficacy, with varying assumptions made about cell cycle timing distribution in the simulations.

sentences: Endoribonuclease activity of XRN-2 is critical for RNA metabolism and survival of Caenorhabditis elegans microRNAs (miRNAs) are known to regulate a vast majority of the eukaryotic genes by post-transcriptional means, and multiple nucleases play critical roles in the biogenesis and turnover of these regulators. A number of studies have indicated that turnover is important for determining the abundance of miRNAs, and thus, in turn govern their functionality. Recent research in Caenorhabditis elegans has revealed an ATP-independent endoribonuclease activity of the  miRNase-XRN-2. Here, we report the characterization of this new enzymatic activity of the fundamentally important XRN-2, and show that it is critical for miRNA turnover and survival of quiescent dauer worms. The dual enzymatic activity of XRN-2 capacitates the mechanism of miRNA turnover to be dynamic, which might confer adaptive advantage to the organism. In continuously growing worms, this new enzymatic activity does not act on miRNAs, but it is important for the generation of mature ribosomal RNAs, which in turn is critical for translation, and thus indispensable for the survival of worms.

sentences: In the back of your mind: Cortical mapping of tactile and proprioceptive paraspinal afferent inputs Persistent pain alters brain-body representations, highlighting their potential pathological significance. In chronic low back pain (LBP), sparse evidence points towards a shift of the cortical representation of sensory afferents of the back. However, systematic investigations of the cortical representation of tactile and proprioceptive paraspinal afferents along the thoracolumbar axis are lacking. Detailed cortical maps of paraspinal afferent input might be crucial to further explore potential relationships between brain changes and the development and maintenance of chronic LBP. We therefore validated a novel and functional magnetic resonance imaging-(fMRI-)compatible method of mapping cortical representations of tactile and proprioceptive afferents of the back, using pneumatic vibrotactile stimulation ("pneuVID") at varying frequencies and paraspinal locations, in conjunction with high-resolution fMRI. We hypothesised that: (i) high (80 Hz) frequency stimulation would lead to increased postural sway compared to low (20 Hz) stimulation, due to differential evoked mechanoreceptor contributions to postural control (proprioceptive vs tactile); and (ii) that high (80 Hz) versus low (20 Hz) frequency stimulation would be associated with neuronal activity in distinct primary somatosensory (S1) and motor (M1) cortical targets of tactile and proprioceptive afferents (N=15, healthy volunteers). Additionally, we expected neural representations to vary spatially along the thoracolumbar axis. We found significant differences between neural representations of low and high frequency stimulation and between representations of thoracic and lumbar paraspinal locations, in several bilateral sensorimotor cortical regions. Proprioceptive (80 Hz) stimulation preferentially activated sub-regions S1 3a and M1 4p, while tactile (20 Hz) stimulation was more encoded in S1 3b and M1 4a. Moreover, in S1, lower back proprioceptive stimulation activated dorsal-posterior representations, compared to ventral-anterior representations activated by upper back stimulation. As per our hypotheses, we found distinct sensorimotor cortical tactile and proprioceptive representations, with the latter displaying clear topographic differences between the upper and lower back. This thus represents the first behavioural and neurobiological validation of the novel pneuVID method for stimulating muscle spindles and mapping cortical representations of paraspinal afferents. Future investigations of detailed cortical maps will be of major importance in elucidating the role of cortical reorganization in the pathophysiology of chronic LBP.

sentences: Next-generation matrices for marine metapopulations: the case of sea lice on salmon farms Classifying habitat patches as sources or sinks and determining metapopulation persistence requires coupling connectivity between habitat patches with local demographic rates. In this paper we show how next-generation matrices, originally popularized in epidemiology to calculate new infections after one generation, can be used in an ecological context to couple connectivity with local demography to calculate sources and sinks as well as metapopulation persistence in marine metapopulations. To demonstrate the utility of the method, we construct a next-generation matrix for a network of sea lice populations on salmon farms in the Broughton Archipelago, BC, an intensive salmon farming region on the west coast of Canada where certain salmon farms are currently being removed under an agreement between local First Nations and the provincial government. We identify the salmon farms which are acting as the largest sources of sea lice and show that in this region the most productive sea lice populations are also the most connected. We find that the farms which are the largest sources of sea lice have not yet been removed from the Broughton Archipelago, and that warming temperatures could lead to increased sea louse growth.

sentences: Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates tissue differentiation, growth and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim to comprehensively identify conserved genes related to fruiting body morphogenesis and distill novel functional hypotheses for functionally poorly characterized genes. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide informed hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defense, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for [~]10% of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi.

sentences: Recycling of nutrient transporters from yeast endosomes is regulated by ubiquitinated Ist1 Trafficking of cell surface membrane proteins to and from the plasma membrane impinges on myriad biological processes and ensures correct cellular function. Upon internalisation, most surface proteins are either sent to the lysosome for degradation or recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labour between interconnected pathways is poorly defined. Furthermore, how well endosomal trafficking mechanisms are conserved across eukaryotes is unclear. In yeast, we show cargo recycling back to the plasma membrane occurs through distinct pathways. In addition to the established retrograde recycling pathways via the late Golgi, which are used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalise to, and recycle from, endosomes marked by Vps4 and the ESCRT-III associated factor Ist1. This endosome population serve as both  early and  recycling endosomes, implying these features of endosomal organisation are evolutionarily conserved. Ist1 has previously been implicated in recycling in yeast and other eukaryotes. We reveal Ist1 ubiquitination regulates its endosomal recruitment and is required for cargo recycling. Additionally, the ubiquitin-binding adaptor Npl4 and the essential ATPase Cdc48 regulate endosomal Ist1 and are required for cargo recycling, suggesting mechanistic features of recycling from endosomes to the plasma membrane are also conserved.

sentences: Atoh8 is a regulator of intestinal microfold cell (M cell) differentiation Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, its development and differentiation remains yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. Increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mouse with increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that the population density of intestinal M-cell in the Peyers patch is regulated by the PRC2 regulated Atoh8.

sentences: Allicin inhibits SARS-CoV-2 replication and abrogates the antiviral host response in the Calu-3 proteome The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is a major health burden. Volatile garlic organosulfur compounds, such as the thiol-reactive allicin (diallyl thiosulfinate) exert strong antimicrobial activity against various respiratory pathogens. Here, we investigated the antiviral activity of allicin against SARS-CoV-2 in infected Vero E6 and Calu-3 lung cells. Calu-3 cells showed greater allicin tolerance due >4-fold increased GSH levels compared to Vero E6. However, biocompatible allicin doses efficiently inhibited viral replication in both cell lines. Proteome analyses of SARS-CoV-2 infected Calu-3 cells revealed a strong induction of the antiviral interferon-stimulated gene (ISG) signature (e.g. cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, 25OAS and ISG15), pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTC1, ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin abrogated the ISG host response and reverted the host cellular pathways to levels of uninfected Calu-3 cells, confirming the antiviral and immunomodulatory activity of allicin in the host proteome. Thus, biocompatible doses of allicin could be promising for protection of lung cells against SARS-CoV-2.

sentences: A solution to temporal credit assignment using cell-type-specific modulatory signals Animals learn and form memories by jointly adjusting the efficacy of their synapses. How they efficiently solve the underlying temporal credit assignment problem remains elusive. Here, we re-analyze the mathematical basis of gradient descent learning in recurrent spiking neural networks (RSNNs) in light of the recent single-cell transcriptomic evidence for cell-type-specific local neuropeptide signaling in the cortex. Our normative theory posits an important role for the notion of neuronal cell types and local diffusive communication by enabling biologically plausible and efficient weight update. While obeying fundamental biological constraints, including separating excitatory vs inhibitory cell types and observing connection sparsity, we trained RSNNs for temporal credit assignment tasks spanning seconds and observed that the inclusion of local modulatory signaling improved learning efficiency. Our learning rule puts forth a novel form of interaction between modulatory signals and synaptic transmission. Moreover, it suggests a computationally efficient learning method for bio-inspired artificial intelligence.

sentences: Assessing the relationships between neurological and psychiatric diseases with astrocyte subtypes and psychotropic medications Astrocytes have many important functions in the brain, but their roles in CNS disorders and their responses to psychotropic medications are still being elucidated. In this study, we used gene enrichment analysis to assess the relationships between different astrocyte subtypes, neurological and psychiatric diseases, and psychotropic medications. We also carried out qPCR analyses and "look-up" studies to further assess the chronic effects of these drugs on astrocyte marker gene expression. Our bioinformatic analysis identified differential gene enrichment of different astrocyte subtypes in CNS disorders. The "common" astrocyte subtype was the most frequently enriched across disorders, but the highest level of enrichment was found in depression, supporting a role for astrocytes in this disorder. We also identified common enrichment of metabolic and signal transduction-related biological processes in astrocyte subtypes and CNS disorders. However, enrichment of different psychotropic medications, including antipsychotics, antidepressants, and mood stabilizers, was limited in astrocyte subtypes. These results were confirmed by "look-up" studies and qPCR analysis, which also reported little effect of common psychotropic medications on astrocyte marker gene expression, suggesting that astrocytes are not a primary target of these medications. Overall, this study provides a unique view of astrocyte subtypes and the effect of medications on astrocytes in disease, which will contribute to our understanding of their role in CNS disorders and offers insights into targeting astrocytes therapeutically.

sentences: Mouse Organ Specific Proteins and Functions Organ specific proteins (OSPs) possess great medical potentials both in clinics and in biomedical research. Applications of them -- such as alanine transaminase, aspartate transaminase, and troponins -- in clinics have raised certain concerns of their organ specificity. The dynamics and diversity of protein expression in heterogeneous human population are well known, yet their effects on OSPs are less addressed. Here we use mouse as a model and implemented a scheme of breadth study to examine the pan-organ proteome for potential variations of organ specificity in different genetic backgrounds. Using reasonable resources, we generated pan-organ proteomes of four in-bred mouse strains. The results revealed a large diversity that is more profound among OSPs than the overall proteomes. We defined a robustness score to quantify such variation and derived three sets of OSPs with different stringencies. In the meantime, we found that the enriched biological functions of OSPs are also organ specific that are sensitive and useful to assess the quality of OSPs. We hope our breadth study can open doors to explore the molecular diversity and dynamics of organ specificity at the protein level.

sentences: Label-free super-resolution chemical imaging of biomedical specimens Raman microscopy provides chemically selective imaging by exploiting intrinsic vibrational properties of specimens. Yet, a fast acquisition, low phototoxicity, and non-specific (to a vibrational/electronic mode) super-resolution method has been elusive for tissue imaging. We demonstrate a single-pixel-based approach, combined with robust structured illumination, that enables fast super-resolution in stimulated Raman scattering microscopy at low power levels. The methodology is straightforward to implement and compatible with thick biological specimens, therefore paving the way for probing complex biological systems when exogenous labelling is challenging.

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Prefrontally Modulated Vagal Tone Inhibits Inflammatory Responses to Prevent Telomere Damage in Healthy Participants BackgroundAccumulated senescent cells are proposed to be one of the main drivers of age-related pathology through disruption of tissue structure and function. We recently proposed the Neuro-Immuno-Senescence Integrative Model (NISIM; Ask et al., 2018) which relates prefrontally modulated vagal tone and subsequent balance between vagal and sympathetic input to the spleen to inflammatory responses leading to generation of reactive oxygen species and oxidative telomere damage. The NISIM is based on converging evidence and argues for the existence of a prefrontal cortex-autonomic nervous system-spleen (PAS) axis, suggesting that the inflammation that induces reactive oxygen species-generation is downstream of this axis.

AimIn this study, we aim to assess inflammation as a mediator in the relationship between prefrontally modulated vagal tone and leukocyte telomere length to test the hypothesis that PAS axis dysregulation accelerates cellular aging. We also assess the relationship between a recently proposed index of vagal immunomodulation (vagal tone/inflammation ratio; NIM index; Gidron et al., 2018) and telomere length, and compare results between the NIM index and vagal tone as predictors of telomere length.

MethodsThis study uses participant data from a large nationally representative longitudinal study since 1974 with a total of 45,000 Norwegian residents so far. A sub-sample of 1372 participants from which vagal tone, C Reactive Protein, and leukocyte telomere length could be obtained were included in the study. Relationships were analyzed with hierarchical multiple linear regression using either vagal tone and C Reactive Protein or the NIM index to predict telomere length. Sleeping problems, tobacco use status, alcohol use status, time since last meal, and symptoms of depression were included as control variables.

ResultsIn the mediation analysis, vagal tone was a significant positive predictor of telomere length, while C Reactive Protein was a significant negative predictor of telomere length. This relationship remained significant when individually controlling for some but not all confounding variables. The NIM index was a significant positive predictor of telomere length. This relationship remained significant when controlling for all confounding variables except one. In a reduced dataset excluding all participants where confounders were present, the NIM index remained a significant predictor of telomere length.

ConclusionThis is the first study suggesting that PAS axis activity is associated with telomere length thus supporting the NISIM. Results indicate that the NIM index is a more sensitive indicator of PAS axis activity than vagal tone and C Reactive Protein in isolation. Clinical relevance and suggestions for future research are discussed.

sentences: Life-history evolution of class-structured populations in fluctuating environments What is the influence of periodic environmental fluctuations on life-history evolution? We present a general theoretical framework to understand and predict the long-term evolution of lifehistory traits under a broad range of ecological scenarios. Specifically, we investigate how periodic fluctuations affect selection when the population is also structured in distinct classes. This analysis yields time-varying selection gradients that clarify the influence of the fluctuations of the environment on the competitive ability of a specific life-history mutation. We use this framework to analyse the evolution of key life-history traits of pathogens. We examine three different epidemiological scenarios and we show how periodic fluctuations of the environment can affect the evolution of virulence and transmission as well as the preference for different hosts. These examples yield new and testable predictions on pathogen evolution, and illustrate how our approach can provide a better understanding of the evolutionary consequences of time-varying environmental fluctuations in a broad range of scenarios.

sentences: Elucidating the crosstalk between inflammation and DNA damage pathways in the pancreatic beta-cell through the diabetes susceptibility gene, TCF19 TCF19 is a gene that is associated with both type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that TCF19 knockdown impairs {beta}-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 {beta}-cells on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNAseq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in cell stress, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the {beta}-cell, and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways may provide potential targets for {beta}-cell survival.

sentences: Inferring long-term effective population size with Mutation-Selection models Mutation-selection phylogenetic codon models are grounded on population genetics first principles and represent a principled approach for investigating the intricate interplay between mutation, selection and drift. In their current form, mutation-selection codon models are entirely characterized by the collection of site-specific amino-acid fitness profiles. However, thus far, they have relied on the assumption of a constant genetic drift, translating into a unique effective population size (Ne) across the phylogeny, clearly an unreasonable hypothesis. This assumption can be alleviated by introducing variation in Ne between lineages. In addition to Ne, the mutation rate () is susceptible to vary between lineages, and both should co-vary with life-history traits (LHTs). This suggests that the model should more globally account for the joint evolutionary process followed by all of these lineage-specific variables (Ne, , and LHTs). In this direction, we introduce an extended mutation-selection model jointly reconstructing in a Bayesian Monte Carlo framework the fitness landscape across sites and long-term trends in Ne,  and LHTs along the phylogeny, from an alignment of DNA coding sequences and a matrix of observed LHTs in extant species. The model was tested against simulated data and applied to empirical data in mammals, isopods and primates. The reconstructed history of Ne in these groups appears to correlate with LHTs or ecological variables in a way that suggests that the reconstruction is reasonable, at least in its global trends. On the other hand, the range of variation in Ne inferred across species is surprisingly narrow. This last point suggests that some of the assumptions of the model, in particular concerning the assumed absence of epistatic interactions between sites, are potentially problematic.

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Automated Feature Extraction from Large Cardiac Electrophysiological Data Sets RationaleA new multi-electrode array-based application for the long-term recording of action potentials from electrogenic cells makes possible exciting cardiac electrophysiology studies in health and disease. With hundreds of simultaneous electrode recordings being acquired over a period of days, the main challenge becomes achieving reliable signal identification and quantification.

ObjectiveWe set out to develop an algorithm capable of automatically extracting regions of high-quality action potentials from terabyte size experimental results and to map the trains of action potentials into a low-dimensional feature space for analysis.

Methods and ResultsOur automatic segmentation algorithm finds regions of acceptable action potentials in large data sets of electrophysiological readings. We use spectral methods and support vector machines to classify our readings and to extract relevant features. We are able to show that action potentials from the same cell site can be recorded over days without detrimental effects to the cell membrane. The variability between measurements 24 h apart is comparable to the natural variability of the features at a single time point.

ConclusionsOur work contributes towards a non-invasive approach for cardiomyocyte functional maturation, as well as developmental, pathological and pharmacological studies. As the human-derived cardiac model tissue has the genetic makeup of its donor, a powerful tool for individual drug toxicity screening emerges.

sentences: Discovery of μ,δ-Opioid receptor dual biased agonists that overcome the limitation of prior biased agonists Morphine is widely used to manage pain in patients, although the risk of side effects is significant. The use of biased agonists to the G protein of -opioid receptors has been suggested as a potential solution, although Oliceridine and PZM21 have previously failed to demonstrate benefits in clinical studies. An amplification-induced confusion in the process of comparing G protein and beta-arrestin pathways may account for previous biased agonist mis-identification. Here, we have devised a strategy to discover biased agonists with intrinsic efficacy. We computationally simulated 430,000 molecular dockings to the -opioid receptor to construct a compound library. Hits were then verified by experiment. Using the verified compounds, we performed simulations to build a second library with a common scaffold, and selected compounds which show biased features to  and {delta}-opioid receptors through a cell-based assay. Three compounds (ID110460001, ID110460002, and ID110460003) with a dual biased agonistic effect for  and {delta}-opioid receptors were identified. These candidates are full agonists for the -opioid receptor, and they show specific binding modes. Based on our findings, we expect our novel compound to act as a biased agonist than conventional drugs such as Oliceridine.

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A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes.

ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

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The use of high-resolution computed tomography to explore age-related trabecular change in human ribs High-resolution computed tomography was used to explore age-related trabecular change in male human ribs ranging in age from 20-95 years (Mean=55 years, SD=21.634 years) from the Texas State Donated Skeletal Collection (TXSTDSC). Two regions of interest (ROIs), midshaft (50%) and anterior (75%) were extracted from each scan to analyze age-related trabecular change. Dragonfly V4.1 was used to isolate cortical bone volumes of interest (VOIs) and three trabecular VOIs for each ROI; one each along the cutaneous cortex, the center of the medullary cavity, and the pleural cortex. Each trabecular VOI was analyzed for bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular spacing (TbSp), connectivity density (Conn.D), and degree of anisotropy (DA), within and between the 50 and 75% ROIs. Overall, the cutaneous VOIs at both the 50% and 75% ROIs exhibited greater BV/TV, TbTh, and Conn.D when compared to the center and pleural VOIs. All results are consistent with expected biomechanical strain on human ribs. Both trabecular variables and cortical bone volume are only weakly associated with age. These results show that 3D analysis of trabecular bone volume does not improve visualization or understanding of trabecular bone changes with age over traditional 2D methods. Future research should incorporate female samples to explore sex-related trabecular change variation.

HIGHLIGHTSO_LI3D analysis of change in trabecular structure along the rib length and with age
C_LIO_LITrabecular spacing at midshaft shows highest correlations with age-at-death
C_LIO_LIRelative cortical area is more strongly correlated with age in anterior ribs than at midshaft
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sentences: Age-related susceptibility of ferrets to SARS-CoV-2 infection Susceptibility to SARS-CoV-2 and the outcome of COVID-19 have been linked to underlying health conditions and the age of affected individuals. Here we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18-to-39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is [~]32 plaque forming units (PFU) per animal while in young animals it was estimated to be [~]100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets when compared to young animals. Similar to observations in humans, this was associated with higher expressions levels of two key viral entry factors - ACE2 and TMPRSS2 - in the upper respiratory tract of aged ferrets.

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`rbioacc`: an R-package to analyse toxicokinetic data O_LI rbioacc is an  R-package dedicated to the analysis of experimental data collected from bioaccumulation tests during which organisms are exposed to a chemical (exposure phase) and then put into a clean media (depuration phase). Internal concentrations are regularly measured over time all along the experiment.
C_LIO_LI rbioacc provides ready-to-use functions to visualize and fully analyze such data. Under a Bayesian framework, this package fits a generic one-compartment toxicokinetic (TK) model automatically built from the data. It provides TK parameter estimates (appropriate uptake and elimination rates) and bioaccumulation metrics (e.g., BCF, BSAF, BMF). All parameter estimates, bioaccumulation metrics as well as predictions of internal concentrations into organisms are delivered with their uncertainty.
C_LIO_LIBioaccumulation metrics are primarily provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve marketing applications of chemical substances.
C_LIO_LIThis paper gives brief worked examples of the use of  rbioacc from data collected through standard bioaccumulation tests, and publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyze any new data set, uploaded in the right format.
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sentences: Fungal community dynamics and carbon mineralization in coarse woody debris across decay stage, tree species, and stand development stage in northern boreal forests Fungi are primary agents of coarse woody debris (CWD) decay in forests, playing an essential role in nutrient cycling and carbon storage. Characterizing fungal communities within CWD will promote further understanding of the fungal controls on CWD decomposition. We compared fungal community assemblages using alpha and beta diversity metrics, carbon mineralization, and physical and chemical properties of CWD across 3 tree species (trembling aspen [Populus tremuloides], black spruce [Picea mariana], and jack pine [Pinus banksiana]), 5 decay classes, and 2 stand development stages, differing in time-since-stand replacing disturbance (i.e., young/self-thinning and mature/steady-state) in Ontarios boreal forest region. In total, we sampled 180 individual CWD logs from 6 independent stands, with 3 replicates per each species x decay class combination at each site. We found that fungal community structure significantly differed across tree species, decay stage, and stand age. Higher proportions of white rot fungi were found in trembling aspen CWD, whereas higher proportions of brown rot fungi were found in black spruce and jack pine CWD. Proportions of specialized wood decay fungi increased with decay stage and were higher in CWD located in mature forest stands. Fungal diversity was highest in decay class 4 CWD. We found that Mn and K concentrations, total carbon, C/N ratio, carbon mineralization (mg CO2 g dry CWD-1 d-1), and moisture content were important predictors of fungal composition across CWD species and/or decay stage, though how CWD chemistry influences fungal species composition (and vice versa) is unknown. Carbon mineralization was highest in trembling aspen CWD and increased with decay stage, perhaps facilitated by increased N concentrations. This study suggests that forest management guidelines that consider both deadwood quantity and quality will support a broader range of fungal species and communities through post-disturbance stand development, thereby conserving biodiversity over the longer-term in our managed forest systems.

sentences: Electrophysiological measures from human iPSC-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRS) for schizophrenia and a clinical diagnosis of schizophrenia, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures associated with diagnosis that implicated altered Na+ channel function and GABA-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these schizophrenia patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.

sentences: eQTL mapping using allele-specific gene expression Using information from allele-specific gene expression (ASE) can sub-stantially improve the power to map gene expression quantitative trait loci (eQTLs). However, such practice has been limited, partly due to high computational cost and the requirement to access raw data that can take a large amount of storage space. To address these computational challenges, we have developed a computational framework that uses a statistical method named TReCASE as its computational engine, and it is computationally feasible for large scale analysis. We applied it to map eQTLs in 28 human tissues using the data from the Genotype-Tissue Expression (GTEx) project. Compared with a popular linear regression method that does not use ASE data, TReCASE can double the number of eGenes (i.e., genes with at least one significant eQTL) when sample size is relatively small, e.g., n = 200. We also demonstrated how to use the ASE data that we have collected to study dynamic eQTLs whose effect sizes vary with respect to another variable, such as age. We find the majority of such dynamic eQTLs are due to some underlying latent factors, such as cell type proportions. We further compare TReCASE versus another method RASQUAL. TReCASE is ten times or more faster than RASQUAL and it provides more robust type I error control.

sentences: Impact of Salmonella genome rearrangement on gene expression In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to genome structure (GS) identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved towards or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation.

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Genetic architecture underlying HPPD-inhibitor resistance in a Nebraska Amaranthus tuberculatus population BACKGROUNDAmaranthus tuberculatus is a primary driver weed species throughout the American Midwest. Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) are an important chemistry for weed management in numerous cropping systems. Here, we characterize the genetic architecture underlying the HPPD-inhibitor resistance trait in an A. tuberculatus population (NEB).

RESULTSDose-response studies of an F1 generation identified HPPD-inhibitor resistance as a dominant trait with a resistance/sensitive ratio of 15.0-21.1. Segregation analysis in a pseudo-F2 generation determined the trait is moderately heritable (H2 = 0.556), and complex. Bulk segregant analysis and validation with molecular markers identified two quantitative trait loci (QTL), one on each of Scaffold 4 and 12.

CONCLUSIONSResistance to HPPD-inhibitors is a complex, largely dominant trait within the NEB population. Two large-effect QTL were identified controlling HPPD-inhibitor resistance in A. tuberculatus. This is the first QTL mapping study to characterize herbicide resistance in a weedy species.

sentences: The ASC Speck And NLRP3 Inflammasome Function Are Spatially And Temporally Distinct Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation, but facilitates and maximizes IL-1{beta} processing. Further, the NLRP3 agonists H2O2 and MSU elicited IL-1{beta} maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size while speck numbers and IL-1{beta} processing are negatively correlated, cyclical, and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.

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Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy BackgroundIn the U.S., more than 50% of ovarian cancer patients die within 5 years of diagnosis, highlighting the need for innovations such as engineered T cell therapies. Mesothelin (Msln) is an attractive immunotherapy target for this cancer, as it is overexpressed by the tumor and contributes to malignant and invasive phenotypes, making antigen loss disadvantageous to the tumor. We previously showed that adoptively transferred T cells engineered to be Msln-specific (TCR1045) preferentially accumulate within established ovarian tumors, delay tumor growth and significantly prolong survival in the ID8VEGF mouse model. However, T cell persistence and anti-tumor activity were not sustained, and we and others have previously detected FasL in the tumor vasculature and the tumor microenvironment (TME) of human and murine ovarian cancers, which can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas).

MethodsTo concurrently overcome this mechanism for potential immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. T cells engineered to express TCR1045 alone or in combination with the IFP were transferred into ID8VEGF-tumor bearing mice and evaluated for persistence, proliferation, anti-tumor cytokine production, and therapeutic efficacy.

ResultsRelative to T cells modified only to express TCR1045, T cells engineered to express both TCR1045 and a Fas IFP preferentially persisted in the TME of tumor-bearing mice due to improved T cell proliferation and survival. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCR1045 T cells lacking the IFP.

ConclusionsFas/FasL signaling can mediate T cell death in the ovarian cancer microenvironment, as well as induce activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature represents a mechanism for protecting growing tumors from attack by tumor-infiltrating lymphocytes. As many solid tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

sentences: Kinetic Monitoring Of Neuronal Stress Response To Proteostasis Dysfunction Proteostasis dysfunction and activation of the unfolded protein response (UPR) are characteristic of all major neurodegenerative diseases. Nevertheless, although the UPR and proteostasis dysfunction has been studied in great detail in model organisms like yeast and mammalian cell lines, it has not yet been examined in neurons. In this study, we applied a viral vector-mediated expression of a reporter protein based on a UPR transcription factor, ATF4, and time-lapse fluorescent microscopy to elucidate how mouse primary neurons respond to pharmacological and genetic perturbations to neuronal proteostasis. In in vitro models of endoplasmic reticulum (ER) stress and proteasome inhibition, we used the ATF4 reporter to reveal the time course of the neuronal stress response relative to neurite degeneration and asynchronous cell death. We showed how potential neurodegenerative disease co-factors, ER stress and mutant -synuclein overexpression, impacted neuronal stress response and overall cellular health. This work therefore introduces a viral vector-based reporter that yields a quantifiable readout suitable for non-cell destructive kinetic monitoring of proteostasis dysfunction in neurons by harnessing ATF4 signaling as part of the UPR activation.

sentences: Pharmacokinetics of Orally Administered GS-441524 in Dogs Despite being FDA-approved for COVID-19, the clinical efficacy of remdesivir (Veklury(R)) remains contentious. We previously pointed out pharmacokinetic, pharmacodynamic and toxicology reasons for why its parent nucleoside GS-441524, is better suited for COVID-19 treatment. Here, we assess the oral bioavailability of GS-441524 in beagle dogs and show that plasma concentrations [~]24-fold higher than the EC50 against SARS-CoV-2 are easily and safely sustained. These data support translation of GS-441524 as an oral agent for COVID-19.

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ksrates: positioning whole-genome duplications relative to speciation events using rate-adjusted mixed paralog-ortholog KS distributions SummaryWe present ksrates, a user-friendly command-line tool to position ancient whole-genome duplication (WGD) events with respect to speciation events in a phylogeny by comparing paralog and ortholog KS distributions derived from genomic or transcriptomic sequences, while adjusting for substitution rate differences among the lineages involved.

Availability and implementationksrates is implemented in Python 3 and as a Nextflow pipeline. The source code, Singularity and Docker containers, documentation and tutorial are available via https://github.com/VIB-PSB/ksrates.

Contactsteven.maere@ugent.vib.be, rolf.lohaus@ugent.vib.be

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Accurate self-assessment in 2D: protein contact map quality estimation by deep evolutionary reconciliation MotivationProtein contact maps have proven to be a valuable tool in the deep learning revolution of protein structure prediction, ushering in the recent breakthrough by AlphaFold2. However, self-assessment of the quality of predicted structures is typically performed at the granularity of 3D coordinates as opposed to directly exploiting the rotation- and translation-invariant 2D contact maps.

ResultsHere we present rrQNet, a deep learning method for self-assessment in 2D by contact map quality estimation. Our approach is based on the intuition that for a contact map to be of high quality, the residue pairs predicted to be in contact should be mutually consistent with the evolutionary context of the protein. The deep neural network architecture of rrQNet implements this intuition by cascading two deep modules--one encoding the evolutionary context and the other performing evolutionary reconciliation. The penultimate stage of rrQNet estimates the quality scores at the interacting residue-pair level, which are then aggregated for estimating the quality of a contact map. This design choice offers versatility at varied resolutions from individual residue pairs to full-fledged contact maps. Trained on multiple complementary sources of contact predictors, rrQNet facilitates generalizability across various contact maps. By rigorously testing using publicly available datasets and comparing against several in-house baseline approaches, we show that rrQNet accurately reproduces the true quality score of a predicted contact map and successfully distinguishes between accurate and inaccurate contact maps predicted by a wide variety of contact predictors.

AvailabilityThe open-source rrQNet software package is freely available at https://github.com/Bhattacharya-Lab/rrQNet.

Contactdbhattacharya@vt.edu

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Quantitative Measurement of Diffusion-weighted Imaging Signal Using Expression-controlled Aquaporin-4 Cells: Comparative Study of 2-compartment and Diffusion Kurtosis Imaging Models PurposeThe purpose of this study was to compare parameter estimates for the 2-compartment (2Comp) and diffusion kurtosis imaging (DKm) models obtained from diffusion-weighted imaging (DWI) of aquaporin-4 (AQP4) expression-controlled cells, and to look for biomarkers that indicate differences in the cell membrane water permeability.

MethodsDWI was performed on AQP4-expressing and non-expressing cells and the signal was analyzed with the 2Comp and DKm models. For the 2Comp model, the diffusion coefficients (Df, Ds) and volume fractions (Ff, Fs, Ff=1-Fs) of the fast and slow compartments were estimated. For the DKm model, estimates of the diffusion kurtosis (K) and corrected diffusion coefficient (D) were obtained.

ResultsFor the 2Comp model, Ds and Fs showed clear differences between AQP4-expressing and non-expressing cells. Fs was also sensitive to cell density. There was no clear relationship with the cell type for the DKm parameters.

ConclusionsChanges to cell membrane water permeability due to AQP4 expression affected DWI of cell suspensions. For the 2Comp and DKm models, Ds was the parameter most sensitive to differences in AQP4 expression.

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Phenotypic melanoma heterogeneity is regulated through cell-matrix interaction-dependent changes in tumor microarchitecture Phenotypic heterogeneity of cancer cells plays a critical role in shaping treatment response. This type of heterogeneity is organized spatially with specific phenotypes, such as sharply demarcated clusters of proliferating and cell cycle-arrested cells, predominating within discrete domains within a tumor. What determines the occurrence of specific tumor cell phenotypes in distinct microdomains of solid cancers is poorly understood. Here, we show that in melanoma spatial organization of phenotypic heterogeneity is dictated by the expression and activity of MITF. We reveal that this lineage survival oncogene controls ECM composition and organization, and ROCK-driven mechanotransduction through focal adhesion maturation and actin cytoskeleton functionality. In turn, altered tumor microarchitecture and structural integrity impact tumor solid stress which then mediates phenotypic heterogeneity through p27Kip1. Rho-ROCK-myosin signaling is necessary to transmit the effect of the reciprocal cell-ECM regulation into phenotypic heterogeneity. Our findings place cell-ECM crosstalk as a central driver of phenotypic tumor heterogeneity.

SignificancePhenotypic heterogeneity is a major culprit of cancer therapy failure. We demonstrate that phenotypic heterogeneity is controlled through tumor cell-ECM crosstalk resulting in altered tumor microarchitecture, mechanotransduction and Rho-ROCK-myosin signaling. Melanoma shares these physical properties with any solid cancer underscoring the importance of our findings for therapeutically targeting this phenomenon.

sentences: The clinical and functional effects of TERT variants in myelodysplastic syndrome Germline pathogenic TERT variants are associated with short telomeres and an increased risk of developing myelodysplastic syndrome (MDS) among patients with a telomere biology disorder. We identified TERT rare variants in 41 of 1514 MDS patients (2.7%) without a clinical diagnosis of telomere biology disorder who underwent allogeneic transplantation. Patients with TERT rare variants had shorter telomere length (p<0.001) and younger age at MDS diagnosis (52 vs. 59 years, p=0.03) than patients without a TERT rare variant. In multivariable analyses, TERT rare variants were associated with inferior overall survival (p=0.034) driven by an increased incidence of non-relapse mortality (NRM) (p=0.015). Death from a non-infectious pulmonary cause was more frequent among patients with a TERT rare variant. According to ACMG/AMP guidelines and Sherloc criteria, 39 TERT rare variants were classified as VUS and one as likely pathogenic. Therefore, we cloned all rare missense variants and quantified their impact on telomere elongation in a cell-based assay. We found that 36 of 40 variants had severe or intermediate impairment in their capacity to elongate telomeres. Using a homology model of human TERT bound to the shelterin protein TPP1, we inferred that TERT rare variants disrupt domain-specific functions, including catalysis, protein-RNA interactions, and recruitment to telomeres. Our results indicate that the contribution of TERT rare variants to MDS pathogenesis and NRM risk is underrecognized and routine screening for TERT rare variants in MDS patients regardless of age or clinical suspicion could identify clinically inapparent telomere biology disorders and improve transplant outcomes through risk-adapted approaches.

sentences: Serotonin modulates learning content-specific neuroplasticity of functional brain networks Learning-induced neuroplastic changes, further modulated by content and setting, are mirrored in brain functional connectivity (FC). In animal models, serotonergic agents were shown to facilitate neuroplasticity. This is especially prominent during emotional relearning, such as fear extinction, which may translate to clinical improvements in human patients. To investigate this assumption, 99 healthy subjects underwent six weeks of emotional or non-emotional learning and subsequent relearning. Resting-state functional magnetic resonance imaging was performed before and after the learning phases to investigate changes in FC. During relearning, subjects received either a daily dose of the selective serotonin reuptake inhibitor (SSRI) escitalopram or placebo. Escitalopram intake modulated FC changes in a network comprising Brocas area, the medial prefrontal cortex, the right inferior temporal and left lingual gyrus. More specifically, escitalopram increased the bidirectional connectivity between medial prefrontal cortex and lingual gyrus for non-emotional and additionally the connectivity from medial prefrontal cortex to Brocas area for emotional relearning. The context-dependence of these effects supports the assumption that SSRIs in clinical practice might improve neuroplasticity rather than psychiatric symptoms per se. Correlations with learning behavior further point towards a relationship with extinction processes in relearning. These results demonstrate that escitalopram intake during relearning results in content-dependent network adaptations and support the conclusion that enhanced neuroplasticity might be the major underlying mechanism also in humans. Beyond expanding the complexities of learning, these findings emphasize the influence of external factors on serotonin-facilitated neuroplasticity of the human brain.

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Motor outcomes congruent with intentions may sharpen metacognitive representations We can monitor our intentional movements, in order to describe how we move our bodies. But it is unclear which information this metacognitive monitoring relies on. For example, when throwing a ball to hit a target, we might use the visual information about how the ball flew to metacognitively assess our performance. Alternatively, we might disregard the ball trajectory -- given that it is not directly relevant to our goal -- and metacognitively assess our performance based solely on whether we reached the goal of hitting the target. In two experiments we aimed to distinguish between these two alternatives and asked whether the distal outcome of a goal-directed action (hitting or missing a target) informs the metacognitive representations of our own movements. Participants performed a semi-virtual task where they moved their arm to throw a virtual ball at a target. After each throw, participants discriminated which of two ball trajectories displayed on the screen corresponded to the flight path of their throw and then rated their confidence in this decision. The task included two conditions that differed on whether the distal outcome of the two trajectories shown matched (congruent) or differed (incongruent). Participants were significantly more accurate in discriminating between the two trajectories, and responded faster in the incongruent condition and, accordingly, were significantly more confident on these trials. Crucially, we found significant differences in metacognitive performance (measured as meta-d/d) between the two conditions only on successful trials, where the virtual ball had hit the target. These results indicate that participants successfully incorporated information about the outcome of the movement into both their discrimination and confidence responses. However, information about the outcome selectively sharpened the precision of confidence ratings only when the outcome of their throw matched their intention. We argue that these findings underline the separation between the different levels of information that may contribute to body monitoring, and we provide evidence that intentions might play a central role in metacognitive motor representations.

HighlightsO_LIParticipants threw a virtual ball to hit a target
C_LIO_LIFollowing their throw participants selected between two plausible trajectories
C_LIO_LIOn half the trials, the two alternative trajectories differed in their distal outcome.
C_LIO_LIDiscrimination and confidence were higher in trials with different outcome.
C_LIO_LIMetacognitive performance was best in hit trials when the alternative missed.
C_LI

sentences: High Fat Diet Blunts Stress-Induced Hypophagia and Activation of Glp1r Dorsal Lateral Septum Neurons in Male but not in Female Mice While stress typically reduces caloric intake (hypophagia) in chow-fed rodents, presentation of palatable, high calorie substances during stress can increase caloric consumption (i.e. "comfort feeding") and promote obesity. However, little is known about how obesity itself affects feeding behavior in response to stress and the mechanisms that can influence stress-associated feeding in the context of obesity. We show that lean male mice display the expected hypophagic response following acute restraint stress, but obese male mice are resistant to this acute stress-induced hypophagia. Activation of the glucagon-like peptide-1 (Glp1) receptor (Glp1r) in various brain regions leads to hypophagia in response to stress. Here we show that Glp1r-positive neurons in the dorsal lateral septum (dLS) are robustly activated during acute restraint stress in lean but not in obese male mice. This raises the possibility that activation of dLS Glp1r neurons during restraint stress contributes to subsequent hypophagia. Supporting this, we show that chemogenetic inhibition of dLS Glp1r neurons attenuates acute restraint stress hypophagia in male mice. Surprisingly, we show that both lean and obese female mice are resistant to acute restraint stress-induced hypophagia and activation of dLS Glp1r neurons. Taken together, these results suggest that dLS Glp1r neurons contribute to the hypophagic response to acute restraint stress in male mice, but not in female mice, and that obesity disrupts this response in male mice. Broadly, these findings show sexually dimorphic mechanisms and feeding behaviors in lean vs. obese mice in response to acute stress.

sentences: The phosphate starvation response recruits the TOR pathway to regulate growth in Arabidopsis cell cultures AO_SCPLOWBSTRACTC_SCPLOWIn eukaryotes, TOR (Target Of Rapamycin) is a conserved regulator of growth that integrates both endogenous and exogenous signals. These signals include the internal nutritional status, and in plants, TOR has been shown to be regulated by carbon, nitrogen and sulfur availability. In this study, we show that in Arabidopsis the TOR pathway also integrates phosphorus availability to actively modulate the cell cycle, which in turn regulates the intracellular content of amino acids and organic acids. We observed a substantial overlap between the phenotypic, metabolic and transcriptomic responses of TOR inactivation and phosphorus starvation in Arabidopsis cell culture. Although phosphorus availability modulates TOR activity, changes in the levels of TOR activity do not alter the expression of marker genes for phosphorus status. These data prompted us to place the sensing of phosphorus availability upstream of the modulation of TOR activity which, in turn, regulates the cell cycle and primary metabolism to adjust plant growth in plants.

sentences: Bayesian Parameter Estimation for Dynamical Models in Systems Biology Dynamical systems modeling, particularly via systems of ordinary differential equations, has been used to effectively capture the temporal behavior of different biochemical components in signal transduction networks. Despite the recent advances in experimental measurements, including sensor development and  -omics studies that have helped populate protein-protein interaction networks in great detail, systems biology modeling lacks systematic methods to estimate kinetic parameters and quantify associated uncertainties. This is because of multiple reasons, including sparse and noisy experimental measurements, lack of detailed molecular mechanisms underlying the reactions, and missing biochemical interactions. Additionally, the inherent nonlinearities with respect to the states and parameters associated with the system of differential equations further compound the challenges of parameter estimation. In this study, we propose a comprehensive framework for Bayesian parameter estimation and complete quantification of the effects of uncertainties in the data and models. We apply these methods to a series of signaling models of increasing mathematical complexity. Systematic analysis of these dynamical systems showed that parameter estimation depends on data sparsity, noise level, and model structure, including the existence of multiple steady states. These results highlight how focused uncertainty quantification can enrich systems biology modeling and enable additional quantitative analyses for parameter estimation.

sentences: Analysis of variability of functionals of recombinant protein production trajectories based on limited data We propose a method for analyzing the variability in smooth, possibly nonlinear, functionals associated with a set of product production trajectories measured under different experimental conditions. The key challenge is to make meaningful inference on these parameters across different experimental conditions when only a limited number of measurements in time are collected for each treatment, and when there are only a few, or no, replicates available. For this purpose we adopt a modeling approach by representing the production trajectories in a B-spline basis, and develop a bootstrap-based inference procedure for the parameters of interest, also accounting for multiple comparisons. The methodology is applied to study two types of quantities of interest - "time to harvest" and "maximal productivity" in the context of an experiment on the production of certain recombinant proteins under laboratory conditions. We complement the findings by extensive numerical experiments that look into the effects of different types of bootstrap procedures and associated schemes for computing p-values for tests of hypotheses.

sentences: Unravelling complex molecular mechanisms in pine with genome-wide association analysis and gene co-expression networks The authors have withdrawn this manuscript because they decided to change the phenotypic characteristics to be evaluated, considering the more significant contribution to the field. Therefore, the authors do not want this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

sentences: Using bootstrap procedures for testing the modular partition inferred via leading eigenvector community detection algorithm Modularity and modular structures can be recognized at various levels of biological organization and in various domains of studies. Recently, algorithms based on network analysis came into focus. And while such a framework is a powerful tool in studying modular structure, those methods usually pose a problem of assessing statistical support for the obtained modular structures. One of the widely applied methods is the leading eigenvector, or Newmans spectral community detection algorithm. We conduct a brief overview of the method, including a comparison with some other community detection algorithms and explore a possible fine-tuning procedure. Finally, we propose an adapted bootstrap-based procedure based on Shimodairas multiscale bootstrap algorithm to derive approximately unbiased p-values for the module partitions of observations datasets. The proposed procedure also gives a lot of freedom to the researcher in constructing the network construction from the raw numeric data, and can be applied to various types of data and used in diverse problems concerning modular structure. We provide an R language code for all the calculations and the visualization of the obtained results for the researchers interested in using the procedure.

sentences: Network Analysis Reveals Different Cellulose Degradation Strategies across Trichoderma harzianum Strains Associated with XYR1 and CRE1 Trichoderma harzianum, whose gene expression is tightly controlled by the transcription factors (TFs) XYR1 and CRE1, is a potential candidate for hydrolytic enzyme production. Here, we performed a network analysis of T. harzianum IOC-3844 and T. harzianum CBMAI-0179 to explore how the regulation of these TFs varies between these strains. In addition, we explored the evolutionary relationships of XYR1 and CRE1 protein sequences among Trichoderma spp. The results of the T. harzianum strains were compared with those of Trichoderma atroviride CBMAI-0020, a mycoparasitic species. Although transcripts encoding carbohydrate-active enzymes (CAZymes), TFs, transporters, and proteins with unknown functions were coexpressed with cre1 or xyr1, other proteins indirectly related to cellulose degradation were identified. The enriched GO terms describing the transcripts of these groups differed across all strains, and several metabolic pathways with high similarity between both regulators but strain-specific differences were identified. In addition, the CRE1 and XYR1 subnetworks presented different topology profiles in each strain, likely indicating differences in the influences of these regulators according to the fungi. The hubs of the cre1 and xyr1 groups included transcripts not yet characterized or described as being related to cellulose degradation. The first-neighbor analyses confirmed the results of the profile of the coexpressed transcripts in cre1 and xyr1. The analyses of the shortest paths revealed that CAZymes upregulated under cellulose degradation conditions are most closely related to both regulators, and new targets between such signaling pathways were discovered. Although the evaluated T. harzianum strains are phylogenetically close and their amino acid sequences related to XYR1 and CRE1 are very similar, the set of transcripts related to xyr1 and cre1 differed, suggesting that each T. harzianum strain used a specific regulation strategy for cellulose degradation. More interestingly, our findings may suggest that XYR1 and CRE1 indirectly regulate genes encoding proteins related to cellulose degradation in the evaluated T. harzianum strains. An improved understanding of the basic biology of fungi during the cellulose degradation process can contribute to the use of their enzymes in several biotechnological applications and pave the way for further studies on the differences across strains of the same species.

sentences: Insights into biotic and abiotic modulation of ocean mesopelagic communities Marine plankton mitigate anthropogenic greenhouse gases, modulate biogeochemical cycles, and provide fishery resources. Plankton is distributed across a stratified ecosystem of sunlit surface waters and a vast, though understudied, mesopelagic  dark ocean. In this study, we mapped viruses, prokaryotes, and pico-eukaryotes across 32 globally-distributed cross-depth samples collected during the Tara Oceans Expedition, and assessed their ecologies. Based on depth and O2 measurements, we divided the marine habitat into epipelagic, oxic mesopelagic, and oxygen minimum zone (OMZ) eco-regions. We identified specific communities associated with each marine habitat, and pinpoint environmental drivers of dark ocean communities. Our results indicate that water masses primarily control mesopelagic community composition. Through co-occurrence network inference and analysis, we identified signature communities strongly associated with OMZ eco-regions. Mesopelagic communities appear to be constrained by a combination of factors compared to epipelagic communities. Thus, variations in a given abiotic factor may cause different responses in sunlit and dark ocean communities. This study expands our knowledge about the ecology of planktonic organisms inhabiting the mesopelagic zone.

sentences: Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

sentences: Chromodomain protein regulates the expression of a subset of RIFINs in P. falciparum Plasmodium falciparum expresses clonally variant proteins on the surface of infected erythrocytes to evade the host immune system. The clonally variant multigenes include var, rifin, and stevor, which express EMP1, RIFIN, and STEVOR proteins, respectively. The rifins are the largest multigene family and are essentially involved in the RBC rosetting, the hallmark of severe malaria. The regulators that control the RIFINs expression in P. falciparum have not been reported so far. This study reports a chromodomain-containing protein (PfCDP) that binds to H3K9me3 modification on P. falciparum chromatin. The ChIP- sequencing analysis revealed that the PfCDP is majorly associated with clonally variant gene families, primarily rifins in P. falciparum. Conditional deletion of the chromodomain (CD) gene in P. falciparum leads to the up-regulation of a subset of virulence genes, including rifins, a few var, and stevor genes. Further, we show that Pf{Delta}CDP P. falciparum lines promote the RBC rosetting. This study provides evidence of an epigenetic regulator mediated control on a subset of RIFINs expression and RBC rosetting by P. falciparum.

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A Scenic River in Rural Ohio Shows Elevated Antibiotic Resistance Genes, Including Acinetobacter Tetracycline and Macrolide Resistance, Downstream of Wastewater Treatment Plant Effluent The entry of antibiotic resistance genes (ARGs) into aquatic systems has been documented for large municipal wastewater treatment plants, but there is less study of the impact of smaller plants that are situated on small rural rivers. We sampled water metagenomes for ARG and taxa composition from the Kokosing River, a small rural river in Knox County, Ohio, which has been designated an Ohio State Scenic River for retention of natural character. Samples were obtained 1.0 km upstream, 120 m downstream, and 6.4 km downstream from the effluent release of the Mount Vernon wastewater treatment plant (WWTP). ARGS were identified in metagenomes using ShortBRED markers from the CARD database screened against UniPROT. Through all seasons, the metagenome just downstream of the WWTP effluent showed a substantial elevation of at least 15 different ARGs, including 6 ARGs commonly associated with Acinetobacter baumannii such as msrE, mphE (macrolide resistance) and tet(39) (tetracycline resistance). The ARGs most prevalent near the effluent pipe persisted 6.4 km downriver. Using MetaPhlAn2 clade-specific marker genes, the taxa distribution near the effluent showed elevation of reads annotated as Acinetobacter species as well as gut-associated taxa, Bacteroides and Firmicutes. The ARG levels and taxa prevalence showed little dependence on seasonal chlorination of the effluent. Nitrogen and phosphorus were elevated near the effluent pipe but had no consistent correlation with ARG levels. We show that in a rural river microbiome, year-round wastewater effluent substantially elevates ARGs including those associated with multidrug-resistant A. baumanii.

IMPORTANCEAntibiotic resistance is a growing problem worldwide, with frequent transmission between pathogens and environmental organisms. Rural rivers can support high levels of recreational use by people unaware of inputs from treated wastewater, while WWTPs can generate a small but significant portion of flow volume into a river surrounded by forest and agriculture. There is little information on the rural impacts of WWTP effluent on the delivery and transport of antibiotic resistance genes. In our study, the river water proximal to wastewater effluent shows evidence for the influx of multidrug-resistant Acinetobacter baumanii, an opportunistic pathogen of concern for hospitals but also widespread in natural environments. Our work highlights the importance of wastewater effluent in management of environmental antibiotic resistance, even in high quality, rural river systems.

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Graphene Oxide reinforced Agarose-Hydroxyapatite Bioprinted 3-D Scaffolds for Bone Regeneration Three-dimensional (3D) bioprinting is an emerging technology for fabricating cells, biomaterials and extracellular matrix (ECM) into customized shapes and patterns. Here, we report additive manufacturing to create a customized 3D bioactive constructs for regenerative medicine. We have attempted to emphasize the use of agarose and graphene oxide as a promising material for the conceptualization of bioink unpaid to its unique physicochemical properties. The 3D printed structure is able to regenerating bone tissues and regulates the cellular differentiation without any significant morphological changes. The presence of graphene oxide enhances the osteoinductive behavior of the developed scaffolds, which is further supplemented by encapsulating human mesenchymal stem cells (hMSCs) on the 3D printed scaffolds.

A significant enhanced expression of early osteogenic markers like morphogenetic protein (BMP), Runx-2, collagen-1, osteopontin, osteocalcin as well as mineralized ECM are observed on agarose-hydroxyapatite and graphene oxide 3D printed scaffolds compared to agarose-hydroxyapatite 3D printed scaffolds. Thus, the outcomes of the developed 3D bioprinted scaffolds provide a promising strategy for development of personalized bone grafts for tissue regeneration.

sentences: A human monoclonal antibody potently pan-neutralizes SARS-CoV-2 VOCs by targeting RBD invariant sites The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). The neutralizing monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are among the most promising strategies to prevent and treat COVID-19. However, SARS-CoV-2 variants of concern (VOCs) profoundly reduced the efficacies of most of mAbs and vaccines approved for clinical use. Herein, we demonstrated mAb 35B5 efficiently neutralizes both wild-type (WT) SARS-CoV-2 and VOCs, including B.1.617.2 (delta) variant, in vitro and in vivo. Cryo-electron microscopy (cryo-EM) revealed that 35B5 neutralizes SARS-CoV-2 by targeting a unique epitope that avoids the prevailing mutation sites on RBD identified in circulating VOCs, providing the molecular basis for its pan-neutralizing efficacy. The 35B5-binding epitope could also be exploited for the rational design of a universal SARS-CoV-2 vaccine.

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Toolkit for Oscillatory Real-time Tracking and Estimation (TORTE) BackgroundClosing the loop between brain activity and behavior is one of the most active areas of development in neuroscience. There is particular interest in developing closed-loop control of neural oscillations. Many studies report correlations between oscillations and functional processes. Oscillation-informed closed-loop experiments might determine whether these relationships are causal and would provide important mechanistic insights which may lead to new therapeutic tools. These closed-loop perturbations require accurate estimates of oscillatory phase and amplitude, which are challenging to compute in real time.

New MethodWe developed an easy to implement, fast and accurate Toolkit for Oscillatory Real-time Tracking and Estimation (TORTE). TORTE operates with the open-source Open Ephys GUI (OEGUI) system, making it immediately compatible with a wide range of acquisition systems and experimental preparations.

ResultsTORTE efficiently extracts oscillatory phase and amplitude from a target signal and includes a variety of options to trigger closed-loop perturbations. Implementing these tools into existing experiments is easy and adds minimal latency to existing protocols.

Comparison with Existing MethodsMost labs use in-house lab-specific approaches, limiting replication and extension of their experiments by other groups. Accuracy of the extracted analytic signal and accuracy of oscillation-informed perturbations with TORTE match presented results by these groups. However, TORTE provides access to these tools in a flexible, easy to use toolkit without requiring proprietary software.

ConclusionWe hope that that the availability of a high-quality, open-source, and broadly applicable toolkit will increase the number of labs able to perform oscillatory closed-loop experiments, and will improve the replicability of protocols and data across labs.

HighlightsO_LITORTE provides a toolkit to investigate closed loop oscillation-informed experiments.
C_LIO_LIThe toolkit is versatile and open-source promoting replicability across scientists.
C_LIO_LIThe analytic signal algorithm within TORTE preforms equally to existing algorithms.
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sentences: Visual space curves before eye movements In most experiments within the field of cognitive and systems neuroscience, fixation is often required prior to the onset of an experimental trial. However, the term "fixation" is rather misleading since our eyes are constantly moving. One type of transient miniature movement ubiquitously observed during fixation is commonly referred to as fixational eye movements or microsaccades. Perimicrosaccadic compression of visual space -- the ability of retinotopic cells to transiently exhibit predictive spatiotemporal retinotopic compressive shifts toward the target of an impending microsaccade -- is known to dramatically alter visual perception. However, whether perimicrosaccadic compressive shifts can become spatially asymmetric, that is, continuously directed toward a specific foveal region over another (e.g., an upper over a lower region in the fovea) and for what purpose, remains poorly understood. Assuming that these transient shifts are indeed asymmetric under certain conditions, the perceptual and oculomotor consequences such asymmetricity might accompany across visual space is poorly understood. Equally unaccounted for is a mechanistic account of the neural computation and architecture that could support these transient asymmetric shifts while the visual system actively maintains retinotopic organization. Here, we systematically measured visual sensitivity in human subjects to faint probes presented during fixation and around the time of saccadic eye movement at geometrically symmetric retinotopic locations in the foveal, parafoveal, and peripheral regions of visual space. Remarkably, we observed transient local asymmetric visual sensitivity differences between these symmetric retinotopic locations where none should be observed. Equally surprising, we observed the trajectories of saccadic eye movements, which are expected to travel along a linear path, routinely deviate along a curved path toward orthogonal eccentric locations. To provide a mechanistic account of the neural computation and architecture that may explain our results, we proposed a novel neurobiologically inspired phenomenological force field model in which underlying attentional and oculomotor signals are modulated by transient eccentric error signals that manifest as temporally overlapping predictive forces and impinge on the retinotopic visual cortex. These forces, which transiently bias putative population sensitivity toward an orthogonal retinotopic foveal region and, around the time of a saccadic eye movement, along an axis orthogonal to the saccade direction toward a mislocalized peripheral region, succinctly capture the essence of our empirical observations.

sentences: Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators. Transcriptional regulators based on CRISPR architecture expand our ability of reprogramming endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely naringenin, eriodictyol, kaempferol and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programs for each of the four metabolites, each program activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation program showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples in five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for custom-made design of metabolic contents in plants.

sentences: Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated with Anti-Tumor Immunity Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1- PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

sentences: Antisense-mediated repression of SAGA-dependent genes involves the HIR histone chaperone Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae (S. cerevisiae), TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.

sentences: Genomic Mutations and Changes in Protein Secondary Structure and Solvent Accessibility of SARS-CoV-2 (COVID-19 Virus) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic virus that has caused the global COVID-19 pandemic. Tracing the evolution and transmission of the virus is crucial to respond to and control the pandemic through appropriate intervention strategies. This paper reports and analyses genomic mutations in the coding regions of SARS-CoV-2 and their probable protein secondary structure and solvent accessibility changes, which are predicted using deep learning models. Prediction results suggest that mutation D614G in the virus spike protein, which has attracted much attention from researchers, is unlikely to make changes in protein secondary structure and relative solvent accessibility. Based on 6,324 viral genome sequences, we create a spreadsheet dataset of point mutations that can facilitate the investigation of SARS-CoV-2 in many perspectives, especially in tracing the evolution and worldwide spread of the virus. Our analysis results also show that coding genes E, M, ORF6, ORF7a, ORF7b and ORF10 are most stable, potentially suitable to be targeted for vaccine and drug development.

sentences: Testing a theoretical framework for the environment-species abundance paradigm: a new approach to the Abundant Centre Hypothesis The linkage between environment, a species fitness and its abundance is central to the theory of evolution. So far, all studies of this linkage have been heuristic and empirical due to an inability to determine fitness either experimentally (independent of abundance) or theoretically (from species-environment interaction). One category of such studies involves the Abundant Centre Hypothesis which posits that a species abundance rises to a maximum at the centre of its range. We argue that the confusing mix of results from ACH studies arises from ignoring the central premise that the abundance distribution cannot be independent of the environment. First, we employed a theoretical framework to identify an environmental context (an elevational transect; 200-2800 m in the eastern Himalayas) likely to favour ACH. We then improved upon some previously identified conceptual and methodological shortcomings of ACH studies. Using systematically collected bird data (245 species; 15867 records) from that transect we found that the community average abundance profile is symmetric, as expected by ACH. Notwithstanding which, the abundance profiles of individual species showed a small degree of asymmetry which was correlated with elevation. This elevational dependence may be due to the hard elevational limits at the lower and upper ends of the mountain, as expected from theoretical considerations. We also showed that the average abundance profile shape is close to gaussian, while ruling out uniform and inverted-quadratic shapes. This work demonstrates that selecting a particular category of environmental contexts can help in integrating theoretical tools into a field dominated by empirical studies. Such a union should spur the development of more detailed and testable theoretical models for better insights in an important field.

sentences: Biologically informed deep learning to infer gene program activity in single cells The increasing availability of large-scale single-cell datasets has enabled the detailed description of cell states across multiple biological conditions and perturbations. In parallel, recent advances in unsupervised machine learning, particularly in transfer learning, have enabled fast and scalable mapping of these new single-cell datasets onto reference atlases. The resulting large-scale machine learning models however often have millions of parameters, rendering interpretation of the newly mapped datasets challenging. Here, we propose expiMap, a deep learning model that enables interpretable reference mapping using biologically understandable entities, such as curated sets of genes and gene programs. The key concept is the substitution of the uninterpretable nodes in an autoencoders bottleneck by labeled nodes mapping to interpretable lists of genes, such as gene ontologies, biological pathways, or curated gene sets, for which activities are learned as constraints during reconstruction. This is enabled by the incorporation of predefined gene programs into the reference model, and at the same time allowing the model to learn de novo new programs and refine existing programs during reference mapping. We show that the model retains similar integration performance as existing methods while providing a biologically interpretable framework for understanding cellular behavior. We demonstrate the capabilities of expiMap by applying it to 15 datasets encompassing five different tissues and species. The interpretable nature of the mapping revealed unreported associations between interferon signaling via the RIG-I/MDA5 and GPCRs pathways, with differential behavior in CD8+ T cells and CD14+ monocytes in severe COVID-19, as well as the role of annexins in the cellular communications between lymphoid and myeloid compartments for explaining patient response to the applied drugs. Finally, expiMap enabled the direct comparison of a diverse set of pancreatic beta cells from multiple studies where we observed a strong, previously unreported correlation between the unfolded protein response and asparagine N-linked glycosylation. Altogether, expiMap enables the interpretable mapping of single cell transcriptome data sets across cohorts, disease states and other perturbations.

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Graded variation in cortical T1w/T2w myelination during adolescence Myelination influences brain connectivity during sensitive periods of development by enhancing neural signaling speed and regulating synapse formation to reduce plasticity. However, in vivo studies characterizing the maturational timing of cortical myelination during human development remain scant. Here, we take advantage of recent advances in high-resolution cortical T1w/T2w myelin mapping methods, including principled correction of B1+ transmit field effects, using data from the Human Connectome Project in Development (N=628, ages 8-21) to characterize the maturational timing of myelination from childhood through early adulthood throughout the cerebral neocortex. We apply Bayesian spline models and functional latent clustering analysis to demonstrate graded variation in the rate of cortical T1w/T2w myelin growth in neocortical areas that is strongly correlated with the sensorimotor-association (S-A) axis of cortical organization reported by others. In sensorimotor areas T1w/T2w myelin starts at high levels at early ages, increases at a fast pace, and decelerates at later ages (18-21). In intermediate multimodal areas along the S-A axis, T1w/T2w myelin tends to start at intermediate levels and increase linearly at an intermediate pace. In transmodal/paralimbic association areas high along the S-A axis, T1w/T2w myelin tends to start at low levels and increase linearly at the slowest pace. These data provide evidence for graded variation along the S-A axis in the rate of cortical myelination during adolescence, which could reflect ongoing plasticity underlying the development of complex information processing and psychological functioning.

Significance StatementMyelin is a lipid membrane that is essential to healthy brain function. Myelin wraps axons to increase neural signaling speed, enabling complex neuronal functioning underlying learning and cognition. Here we characterize the developmental timing of myelination across the cerebral cortex during adolescence using recent advances in non-invasive myelin mapping. Our results provide new evidence demonstrating graded variation across the cortex in the timing of myelination during adolescence, with rapid myelination in lower-order sensory areas and gradual myelination in higher-order association areas. This spatial pattern of microstructural brain development closely parallels the sensorimotor-to-association axis of cortical organization and plasticity during ontogeny.

sentences: MacroH2A impedes metastatic growth by enforcing a discrete dormancy program in disseminated cancer cells AO_SCPLOWBSTRACTC_SCPLOWMacroH2A variants have been associated with tumor suppression through inhibition of proliferation and metastasis, as well as their role in cellular senescence. However, their role in regulating the dormant state of disseminated cancer cells (DCCs) remains unclear. Here we reveal that solitary dormant DCCs display increased levels of macroH2A variants in head and neck squamous cell carcinoma PDX models and patient samples compared to proliferating primary or metastatic lesions. We further demonstrate that microenvironmental and stress adaptive signals such as TGF{beta}2 and p38/{beta}, which induce DCC dormancy, upregulate macroH2A expression. Functionally, we find that overexpression of macroH2A variants is sufficient to induce tumor cells into dormancy and notably, inducible expression of the macroH2A2 variant suppresses the growth of DCCs into overt metastasis. However, this dormant state does not require well-characterized dormancy factors such as DEC2 and NR2F1, suggesting alternate pathways. Our transcriptomic analyses reveal that macroH2A2 overexpression inhibits E2F, RAS and MYC signaling programs, while upregulating inflammatory cytokines commonly secreted by senescent cells. Taken together, our results demonstrate that macroH2A2 enforces a stable dormant phenotype in DCCs by activating a select subset of dormancy and senescence genes that limit metastasis initiation.

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Chronic Cochlear Implantation with and without Electric Stimulation in a Mouse Model Induces Robust Cochlear Influx of CX3CR1+/GFP Macrophages. BackgroundCochlear implantation is an effective auditory rehabilitation strategy for those with profound hearing loss, including those with residual low frequency hearing through use of hybrid cochlear implantation techniques. Post-mortem studies demonstrate the nearly ubiquitous presence of intracochlear fibrosis and neo-ossification following cochlear implantation. Current evidence suggests post-implantation intracochlear fibrosis is associated with delayed loss of residual acoustic hearing in hybrid cochlear implant (CI) recipients and may also negatively influence outcomes in traditional CI recipients. This study examined the contributions of surgical trauma, foreign body response and electric stimulation to intracochlear fibrosis and the innate immune response to cochlear implantation and the hierarchy of these contributions.

MethodsNormal hearing CX3CR1+/GFP mice underwent either round window opening (sham), acute CI insertion or chronic CI insertion with no, low- or high-level electric stimulation. Electric stimulation levels were based on neural response telemetry (NRT), beginning post-operative day 7 for 4 hours per day. Subjects (n=3 per timepoint) were sacrificed at 4 hours, 1,4,7,8,11,14 and 21 days. An unimplanted group (n=3) served as controls. Cochleae were harvested at each time-point and prepared for immunohistochemistry with confocal imaging. The images were analyzed to obtain CX3CR1+ macrophage cell number and density in the lateral wall (LW), scala tympani (ST) and Rosenthals canal (RC).

ResultsA ST peri-implant cellular infiltrate and fibrosis occurred exclusively in the chronically implanted groups starting on day 7 with a concurrent infiltration of CX3CR1+ macrophages not seen in the other groups. CX3CR1+ macrophage infiltration was seen in the LW and RC in all experimental groups within the first week, being most prominent in the 3 chronically implanted groups during the second and third week. There were no significant differences in macrophage infiltration related to levels of electric stimulation.

ConclusionsThe cochlear immune response was most prominent in the presence of chronic cochlear implantation, regardless of electric stimulation level. Further, the development of intracochlear ST fibrosis was dependent on the presence of the indwelling CI foreign body. An innate immune response was evoked by surgical trauma alone (sham and acute CI groups) to a lesser degree. These data suggest that cochlear inflammation and intrascalar fibrosis after cochlear implantation are largely dependent on the presence of a chronic indwelling foreign body and are not critically dependent on electrical stimulation. Also, these data support a role for surgical trauma in inciting the initial innate immune response.

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Unifying adaptive stress and adaptive habitat selection hypotheses through movement ecology O_LIGlucocorticoid production is often indicative of internal state that underpins movement and resulting habitat selection. Habitat selection is adaptive behaviour, but habitats where animals exhibit atypical glucocorticoid production are often associated with low fitness. Discrepancies between poor fitness outcomes and purported adaptive behaviour have contributed to a lack of consensus about the ecological and evolutionary implications of glucocorticoid production.
C_LIO_LIWe aim to demonstrate the adaptive ecological and evolutionary roles of glucocorticoids by integrating stress physiology with habitat selection theory.
C_LIO_LIFirst, we show how habitat selection theory can benefit from a mechanistic understanding of the relationships between stressors, internal state, and habitat selection. We show that glucocorticoids drive everything from short-term locomotion to large-scale movements. In turn, these movements produce familiar patterns of adaptive habitat selection like habitat specialization and migration. Populations arrive at these evolutionarily stable habitat selection strategies by responding to stressors like predation and food availability. To quantify responses to stressors, we show how to parameterize glucocorticoid data in habitat selection models. Modelling the ecological and evolutionary patterns that make habitat selection adaptive also help clarify relationships between glucocorticoids, stress, and fitness.
C_LIO_LIOur approach delivers a practical framework for testing existing hypotheses about habitat selection and stress physiology. Our framework can also be applied to new questions that integrate both, with the potential to understand adaptations of populations to their environments, and implications of stressors and habitat selection for ecosystem functioning.
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sentences: Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in neurons in the central nervous system. It has been documented that HCN channels regulate the intrinsic excitability of pyramidal cells in the medial prefrontal cortex (mPFC) of rats. Here, we report that HCN channels limited GABAergic transmission onto pyramidal cells in the mPFC. Pharmacological block of HCN channels resulted in a significant increase in the frequency of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs) in mPFC pyramidal cells. Such facilitation effect on mIPSCs required presynaptic Ca2+ influx and reversed by high-dose cAMP. Such facilitation did not exist in the presence of the T-type Ca2+ channel selective blockers. Immunofluorescence staining revealed that HCN channels expressed in presynaptic GABAergic terminals, as well as in both soma and neurite of parvalbumin-expressing (PV-expressing) basket cells in the mPFC. The present results indicate that HCN channels in GABAergic interneurons, most likely PV-expressing basket cells, constrain inhibitory control over layer 5-6 pyramidal cells through restricting presynaptic Ca2+ entry.

sentences: Peptide-centric analyses of human plasma enable increased resolution of biological insights into non-small cell lung cancer relative to protein-centric analysis Comprehensive assessment of the human proteome remains challenging due to multiple forms of a protein, or proteoforms, arising from alternative splicing, allelic variation, and protein modifications. As proteoforms can serve distinct functions and act as functional links between genotype and phenotype, proteoform-level knowledge is critical in understanding the molecular mechanisms underlying health and disease. However, identification of proteoforms requires unbiased protein coverage at amino acid resolution. Scalable, deep, and unbiased proteomics studies have been impractical due to cumbersome and lengthy workflows required for complex samples, like blood plasma. Here, we demonstrate the power of the Proteograph Product Suite in enabling unbiased, deep, and rapid proteomics at scale in a proof-of-concept proteoform analysis to dissect differences between protein isoforms in plasma samples from 80 healthy controls and 61 patients with early-stage non-small-cell lung cancer (NSCLC). Processing the 141 plasma samples with Proteograph yielded 22,993 peptides corresponding to 2,569 protein groups at a confidence of 1% false discovery rate. We extracted four proteins with peptides with significant abundance differences (p < 0.05; Benjamini-Hochberg corrected) in healthy control and cancer plasma samples. For one, the abundance variation can be explained by underlying annotated protein isoforms. For a second, we find evidence for differentially transcribed isoforms in the broader sequence data, but not in the known annotated protein isoforms. The others may be explained by novel isoforms or post-translational modifications. In addition, we sought to identify protein variants arising from allelic variation. To this end, we first performed whole exome sequencing on buffy coat samples from 29 individuals in the NSCLC study. Then, we created personalized mass spectrometry search databases for each individual subject from the exome sequences. From these libraries, we identified 422 protein variants, one of which has previously been shown to relate to lung cancer. In conclusion, our results demonstrate that Proteograph can generate unbiased and deep plasma proteome profiles that enable identification of proteoforms present in plasma at a scale sufficient to enable population-scale proteomic studies powered to reveal novel mechanistic and biomedical insights.

sentences: Nuclear genome of a pedinophyte pinpoints genomic innovation and streamlining in the green algae The genomic diversity underpinning high ecological and species diversity in the green algae (Chlorophyta) remains little known. Here, we aimed to track genome evolution in the Chlorophyta, focusing on loss and gain of homologous genes, and lineage-specific innovations of the Core Chlorophyta. We generated a high-quality nuclear genome for pedinophyte YPF701, a sister lineage to others in the Core Chlorophyta, and incorporated this genome in a comparative analysis with 25 other genomes from diverse Viridiplantae taxa. The nuclear genome of pedinophyte YPF701 has an intermediate size and gene number between those of most early-diverging prasinophytes and the remainder of the Core Chlorophyta. Our results suggest positive selection for genome streamlining in Pedinophyceae, independent from genome minimisation observed among prasinophyte lineages. Genome expansion was predicted along the branch leading to the UTC clade (classes Ulvophyceae, Trebouxiophyceae and Chlorophyceae) after divergence from their common ancestor with pedinophytes, with genomic novelty implicated in a range of basic biological functions. These results emphasise multiple independent signals of genome minimisation within the Chlorophyta, as well as the genomic novelty arising prior to diversification in the UTC clade, which may underpin the success of this species-rich clade in a diversity of habitats.

sentences: Decoding shared versus divergent transcriptomic signatures across cortico-amygdala circuitry in PTSD and depressive disorders Post-traumatic stress disorder (PTSD) is a debilitating neuropsychiatric disease with a projected lifetime risk of 8.7%. PTSD is highly comorbid with depressive disorders including major depressive disorder (MDD) and bipolar disorder (BD). It is hypothesized that the overlap in symptoms stems from partially shared underlying neurobiological mechanisms. To better understand shared and unique transcriptional patterns of PTSD and MDD we performed RNA-sequencing in the postmortem brain of two prefrontal cortex (PFC) regions and two amygdala (AMY) regions, from neurotypical donors (N=109) as well as donors with diagnoses of PTSD (N=107) or MDD (N=109) across 1285 RNA-seq samples. We identified a small number of differentially expressed genes (DEGs) specific to PTSD, mostly in the cortex compared to amygdala. PTSD-specific DEGs were preferentially enriched in cortistatin-expressing cells, a subpopulation of somatostatin interneurons. These PTSD DEGs also showed strong enrichment for gene sets associated with immune-related pathways and microglia, largely driven by decreased expression of these genes in PTSD donors. While we identified a greater number of DEGs for MDD, there were only a few that were specific to MDD as they showed high overlap with PTSD DEGs. Finally, we used weighted gene co-expression network analysis (WGCNA) as an orthogonal approach to confirm the observed cellular and molecular associations. These findings highlight the sub-population of cortistatin-expressing interneurons as having potential functional significance in PTSD and provide supporting evidence for dysregulated neuroinflammation and immune signaling in MDD and PTSD pathophysiology.

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A new species of the genus Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from eastern India with notes on Indian species A new species of the genus Microhyla, Microhyla bengalensis sp. nov., described from West Bengal state, India. The new species is distinguished from its congeners by a combination of the following morphological characters: 1) Small in size (SVL= 16.2 mm. in male); 2) truncated snout in dorsal view; 3) head wider than long (HW: HL= 1.36); 4) canthus rostralis and tympanum are indistinct; 5) nostril placed on the dorsal side of the snout; 6) tibiotarsal articulation not reaching the eye; 7) fingers and toes without disc; 8) toe webbing basal; 9) thigh and foot length are equal and smaller than shank; 10) skin tuberculated on dorsum; 11)  teddy bear dark brown mark on dorsum; 12) an inverted  V-shaped dark brown mark above the vent.

A comparative morphological data of all the 14 Indian species of Microhyla is also provided.

sentences: Reconstructing co-dependent cellular crosstalk in lung adenocarcinoma using REMI Cellular crosstalk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCI) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA-seq dataset, then leverage single-cell transcriptomics data to increase its resolution and identify LR prognostic signatures. We experimentally confirmed colocalization of CTGF:LRP6 as an interaction predicted to be associated with LUAD progression. Our work presents a novel way to reconstruct interactomes and a new approach to identify clinically-relevant cell-cell interactions.

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HyMM: Hybrid method for disease-gene prediction by integrating multiscale module structures MotivationIdentifying disease-related genes is important for the study of human complex diseases. Module structures or community structures are ubiquitous in biological networks. Although the modular nature of human diseases can provide useful insights, the mining of information hidden in multiscale module structures has received less attention in disease-gene prediction.

ResultsWe propose a hybrid method, HyMM, to predict disease-related genes more effectively by integrating the information from multiscale module structures. HyMM consists of three key steps: extraction of multiscale modules, gene rankings based on multiscale modules and integration of multiple gene rankings. The statistical analysis of multiscale modules extracted by three multiscale-module-decomposition algorithms (MO, AS and HC) shows that the functional consistency of the modules gradually improves as the resolution increases. This suggests the existence of different levels of functional relationships in the multiscale modules, which may help reveal disease-gene associations. We display the effectiveness of multiscale module information in the disease-gene prediction and confirm the excellent performance of HyMM by 5-fold cross-validation and independent test. Specifically, HyMM with MO can more effectively enhance the ability of disease-gene prediction; HyMM (MO, RWR) and HyMM (MO, RWRH) are especially preferred due to their excellent comprehensive performance, and HyMM (AS, RWRH) is also good choice due to its local performance. We anticipate that this work could provide useful insights for disease-module analysis and disease-gene prediction based on multi-scale module structures.

Availabilityhttps://github.com/xiangiu0208/HvMM

Contactlimin@mail.csu.edu.cn

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Acute SARS-CoV-2 infection in pregnancy is associated with placental ACE-2 shedding Human placental tissues have variable rates of SARS-CoV-2 invasion resulting in consistently low rates of fetal transmission suggesting a unique physiologic blockade against SARS-CoV-2. Angiotensin-converting enzyme (ACE)-2, the main receptor for SARS-CoV-2, is expressed as cell surface and soluble forms regulated by a metalloprotease cleavage enzyme, ADAM17. ACE-2 is expressed in the human placenta, but the regulation of placental ACE-2 expression in relation to timing of maternal SARS-CoV-2 infection in pregnancy is not well understood. In this study, we evaluated ACE-2 expression, ADAM17 activity and serum ACE-2 abundance in a cohort of matched villous placental and maternal serum samples from Control pregnancies (SARS-CoV-2 negative, n=8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the 2nd trimester ("2ndTri COVID", n=8) and 3rd trimester ("3rdTri COVID", n=8). In 3rdTri COVID as compared to control and 2ndTri-COVID villous placental tissues ACE-2 mRNA expression was remarkably elevated, however, ACE-2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE-2 was also significantly increased in the maternal serum from 3rdTri COVID infections as compared to control and 2ndTri-COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE-2 protein may be the result of ACE-2 shedding. Overall, this work highlights the importance of ACE-2 for ongoing studies on SARS-CoV-2 responses at the maternal-fetal interface.

sentences: Scarcity of hosts for gregarious parasitoids indicates an increase of individual offspring fertility by reducing their own fertility The gregarious parasitoid strategy allows multiple larvae to complete development in a single host due to their tolerance and/or lower mobility and thus flexibly adjust their reproductive potential amidst changing environmental conditions. Reproductive success is generally measured as the number of each mothers offspring. We propose that with scarcity of host, for gregarious parasitoids is important the view on the fertility not only of a mother but also of her offspring (F1 generation). Due to the body size-fitness correlation, each female deliberately adjusts the clutch size, determining the offspring body size and their reproductive potential. In our study, using Anaphes flavipes as a model species, we showed that under a limited number of hosts, the females reduced their fertility. We propose that the lower fertility of mothers can cause higher fertility in the F2 generation using a larger offspring body while halving fertility. The females increase their individual offsprings fertility by reducing their own fertility. Moreover, we showed that with a scarcity of hosts, the mothers increased the number of their female offspring, and thus, they obtained more offspring in the F2 generation. Additionally, other costs and benefits of the gregarious strategy in relation to superparasitism were tested and discussed.

sentences: Neural dynamics and architecture of the heading direction circuit in a vertebrate brain Animals can use different strategies to navigate. They may guide their movements by relying on external cues in their environment or, alternatively, by using an internal cognitive map of the space around them and their position within it. An essential part of this representation are heading cells, neurons whose activity depends on the heading direction of the animal. Although those cells have been found in vertebrates, the full network has never been observed and there is very little mechanistic understanding of how these cells acquire their response properties. In this study, we use volumetric functional imaging in larval zebrafish to observe, for the first time in a vertebrate, a full network that encodes allocentric heading direction. This network of approximately one hundred inhibitory neurons is arranged in an anatomical circle in the anterior hindbrain. Its activity is driven purely by the integration of internally generated signals, indicating that a simple vertebrate brain can encode maps of how an animal moves within its surroundings. Single cell reconstructions of electron micrographs allow us to uncover how the connectivity pattern of neurons within the network supports the implementation of a ring attractor network. The neurons we identify share features with neurons in the dorsal tegmentum nucleus of rodents and the fly central complex, showing that similar connectivity and mechanistic principles underlie the generation of cognitive maps of heading direction across the animal kingdom.

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

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

sentences: State Dependent Coupling of Hippocampal Oscillations Oscillations occurring simultaneously in a given area represent a physiological unit of brain states. They allow for temporal segmentation of spikes and support distinct behaviors. To establish how multiple oscillatory components co-varies simultaneously and influence neuronal firing during sleep and wakefulness, we describe a multi-variate analytical framework for constructing the state space of hippocampal oscillations. Examining the co-occurrence patterns of oscillations on the state space, across species, uncovered the presence of network constraints and distinct set of cross-frequency interactions during wakefulness as compared to sleep. We demonstrated how the state space can be used as a canvas to map the neural firing and found that distinct neurons during navigation were tuned to different set of simultaneously occurring oscillations during sleep. This multivariate analytical framework provides a window to move beyond classical bivariate pipelines, for investigating oscillations and neuronal firing, thereby allowing to factor-in the complexity of oscillation-population interactions.

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Genome-wide analysis of Schistosoma mansoni reveals population structure and praziquantel drug selection pressure within Ugandan hot-spot communities Populations within schistosomiasis control areas, especially those in Africa, are recommended to receive regular mass drug administration (MDA) with praziquantel (PZQ) as the main strategy for controlling the disease. The impact of PZQ treatment on schistosome genetics remains poorly understood, and is limited by a lack of high-resolution genetic data on the population structure of parasites within these control areas. We generated whole-genome sequence data from 174 individual miracidia collected from both children and adults from fishing communities on islands in Lake Victoria in Uganda that had received either annual or quarterly MDA with PZQ over four years, including samples collected immediately before and four weeks after treatment. Genome variation within and between samples was characterised and we investigated genomic signatures of natural selection acting on these populations that could be due to PZQ treatment. The parasite population on these islands was more diverse than found in nearby villages on the lake shore. We saw little or no genetic differentiation between villages, or between the groups of villages with different treatment intensity, but slightly higher genetic diversity within the pre-treatment compared to post-treatment parasite populations. We identified classes of genes significantly enriched within regions of the genome with evidence of recent positive selection among post-treatment and intensively treated parasite populations. The differential selection observed in post-treatment and pre-treatment parasite populations could be linked to any reduced susceptibility of parasites to praziquantel treatment.

Author summarySchistosomiasis is caused by parasitic helminths of the genus Schistosoma. Schistosoma mansoni is the primary cause of intestinal schistosomiasis, a devastating and widespread parasitic infection that causes morbidity, death and socio-economic impact on endemic communities across the world and especially sub-Saharan Africa. Using whole-genome sequencing, we were able to elucidate the parasite population within Lake Victoria island fishing communities in Uganda which are among the major hotspots for schistosomiasis. We further assessed genetic markers that might be linked to recent observations concerning reduced susceptibility to praziquantel, the major drug used in the treatment of this disease. Whole-genome data on the population genetics of S. mansoni in an African setting will provide a strong basis for future functional genomics or transcriptomic studies that will be key to identifying drug targets, improving existing drugs or developing new therapeutic interventions.

sentences: 31P Transversal Relaxation Times and Metabolite Concentrations in the Human Brain at 9.4T A method to estimate phosphorus (31P) transversal relaxation times (T2) of coupled spin systems is demonstrated. Additionally, intracellular and extracellular pH (pHext, pHint) and relaxation corrected metabolite concentrations are reported. Echo time (TE) series of 31P metabolite spectra were acquired using STEAM localization. Spectra were fitted using LCModel with accurately modeled Vespa basis sets accounting for J-evolution of the coupled spin systems. T2s were estimated by fitting a single exponential two-parameter model across the TE series. Fitted inorganic phosphate frequencies were used to calculate pH, and relaxation times were used to determine the brain metabolite concentrations. The method was demonstrated in the healthy human brain at a field strength of 9.4T. T2 relaxation times of ATP and NAD are the shortest between 8 ms and 20 ms, followed by T2s of inorganic phosphate between 25 ms and 50 ms, and PCr with a T2 of 100 ms. Phosphomonoesters and -diesters have the longest T2s of about 130 ms. Measured T2s are comparable to literature values and fit in a decreasing trend with increasing field strengths. Calculated pHs and metabolite concentrations are also comparable to literature values.

sentences: A Multigraph model of the 3D genome Spatial organisation of the genome has a fundamental effect on its biological functions. Chromatin-chromatin interactions and 3D spatial structures are involved in transcriptional regulation and have a decisive role in DNA replication and repair. To understand how individual genes and their regulatory elements function within the larger genomic context, and how the genome reacts as a whole to environmental stimuli, the linear sequence information needs to be interpreted in 3-dimensional space. While recent advances in chromatin conformation capture technologies including Hi-C, considerably advanced our understanding of the genomes, defining the DNA, as it is organized in the cell nucleus is still a challenging task. 3D genome modelling needs to reflect the DNA as a flexible polymer, which can wind up to the fraction of its total length and greatly unwind and stretch to implement a multitude of functions. Here we propose a novel approach to model genomes as a multigraph based on Hi-C contact data. Multigraph-based 3D genome modelling of barley and rice revealed the well-known Rabl and Rosetta chromatin organizations, respectively, as well as other higher order structures. Our results shows that the well-established toolset of Graph theory is highly valuable in modelling large genomes in 3D.

sentences: Dissecting the co-transcriptome landscape of plants and microbiota members Interactions between plants and neighboring microbial species are fundamental elements that collectively determine the structure and function of the plant microbiota. However, the molecular basis of such interactions is poorly characterized. Here, we monocolonized Arabidopsis leaves with nine plant- associated bacteria from all major phyla of the plant microbiota and profiled co- transcriptomes of plants and bacteria. We detected both common and distinct co- transcriptome signatures among plant-commensal pairs. In planta responses of commensals were similar to those of a disarmed pathogen characterized by the suppression of genes involved in general metabolism in contrast to a virulent pathogen. We identified genes that are enriched in the genome of plant-associated bacteria and induced in planta, which may be instrumental for bacterial adaptation to the host environment and niche separation. This study provides insights into how plants discriminate among bacterial strains and lays the foundation for in- depth mechanistic dissection of plant-microbiota interactions.

sentences: SBGNview: Data Analysis, Integration and Visualization on All Pathways Pathway analysis is widely used in genomics and omics research, but the data visualization has been highly limited in function, pathway coverage and data format. Here we develop SBGNview a comprehensive solution to address these needs. By adopting the standard SBGN format, SBGNview greatly extend the coverage of pathway based analysis and data visualization to essentially all major pathway databases beyond KEGG, including 5200 reference pathways and over 3000 species. In addition, SBGNview substantially extends current tools in both design and function, including standard coherent input/output formats, high quality graphics convenient for both computational and manual analysis, and flexible and open-end workflow. In addition to pathway analysis and data visualization, SBGNview provides essential infrastructure for SBGN data manipulation and processing. SBGNview is available online: https://github.com/datapplab/SBGNview.

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A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.

We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-{beta}H1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.

We identified a total of 264 genes stably expressed in EndoC-{beta}H1 cells and human islets following cytokine- or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

sentences: Daily torpor reduces the energetic consequences of habitat selection for a widespread bat Homeothermy requires increased metabolic rates as temperatures decline below the thermoneutral zone, so homeotherms typically select microhabitats within or near their thermoneutral zones during periods of inactivity. However, many mammals and birds are heterotherms that relax internal controls on body temperature when maintaining a high, stable body temperature is energetically costly. Such heterotherms should be less tied to microhabitats near their thermoneutral zones, and because heterotherms spend more time in torpor and expend less energy at colder temperatures, heterotherms may even select microhabitats in which temperatures are well below their thermoneutral zones. We studied how temperature and daily torpor influence selection of diurnal roosts by a heterothermic bat (Myotis thysanodes). We (1) quantified the relationship between ambient temperature and daily duration of torpor, (2) simulated daily energy expenditure over a range of microhabitat (roost) temperatures, and (3) quantified the influence of roost temperature on roost selection. While warm roosts substantially reduced energy expenditure of simulated homeothermic bats, heterothermic bats modulated their use of torpor to maintain a constant level of energy expenditure over the course of a day. Daily torpor expanded the range of energetically economical microhabitats, such that roost selection was independent of roost temperature. Our work adds to a growing literature documenting functions of torpor beyond its historical conceptualization as a last-resort measure to save energy during extended or acute energetic stress.

sentences: DNA methylation aging and transcriptomic studies in horses Human DNA methylation profiles have been used successfully to develop highly accurate biomarkers of aging ("epigenetic clocks"). Here, we describe epigenetic clocks for horses, based on methylation profiles of CpGs with flanking DNA sequences that are highly conserved between multiple mammalian species. Methylation levels of these CpGs were measured using a custom-designed Infinium array (HorvathMammalMethylChip40). We generated 336 DNA methylation profiles from 42 different horse tissues and body parts, which we used to develop five epigenetic clocks for horses: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. Epigenetic age measured by these clocks show that while castration affects the basal methylation levels of individual cytosines, it does not exert a significant impact on the epigenetic aging rate of the horse. We observed that most age-related CpGs are adjacent to developmental genes. Consistently, these CpGs reside in bivalent chromatin domains and polycomb repressive targets, which are elements that control expression of developmental genes. The availability of an RNA expression atlas of these tissues allowed us to correlate CpG methylation, their corresponding contextual chromatin features and gene expression. This analysis revealed that while increased methylation of CpGs in enhancers is likely to repress gene expression, methylation of CpGs in bivalent chromatin domains on the other hand is likely to stimulate expression of the corresponding downstream loci, which are often developmental genes. This supports the notion that aging may be accompanied by increased expression of developmental genes. It is expected that the epigenetic clocks will be useful for identifying and validating anti-aging interventions for horses.

sentences: Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

sentences: Fast and memory-efficient mapping of short bisulfite sequencing reads using a two-letter alphabet DNA cytosine methylation is an important epigenomic mark with a wide range of functions across many organisms. Whole genome bisulfite sequencing (WGBS) is the gold standard to interrogate cyto-sine methylation genome-wide. Algorithms used to map WGBS reads often encode the four-base DNA alphabet with three letters by reducing two bases to a common letter. This encoding substantially reduces the entropy of nucleotide frequencies in the resulting reference genome. Within the paradigm of read mapping by first filtering possible candidate alignments, reduced entropy of the reference can increase the required computing effort. We introduce another bisulfite mapping algorithm (abismal), based on the idea of encoding a four-letter DNA sequence as only two letters, one for purines and one for pyrimidines. We show that this encoding has greater specificity when subsequences are selected from reads for filtration. Through the two-letter encoding, the abismal software tool maps reads in less time and using less memory than most WGBS read mapping software tools, while attaining similar accuracy. This allows in silico methylation analysis to be performed in a wider range of computing machines with limited hardware settings.

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DNA reconciles morphology and colouration in the drunk blenny genus Scartichthys (Teleostei: Blenniidae) and provides insights into their evolutionary history The blenniids of the genus Scartichthys are one of the most common fishes of Central and South American Pacific coastal reefs. This being said, Scartichthys spp. remain difficult to identify in the field, and identification is particularly challenging across the ca. 6000 km where three of the four currently accepted species are known to occur in sympatry. A reason for this is that the main taxonomic characters from traditional taxonomy are indeed elusive. Additionally, At the same time, species can display multiple colour patterns in the field, depending on their ontogenetic stage, habitat association, and reproductive behaviour. Overall, molecular characterization is warranted to help address these issues. Here, we have used a novel approach to revise the genus by combining colouration, morphological, and molecular data of representative specimens of the four currently valid species and seven described colour patterns. From this, we show that only three of the four species should be considered as valid; Scartichthys gigas (Steindachner, 1876), S. variolatus (Valenciennes, 1836), and S. viridis (Valenciennes, 1836); while S. crapulatus Williams 1990 should be synonymized with S. viridis. In the same way, our analyses show that one of the colour patterns attributed so far only to S. gigas is characteristic of the juvenile stages of S. viridis. Our time-calibrated phylogeny shows that this genus is relatively young and that the estimated time of divergence between Scartichthys gigas and S. viridis is around 1.71 Ma. In comparison, the Desventuradas and Juan Fernandez Islands endemic S. variolatus diverged about 1.95 Ma. Our results help to clarify the taxonomy of Scartichthys.

SIGNIFICANCE STATEMENTThe blenniids of the genus Scartichthys are one of the most common fishes of Central and South American Pacific coastal reefs. Here we provide an updated phylogeny of this genus, comparing for the first time morphological, coloration, and molecular data in combination to resolve a 30-year-old discord among ecologists and taxonomists and discuss the potential underlying evolutionary processes that led to their presentday distribution.

sentences: Hsp40s display class-specific binding profiles, serving complementary roles in the prevention of tau amyloid formation The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles, forming insoluble, amyloid-type aggregates associated with neurodegenerative conditions, such as Alzheimers disease. Tau aggregation, however, can be prevented in the cell by a class of proteins known as molecular chaperones, which play important roles in maintaining protein homeostasis. While numerous chaperones are known to interact with tau, though, little is known about the detailed mechanisms by which these prevent tau aggregation. Here, we describe the effects of the ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid fiber formation and compare these to the well-studied small heat shock protein HSPB1. We find that each chaperone prevents tau aggregation differently, by interacting with distinct sets of tau species along the aggregation pathway and thereby affecting their incorporation into fibers. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone tau conformers and even mature fibers, thus efficiently preventing formation of tau amyloids. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, different site in CTDI. These results provide important insight into the molecular mechanism by which the different members of the Hsp40 chaperone family counteract the formation, propagation, and toxicity of tau aggregates. Furthermore, our findings highlight the fact that chaperones from different families and different classes play distinct, but complementary roles in preventing pathological protein aggregation.

sentences: Connectomic Analysis of the Drosophila Lateral Neuron Clock Cells Reveals the Synaptic Basis of Functional Pacemaker Classes. The circadian clock orchestrates daily changes in physiology and behavior to ensure internal temporal order and optimal timing across the day. In animals, a central brain clock coordinates circadian rhythms throughout the body and is characterized by a remarkable robustness that depends on synaptic connections between constituent neurons. The clock neuron network of Drosophila, which shares network motifs with clock networks in the mammalian brain yet is built of many fewer neurons, offers a powerful model for understanding the network properties of circadian timekeeping. Here we report an assessment of synaptic connectivity within a clock network, focusing on the critical lateral neuron (LN) clock neuron classes. Our results reveal that previously identified anatomical and functional subclasses of LNs represent distinct connectomic types. Moreover, we identify a small number of clock cell subtypes representing highly synaptically coupled nodes within the clock neuron network. This suggests that neurons lacking molecular timekeeping likely play integral roles within the circadian timekeeping network. To our knowledge, this represents the first comprehensive connectomic analysis of a circadian neuronal network.

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Coding Recognition of the Dose-Effect Interdependence of Small Biomolecules Encrypted on Paired Chromatographic-Based Microassay Arrays The discovery of small biomolecules suffers from the lack of a comprehensive framework to express the intrinsic correlation between bioactivity and contributing small molecules in complex samples with molecular and bioactivity diversity. Here, by mapping a samples 2D-HPTLC fingerprint to microplates, the paired chromatographic-based microassay arrays are created, which can be used as quasi-chip to characterize multiple attributes of chromatographic components; and as the array differential expression of the bioactivity and molecular attributes of irregular chromatographic spots for dose-effect interdependent encoding; and also as the automatic-collimated array mosaics of the multi-attributes of each component itself encrypted by its chromatographic fingerprint. Based on this homologous framework, we propose a correlating recognition strategy for small-biomolecules through their self-consistent chromatographic behavior characteristics. In the approach, the small-biomolecule recognition in diverse compounds is transformed into a constraint satisfaction problem, which is addressed through examining the dose-effect interdependence of the homologous 2D code pairs by array matching algorithm, instead of preparing diverse compound monomers of complex test sample for identifying item-by-item. Furtherly, considering the dose-effect interdependent 2D code pairs as links and the digital-specific quasimolecular ions as nodes, an extendable self-consistent framework that correlates mammalian cell phenotypic and target-based bioassays with small biomolecules is established. Therefore, the small molecule contributions and the correlations of bioactivities, as well as their pathway can be comprehensively revealed, so as to improve the reliability and efficiency of screening. This strategy was successfully applied to galangal, and practiced the high-throughput digital preliminary screening of small-biomolecules in a natural product.

HighlightRapid self-recognition of small biomolecules in diverse samples without pre-isolation Recognized by dose-effect interdependency developed from homologous TLC fingerprint Matching of HPTLC-based molecular imprinting and bioautography on microassay arrays Microarray-based differential expression of substance attributes instead of spot scan An array framework for combining phenotype-based and target-based assays with TLC-MS

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sentences: Dendritic domain-specific sampling of long-range axons shapes feedforward and feedback connectivity of L5 neurons Feedforward and feedback pathways interact in specific dendritic domains to enable cognitive functions such as predictive processing and learning. Based on axonal projections, hierarchically lower areas are thought to form synapses primarily on dendrites in middle cortical layers, while higher-order areas are posited to target dendrites in layer 1 and in deep layers. However, the extent to which functional synapses form in regions of axo-dendritic overlap has not been extensively studied. Here, we use viral tracing in the secondary visual cortex of mice to map brain-wide inputs to thick-tufted layer 5 pyramidal neurons. Furthermore, we provide a comprehensive map of input locations through subcellular optogenetic circuit mapping. We show that input pathways target distinct dendritic domains with far greater specificity than appears from their axonal branching, often deviating substantially from the canonical patterns. Common assumptions regarding the dendrite-level interaction of feedforward and feedback inputs may thus need revisiting.

sentences: Modelling the collective mechanical regulation of the structure and morphology of epithelial cell layers The morphology and function of epithelial sheets play an important role in healthy tissue development and cancer progression. The maintenance of structure of closely packed epithelial layers requires the coordination of various mechanical forces within the cells and others resulting from interactions with other cells and other tissues or substrates. However, a general model for the combination of mechanical properties which determine the cell shape and the overall structure of epithelial layers remains elusive. Here, we propose a computational model, based on the Cellular Potts Model, to study the interplay between mechanical properties of cells and dynamical transitions in epithelial structures and cell shapes. We map out phase diagrams as functions of cellular properties and the orientation of cell division. Monolayers of squamous, cuboidal, and columnar cells are found when the axis of cell proliferation is perpendicular to the substrate. Monolayer-to-multilayer transition is promoted via cell extrusion, depending on the mechanical properties of cells and the orientation of cell division. The results and model predictions are discussed in the context of experimental observations.

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Comprehensive machine-learning-based analysis of microRNA-target interactions reveals variable transferability of interaction rules across species BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally via base-pairing with complementary sequences on messenger RNAs (mRNAs). Due to the technical challenges involved in the application of high-throughput experimental methods, datasets of direct bona-fide miRNA targets exist only for a few model organisms. Machine learning (ML) based target prediction methods were successfully trained and tested on some of these datasets. There is a need to further apply the trained models to organisms where experimental training data is unavailable. However, it is largely unknown how the features of miRNA-target interactions evolve and whether there are features that have been fixed during evolution, questioning the general applicability of these ML methods across species.

ResultsIn this paper, we examined the evolution of miRNA-target interaction rules and used data science and ML approaches to investigate whether these rules are transferable between species. We analyzed eight datasets of direct miRNA-target interactions in four organisms (human, mouse, worm, cattle). Using ML classifiers, we achieved high accuracy for intra-dataset classification and found that the most influential features of all datasets significantly overlap. To explore the relationships between datasets we measured the divergence of their miRNA seed sequences and evaluated the performance of cross-datasets classification. We showed that both measures coincide with the evolutionary distance of the compared organisms.

ConclusionsOur results indicate that the transferability of miRNA-targeting rules between organisms depends on several factors, the most associated factors being the composition of seed families and evolutionary distance. Furthermore, our feature importance results suggest that some miRNA-target features have been evolving while some have been fixed during evolution. Our study lays the foundation for the future developments of target prediction tools that could be applied to "non-model" organisms for which minimal experimental data is available.

Availability and implementation The code is freely available at https://github.com/gbenor/TPVOD

sentences: The salmon louse genome: copepod features and parasitic adaptations. Copepods encompass a range of ecological roles from parasites to phytoplankton grazers linking primary producers to higher trophic levels. Despite these important roles, copepod genome assemblies are scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod. We present the 695.4 Mbp L. salmonis genome assembly containing {approx}60% repetitive regions and 13081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics validated annotation and revealed a marked shift in gene expression after host attachment, including downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of peroxisomes, numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite large capacity to develop resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.

sentences: Asynchronous glutamate exocytosis is enhanced in low release probability synapses and is widely dispersed across the active zone The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs (presynaptic boutons) from single pyramidal cells in culture with 4 millisecond temporal resolution, and localise vesicular release sites with ~ 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among presynaptic boutons supplied by the same axon, and that asynchronous release fraction is elevated in parallel with short-term facilitation at synapses with low release probability. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the presynaptic release area than synchronous sites. These findings are consistent with a model in which functional presynaptic properties are regulated via a synapsespecific adjustment of the coupling distance between presynaptic Ca2+ channels and releaseready synaptic vesicles. Together our results reveal a universal relationship between the two major functional properties of synapses - the timing and the overall probability of neurotransmitter release.

sentences: Coupling of pupil- and neuronal population dynamics reveals diverse influences of arousal on cortical processing Fluctuations in arousal, controlled by subcortical neuromodulatory systems, continuously shape cortical state, with profound consequences for information processing. Yet, how arousal signals influence cortical population activity in detail has so far only been characterized for a few selected brain regions. Traditional accounts conceptualize arousal as a homogeneous modulator of neural population activity across the cerebral cortex. Recent insights, however, point to a higher specificity of arousal effects on different components of neural activity and across cortical regions. Here, we provide a comprehensive account of the relationships between fluctuations in arousal and neuronal population activity across the human brain. Exploiting the established link between pupil size and central arousal systems, we performed concurrent magnetoencephalographic (MEG) and pupillographic recordings in a large number of participants, pooled across three laboratories. We found a cascade of effects relative to the peak timing of spontaneous pupil dilations: Decreases in low-frequency (2-8 Hz) activity in temporal and lateral frontal cortex, followed by increased high-frequency (>64 Hz) activity in mid-frontal regions, followed by monotonic and inverted-U relationships with intermediate frequency-range activity (8-32 Hz) in occipito-parietal regions. Pupil-linked arousal also coincided with widespread changes in the structure of the aperiodic component of cortical population activity, indicative of changes in the excitation-inhibition balance in underlying microcircuits. Our results provide a novel basis for studying the arousal modulation of cognitive computations in cortical circuits.

sentences: Identification of a filamentous form of kunitz protease inhibitor in Asteraceae Filamentous structures were observed in purified extracts from chrysanthemum, gerbera, sunflower and zinnia. When purified filament proteins were subjected to SDS-PAGE, the major protein associated with filaments from all three species has an apparent molecular mass of {approx}25 kDa. Protein bands from chrysanthemum, gerbera, and zinnia were subjected to N-terminal protein sequencing while proteins from sunflower were sequenced by CID MS/MS. All of the sequences shared highest similarity to the kunitz trypsin inhibitor family. The sequencing results indicated that the proteins lacked the signal sequences. We tested the gerbera filament protein for glycosylation and found that it was a glycoprotein. Together these results indicate that the filaments are composed of mature KTI protein. This is the first report of a KTI assembling into filaments and the first report of a filament forming Asteraceae enzyme.

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DAF-2/insulin IGF-1 receptor regulates mobility during ageing by integrating opposite inputs from muscle and neuronal tissues During ageing, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In C. elegans, mutants of the insulin-IGF-1 receptor DAF-2/IIRc represent a paradigm of healthy ageing, as their increased lifespan is accompanied by a delay in age-related loss of motility. However, these animals are less mobile than wild-type animals in early adulthood. Here we investigated the DAF-2/IIRc- dependent relationship between longevity and motility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2/IIRc. As previously reported, inactivation of DAF-2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2/IIRc prevented the age-related loss of motility. In 1-day-old adults, DAF-2/IIRc depletion in neurons reduced motility in a DAF-16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middleage animals improved their motility independently of DAF-16/FOXO but required UNC-120/SRF.

Yet, neuronal or muscle DAF-2/IIRc depletion both preserved the mitochondria network in ageing muscle. Overall, these results show that the motility pattern of daf-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.

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Parallel CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrhoeal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by sgRNA to introduce targeted CT transitions, resulting in internal STOP codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter assay, we were able to generate loss-of-function mutants in both E. coli and Shigella with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion is in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Further, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella genes and found the mutant phenotype to be similar to that of the reported gene deletion mutants, with no apparent polar effects. This method can be used in a 96-well plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days.

ImportanceLoss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches system biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen - Shigella. This pathogen causes high morbidity and mortality in children and antibiotic resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method can be programmed to generated multiple mutants in a single, massively parallel, reaction. By the virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

sentences: Strong basal/tonic TCR signals are associated with negative regulation of naive CD4+ T cells Tonic TCR signaling occurs constitutively in response to self-peptides presented by MHC (pMHC). Tonic TCR signal intensity correlates with Nur77-GFP reporter transgene expression. A broad range of Nur77-GFP is first detectable in post-selection thymocytes and persists in mature T cells. Nur77-GFPHI Ly6C- CD4+ T cells experience the strongest tonic TCR signaling and exhibit functional hypo-responsiveness to foreign pMHC stimulation. Gene expression analyses suggest similarities between the programs induced by strong tonic TCR signaling and T cell activation. However, the strongest tonic TCR signals also appear to induce expression of negative regulators, including coinhibitory receptors. Analysis of chromatin accessibility similarly suggest that strong tonic TCR signaling correlates with differentially higher accessibility of over 3000 chromatin regions in or near genes that encode positive and negative regulators of T cell activation. We propose that very strong tonic TCR signaling induces mechanisms of negative feedback to recalibrate T cell sensitivity.

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Looking at the BiG picture: Incorporating bipartite graphs in drug response prediction MotivationThe increasing number of publicly available databases containing drugs chemical structures, their response in cell lines, and molecular profiles of the cell lines has garnered attention to the problem of drug response prediction. However, many existing methods do not fully leverage the information that is shared among cell lines and drugs with similar structure. As such, drug similarities in terms of cell line responses and chemical structures could prove to be useful in forming drug representations to improve drug response prediction accuracy.

ResultsWe present two deep learning approaches, BiG-DRP and BiG-DRP+, for drug response prediction. Our models take advantage of the drugs chemical structure and the underlying relationships of drugs and cell lines through a bipartite graph and a heterogenous graph convolutional network that incorporate sensitive and resistant cell line information in forming drug representations. Evaluation of our methods and other state-of-the-art models in different scenarios shows that incorporating this bipartite graph significantly improves the prediction performance. Additionally, genes that contribute significantly to the performance of our models also point to important biological processes and signaling pathways. Analysis of predicted drug response of patients tumors using our model revealed important associations between mutations and drug sensitivity, illustrating the utility of our model in pharmacogenomics studies.

Availability and ImplementationAn implementation of the algorithms in Python is provided in github.com/ddhostallero/BiG-DRP.

Contactamin.emad@mcgill.ca

Supplementary InformationOnline-only supplementary data is available at the journals website.

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Accounting for fragments of unexpectedorigin improves transcript quantification inRNA-seq simulations focused on increased realism Transcript and gene quantification is the first step in many RNA-seq analyses. While many factors and properties of experimental RNA-seq data likely contribute to differences in accuracy between various approaches to quantification, it has been demonstrated (1) that quantification accuracy generally benefits from considering, during alignment, potential genomic origins for sequenced fragments that reside outside of the annotated transcriptome.

Recently, Varabyou et al. (2) demonstrated that the presence of transcriptional noise leads to systematic errors in the ability of tools -- particularly annotation-based ones -- to accurately estimate transcript expression. Here, we confirm the findings of Varabyou et al. (2) using the simulation framework they have provided. Using the same data, we also examine the methodology of Srivastava et al.(1) as implemented in recent versions of salmon (3), and show that it substantially enhances the accuracy of annotation-based transcript quantification in these data.

sentences: Recruitment of KRAS downstream target ARL4C to membrane protrusions accelerates pancreatic cancer cell invasion. Pancreatic cancer (PC) has a high mortality rate due to metastasis. Whereas KRAS is mutated in most PC patients, controlling KRAS or its downstream effectors has not been succeeded clinically. ARL4C is a small G protein whose expression is induced by the Wnt and EGF-RAS pathways. In the present study, we found that ARL4C is frequently overexpressed in PC patients and showed that its unique localization to membrane protrusions is required for cancer cell invasion. IQGAP1 was identified as a novel interacting protein for ARL4C. ARL4C recruited IQGAP1 and its downstream effector, MMP14, to membrane protrusions. Specific localization of ARL4C, IQGAP1, and MMP14 was the active site of invasion, which induced degradation of the extracellular matrix. Moreover, subcutaneously injected antisense oligonucleotide (ASO) against ARL4C into tumor-bearing mice suppressed metastasis of PC. These results suggest that ARL4C-IQGAP1-MMP14 signaling is activated at membrane protrusions of PC cells.

sentences: Proteomic phenotyping of stimulated Müller cells uncovers profound pro-inflammatory signaling and antigen-presenting capacity Muller cells are the main macroglial cells of the retina exerting a wealth of functions to maintain retinal homoeostasis. Upon pathological changes in the retina, they become gliotic with both protective and detrimental consequences. Accumulating data also provide evidence for a pivotal role of Muller cells in the pathogenesis of diabetic retinopathy (DR). While microglial cells, the resident immune cells of the retina are considered as main players in inflammatory processes associated with DR, the implication of activated Muller cells in chronic retinal inflammation remains to be elucidated. In order to assess the signaling capacity of Muller cells and their role in retinal inflammation, we performed in-depth proteomic analysis of Muller cell proteomes and secretomes after stimulation with INF{gamma}, TNF, IL-4, IL-6, IL-10, VEGF, TGF{beta}1, TGF{beta}2 and TGF{beta}3. We used both, primary porcine Muller cells and the human Muller cell line MIO-M1 for our hypothesis generating approach. Our results point towards an intense signaling capacity of Muller cells, which reacted in a highly discriminating manner upon treatment with different cytokines. Stimulation of Muller cells resulted in a primarily pro-inflammatory phenotype with secretion of cytokines and components of the complement system. Furthermore, we observed evidence for mitochondrial dysfunction, implying oxidative stress after treatment with the various cytokines. Finally, both MIO-M1 cells and primary porcine Muller cells showed several characteristics of atypical antigen-presenting cells, as they are capable of inducing MHC class I and MHC class II with co-stimulatory molecules. In line with this, they express proteins associated with formation and maturation of phagosomes. Thus, our findings underline the importance of Muller cell signaling in the inflamed retina, indicating an active role in chronic retinal inflammation underlying the pathogenesis of diabetic retinopathy.

sentences: Feature-based attention enables robust, long-lasting location transfer in human perceptual learning Visual perceptual learning (VPL) is typically specific to the trained location and feature. However, the degree of specificity depends upon particular training protocols. Manipulating covert spatial attention during training facilitates learning transfer to other locations. Here we investigated whether feature-based attention (FBA), which enhances the representation of particular features throughout the visual field, facilitates VPL transfer, and how long such an effect would last. To do so, we implemented a novel task in which observers discriminated a stimulus orientation relative to two reference angles presented simultaneously before each block. We found that training with FBA enabled remarkable location transfer, reminiscent of its global effect across the visual field, but preserved orientation specificity in VPL. Critically, both the perceptual improvement and location transfer persisted after one year. Our results reveal robust, long-lasting benefits induced by FBA in VPL, and have translational implications for improving generalization of training protocols in visual rehabilitation.

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Molecular diversity maintained by long-term balancing selection in mating loci defines multiple mating types in fungi Balancing selection, an evolutionary force that retains genetic diversity, has been detected in multiple genes and organisms, such as the sexual mating loci in fungi. However, to quantify the strength of balancing selection and define the mating-related genes require a large number of specimens. In tetrapolar basidiomycete fungi, sexual type is determined by two unlinked loci, MATA and MATB. Genes in both loci defines mating type identity, control successful mating and completion of the life cycle. These loci are usually highly diverse. Previous studies have speculated, based on culture crosses, that species of the non-model genus Trichaptum (Hymenochaetales, Basidiomycota) possess a tetrapolar mating system, with multiple alleles. Here, we sequenced a hundred and eighty specimens of three Trichaptum species. We characterized the chromosomal location of MATA and MATB, the molecular structure of MAT regions and their allelic richness. Our sequencing effort was sufficient to molecularly characterize multiple MAT alleles segregating before the speciation event of Trichaptum species. Our analyses suggested that long-term balancing selection has generated trans-species polymorphisms. Mating sequences were classified in different allelic classes based on an amino acid identity (AAI) threshold supported by phylogenetics. The inferred allelic information mirrored the outcome of in vitro crosses, thus allowing us to support the degree of allelic divergence needed for successful mating. Even with the high amount of divergence, key amino acids in functional domains are conserved. The observed allelic classes could potentially generate 14,560 different mating types. We conclude that the genetic diversity of mating in Trichaptum loci is due to long-term balancing selection, with limited recombination and duplication activity. Our large number of sequenced specimens highlighted the importance of sequencing multiple individuals from different species to detect the mating-related genes, the mechanisms generating diversity and the evolutionary forces maintaining them.

Author summaryFungi have complex mating systems, and basidiomycete fungi can encode thousands of mating types. Individuals with the same mating type cannot mate. This sexual system has evolved to facilitate sexual mating, increasing the chances to recombine into advantageous allelic combination and prune deleterious alleles. We explored the genomes of hundred and eighty specimens, combined with experimental mating studies of selected specimens, from a non-model organism (Trichaptum). We characterized the genomic regions controlling sex. The mating ability of the specimens confirmed the role of the mating alleles observed in the genomic data. The detailed analyses of many specimens allowed us to observe gene duplication and rearrangements within the mating loci, increasing the diversity within these loci. We supported previous suggestions of balancing selection in this region, an evolutionary force that maintains genomic diversity. These results supports that our fungal specimens are prone to outcross, which might facilitate the adaptation to new conditions.

sentences: Action sequence dynamics rely on accessory hyperdirect pathway-related targets in the striatum Performing several actions in swift succession is often necessary to exploit known contingencies in the environment. However, after a change in the contingency rules, the ability to appropriately adapt rapid action sequences must be procured for continued success. By combining analyses of behavioural microstructure with circuit-specific tracing in mice, we report on a relationship between action timing-variability and successful adaptation that relies on post-synaptic targets of primary motor cortical (M1) projections to dorsolateral striatum (DLS). We found that M1 hyperdirect pathway projections to the STN also send dense axonal collaterals to external globus pallidus and dorsal striatum, with the highest synaptic volumes found in the DLS. Specific interruption of the M1[->]DLS circuit reduced the proportion of successful sequences while speeding-up and reducing action timing-variability, revealing a role for M1[->]DLS circuitry in setting the exploration/exploitation balance that is required for adaptively guiding the timing and success of instrumental action.

sentences: An efficient algorithm for estimating population history from genetic data The Legofit statistical package uses genetic data to estimate parameters describing population history. Previous versions used computer simulations to estimate probabilities, an approach that limited both speed and accuracy. This article describes a new deterministic algorithm, which makes Legofit faster and more accurate. The speed of this algorithm declines as model complexity increases. With very complex models, the deterministic algorithm is slower than the stochastic one. In an application to simulated data sets, the estimates produced by the deterministic and stochastic algorithms were essentially identical. Reanalysis of a human data set replicated the findings of a previous study and provided increased support for the hypotheses that (a) early modern humans contributed genes to Neanderthals, and (b) a "superarchaic" population (which separated from all other humans early in the Pleistocene) was either large or deeply subdivided.

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Towards a multi-stressor theory for coral reefs in a changing world Coral reefs are facing a constant barrage of human impacts, including eutrophication, overharvesting and climate change. However, research and management are just beginning to depart from a single-dominant-stressor paradigm and a holistic ecosystem-based understanding of these systems is still in its infancy. We expand on a well-known theoretical model to motivate an integrated multi-stressor framework for coral reefs by incorporating empirical evidence of multi-stressor impacts (overfishing, eutrophication and climate-driven mortality) as well as general ecological and theoretical concepts. We show that: i) the geometry of a simple, empirically-motivated model suggests nutrients and harvesting can operate similarly in driving shifts from coral- to algae-dominated reefs; ii) these impacts increase nonlinearly when acting concurrently, resulting in clear context-dependent management implications; and iii) this same geometry suggests climate-driven coral mortality (temperature-stress, cyclonic storms) can drive the presence of long transients and climate-driven alternate states, even in moderately-impacted ecosystems. These results imply that reefs that appear to be in a "safe space" may in fact be in danger of being pushed into a degraded algae-dominated state as storms and bleaching events are increasing in frequency and magnitude. Altogether, we find that responses in benthic composition as "signatures of change" to multi-stressors allows us to develop a predictive multi-stressor framework for coral reefs. In line with this theory, we detail empirical evidence from Barbados that highlights the context-dependent nature of coral reefs in a changing world. Our results present novel and generalizable insights into the functioning of coral reefs, that draw from classic theoretical and ecological concepts such as keystone predation theory, ecological succession and life history theory, as well as the emerging fields of long transients and early warning signals. By bridging coral reef ecology and general ecological concepts, we can better understand ecosystem functioning and resilience in these important yet highly threatened systems.

Open Research Statement: Data used in this manuscript are already published and publicly available. Sources can be found within the manuscript and in Supplement B. Mathematical analyses and simulations were performed in Wolfram Mathematica (version 12.1.0.0); code is available at: https://github.com/carlingbieg/corals

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Increased amphetamine-induced hyperactivity in translin (Tsn) KO mice is driven by elevated adiposity The translin/trax microRNA-degrading enzyme mediates activity-induced changes in translation that underlie several long-lasting forms of cellular plasticity. As translin and trax are expressed in dopaminergic and striatal neurons, we proceeded to investigate whether deletion of Tsn blocks amphetamine sensitization, a long-lasting, translation-dependent form of behavioral plasticity,

Although we expected constitutive Tsn deletion to impair amphetamine sensitization, we found, instead, that it enhances the hyperlocomotion produced by the initial dose of amphetamine. Since these mice display elevated adiposity, which alters pharmacokinetics of many drugs, we measured brain amphetamine levels in Tsn knockout mice and found that these are elevated. We also found that diet-induced increases in adiposity in WT mice correlate with elevated brain amphetamine levels. As amphetamine and its analogues are widely used to treat attention deficit disorder, which is associated with obesity, further studies are needed to assess the impact of adiposity on amphetamine levels in these patients.

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Genome structures resolve the early diversification of teleost fishes Accurate species phylogenies are a prerequisite for evolutionary research. Teleosts are by far the largest and the most diversified group of extant vertebrates, but relationships among the three oldest lineages of extant teleosts remain unresolved. Based on seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (bony-tongues) in a monophyletic group sister to all other teleosts, i.e., the Clupeocephala lineage. This finding resolves over 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.

One-Sentence SummaryWhole-genome analyses place Elopomorpha (tarpons, eels) and Osteoglossomorpha (bony-tongues) as sister groups at the deepest branching of crown teleosts.

sentences: Hyperactive WNT/CTNNB1 signaling induces a competing cell proliferation and epidermal differentiation response in the mouse mammary epithelium In the past forty years, the WNT/CTNNB1 signaling pathway has emerged as a key player in mammary gland development and homeostasis. While also evidently involved in breast cancer, much unclarity continues to surround its precise role in mammary tumor formation and progression. This is largely due to the fact that the specific and direct effects of hyperactive WNT/CTNNB1 signaling on the mammary epithelium remain unknown. Here we use a primary mouse mammary organoid culture system to close this fundamental knowledge gap. We show that hyperactive WNT/CTNNB1 signaling induces competing cell proliferation and differentiation responses. While proliferation is dominant at lower levels of WNT/CTNNB1 signaling activity, higher levels cause reprogramming towards an epidermal cell fate. We show that this involves de novo activation of the epidermal differentiation cluster (EDC) locus and we identify master regulatory transcription factors that likely control the process. This is the first time that the molecular and cellular dose-response effects of WNT/CTNNB1 signaling in the mammary epithelium have been dissected in such detail. Our analyses reveal that the mammary epithelium is exquisitely sensitive to small changes in WNT/CTNNB1 signaling and offer a mechanistic explanation for the squamous differentiation that is observed in some WNT/CTNNB1 driven tumors.

sentences: Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis Pregnancy reprograms the epigenome of mammary epithelial cells (MECs) in a manner that control responses to pregnancy hormone re-exposure and the rate of carcinoma progression. However, the influence of pregnancy on the tissue microenvironment of the mammary gland is less clear. Here, we used single-cell RNA sequencing to comparatively profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis revealed an expansion of {gamma}{delta} Natural Killer T (NKT) immune cells following pregnancy, in association with upregulation of immune signal molecules in post-pregnancy MECs. We show that expansion of NKT cells following pregnancy is due to elevated expression of the antigen presenting molecule CD1d protein, which is known to induce NKT activation. Accordingly, loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKT cells, accompanied the development of mammary oncogenesis in response to cMYC overexpression and loss of Brca1 function. Collectively, our findings illustrate how pregnancy-induced epigenetic changes modulate the communication between MECs and the mammary immune microenvironment, and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

sentences: Patterns of retrieval-related cortico-striatal connectivity are stable across the adult lifespan Memory retrieval effects in the striatum are well documented and robust across a number of experimental paradigms and stimulus materials. However, the functional significance of these effects, and whether they are moderated by age, remains unclear. In the present study, we used fMRI paired with an associative recognition task to examine retrieval effects in the striatum in a large sample of healthy young, middle aged, and older adults. We identified anatomically segregated patterns of enhanced striatal BOLD activity during recollection- and familiarity-based memory judgments. Successful recollection was associated with enhanced BOLD activity in bilateral putamen and nucleus accumbens. Familiarity effects were evident in the head of the caudate nucleus bilaterally. Crucially, none of these effects were reliably moderated by age. Using psychophysiological interaction analyses, we observed a monitoring-related increase in functional connectivity between the caudate and regions of the frontoparietal control network, and between the putamen and bilateral retrosplenial cortex and intraparietal sulcus. In all instances, monitoring-related increases in cortico-striatal connectivity were unmoderated by age. These results suggest that the striatum, and the caudate in particular, couples with the frontoparietal control network to support top-down retrieval monitoring operations, and that the integrity of these effects are preserved in advanced age.

sentences: Community science designed ribosomes with beneficial phenotypes Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

sentences: Measuring expression heterogeneity of single-cell cytoskeletal protein complexes Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock dysregulates F- and G-actin correlation. The assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

sentences: Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

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Dorsal skull meningeal lymphatic vessels drain blood-solutes after intracerebral hemorrhage Drainage of intraparenchymal hematoma is crucial for the treatment of intracerebral hemorrhage (ICH). We investigated here the possible function of the meningeal lymphatic vessels (mLVs) in ICH resolution. Meningeal lymphatics have been reported to be involved in cerebrospinal fluid drainage, but their role in the drainage and clearance of brain parenchyma has not been characterized in details. Using two preclinical models of ICH, mimicking focal cortical ischemic hemorrhage and subcortical extended hemorrhage, we characterized the dynamics of blood drainage through the dorsal mLVs by two-photon real time imaging in awake mice. After ICH induction, we observe the flow of blood-derived components within the mLVs, suggesting that meningeal lymphatics can play a role in facilitating the drainage of the hemorrhage. We also found that local formation of new mLVs is directly correlated with ICH-related neuroinflammation levels. These findings suggest that meningeal lymphatics could provide a valuable therapeutic target for ICH resolution.

SummaryIn vivo awake imaging reveals the direct drainage of extravasated blood-solutes from brain parenchyma into dorsal meningeal lymphatic vessels, following focal or diffuse intracranial hemorrhage

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Objective method reveals minimum time required to quantify net cost of transport across slow to fast walking speeds The U-shaped net cost of transport (COT) curve of walking has helped scientists understand the biomechanical basis that underlies energy minimization during walking. However, to produce an individuals net COT curve, data must be analyzed during periods of steady-rate metabolism. Traditionally, studies analyze the last few minutes of a 6-10 min trial, assuming that steady-rate metabolism has been achieved. Yet, it is possible that an individual achieves steady rates of metabolism much earlier. However, there is no consensus on how to objectively quantify steady-rate metabolism across a range of walking speeds. Therefore, we developed an objective method to determine the minimum time needed for humans to achieve steady rates of metabolism across slow to fast walking speeds. We hypothesized that a shorter time window could be used to produce a net COT curve that is comparable to the net COT curve created using traditional methods. We analyzed metabolic data from twenty-one subjects who completed several 7-min walking trials ranging from 0.50-2.00 m/s. We partitioned the metabolic data for each trial into moving 1-min, 2-min, and 3 min intervals and calculated their slopes. We statistically compared these slope values to values derived from the last 3-min of the 7-min trial, our  gold standard comparison. We found that a minimum of 2 min is required to achieve steady-rate metabolism and that data from 2-4 min yields a net COT curve that is not statistically different from the one derived from experimental protocols that are generally accepted in the field.

New and NoteworthyA simple slope method reveals that 4-min of walking at speeds between 0.50-1.75 m/s is the minimum time required to produce an accurate net cost of transport curve.

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Leukotrienes promote stem cell self-renewal and chemoresistance in acute myeloid leukemia Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

Key PointsO_LIAML blasts overexpress and induce ALOX5 expression in response to daunorubicin
C_LIO_LI5-Lipoxygenase (ALOX5) promotes blast survival and LSC self-renewal.
C_LIO_LIThe effects of ALOX5 on AML blast survival and self-renewal are mediated by the LTB4-BLTR signaling axis.
C_LI

sentences: Reconstitution of ATP-dependent 3'-cleavage of mammalian pre-mRNA reveals a central role of RBBP6 The 3 ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction, endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of fourteen polypeptides essential and two stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, Cleavage Factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.

sentences: The Food Additive Xanthan Gum Drives Adaptation of the Human Gut Microbiota The diets of industrialized countries reflect the increasing use of processed foods, often with the introduction of novel food additives. Xanthan gum is a complex polysaccharide with unique rheological properties that have established its use as a widespread stabilizer and thickening agent1. However, little is known about its direct interaction with the gut microbiota, which plays a central role in digestion of other, chemically-distinct dietary fiber polysaccharides. Here, we show that the ability to digest xanthan gum is surprisingly common in industrialized human gut microbiomes and appears to be contingent on the activity of a single bacterium that is a member of an uncultured bacterial genus in the family Ruminococcaceae. We used a combination of enrichment culture, multi-omics, and recombinant enzyme studies to identify and characterize a complete pathway in this uncultured bacterium for the degradation of xanthan gum. Our data reveal that this keystone degrader cleaves the xanthan gum backbone with a novel glycoside hydrolase family 5 (GH5) enzyme before processing the released oligosaccharides using additional enzymes. Surprisingly, some individuals harbor a Bacteroides species that is capable of consuming oligosaccharide products generated by the keystone Ruminococcaceae or a purified form of the GH5 enzyme. This Bacteroides symbiont is equipped with its own distinct enzymatic pathway to cross-feed on xanthan gum breakdown products, which still harbor the native linkage complexity in xanthan gum, but it cannot directly degrade the high molecular weight polymer. Thus, the introduction of a common food additive into the human diet in the past 50 years has promoted the establishment of a food chain involving at least two members of different phyla of gut bacteria.

sentences: Same role but different actors: genetic regulation of post-translational modification of two distinct proteins Post-translational modifications (PTMs) diversify protein functions and dynamically coordinate their signalling networks, influencing most aspects of cell physiology. Nevertheless, their genetic regulation or influence on complex traits is not fully understood. Here, we compare for the first time the genetic regulation of the same PTM of two proteins - glycosylation of transferrin and immunoglobulin G (IgG). By performing genome-wide association analysis of transferrin glycosylation, we identified 10 significantly associated loci, all novel. Comparing these with IgG glycosylation-associated genes, we note protein-specific associations with genes encoding glycosylation enzymes (transferrin - MGAT5, ST3GAL4, B3GAT1; IgG - MGAT3, ST6GAL1) as well as shared associations (FUT6, FUT8). Colocalisation analyses of the latter suggest that different causal variants in the FUT genes regulate fucosylation of the two proteins. We propose that they affect the binding of different transcription factors in different tissues, with fucosylation of IgG being regulated by IKZF1 in B-cells and of transferrin by HNF1A in liver.

sentences: Nanofibrous PEDOT-Carbon Composite on Flexible Probes for Soft Neural Interfacing In this study, we report a flexible implantable 4-channel microelectrode probe coated with highly porous and robust nanocomposite of poly(3,4-ethylenedioxythiophene) (PEDOT) and carbon nanofiber (CNF) as a solid doping template for high-performance in vivo neuronal recording and stimulation. A simple yet well-controlled deposition strategy was developed via in situ electrochemical polymerization technique to create a porous network of PEDOT and CNFs on a flexible 4-channel gold microelectrode probe. Different morphological and electrochemical characterizations showed that they exhibit remarkable and superior electrochemical properties, yielding microelectrodes combining high surface area, low impedance (16.8 {+/-} 2 M{Omega}.m2 at 1 kHz) and elevated charge injection capabilities (7.6 {+/-} 1.3 mC/cm2) that exceed those of pure and composite PEDOT layers. In addition, the PEDOT-CNF composite electrode exhibited extended biphasic charge cycle endurance, resulting in a negligible physical delamination or degradation for long periods of electrical stimulation. In vitro testing on mouse brain slices showed that they can record spontaneous oscillatory field potentials as well as single-unit action potentials and allow to safely deliver electrical stimulation for evoking field potentials. The combined superior electrical properties, durability and 3D microstructure topology of the PEDOT-CNF composite electrodes demonstrate outstanding potential for developing future neural surface interfacing applications.

sentences: PEN receptor GPR83 in anxiety-like behaviors: differential regulation in global vs amygdalar knockdown Anxiety disorders are prevalent across the United States and result in a large personal and societal burden. Currently, numerous therapeutic and pharmaceutical treatment options exist. However, drugs to classical receptor targets have shown limited efficacy and often come with unpleasant side effects, highlighting the need to identify novel targets involved in the etiology and treatment of anxiety disorders. GPR83, a recently deorphanized receptor activated by the abundant neuropeptide PEN, has also been identified as a glucocorticoid regulated receptor (and named GIR) suggesting that this receptor may be involved in stress-responses that underlie anxiety. Consistent with this, GPR83 null mice have been found to be resistant to stress-induced anxiety. However, studies examining the role of GPR83 within specific brain regions or potential sex differences have been lacking. In this study, we investigate anxiety-related behaviors in male and female mice with global knockout and following local GPR83 knockdown in female mice. We find that a global knockdown of GPR83 has minimal impact on anxiety-like behaviors in female mice and a decrease in anxiety-related behaviors in male mice. In contrast, a local GPR83 knockdown in the basolateral amygdala leads to more anxiety-related behaviors in female mice. Local GPR83 knockdown in the central amygdala or nucleus accumbens showed no significant effect on anxiety-related behaviors. Finally, dexamethasone administration leads to a significant decrease in receptor expression in the amygdala and nucleus accumbens of female mice. Together, our studies uncover a significant, but divergent role for GPR83 in different brain regions in the regulation of anxiety-related behaviors, which is furthermore dependent on sex.

sentences: Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft Microdomains Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains, to develop a set of photoswitchable sphingolipids, with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A ({lambda} = 365 nm) and blue ({lambda} = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine-(Azo-{beta}-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo--Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable Lo lipid raft domains.

sentences: Probabilistic modeling of ambient noise in single-cell omics data Droplet-based single-cell omics, including single-cell RNA sequencing (scRNAseq), single-cell CRISPR perturbations (e.g., CROP-seq), and single-cell protein and transcriptomic profiling (CITE-seq) hold great promise for comprehensive cell profiling and genetic screening at the single-cell resolution. However, these technologies suffer from substantial noise, among which ambient signals present in the cell suspension may be the predominant source. Current models to address this issue are highly technology-specific and relatively scRNAseq-centric. while a universal model to describe the noise across these technologies may reveal this common source, improving the denoising accuracy. To this end, we explicitly examined these unexpected signals in multiple datasets across droplet-based technologies, summarised a predictable pattern, and developed single-cell Ambient Remover (scAR) - a hypothesis-driven machine learning model to predict and remove ambient signals (including mRNA counts, protein counts, and sgRNA counts) at the molecular level. We benchmarked scAR on three technologies - single-cell CRISPR screens, CITE-seq, and scRNAseq along with the state-of-the-art single-technology-specific approaches. scAR showed high denoising accuracy for each type of dataset.

sentences: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 [A]) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 [A]). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

sentences: Bacterial and fungal communities experience rapid secondary succession during the first year following a wildfire in a California chaparral. The rise in wildfire frequency and severity across the globe has increased interest in secondary succession. However, despite the role of soil microbial communities in the regeneration of post-fire vegetation and biogeochemical cycling, the lack of measurements immediately post-fire and at high temporal resolution has resulted in a limited understanding of microbial secondary succession. To fill this knowledge gap, we sampled soils at 17, 25, 34, 67, 95, 131, 187, 286, and 376 days after a Southern California wildfire in fire-adapted chaparral shrublands. We assessed bacterial and fungal biomass with qPCR of 16S and 18S and richness and composition with Illumina MiSeq sequencing of 16S and ITS2 amplicons. Fire severely reduced bacterial biomass by 47% and richness by 46%, but the impacts were stronger for fungi, with biomass decreasing by 86% and richness by 68%. While bacterial biomass and richness oscillated in response to precipitation, the declines in biomass and richness for both groups persisted for the entire year. In contrast to the unburned plots, burned fungal and bacterial communities experienced rapid succession, with 5-6 compositional turnover periods. Analogous to plants, "fire-loving" pyrophilous microbes increased post-fire and drove successional dynamics. Interestingly, many of the same pyrophilous taxa found in forests worldwide also dominated post-fire chaparral and traded off markedly in abundance over time. For fungi, an initial peak in the Basidiomycete yeast Geminibasidium traded off with the filamentous Ascomycetes Pyronema, Aspergillus, and Penicillium. For bacteria, the Proteobacteria genus Massilia dominated burned plots throughout the year, but the following most dominant bacteria, the Firmicute Bacillus and Proteobacteria Noviherbaspirillum, traded off in abundance over time. Our unprecedented high-resolution temporal sampling allowed us to capture post-fire microbial secondary successional dynamics and show that putative tradeoffs in thermotolerance, colonization, and competition among dominant pyrophilous microbes control microbial succession with possible implications for ecosystem function.

sentences: TELSAM polymers accelerate crystallization of fused target proteins by stabilizing minimal crystal contacts and in the absence of direct inter-TELSAM contacts We extend investigation into the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We tested various numbers of the target protein fused per turn of the TELSAM helical polymer and various TELSAM-target connection strategies. We provide definitive evidence that: 1. A TELSAM-target protein fusion can crystallize more rapidly than the same target protein alone, 2. TELSAM-target protein fusions can form well-ordered, diffracting crystals using either flexible or rigid TELSAM-target linkers, 3. Well-ordered crystals can be obtained when either 2 or 6 copies of the target protein are presented per turn of the TELSAM helical polymer, 4. The TELSAM polymers themselves need not directly contact one another in the crystal lattice, and 5. Fusion to TELSAM polymer confers immense avidity to stabilize exquisitely weak inter-target protein crystal contacts. We report features of TELSAM-target protein crystals and outline future work needed to define the requirements for reliably obtaining optimal crystals of TELSAM-target protein fusions.

sentences: Crumbs complex-directed apical membrane dynamics in epithelial cell ingression Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cell lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

sentences: Discovery of TMPRSS2 inhibitors from virtual screening The SARS-CoV-2 pandemic has prompted researchers to pivot their efforts to finding antiviral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received attention for the development of drugs against SARS and MERS. Starting with comparative structural modeling and binding model analysis, we developed an efficient pharmacophore-based approach and applied a large-scale in silico database screening for small molecule inhibitors against TMPRSS2. The hits were evaluated in the TMPRSS2 biochemical assay and the SARS-CoV-2 pseudotyped particle (PP) entry assay. A number of novel inhibitors were identified, providing starting points for further development of drug candidates for the treatment of COVID-19.

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Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir (PF-07321332) The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics showed that these Mpro variants are similarly catalytically competent as the wildtype. We show that nirmatrelvir has similar potency against the variants as against the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.

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sentences: Evidence for continent-wide convergent evolution and stasis throughout 150 years of a biological invasion The extent to which evolution can rescue a species from extinction, or facilitate range expansion, depends critically on the rate, duration, and geographical extent of the evolutionary response to natural selection. Adaptive evolution can occur quickly, but the duration and geographical extent of contemporary evolution in natural systems remains poorly studied. This is particularly true for species with large geographical ranges and for timescales that lie between  long-term field experiments and the fossil record. Here, we introduce the Virtual Common Garden (VCG) to investigate phenotypic evolution in natural history collections while controlling for phenotypic plasticity in response to local growing conditions. Reconstructing 150 years of evolution in Lythrum salicaria (purple loosestrife) as it invaded North America, we analyze phenology measurements of 3,429 herbarium records, reconstruct growing conditions from more than 12 million local temperature records, and validate predictions across three common gardens spanning 10 degrees of latitude. We find that phenology clines have evolved along parallel climatic gradients, repeatedly throughout the range, during the first century of evolution. Thereafter, the rate of microevolution stalls, recapitulating macroevolutionary stasis observed in the fossil record. Our study demonstrates that preserved specimens are a critical resource for investigating limits to evolution in natural populations. Our results show how natural selection and trade-offs measured in field studies predict adaptive divergence observable in herbarium specimens over 15 decades at a continental scale.

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Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme Cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes acting during carbapenem antibiotic biosynthesis carry out radical-mediated methyl transfers that underlie the therapeutic usefulness of these essential medicines. Here we present x-ray crystal structures of TokK, which are representative of this functional class, containing its two metallocofactors and determined in the presence and absence of carbapenam substrate. The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggests that substrate positioning governs the rate of each methylation event.

One Sentence SummaryStructural insight into a cobalamin-dependent radical SAM methylase that performs three sequential radical-mediated methylations to install the C6 side chain of a carbapenem antibiotic.

sentences: Introspection confidence predicts EEG decoding of self-generated thoughts and meta-awareness The neurophysiological bases of mind wandering (MW) - an experiential state wherein attention is disengaged from the external environment in favour of internal thoughts, and state meta-awareness are poorly understood. In parallel, the relationship between introspection confidence in experiential state judgements and neural representations remains unclear. Here, we recorded EEG whilst participants completed a listening task within which they made experiential state judgments and rated their confidence. Alpha power was reliably greater during MW episodes, with unaware MW further associated with greater delta and theta power. Multivariate pattern classification analysis revealed that MW, and meta-awareness can be decoded from the distribution of power in these three frequency bands. Critically, we show that individual decoding accuracies positively correlate with introspection confidence. Our results reaffirm the role of alpha oscillations in MW, implicate lower frequencies in meta-awareness, and are consistent with the proposal that introspection confidence indexes neurophysiological discriminability of representational states.

sentences: Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitors The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with GO pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.

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A base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding While methylation of CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of most transcription factors (TFs), recent in vitro studies have revealed a more complex picture, suggesting that over a third of TFs may preferentially bind to methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences, however, is challenging, as the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. As a result, the in vivo methylation preferences of most TFs remain uncharacterized.

Here, we introduce joint accessibility-methylation-sequence (JAMS) models, which connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. We show that JAMS models quantitatively explain the TF binding strength, recapitulate cell type-specific TF binding, and have high precision for inferring intra-motif methylation effects. Analysis of 2209 ChIP-seq experiments resulted in high-confidence JAMS models for 260 TFs, revealing that 45% of TFs are inhibited by intra-motif methylation in vivo. In contrast, only 16 TFs (6%) preferentially bind to methylated sites, including 11 novel methyl-binding TFs that are mostly from the multi-zinc finger family of TFs.

Our study substantially expands the repertoire of in vivo methyl-binding TFs, but also suggests that most TFs that prefer methylated CpGs in vitro present themselves as methylation agnostic in vivo, potentially due to the balancing effect of competition with other methyl-binding proteins.

sentences: A mechanical G2 checkpoint controls epithelial cell division through E-cadherin-mediated regulation of Wee1-Cdk1 Epithelial cell divisions must be tightly coordinated with cell loss to preserve epithelial integrity. However, it is not well understood how the rate of epithelial cell division adapts to changes in cell number, for instance during homeostatic turnover or upon wounding of epithelia. Here, we show epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell cycle progression. We demonstrate that tensile forces on E-cadherin adhesions are reduced as local cell density increases, which prompts the accumulation of the G2 checkpoint kinase Wee1. This elevated abundance of Wee1 results in inhibitory phoshorylation of Cdk1, and thereby establishes a pool of cells that is temporarily halted in G2-phase. Importantly, these cells are readily triggered to divide upon epithelial wounding, due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data thus demonstrate that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.

sentences: Open-Source Thermometer, Temperature Controller, and Light Meter for Use in Animal Facilities and During Experiments Experiments with biological samples require precise control of environmental conditions. In our work we use zebrafish (Danio rerio) to understand the neurobiology of sleep, which requires precise control of temperature and lighting. Like many labs, lighting and temperature in the animal facility are centrally controlled in the building. During behavioral experiments and microscopy sessions, we use custom-built heating systems and perform occasional manual checks of conditions. However, without a system to precisely record conditions, gradual changes in temperature can go unnoticed for a long time, and temporary failures may be missed entirely. Here we present the design and characterization of affordable open-source tools to record temperature and light conditions during animal experiments using an Arduino microcontroller or a Raspberry Pi compact computer. The waterproof temperature sensor has high stability over 50 days of recording and is precise to 0.1{degrees}C. The Arduino device can be used through a common serial port interface for which we present code in Python and MATLAB. The Raspberry Pi version can be accessed through a web interface, for which we provide an installation guide. We use the device to record and review temperature and lighting conditions in two zebrafish animal facilities. We use our platform to add a water heating system to maintain temperature at 28{degrees}C during in vivo light-sheet imaging of larval zebrafish. We show that a change in temperature from 28{degrees}C to 32{degrees}C affects resting heart rate of the animal, highlighting the importance of maintaining and recording conditions. The protocols presented here do not require advanced engineering, fabrication, or software skills, and provide an approach to accurately record and report experimental conditions.

sentences: Modulation of nuclear and cytoplasmic mRNA fluctuations by time-dependent stimuli: analytical distributions Temporal variation of environmental stimuli leads to changes in gene expression. Since the latter is noisy and since many reaction events occur between the birth and death of an mRNA molecule, it is of interest to understand how a stimulus affects the transcript numbers measured at various sub-cellular locations. Here, we construct a stochastic model describing the dynamics of signal-dependent gene expression and its propagation downstream of transcription. For any time-dependent stimulus and assuming bursty gene expression, we devise a procedure which allows us to obtain time-dependent distributions of mRNA numbers at various stages of its life-cycle, e.g. in its nascent form at the transcription site, post-splicing in the nucleus, and after it is exported to the cytoplasm. We also derive an expression for the error in the approximation whose accuracy is verified via stochastic simulation. We find that, depending on the frequency of oscillation and the time of measurement, a stimulus can lead to cytoplasmic amplification or attenuation of transcriptional noise.

sentences: Simulations of flow over a bio-inspired undulated cylinder with dynamically morphing topography Inspired by the geometric properties of seal whiskers, the addition of spanwise undulations on a cylinder have been shown to lower the mean and oscillatory forces and modify the frequency of flow-induced vibration when compared to smooth cylinders. Previously, computational fluid dynamics (CFD) has been used to characterize the hydrodynamic response with respect to specific geometric features. However simulations are time intensive due to complex three-dimensional meshing and computation time, limiting the number of geometric perturbations explored. A method is proposed in which the specific geometric features of this complex topography can be modified during a simulation thereby decreasing the time per geometric modification and removing the need for manual meshing of the complex structure. The surface of the seal whisker inspired geometry is parameterized into seven defining parameters, each of which is directly controlled in order to morph the surface into any realization within the defined parameter space. Once validated, the algorithm is used to explore the undulation amplitudes in the chord and thickness directions, by varying each independently from 0 to 0.3 thicknesses in increments of 0.05 at a Reynolds number of 500. The force and frequency response are examined for this matrix of geometric parameters, yielding detailed force trends not previously investigated. The impact of the bio-inspired morphing algorithm will allow for further optimization and development of force-mitigating underwater devices and other engineering applications in need of vibration suppression, frequency tuning, or force reduction.

sentences: Hyperoxia Inhibits the Growth of Mouse Forebrain Oligodendrocyte Progenitors NG2 chondroitin sulfate proteoglycan positive oligodendrocyte progenitor cells (OPCs) reside throughout the brain. They divide asymmetrically and differentiate into myelinating oligodendrocytes throughout adulthood. OPCs have been successfully isolated from rodents using several techniques including magnetic beads, immunopanning and exploiting differential centripetal adhesion. Whereas rat OPCs are relatively simple to propagate in vitro, it has been difficult to expand mouse OPCs. Therefore, we evaluated the effects of oxygen levels, growth factors and extracellular matrix components to produce a simple and reproducible method to prepare large numbers of nearly homogenous cultures of primary mouse OPCs from postnatal day 0-2 mouse telencephala. Using the McCarthy and de Vellis mechanical separation method OPCs were separated from mixed culture of glial cells. When the OPCs were plated onto fibronectin coated tissue culture plates in a biochemically defined medium that contained fibroblast growth factor-2 (FGF-2) and platelet derived growth factor AA (PDGFAA), and they were maintained in a standard tissue culture incubator, they proliferated very slowly. By contrast, mouse OPCs doubled approximately every 7 days when maintained in a 2% oxygen, nitrogen buffered environment. After 3 passages, greater than 99% of these OPCs were NG2+/PDGFR+. In medium containing only FGF-2, mouse OPCs progressed to late stage OPCs whereupon A2B5 expression decreased and O4 expression increased. When these cells were differentiated between passages 1 and 3, the majority of the OPCs differentiated into MBP+ mature oligodendrocytes However, cells that were repeatedly passaged beyond 4 passages progressed to a late O4+ OPC (even with mitogens present) and when differentiated by mitogen removal a minority of the OPCs differentiated into MBP+ cells. These studies reveal significant differences between mouse and rat OPCs and an inhibitory role for oxygen in mouse OPC proliferation.

sentences: SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

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Machine Learning to Summarize and Provide Context for Sleep and Eating Schedules. The relative timing of sleep and of eating within the circadian day is important for human health. Despite much data on sleep, and a growing data set for eating, there remains a need for an interpretative framework for the understanding of this data for health decisions. This study provides a new statistical and machine learning analysis of more than 500 participants in the Daily24 project. From their data, and the analysis, we propose a framework for determining the classification of participants into different chronotypes and with that the ability to realize the potential impact of daily circadian habits on health. We propose that our resulting distribution curves could be used, similar to NHANES (National Health and Nutrition Examination Survey) data for pediatric growth, as a measure for circadian misalignment and used to help guide re-entrainment schedules.

Author summaryDaily habits can be positive, negative or neutral for human health. Generally sleep and eating schedules are assumed without thought for their potential to help or interfere with health. In this study we propose a framework, based on data from more than 500 participants, for evaluating the relative timing of meals and sleep schedules. This evaluation, similar to pediatric growth charts, can guide clinical suggestions for those at the extremes, while helping others to realize that they are unusual relative to the population average

sentences: Structural basis for Parkinson's Disease-linked LRRK2's binding to microtubules Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinsons Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.

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5-aminolevulinic acid-photodynamic therapy ameliorates cutaneous granuloma by killing drug-resistant Mycobacterium marinum BackgroundAlthough 5-aminolevulinic acid photodynamic therapy (ALA-PDT) has been extensively used to treat to various skin disease, the application of ALA-PDT on cutaneous infection caused by Mycobacterium marinum (M. marinum), especially by drug-resistant M. marinum is still not clear.

ObjectivesWe evaluated the therapeutic efficacy of ALA-PDT on M. marinum in a mouse infection model and tested its killing effect on M. marinum in vitro. Finally, we investigated the clinical effect of ALA-PDT in treating cutaneous granuloma caused by drug-resistant M. marinum.

Materials and methodsWe isolated total 9 strains of M. marinum from patients and confirmed by morphological and molecular approaches. The strains were identified by anti-mycobacterial susceptibility. Then we evaluated the killing effect of ALA-PDT on M. marinum in vitro and in a mouse model to observe the antimycobacterial effect of ALA-PDT. Therapeutic efficacy was further assessed in patients with cutaneous granuloma caused by drug-resistant M. marinum.

ResultsWe demonstrated that the ALA-PDT directly killed M. marinum in vitro. The paws cutaneous lesions of mice caused by M. marinum were fully recovered 2 weeks after ALA-PDT treatment. However, there was no significant difference for immune cells in peripheral blood before and after ALA-PDT therapy. Finally, ALA-PDT proved to be effective in treat two patients with cutaneous infection caused by drug-resistant M. marinum.

ConclusionsThe results suggest that ALA-PDT is effective in treating M. marinum cutaneous infections by killing M. marinum directly, independent of systemic immune responses. The data highlight the ALA-PDT as a promising therapeutic choice for M. marinum infection, especially for drug-resistant strains.

sentences: Generalization of motor learning in psychological space To generate adaptive movements, we must generalize what we have previously learned to novel situations. The generalization of learned movements has typically been framed as a consequence of neural tuning functions that overlap for similar movement kinematics. However, as is true in many domains of human behavior, situations that require generalization can also be framed as inference problems. Here, we attempt to broaden the scope of theories about motor generalization, hypothesizing that part of the typical motor generalization function can be characterized as a consequence of top-down decisions about different movement contexts. We tested this proposal by having participants make explicit similarity ratings over traditional contextual dimensions (movement directions) and abstract contextual dimensions (target shape), and perform a visuomotor adaptation generalization task where trials varied over those dimensions. We found support for our predictions across five experiments, which revealed a tight link between subjective similarity and motor generalization. Our findings suggest that the generalization of learned motor behaviors is influenced by both low-level kinematic features and high-level inferences.

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Natural reassortment of a segmented RNA arbovirus illustrates plasticity of phenotype in the arthropod vector and mammalian host in vivo Segmented RNA viruses are a taxonomically diverse group of 11 families that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during co-infection of a host by a process termed  reassortment. Reassortment enables rapid evolutionary change, but in the case of segmented RNA viruses utilising an arthropod vector is set against the constraint of purifying selection and genetic bottlenecks imposed by replication in two evolutionarily distant hosts. In this study, we use an in vivo host: arbovirus: vector model to investigate the impact of reassortment on two phenotypic traits: vector competence and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV is comprised of 10 linear segments of dsRNA and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4 and BTV-8) were isolated from naturally infected ruminants in Europe and parental/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to infect Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the parental virus strains which allowed inferences of the genome segments conferring this phenotypic trait.

IMPORTANCEReassortment between strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses with consequences for spread, persistence and impact. The ability of these pathogens to change their phenotypes rapidly in response to selection pressure in new environments presents a major challenge in understanding factors driving emergence. Utilising a natural mammalian host-insect vector infection and transmission model, we demonstrated for the first time the genetic basis for a phenotypic trait of BTV within strains directly isolated from the field and, hence, selected and relevant for natural transmission.

sentences: Fast, live-cell imaging of 15 intracellular compartments by deep learning segmentation of super-resolution data The number of colors that can be used in fluorescence microscopy to image the live-cell anatomy and organelles interactions is far less than the number of intracellular organelles and compartments. Here, we report that deep convolutional neuronal networks can predict 15 subcellular structures from super-resolution spinning-disk microscopy images using only one dye, one laser excitation, and two detection channels. Comparing to the colocalization images, this method achieves pixel accuracies of over 91.7%, which not only bypasses the fundamental limitation of multi-color imaging but also accelerates the imaging speed by more than one order of magnitude.

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Combined use of Oxford Nanopore and Illumina sequencing yields insights into soybean structural variation biology BackgroundStructural variant (SV) discovery based on short reads is challenging due to their complex signatures and tendency to occur in repeated regions. The increasing availability of long-read technologies has greatly facilitated SV discovery, however these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars.

ResultsWe used Oxford Nanopore sequencing data ([~]12X mean coverage) for 17 samples to both benchmark SV calls made from the Illumina data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped SVs for population structure analysis and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of SVs are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency SVs are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the SVs and report evidence of the recent activity of a Stowaway MITE.

ConclusionsOur results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species.

sentences: LOSS OF PLASMA MEMBRANE LIPID ASYMMETRY CAN INDUCE ORDERED DOMAIN (RAFT) FORMATION In some cases lipids in one leaflet of an asymmetric artificial lipid vesicle can suppress formation of ordered lipid domains (rafts) in the opposing leaflet. Whether suppression of domain formation might occur in plasma membranes was studied using plasma membrane vesicles (PMVs) from RBL-2H3 cells. Membrane domain formation and order was assessed by FRET and fluorescence anisotropy. Ordered domains in PMV prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ~37{degrees}C, while ordered domains in symmetric vesicles formed from extracted PMV lipids were stable to 55{degrees}C, indicating that stability of ordered domains was substantially less in intact PMV. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting that intact PMV have some degree of lipid asymmetry. This was confirmed by annexin binding showing that NEM PMV are much more asymmetric than PMV formed by dithiothreitol/paraformaldehyde treatment. Stabilization of ordered domain formation, and increased membrane order at low temperature was also observed after detergent solubilization of PMV followed by membrane reconstitution via dilution from detergent, which also should destroy asymmetry, even though membrane proteins remained associated with the reconstituted vesicles. Similar changes in domain formation and membrane order were observed after detergent reconstitution of artificial asymmetric lipid vesicles. PMV ordered domain stability was not increased by digesting peripheral domains of PMV proteins with proteinase K. We conclude loss of PMV lipid asymmetry can induce ordered domain formation. Dynamic control of asymmetry may regulate ordered domain formation in plasma membranes.

sentences: Harringtonine has the effects of double blocking SARS-CoV-2 membrane fusion Fusion with host cell membrane is the main mechanism of infection of SARS-CoV-2. Here, we propose a new strategy to double block SARS-CoV-2 membrane fusion by using Harringtonine (HT), a small-molecule antagonist. By using cell membrane chromatography (CMC), we found that HT specifically targeted the SARS-CoV-2 S protein and host cell TMPRSS2, and then confirmed that HT can inhibit pseudotyped virus membrane fusion. Furthermore, HT successfully blocked SARS-CoV-2 infection, especially in the delta and Omicron mutant. Since HT is a small-molecule antagonist, it is minimally affected by the continuous variation of SARS-CoV-2. Our findings show that HT is a potential small-molecule antagonist with a new mechanism of action against SARS-CoV-2 infection, and thus HT mainly targets the S protein, and thus, greatly reduces the damage of the S proteins autotoxicity to the organ system, has promising advantages in the clinical treatment of COVID-19.

sentences: Modeling the SARS-CoV-2 mutation based on geographical regions and time The Coronavirus Disease 2019 (COVID-19) epidemic was first detected in late-December 2019. So far, it has caused 203,815,431 confirmed cases and 4,310,623 deaths in the world. We collected sequences from 150,659 COVID-19 patients. Based on the previous phylogenomic analysis, we found three major branches of the virus RNA genomic mutation located in Asia, America, and Europe which is consistent with other studies. We selected sites with high mutation frequencies from Asia, America, and Europe. There are only 13 common mutation sites in these three regions. It infers that the viral mutations are highly dependent on their location and different locations have specific mutations. Most mutations can lead to amino acid substitutions, which occurred in 3/5UTR, S/N/M protein, and ORF1ab/3a/8/10. Thus, the mutations may affect the pathogenesis of the virus. In addition, we applied an ARIMA model to predict the short-term frequency change of these top mutation sites during the spread of the disease. We tested a variety of settings of the ARIMA model to optimize the prediction effect of three patterns. This model can provide good help for predicting short-term mutation frequency changes.

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Metabolic differentiation of co-occurring Accumulibacter clades revealed through genome-resolved metatranscriptomics Natural microbial communities consist of closely related taxa that may exhibit phenotypic differences and inhabit distinct niches. However, connecting genetic diversity to ecological properties remains a challenge in microbial ecology due to the lack of pure cultures across the microbial tree of life.  Candidatus Accumulibacter phosphatis is a polyphosphate-accumulating organism that contributes to the Enhanced Biological Phosphorus Removal (EBPR) biotechnological process for removing excess phosphorus from wastewater and preventing eutrophication from downstream receiving waters. Distinct Accumulibacter clades often co-exist in full-scale wastewater treatment plants and lab-scale enrichment bioreactors, and have been hypothesized to inhabit distinct ecological niches. However, since individual strains of the Accumulibacter lineage have not been isolated in pure culture to date, these predictions have been made solely on genome-based comparisons and enrichments with varying strain composition. Here, we used genome-resolved metagenomics and metatranscriptomics to explore the activity of co-existing Accumulibacter strains in an engineered bioreactor environment. We obtained four high-quality genomes of Accumulibacter strains that were present in the bioreactor ecosystem, one of which is a completely contiguous draft genome scaffolded with long Nanopore reads. We identified core and accessory genes to investigate how gene expression patterns differed among the dominating strains. Using this approach, we were able to identify putative pathways and functions that may confer distinct functions to Accumulibacter strains and provide key functional insights into this biotechnologically significant microbial lineage.

IMPORTANCE Candidatus Accumulibacter phosphatis is a model polyphosphate accumulating organism that has been studied using genome-resolved metagenomics, metatranscriptomics, and metaproteomics to understand the EBPR process. Within the Accumulibacter lineage, several similar but diverging clades are defined by shared sequence identity of the polyphosphate kinase (ppk1) locus. These clades are predicted to have key functional differences in acetate uptake rates, phage defense mechanisms, and nitrogen cycling capabilities. However, such hypotheses have largely been made based on gene-content comparisons of sequenced Accumulibacter genomes, some of which were obtained from different systems. Here, we performed time-series genome-resolved metatranscriptomics to explore gene expression patterns of co-existing Accumulibacter clades in the same bioreactor ecosystem. Our work provides an approach for elucidating ecologically relevant functions based on gene expression patterns between closely related microbial populations.

sentences: Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information is available on the complex and a detailed understanding of its mechanism is lacking. We investigated the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1N-HDAC1-MBD3GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2 (NuDe) using Bayesian integrative structure determination with IMP (Integrative Modeling Platform), drawing on information from SEC-MALLS, DIA-MS, XLMS, negative-stain EM, X-ray crystallography, NMR spectroscopy, secondary structure and homology predictions. The structures were corroborated by independent cryo-EM maps, biochemical assays, and known cancer-associated mutations. MBD3 is a demethylase that connects the deacetylase and chromatin-remodeling modules in NuRD. Localization of the full-length MBD3 in NuRD was not previously characterized. Our models indicate two different localizations for MBD3 in NuRD, suggesting a mechanism by which MBD3 in the presence of GATAD2 asymmetrically bridges the two modules in NuRD. Further, our models indicate three previously unrecognized subunit interfaces in NuDe: HDAC1C-MTA1BAH, MTA1BAH-MBD3, and HDAC160-100-MBD3. We observed that a significant number of cancer-associated mutations mapped to protein-protein interfaces in NuDe. Our approach also allows us to localize regions of unknown structure, such as HDAC1C and MBD3IDR, thereby resulting in the most complete structural characterization of these NuRD sub-complexes so far.

sentences: Quantifying concordant genetic effects of de novo mutations on multiple disorders Exome sequencing on tens of thousands of parent-proband trios has identified numerous deleterious de novo mutations (DNMs) and implicated risk genes for many disorders. Recent studies have suggested shared genes and pathways are enriched for DNMs across multiple disorders. However, existing analytic strategies only focus on genes that reach statistical significance for multiple disorders and require large trio samples in each study. As a result, these methods are not able to characterize the full landscape of genetic sharing due to polygenicity and incomplete penetrance. In this work, we introduce EncoreDNM, a novel statistical framework to quantify shared genetic effects between two disorders characterized by concordant enrichment of DNMs in the exome. EncoreDNM makes use of exome-wide, summary-level DNM data, including genes that do not reach statistical significance in single-disorder analysis, to evaluate the overall and annotation-partitioned genetic sharing between two disorders. Applying EncoreDNM to DNM data of nine disorders, we identified abundant pairwise enrichment correlations, especially in genes intolerant to pathogenic mutations and genes highly expressed in fetal tissues. These results suggest that EncoreDNM improves current analytic approaches and may have broad applications in DNM studies.

sentences: iRGD-liposomes enhance tumor delivery and therapeutic efficacy of antisense oligonucleotide drugs against primary prostate cancer and bone metastasis Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.

sentences: SnoRNA guide activities-real and ambiguous In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified posttranscriptionally. Pseudouridylation and 2-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-{Psi}609, despite some non-canonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, posttranscriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.

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SAR11 Cells Rely on Enzyme Multifunctionality to Transport and Metabolize a Range of Polyamine Compounds In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells transport and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, Ca. Pelagibacter st. HTCC7211 and C. P. ubique st. HTCC1062. Both strains transported all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but we did not find evidence of use as carbon sources. We propose potABCD transports cadaverine, agmatine, and norspermidine, in addition to its usual substrates of spermidine and putrescine, and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they oxidize.

ImportanceGenome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality is hypothesized to be an adaptation that increases the range of organic compounds oxidized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells use multiple polyamine compounds and propose that a small set of multifunctional genes catalyze this metabolism. We also report polyamine uptake rates can exceed metabolism, resulting in high intracellular concentrations of these nitrogen-rich compounds and an increase in cell size. Increases in cytoplasmic solute concentrations during transient episodes of high nutrient exposure has previously been observed in SAR11 cells and may be a feature of their strategy for maximizing the share of labile DOM acquired when in competition with other cell types.

sentences: Divergent effects of absolute evidence magnitude on decision accuracy and confidence in perceptual judgements Whether people change their mind after making a perceptual judgement may depend on how confident they are in their decision. Recently, it was shown that, when making perceptual judgements about stimuli containing high levels of  absolute evidence (i.e., the overall magnitude of sensory evidence across choice options), people make less accurate decisions and are also slower to change their mind and correct their mistakes. Here we report two studies that investigated whether high levels of absolute evidence also lead to increased decision confidence. We used a luminance judgment task in which participants decided which of two dynamic, flickering stimuli was brighter. After making a decision, participants rated their confidence. We manipulated relative evidence (i.e., the mean luminance difference between the two stimuli) and absolute evidence (i.e., the summed luminance of the two stimuli). In the first experiment, we found that higher absolute evidence was associated with decreased decision accuracy but increased decision confidence. In the second experiment, we additionally manipulated the degree of luminance variability to assess whether the observed effects were due to differences in perceived evidence variability. We replicated the results of the first experiment but did not find substantial effects of luminance variability on confidence ratings. Our findings support the view that decisions and confidence judgments are based on partly dissociable sources of information, and suggest that decisions initially made with higher confidence may be more resistant to subsequent changes of mind.

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Rate-of-change analysis in palaeoecology revisited: a new approach AO_SCPLOWBSTRACTC_SCPLOWDynamics in the rate of compositional change (rate-of-change; RoC) of biotic or abiotic assemblages preserved in palaeoecological sequences, are thought to reflect changes due to exogenous drivers such as climate and human forcing as well as endogenous factors linked to local dynamics and biotic interactions. However, changes in sedimentation rates and sampling strategies can result in an uneven distribution of time intervals and are known to affect RoC estimates. Furthermore, there has been relatively little exploration of the implications of these challenges in quantifying RoC in palaeoecology.

Here, we introduce R-Ratepol - an easy-to-use R package - that provides a robust numerical technique for detecting and summarising RoC patterns in complex multivariate time-ordered stratigraphical sequences. First, we compare the performance of common methods of estimating RoC and detecting periods of high RoC (peak-point) using simulated pollen-stratigraphical data with known patterns of compositional change and temporal resolution. In addition, we propose a new method of binning with a moving window, which shows a more than 5-fold increase in the correct detection of peak-points compared to the more traditional way of using individual levels.

Next, we apply our new methodology to four representative European pollen sequences and show that our approach also performs well in detecting periods of significant compositional change during known onsets of human activity, early land-use transformation, and changes in fire frequency.

Expanding the approach using R-Ratepol to open-access paleoecological datasets in global databases, such as Neotoma, will allow future palaeoecological and macroecological studies to quantify major changes in biotic composition or in sets of abiotic variables across broad spatio-temporal scales.

sentences: Systemic and mucosal mobilization of granulocyte subsets during lentivirus infection Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentivirus infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood from naive, and chronically SHIVsf162p3-infected RM were analyzed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils - CD45+CD66+CD49d+; neutrophils - CD45+CD66+CD14+; and basophils - CD45+CD123+FcR{varepsilon}+. Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcR{gamma}II) but did not express CD16 (FcR{gamma}III). Additionally, constitutive expression of CD64 (FcR{gamma}I) on neutrophils and FcR{varepsilon} on basophils, indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naive whole blood ranged 25.4-81.5% neutrophils, 0.59-13.3% eosinophils and 0.059-1.8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils, and colorectal eosinophils were all observed in chronic lentivirus disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils, and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentivirus infection, most notably in the gastrointestinal mucosae where significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.

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Reading-related brain changes in audiovisual processing: cross-sectional and longitudinal MEG evidence The ability to establish associations between visual objects and speech sounds is essential for human reading. Understanding the neural adjustments required for acquisition of these arbitrary audiovisual associations can shed light on fundamental reading mechanisms and help reveal how literacy builds on pre-existing brain circuits. To address these questions, the present longitudinal and cross-sectional MEG studies characterize the temporal and spatial neural correlates of audiovisual syllable congruency in children (4-9 years old, 22 males and 20 females) learning to read. Both studies showed that during the first years of reading instruction children gradually set up audiovisual correspondences between letters and speech sounds, which can be detected within the first 400 ms of a bimodal presentation and recruit the superior portions of the left temporal cortex. These findings suggest that children progressively change the way they treat audiovisual syllables as a function of their reading experience. This reading-specific brain plasticity implies (partial) recruitment of pre-existing brain circuits for audiovisual analysis.

Significance StatementLinking visual and auditory linguistic representations is the basis for the development of efficient reading, while dysfunctional audiovisual letter processing predicts future reading disorders. Our developmental MEG project included a longitudinal and a cross-sectional study; both studies showed that childrens audiovisual brain circuits progressively change as a function of reading experience. They also revealed an exceptional degree of neuroplasticity in audiovisual neural networks, showing that as children develop literacy, the brain progressively adapts so as to better detect new correspondences between letters and speech sounds.

sentences: Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as identified by sCRilecs-seq Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the {beta}-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

sentences: Shaping nanoparticle diffusion through biological barriers to drug delivery Nanoparticle drug delivery systems encounter many biological barriers, such as the extracellular matrix and mucus gels, that they must bypass to gain access to target cells. A design parameter that has recently gained attention is nanoparticle shape, as it has been shown elongated rod-shaped nanoparticles achieve higher diffusion rates through biological gels. However, the optimal dimensions of rod-shaped nanoparticles to enhance this effect has yet to be established. To systematically approach this, rod-shaped nanoparticles were synthesized by mechanically stretching 100 nm, 200 nm, and 500 nm spherical nanoparticles. Transmission electron microscopy confirmed this procedure yields a significant fraction of elongated rods and remaining spheres could be removed by centrifugation. Fluorescent microscopy and multiple particle tracking analysis was then used to characterize rod-shaped and spherical nanoparticle diffusion in MaxGel(R), a model extracellular matrix hydrogel. When dispersed in MaxGel, we found rod-shaped nanoparticles exhibited the greatest enhancement in diffusion rate when their length far exceeds the average hydrogel network size. These results further establish the importance of shape as a design criterion to improve nanoparticle-based drug delivery systems.

sentences: Disentangling the flow of signals between populations of neurons Technological advances now allow us to record from large populations of neurons across multiple brain areas. These recordings may illuminate how communication between areas contributes to brain function, yet a substantial barrier remains: How do we disentangle the concurrent, bidirectional flow of signals between populations of neurons? We therefore propose here a novel dimensionality reduction framework: Delayed Latents Across Groups (DLAG). DLAG disentangles signals relayed in each direction, identifies how these signals are represented by each population, and characterizes how they evolve within and across trials. We demonstrate that DLAG performs well on synthetic datasets similar in scale to current neurophysiological recordings. Then we study simultaneously recorded populations in primate visual areas V1 and V2, where DLAG reveals signatures of bidirectional yet selective communication. Our framework lays a foundation for dissecting the intricate flow of signals across populations of neurons, and how this signaling contributes to cortical computation.

sentences: Metal-binding propensity in the mitochondrial dynamin-related protein 1 Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Metal-binding therefore represents a potential strategy to deplete or purify Drp1 from native tissue lysates. Importantly, high concentrations of the metal in solution inhibit GTP hydrolysis and renders Drp1 inactive in membrane fission. Together, our results emphasize a metal-binding propensity, which could significantly impact Drp1 functions.

sentences: The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic ELMODs are a family of three mammalian paralogs that display GTPase activating protein (GAP) activity towards a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogs ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon expression of activating mutants of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions. Thus, we believe that ELMOD1 and ELMOD3 perform multiple functions in cells, most prominently linked to ciliary biology and Golgi-ciliary traffic, and likely acting from more than one cellular location.

sentences: Identification of Gip as a novel phage-encoded gyrase inhibitor protein featuring a broad activity profile Bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. In this study, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum, resulted in the identification of the novel gyrase-inhibiting protein Cg1978 (Gip), which shows a direct interaction with the gyrase subunit A (GyrA). In vitro supercoiling assays further suggest a stabilization of the cleavage complex by Gip. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. The cells adapted to gip overexpression by increasing expression levels of gyrAB and by reducing topA expression reflecting the homeostatic control of DNA topology. Interestingly, Gip features a similar activity profile towards gyrases of Escherichia coli, Mycobacterium tuberculosis and C. glutamicum. Therefore, the detailed elucidation of the mechanism of Gip action may provide novel directions for the design of drugs targeting DNA gyrase.

sentences: Morphognostic honey bees communicating nectar location through dance movements AO_SCPLOWBSTRACTC_SCPLOWHoney bees are social insects that forage for flower nectar cooperatively. When an individual forager discovers a flower patch rich in nectar, it returns to the hive and performs a "waggle dance" in the vicinity of other bees that consists of movements communicating the direction and distance to the nectar source. The dance recruits "witnessing" bees to fly to the location of the nectar to retrieve it, thus cooperatively exploiting the environment. Replicating such complex animal behavior is a step forward on the path to artificial intelligence. This project simulates the bee foraging behavior in a cellular automaton using the Morphognosis machine learning model. The model features hierarchical spatial and temporal contexts that output motor responses from sensory inputs. Given a set of bee foraging and dancing exemplars, and exposing only the external input-output of these behaviors to the Morphognosis learning algorithm, a hive of artificial bees can be generated that forage as their biological counterparts do. A comparison of Morphognosis foraging performance with that of an artificial recurrent neural network is also presented.

sentences: Predicting drug polypharmacology from cell morphology readouts using variational autoencoder latent space arithmetic A variational autoencoder (VAE) is a machine learning algorithm, useful for generating a compressed and interpretable latent space. These representations have been generated from various biomedical data types and can be used to produce realistic-looking simulated data. However, standard vanilla VAEs suffer from entangled and uninformative latent spaces, which can be mitigated using other types of VAEs such as {beta}-VAE and MMD-VAE. In this project, we evaluated the ability of VAEs to learn cell morphology characteristics derived from cell images. We trained and evaluated these three VAE variants--Vanilla VAE, {beta}-VAE, and MMD-VAE--on cell morphology readouts and explored the generative capacity of each model to predict compound polypharmacology (the interactions of a drug with more than one target) using an approach called latent space arithmetic (LSA). To test the generalizability of the strategy, we also trained these VAEs using gene expression data of the same compound perturbations and found that gene expression provides complementary information. We found that the {beta}-VAE and MMD-VAE disentangle morphology signals and reveal a more interpretable latent space. We reliably simulated morphology and gene expression readouts from certain compounds thereby predicting cell states perturbed with compounds of known polypharmacology. Inferring cell state for specific drug mechanisms could aid researchers in developing and identifying targeted therapeutics and categorizing off-target effects in the future.

sentences: Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3'UTRs. Although widespread, the role of APA in the biology of cells, tissues and organisms has been controversial. We identified over 500 Drosophila genes that express mRNA isoforms with a long 3'UTR in proliferating spermatogonia but a short 3'UTR in differentiating spermatocytes due to APA. We show that the stage- specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3'UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed over 250 genes where the long 3'UTR versus short 3'UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3'end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

sentences: Assembly and Validation of Two Gap-free Reference Genomes for Xian/indica Rice Reveals Insights into Plant Centromere Architecture Rice (Oryza sativa), a major staple throughout the world and a model system for plant genomics and breeding, was the first crop genome completed almost two decades ago. However, all sequenced genomes to date contain gaps and missing sequences. Here, we report, for the first time, the assembly and analyses of two gap-free reference genome sequences of the elite O. sativa xian/indica rice varieties  Zhenshan 97 (ZS97) and  Minghui 63 (MH63) that are being used as a model system to study heterosis. Gap-free reference genomes also provide global insights into the structure and function of centromeres. All rice centromeric regions share conserved centromere-specific satellite motifs but with different copy numbers and structures. Importantly, we demonstrate that >1,500 genes are located in centromere regions, of which ~15.6% are actively transcribed. The generation and release of both the ZS97 and MH63 gap-free genomes lays a solid foundation for the comprehensive study of genome structure and function in plants and breed climate resilient varieties for the 21st century.

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Biological responses to terahertz radiation with different power density in primary hippocampal neurons BackgroundTerahertz (THz) radiation is a valuable imaging and sensing tool which is widely used in industry and medicine. However, it biological effects including genotoxicity and cytotoxicity are not clear, particularly on the nervous system. In this study, we investigated how THz radiation with different frequency and intensity would affect the morphology, cell growth and function of rat hippocampal neurons in vitro.

MethodsPrimary hippocampal neuronal cells were isolated from newborn Wistar rat pups and were exposed to THz radiation with the frequencies of 0.12 THz and 0.141 THz, at an power intensities of 10 mW/cm2 and 30 mW/cm2 respectively. The cellular activities were evaluated by Cell Counting Kit-8 after exposure for 10 min and 30 min. The cellular apoptosis was examined by Annexin V staining using flow cytometry and the ultrastructure was detected by electron microscopy. Moreover, the amount of amino acid in the cultured neurons was measured by high performance liquid chromatography (HPLC).

ResultsNo obvious morphological changes, such as axon length and axon number, could be detected in primary neurons after exposure to 10mW/cm2 or 50 mW/cm2 THz for 30 min. However, the overall cellular activities were differentially affected by 10mW/cm2 and 50 mW/cm2 THz. Obvious cellular apoptosis was induced by both 10mW/cm2 and 50 mW/cm2 exposure. We also found that the amounts of most amino acids did not change significantly after exposure to these two types of THz radiation.

ConclusionsThe effects of THz radiation on hippocampal neurons depend on frequency, power, exposure time as well as the measurement time after the exposure. These results will provide important information for further investigation of the effect of THz radiation on the nervous system.

sentences: Characterizing interactions between the microtubule-binding protein CLIP-170 and F-actin The cooperation between the actin and microtubule (MT) cytoskeletons is important for cellular processes such as cell migration and muscle cell development. Full understanding of how this cooperation occurs has yet to be sufficiently developed. The MT plus-end tracking protein (+TIP) CLIP-170 has been implicated in this actin-MT coordination by associating with the actin-binding signaling protein IQGAP1, and by promoting actin polymerization through binding with formins. Thus far, CLIP-170s interactions with actin were assumed to be indirect. Here, we demonstrate that CLIP-170 can bind to filamentous actin (F-actin) directly. The affinity is relatively weak, but is strong enough to be significant in the actin-rich cortex, where actin concentrations can be extremely high. Using CLIP-170 fragments and mutants, we show that the direct CLIP-170:actin interaction is independent of the FEED domain, the region that mediates formin-dependent actin polymerization, and that the CLIP-170 F-actin-binding region overlaps with the MT-binding region. Consistent with these observations, in vitro competition assays indicate that CLIP-170:F-actin and CLIP-170:MT interactions are mutually exclusive. Taken together, these observations lead us to speculate that direct CLIP-170:F-actin interactions may function to reduce the stability of MTs in actin-rich regions of the cell, as previously proposed for EB1.

sentences: Correction of amblyopia in cats and mice after the critical period Monocular deprivation early in development causes amblyopia, a severe visual impairment. Prognosis is poor if therapy is initiated after an early critical period. However, clinical observations have shown that recovery from amblyopia can occur later in life when the non-deprived (fellow) eye is removed. The traditional interpretation of this finding is that vision is improved by relieving interocular suppression in primary visual cortex. However, an alternative explanation is that elimination of activity in the fellow eye establishes conditions in visual cortex that enable the weak connections from the amblyopic eye to gain strength. Here we show in cats and mice that temporary inactivation of the fellow eye is sufficient to promote a full and enduring recovery from amblyopia at ages when conventional treatments fail. Thus, connections serving the amblyopic eye are capable of substantial plasticity beyond the critical period, and this potential is unleashed by reversibly silencing the fellow eye.

sentences: Geometric and Topological Approaches to Shape Variation in Ginkgo Leaves Leaf shape is a key plant trait that varies enormously. The diversity of leaf shape, and the range of applications for data on this trait, requires frequent methodological developments so that researchers have an up-to-date toolkit with which to quantify leaf shape. We generated a dataset of 468 leaves produced by Ginkgo biloba, and 24 fossil leaves produced by evolutionary relatives of extant Ginkgo. We quantified the shape of each leaf by developing a geometric method based on elastic curves and a topological method based on persistent homology. Our geometric method indicates that shape variation in our modern sample is dominated by leaf size, furrow depth, and the angle of the two lobes at the base of the leaf that is also related to leaf width. Our topological method indicates that shape variation in our modern sample is dominated by leaf size and furrow depth. We have applied both methods to modern and fossil material: the methods are complementary, identifying similar primary patterns of variation, but also revealing some different aspects of morphological variation. Our topological approach distinguishes long-shoot leaves from short-shoot leaves and both methods indicate that leaf shape influences or is at least related to leaf area.

sentences: RIG-I uses its intrinsically disordered CARDs-Helicase linker in RNA proofreading functions The innate immune receptor RIG-I provides the first line of defense against viral infections. Viral RNAs are recognized by the C-terminal domain (CTD) of RIG-I, but the RNA must engage the helicase domain to release the signaling domain CARDs from their autoinhibitory CARD2:Hel2i interactions. Because the helicase lacks RNA specificity, there must be mechanisms to proofread RNAs entering the helicase domain. Although such mechanisms are crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely unknown. This study reveals a previously unknown proofreading mechanism that RIG-I uses to ensure the helicase engages RNAs chosen explicitly by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which uses its negatively charged regions to electrostatically antagonize incoming RNAs. In addition to RNA gating, CHL is essential in stabilizing the CARD2:Hel2i interface. The CHL and CARD2:Hel2i interface work together, establishing a tunable gating mechanism that allows CTD-chosen RNAs to bind into the helicase while blocking non-specific RNAs. With its critical regulatory functions, CHL represents a novel target for RIG-I-based therapeutics.

sentences: Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

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Non-linear Dimensionality Reduction on Extracellular Waveforms Reveals Physiological, Functional, and Laminar Diversity in Premotor Cortex Cortical circuits are thought to contain a large number of cell types that coordinate to produce behavior. Current in vivo methods rely on clustering of specified features of extracellular waveforms to identify putative cell types, but these capture only a small amount of variation. Here, we develop a new method (WaveMAP) that combines non-linear dimensionality reduction with graph clustering to identify putative cell types. We apply WaveMAP to extracellular waveforms recorded from dorsal premotor cortex of macaque monkeys performing a decision-making task. Using WaveMAP, we robustly establish eight waveform clusters and show that these clusters recapitulate previously identified narrow- and broad-spiking types while revealing previously unknown diversity within these subtypes. The eight clusters exhibited distinct laminar distributions, characteristic firing rate patterns, and decision-related dynamics. Such insights were weaker when using feature-based approaches. WaveMAP therefore provides a more nuanced understanding of the dynamics of cell types in cortical circuits.

SignificanceHow different cell types sculpt activity patterns in brain areas associated with decision-making is a fundamentally unresolved problem in neuroscience. In monkeys, and other species where transgenic access is not yet possible, identifying physiological types in vivo relies on only a few discrete user-specified features of extracellular waveforms to identify cell types. Here, we show that non-linear dimensionality reduction with graph clustering applied to the entire extracellular waveform can delineate many different putative cell types and does so in an interpretable manner. We show that this method reveals previously undocumented physiological, functional, and laminar diversity in the dorsal premotor cortex of monkeys, a key brain area implicated in decision-making.

sentences: Talaromyces marneffei promotes M2 polarization of human macrophages by downregulating SOCS3 expression and activating TLR9 pathway Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1 and fungal-promoting M2 types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them towards M2 polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2 polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1 polarization and plays an important role in limiting M2 polarization. Furthermore, we found that inhibition of TLR9 pathway partially blocked T. marneffei-induced M2 polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

sentences: An Experimental Test of the Effects of Redacting Grant Applicant Identifiers on Peer Review Outcomes Blinding reviewers to applicant identity has been proposed to reduce bias in peer review. This experimental test used 1200 NIH grant applications, 400 from Black investigators, 400 matched applications from White investigators, and 400 randomly selected applications from White investigators. Applications were reviewed by mail in standard and redacted formats. Redaction reduced, but did not eliminate, reviewers ability to correctly guess features of identity. The primary, pre-registered analysis hypothesized a differential effect of redaction according to investigator race in the matched applications. A set of secondary analyses (not pre-registered) used the randomly selected applications from White scientists and tested the same interaction. Both analyses revealed similar effects: Standard format applications from White investigators scored better than those from Black investigators; redaction reduced the size of the difference by about half (e.g. from a Cohens d of 0.20 to 0.10 in matched applications); redaction caused applications from White scientists to score worse but had no effect on scores for Black applications. The primary statistical test of the study hypothesis was not significant; the secondary analysis was significant. The findings support further evaluation of peer review models that diminish the influence of applicant identity.

sentences: An Efficient and Cost-effective Purification Methodology for Cas9 Nuclease With an ever-increasing demand for laboratory-grade Cas9 proteins by many groups advancing the use of CRISPR technology, a more efficient and scalable process for generating the proteins, coupled with rapid purification methods is in urgent demand. Here, we introduce a modified methodology for rapid purification of active SaCas9 protein within 24 hours. The product has over 90% protein purity. The simplicity and cost-effectiveness of such methodology will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating the advancement of CRISPR/Cas9-based research.

sentences: Engineered bacteria detect tumor DNA in vivo Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to diseases such as gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can easily access the entire gastrointestinal tract via oral administration8, and they can produce outputs that can be noninvasively measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangement11, has been used to allow bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells and organoids. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered orally or rectally into mice injected with orthotopic donor CRC organoids. We observe horizontal gene transfer from the tumor to the sensor bacteria in vivo, allowing their detection in stool. The sensor bacteria achieved 100% discrimination between mice with and without CRC using both delivery methods. Our findings establish a framework for biosensing applications that require the detection of mutations or organisms within environments that are difficult to sample. In addition, the platform can be readily expanded to include in situ production and delivery of therapeutic payloads at the detection site.

sentences: Genotype by microbiome interactions have large effects on growth in Lotus japonicus Biological interactions between plants and their root microbiomes are pivotal for plant growth. Even though the plant genotype [G], soil microbiome [C], and growth conditions (environment) [E] are core factors shaping the root microbiome, their relationships remain unclear. We disentangled the effects of G, C, E, and their interactions on the Lotus root microbiome and plant growth using a cross-inoculation approach that reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. We found that a large proportion of the root microbiome composition was determined by C and E and that G-related (G, G x C, and G x E) effects were significant but small. In contrast, the interactions between G and C had a more pronounced effect on plant shoot growth than C alone. Our findings indicate that most microbiome variations controlled by C have little effect on the plant phenotype, whereas G x C interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention in efforts to optimize crop yield and resilience.

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A novel subset of follicular helper-like MAIT cells has capacity for B cell help and antibody production in the mucosa. Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against bacterial pathogens at mucosal surfaces via release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests MAIT cells are capable of providing B cell help. In this study, we describe a previously unreported population of CXCR5+ T follicular helper (Tfh)-like MAIT cells, MAITfh, that have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially located near germinal centers in human tonsils and express the classical Tfh-associated transcription factor, B-cell lymphoma 6 (BCL-6), co-stimulatory markers, inducible T cell costimulatory (ICOS) and programmed death receptor 1 (PD-1), and cytokines, interleukin (IL)-21. Furthermore, we demonstrate the ability of MAIT cells to provide B cell help in vivo following mucosal challenge with Vibrio cholerae. Specifically, we show that adoptive transfer of MAIT cells into {beta} T cell-deficient mice promoted B cell differentiation and increased serum V. cholerae-specific IgA responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses, and suggest that MAIT cells may be potential targets for mucosal vaccines.

One Sentence SummaryWe identified and characterized a novel subset of T follicular helper-like MAIT (MAITfh) cells that has the capacity to provide B cell help, and show the sufficiency of MAIT cells to promote production of pathogen-specific IgA antibodies and B cell differentiation in mucosal challenge.

sentences: The efficiency of neutral honey as a tissue fixative in histopathology In the routine laboratory, 10% neutral buffered formalin (NBF) is the fixative of choice. However, formalin is a human carcinogen. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological tissues. This study aimed to examine the efficiency of neutral buffered honey and other types of honey fixatives to fix histological tissues. The most two natural common Omani honey were used as fixatives, namely Sumar and date. We tested samples of rat liver, kidney, and stomach. Nine types of fixatives were used. All tissues were treated equally. The evaluation was performed blindly by three senior biomedical scientists who work in a histopathology laboratory. Hematoxylin and eosin showed adequate staining in all groups when compared to 10% NBF. The intensity and specificity of Jones Methenamine silver stain in 10% Sumer and Date honey and 10% alcoholic Sumer honey showed similar findings of 10% NBF. The specificity and intensity of all groups for Periodic acid-Schiff were comparable with 10% neutral buffered formalin accepts for 10% Sumer honey and 10% Alcoholic Date honey. However, all honey groups showed weak staining for the reticulin fibers using Gordon and Sweets method. Vimentin showed comparable findings with 10% NBF as there were no significant differences. The findings of this study are promising. Further in depth research on honey as a possible safe substitute fixative for formalin should be conducted.

sentences: Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl homoserine lactones that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulations of their initial catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade bacterial signaling molecules via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these biotechnologically well-optimized enzymes to the QQ toolbox. We also observed enzyme- and substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier because its shallow acyl binding site could not accommodate a productive substrate-binding configuration. Conversely, aPGA in complex with both substrates exhibited unfavorable energetics in both reaction steps due to the dynamics of the residues gating the acyl binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297{beta} in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species.

sentences: Visualization and Modeling of Inhibition of IL-1β and TNFα mRNA Transcription at the Single-Cell Level IL-1{beta} and TNF- are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1{beta} and TNF- at the single-cell level. The amount of IL-1{beta} and TNF- mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-{kappa}B signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1{beta} and TNF- mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1{beta} is activated by both NF-{kappa}B and C/EBP, while TNF- is predominantly activated by NF-{kappa}B. Our combined single-cell experimental modeling efforts shows the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

sentences: Quantitative Analysis of Transcriptome Dynamics Provides Novel Insights into Developmental State Transitions During embryogenesis, the developmental potential of initially pluripotent cells becomes progressively restricted as they transit to lineage restricted states. The pluripotent cells of Xenopus blastula-stage embryos are an ideal system in which to study cell state transitions during developmental decision-making, as gene expression dynamics can be followed at high temporal resolution. Here we use transcriptomics to interrogate the process by which pluripotent cells transit to four different lineage-restricted states: neural progenitors, epidermis, endoderm and ventral mesoderm, providing quantitative insights into the dynamics of Waddingtons landscape. Our findings shed light on why the neural progenitor state is the default lineage state for pluripotent cells, and uncover novel components of lineage-specific gene regulation. These data reveal an unexpected overlap in the transcriptional responses to BMP4/7 and activin signaling, and provide mechanistic insight into how the timing of signaling inputs such as BMP are temporally controlled to ensure correct lineage decisions. Together these analyses provide quantitative insights into the logic and dynamics of developmental decision making in early embryos.

sentences: A novel ion conducting route besides the central pore in an inherited mutant of G-protein-gated inwardly rectifying K+ channel G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+, while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+, suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+-selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q sensitive K+ current and the second a TPN-Q resistant Li+ current. The results show that a novel Li+ permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix (PH), S148F and T151A, also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.

sentences: Immunomodulatory Contribution of Mast Cells to the Regenerative Biomaterial Microenvironment Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

sentences: THE LIFESPAN AND OXIDATIVE STRESS BETWEEN FERAL AND MANAGED HONEY BEE COLONIES Molecular damage caused by oxidative stress may lead to organismal aging and resulted in acute mortality in organisms. Oxidative stress resistance and longevity are closely linked. Honey bees are the most important managed pollinator in agriculture but the long-term survival of honey bees is seriously threatened. Feral honey bee colonies displayed persistence to Varroa mites. However, it is unknown whether feral honey bees are stress-resistant or survive longer than managed bee populations. More work is needed to determine the impact of oxidative stress on honey bee health and survival. We used the paired colony design to determine the lifespan and levels of oxidative stress on worker bees from either a feral or a managed colony. Each pair of colonies shared similar foraging resources. Results exhibit longer survival time and lifespans of foragers in feral colonies than the managed colonies. The levels of oxidative stress from the lipid damage of feral colonies are higher than the managed colonies, indicating a tolerant mechanism not a repair mechanism to survive. Our study provided new insights into colony difference of physiology and oxidative stress resistance between feral honey bees and commercial stocks.

sentences: A chloride efflux transporter OsBIRG1 regulates grain size and salt tolerance in rice Grain size is determined by the number of cells and cell size of the grain. Regulation of grain size is crucial for improving crop yield. However, the genes and underlying molecular mechanisms controlling grain size remain elusive. Here we report a member of Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) family transporter, BIG RICE GRAIN 1 (BIRG1), negatively regulates the grain size in rice. BIRG1 is highly expressed in reproductive organs and roots. In birg1 grain, the size of the outer parenchyma layer cells of spikelet hulls is noticeably larger but the cell number is not altered compared with that in the wild-type (WT) grain. When expressed in Xenopus oocytes, BIRG1 exhibits chloride efflux activity. In line with the role of BIRG1 in mediating chloride efflux, the birg1 mutant shows reduced tolerance to salt stress under which the chloride level is toxic. Moreover, the birg1 grains contain higher level of chloride compared to WT grains when grown under normal paddy field. The birg1 roots accumulate more chloride than those of WT under saline condition. Collectively, our findings suggest that BIRG1 functions as a chloride efflux transporter regulating grain size and salt tolerance via controlling chloride homeostasis in rice.

sentences: Deep learning-enhanced morphological profiling predicts cell fate dynamics in real-time in hPSCs Predicting how stem cells become patterned and differentiated into target tissues is key for optimising human tissue design. Here, we established DEEP-MAP - for deep learning-enhanced morphological profiling - an approach that integrates single-cell, multi-day, multi-colour microscopy phenomics with deep learning and allows to robustly map and predict cell fate dynamics in real-time without a need for cell state-specific reporters. Using human pluripotent stem cells (hPSCs) engineered to co-express the histone H2B and two-colour FUCCI cell cycle reporters, we used DEEP-MAP to capture hundreds of morphological- and proliferation-associated features for hundreds of thousands of cells and used this information to map and predict spatiotemporally single-cell fate dynamics across germ layer cell fates. We show that DEEP-MAP predicts fate changes as early or earlier than transcription factor-based fate reporters, reveals the timing and existence of intermediate cell fates invisible to fixed-cell technologies, and identifies proliferative properties predictive of cell fate transitions. DEEP-MAP provides a versatile, universal strategy to map tissue evolution and organisation across many developmental and tissue engineering contexts.

sentences: m6A RNA methylation impairs gene expression variability and reproductive thermotolerance in Arabidopsis Plants are more susceptible to high temperature stress during reproductive development, which can cause drastic yield loss of fruit and seed crops. Unfortunately, the underlying mechanism remains largely unknown. Here we suggest that m6A RNA methylation level increases in the reproductive tissues of Arabidopsis and negatively regulates gene expression variability. It has been suggested that stochasticity of gene expression can be advantageous to fitness of living organisms under environmental challenges. Indeed, reduced gene expression variability in flowers was associated with compromised transcriptional activation of heat-responsive genes. Importantly, disruption of an RNA demethylase AtALKBH10B led to lower gene expression variability, hypo-responsiveness of heat-activated genes, and strong reduction of plant fertility. Overall, our work proposes a novel mechanism that m6A RNA modification mediates the bet-hedging strategy of plants challenged by heat stress.

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A deep learning approach to real-time HIV outbreak detection using genetic data Pathogen genomic sequence data are increasingly made available for epidemiological monitoring. A main interest is to identify and assess the potential of infectious disease outbreaks. While popular methods to analyze sequence data often involve phylogenetic tree inference, they are vulnerable to errors from recombination and impose a high computational cost, making it difficult to obtain real-time results when the number of sequences is in or above the thousands.

Here, we propose an alternative strategy to outbreak detection using genomic data based on deep learning methods developed for image classification. The key idea is to use a pairwise genetic distance matrix calculated from viral sequences as an image, and develop convolutional neutral network (CNN) models to classify areas of the images that show signatures of active outbreak, leading to identification of subsets of sequences taken from an active outbreak. We showed that our method is efficient in finding HIV-1 outbreaks with R0[≥] 3, and overall a specificity exceeding 85% and sensitivity better than 70%. We validated our approach using data from HIV-1 CRF01 in Europe, containing both endemic sequences and a well-known dual outbreak in intravenous drug users. Our model accurately identified known outbreak sequences in the background of slower spreading HIV. Importantly, we detected both outbreaks early on, before they were over, implying that had this method been applied in real-time as data became available, one would have been able to intervene and possibly prevent the extent of these outbreaks. This approach is scalable to processing hundreds of thousands of sequences, making it useful for current and future real-time epidemiological investigations, including public health monitoring using large databases and especially for rapid outbreak identification.

Author summaryThe analysis of pathogen genomic data to analyze epidemics at scale is constrained by the computational cost associated with phylogenetic tree reconstruction. As a fast and efficient alternative, we employed convolutional neural networks to analyze evolutionary pairwise distance matrices as images to perform classifications of the current epidemiological situation of a growing public health sequence database. We used simulated data to train and test our model, and as validation we accurately mapped the start and end of two linked well-documented HIV-1 outbreaks in the backdrop of ongoing slower HIV spread. Thus, our new approach is efficient, accurate, scalable, and can analyze data in real time.

sentences: Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are eliminated via cell competition by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also relies on the activities of the transcription factors Irbp18 and Xrp1, however how these contribute to the loser status is partially understood. Here, we show that Irbp18 and Xrp1 induce the loser status by promoting proteotoxic stress. We find that Xrp1 is necessary for Rp/+ - induced proteotoxic stress and is sufficient to induce proteotoxic stress in otherwise wild-type cells. Xrp1 is also induced downstream of proteotoxic stress and required for the competitive elimination of cells suffering from proteotoxic stress. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.

sentences: Complex Systems Analysis Informs on the Spread of COVID-19 The non-linear progression of new infection numbers in a pandemic poses challenges to the evaluation of its management. The tools of complex systems research may aid in attaining information that would be difficult to extract with other means. To study the COVID-19 pandemic, we utilize the reported new cases per day for the globe, nine countries and six US states through October 2020. Fourier and univariate wavelet analyses inform on periodicity and extent of change. Evaluating time-lagged data sets of various lag lengths, we find that the autocorrelation function, average mutual information and box counting dimension represent good quantitative readouts for the progression of new infections. Bivariate wavelet analysis and return plots give indications of containment versus exacerbation. Homogeneity or heterogeneity in the population response, uptick versus suppression, and worsening or improving trends are discernible, in part by plotting various time lags in three dimensions. The analysis of epidemic or pandemic progression with the techniques available for observed (noisy) complex data can aid decision making in the public health response.

sentences: The vagus nerve is critical for regulation of hypothalamic-pituitary-adrenal axis responses to acute stress The hypothalamic pituitary adrenal (HPA) axis is a critical regulator of physiologic and psychological responses to acute and chronic stressors. HPA axis function is control by numerous feedback inhibitory mechanisms, disruptions of which can lead to various psychiatric conditions, such as depression, posttraumatic stress disorder, and schizophrenia. Vagus nerve stimulation has been shown to be efficacious in the treatment of in these various mental health issues potentially via modulation of HPA axis function, but the mechanisms by which the vagus nerve may regulate HPA function has not been fully elucidated. In the present studies, we sought to test the hypothesis that the vagus nerve is a critical regulator of HPA function. Neuroendocrine function and neurocircuit changes in corticotropin releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVN) were examined following acute stress after subdiaphragmatic left vagotomy (VX) in adult male Sprague-Dawley rats. We found that VX mimics HPA activation seen in sham surgery animals exposed to acute restraint stress, particularly increased plasma corticosterone levels, elevated PVN CRF mRNA, and increased action potential firing of putative CRF neurons in PVN brain slices. Furthermore, VX animals exposed to acute restraint stress showed increased elevations of plasma corticosterone and PVN CRF mRNA which may be due to lack of compensatory PVN GABAergic signaling in response to acute stress. Both Sham/Stress and VX/no stress conditions increased action potential firing in putative PVN CRF neurons, but this effect was not seen in the VX/stress condition, suggesting that not all forms of stress compensation are lost following VX. Overall, these findings suggest that the vagus nerve may play a critical role in regulating HPA axis function via modulation of local PVN neurocircuit activity.

sentences: Ultra-sensitive nanoLC-MS of sub nanogram protein samples using second generation micro pillar array LC technology with Orbitrap Exploris 480 and FAIMS PRO In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micro pillar dimensions that have been further downscaled (inter pillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5-0.05 ng/L) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

sentences: Probing the influence of SMA and vmPFC on the motor system with dual-site transcranial magnetic stimulation Dual-site transcranial magnetic stimulation (TMS) has been widely exploited to probe the influence of cortical structures on the primary motor cortex (M1). However, several issues remain open, notably regarding the use of this approach on areas of the medial frontal cortex - including the supplementary motor area (SMA) and the ventromedial prefrontal cortex (vmPFC) - known to play a fundamental role in motor behavior. First, the few TMS studies that have targeted SMA have mostly focused on short inter-stimulation intervals (6-8 ms), supposed to recruit cortico-cortical circuits. There is a current lack of data on the nature of the influence (i.e., facilitatory vs. suppressive) of SMA stimulation on M1 when probed with longer intervals (10-15 ms), thought to recruit more indirect cortico-subcortico-cortical circuits. Second, it is unclear whether the facilitatory influence of SMA stimulation previously reported with short intervals reflects the recruitment of cortico-cortical circuits (as commonly assumed) or results from the summation of volleys descending from SMA and M1 at the spinal level. Third, dual-site TMS has never been used to date to probe the influence of the vmPFC on M1, putatively due to the presumed difficulty of reaching the former area with magnetic fields. Here, we show that SMA stimulation facilitates motor activity with a 12 ms interval. Additionally, our data reveal that the facilitatory influence of SMA stimulation observed with short intervals does not result from spinal interactions. Finally, we show that vmPFC stimulation induces a moderate suppressive effect on M1, both with short and with long inter-stimulation intervals.

sentences: Genomic analyses of 10,376 individuals provides comprehensive map of genetic variations, structure and reference haplotypes for Chinese population Here, we initiated the Westlake BioBank for Chinese (WBBC) pilot project with 4,535 whole-genome sequencing individuals and 5,481 high-density genotyping individuals. We identified 80.99 million SNPs and INDELs, of which 38.6% are novel. The genetic evidence of Chinese population structure supported the corresponding geographical boundaries of the Qinling-Huaihe Line and Nanling Mountains. The genetic architecture within North Han was more homogeneous than South Han, and the history of effective population size of Lingnan began to deviate from the other three regions from 6 thousand years ago. In addition, we identified a novel locus (SNX29) under selection pressure and confirmed several loci associated with alcohol metabolism and histocompatibility systems. We observed significant selection of genes on epidermal cell differentiation and skin development only in southern Chinese. Finally, we provided an online imputation server (https://wbbc.westlake.edu.cn/) which could result in higher imputation accuracy compared to the existing panels, especially for lower frequency variants.

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Sharing individualised template MRI data for MEG source reconstruction: a solution for open data while keeping subject confidentiality The increasing requirements for adoption of FAIR data management and sharing original research data from neuroimaging studies can be at odds with protecting the anonymity of the research participants due to the person-identifiable anatomical features in the data. We propose a solution to this dilemma for anatomical MRIs used in MEG source analysis. In MEG analysis, the channel-level data is reconstructed to the source-level using models derived from anatomical MRIs. Sharing data, therefore, requires sharing the anatomical MRI to replicate the analysis. The suggested solution is to replace the individual anatomical MRIs with individualised warped templates that can be used to carry out the MEG source analysis and that provide sufficient geometrical similarity to the original participants MRIs.

First, we demonstrate how the individualised template warping can be implemented with one of the leading open-source neuroimaging analysis toolboxes. Second, we compare results from four different MEG source reconstruction methods performed with an individualised warped template to those using the participants original MRI. While the source reconstruction results are not numerically identical, there is a high similarity between the results for single dipole fits, dynamic imaging of coherent sources beamforming, and atlas-based virtual channel beamforming. There is a moderate similarity between minimum-norm estimates, as anticipated due to this method being anatomically constrained and dependent on the exact morphological features of the cortical sheet.

We also compared the morphological features of the warped template to those of the original MRI. These showed a high similarity in grey matter volume and surface area, but a low similarity in the average cortical thickness and the mean folding index within cortical parcels.

Taken together, this demonstrates that the results obtained by MEG source reconstruction can be preserved with the warped templates, whereas the anatomical and morphological fingerprint is sufficiently altered to protect the anonymity of research participants. In cases where participants consent to sharing anatomical MRI data, it remains preferable to share the original defaced data with an appropriate data use agreement. In cases where participants did not consent to share their MRIs, the individualised warped MRI template offers a good compromise in sharing data for reuse while retaining anonymity for those research participants.

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High-throughput mapping of single-cell molecular and projection architecture of neurons by retrograde barcoded labelling Deciphering mesoscopic connectivity of the mammalian brain is a pivotal step in neuroscience. Most imaging-based conventional neuroanatomical tracing methods identify area-to-area or sparse single neuronal labeling information. Although recently developed barcode-based connectomics has been able to map a large number of single-neuron projections efficiently, there is a missing link in single-cell connectome and transcriptome. Here, combining single-cell RNA sequencing technology, we established a retro-AAV barcode-based multiplexed tracing method called MEGRE-seq (Multiplexed projEction neuRons retroGrade barcodE), which can resolve projectome and transcriptome of source neurons simultaneously. Using the ventromedial prefrontal cortex (vmPFC) as a proof- of-concept neocortical region, we investigated projection patterns of its excitatory neurons targeting five canonical brain regions, as well as corresponding transcriptional profiles. Dedicated, bifurcated or collateral projection patterns were inferred by digital projectome. In combination with simultaneously recovered transcriptome, we find that certain projection pattern has a preferential layer or neuron subtype bias. Further, we fitted single-neuron two-modal data into a machine learning-based model and delineated gene importance by each projection target. In summary, we anticipate that the new multiplexed digital connectome technique is potential to understand the organizing principle of the neural circuit by linking projectome and transcriptome.

HighlightsO_LIMERGE-seq recovers single-neuron transcriptome and projectome simultaneously
C_LIO_LIMERGE-seq partitions projection pattern into neuronal layer and subtype
C_LIO_LIMERGE-seq bridges projection pattern to differentially expressed genes
C_LIO_LIMachine learning models interpret relationship between digital projectome and gene expression
C_LI

sentences: Large scale PVA modelling of insects in cultivated grasslands: the role of dispersal in mitigating the effects of management schedules under climate change In many species, dispersal is decisive for survival in a changing climate. Simulation models for population dynamics under climate change thus need to account for this factor. Moreover, large numbers of species inhabiting agricultural landscapes are subject to disturbances induced by human land use. We included dispersal in the HiLEG model that we previously developed to study the interaction between climate change and agricultural land use in single populations. Here, the model was parameterized for the large marsh grasshopper (LMG) in cultivated grasslands of North Germany to analyze (1) the species development and dispersal success depending on severity of climate change in sub regions, (2) the additional effect of grassland cover on dispersal success, and (3) the role of dispersal in compensating for detrimental grassland mowing. Our model simulated population dynamics in 60-year periods (2020-2079) on a fine temporal (daily) and high spatial (250 x 250 m2) scale in 107 sub regions, altogether encompassing a range of different grassland cover, climate change projections and mowing schedules. We show that climate change alone would allow the LMG to thrive and expand, while grassland cover played a minor role. Some mowing schedules that were harmful to the LMG nevertheless allowed the species to moderately expand its range. Especially under minor climate change, in many sub regions dispersal allowed for mowing early in the year, which is economically beneficial for farmers. More severe climate change could facilitate LMG expansion to uninhabited regions, but would require suitable mowing schedules along the path. These insights can be transferred to other species, given that the LMG is considered a representative of grassland communities. For more specific predictions on the dynamics of other species affected by climate change and land use, the publicly available HiLEG model can be easily adapted to the characteristics of their life cycle.

sentences: Interleukin-17A causes osteoarthritis-like transcriptional changes in human osteoarthritis-derived chondrocytes and synovial fibroblasts in vitro Increased interleukin (IL)-17A has been identified in joints affected by osteoarthritis (OA), but it is unclear how IL-17A, and its family members IL-17AF and IL-17F, can contribute to human OA pathophysiology. Therefore, we aimed to evaluate the gene expression and signalling pathway activation effects of the different IL-17 family members in fibroblasts derived from cartilage and synovium of patients with end-stage knee OA. Immunohistochemistry staining confirmed that IL-17 receptors A (IL-17RA) and IL-17RC are expressed in end-stage OA-derived cartilage and synovium. Chondrocytes and synovial fibroblasts derived from end-stage OA patients were treated with IL-17A, IL-17AF, or IL-17F, and gene expression was assessed with bulk RNA-Seq. Hallmark pathway analysis showed that IL-17 cytokines regulated several OA pathophysiology-related pathways including immune-, angiogenesis-, and complement-pathways in both chondrocytes and synovial fibroblasts derived from end-stage OA patients. While overall IL-17A induced the strongest transcriptional response, followed by IL-17AF and IL-17F, not all genes followed this pattern. Disease-Gene Network analysis revealed that IL-17A-related changes in gene expression in these cells are associated with experimental arthritis, knee arthritis, and musculoskeletal disease gene-sets. Western blot analysis confirmed that IL-17A significantly activates p38 and p65 NF-{kappa}B. Incubation of chondrocytes and synovial fibroblasts with IL-17A antibody secukinumab significantly inhibited IL-17A-induced gene expression. In conclusion, the association of IL-17-induced transcriptional changes with arthritic gene-sets supports a role for IL-17A in OA pathophysiology. Therefore, secukinumab could be investigated as a potential therapeutic option in OA patients.

sentences: Recurrent deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape Zoonotic pandemics, like that caused by SARS-CoV-2, can follow the spillover of animal viruses into highly susceptible human populations. Their descendants have adapted to the human host and evolved to evade immune pressure. Coronaviruses acquire substitutions more slowly than other RNA viruses, due to a proofreading polymerase. In the spike glycoprotein, we find recurrent deletions overcome this slow substitution rate. Deletion variants arise in diverse genetic and geographic backgrounds, transmit efficiently, and are present in novel lineages, including those of current global concern. They frequently occupy recurrent deletion regions (RDRs), which map to defined antibody epitopes. Deletions in RDRs confer resistance to neutralizing antibodies. By altering stretches of amino acids, deletions appear to accelerate SARS-CoV-2 antigenic evolution and may, more generally, drive adaptive evolution.

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Estimation of Average Blood Glucose Values Based on Fructosamine Values IntroductionThe fructosamine is originated of the glycation of plasmatic proteins, especially albumin, in addition to immunoglobulins and proteins diverse. It constitutes an alternative biomarker of glycemic control when glycated hemoglobin is not indicated for this purpose.

ObjectiveTo define the mathematical relationship between fructosamine and average glucose values.

MethodThe study comprised the laboratorial data collected of 1227 diabetic subjects (type 1 and type 2). Fructosamine levels obtained at the end of three weeks and measured were compared with the average glucose levels of the three previous weeks. The average glucose levels were determined by the weighted mean of the daily fasting capillary glucose results performed during the study period, and the plasma glucose taken at the time of the fructosamine.

ResultsA total of 9,450 glucoses were performed. Linear regression analysis between the fructosamine and average glucose levels showed that each increase of 1.0 {micro}mol/L in fructosamine increase 0.5mg/dL in the average glucose levels as evidenced in the equation forward: Average glucose levels = 0.5157 x Fructosamine - 20. According to the coefficient of determination (r2 = 0.353492, P < 0.006881), making it possible to calculate the estimated average glucose according to the frutosamine values.

ConclusionFructosamine levels can be expressed as average glucose levels for assessing the metabolic control of diabetic patients.

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Umibato: estimation of time-varying microbial interaction using continuous-time regression hidden Markov model MotivationAccumulating evidence has highlighted the importance of microbial interaction networks. Methods have been developed for estimating microbial interaction networks, of which the generalized Lotka-Volterra equation (gLVE)-based method can estimate a directed interaction network. The previous gLVE-based method for estimating microbial interaction networks did not consider time-varying interactions.

ResultsIn this study, we developed unsupervised learning based microbial interaction inference method using Bayesian estimation (Umibato), a method for estimating time-varying microbial interactions. The Umibato algorithm comprises Gaussian process regression (GPR) and a new Bayesian probabilistic model, the continuous-time regression hidden Markov model (CTRHMM). Growth rates are estimated by GPR, and interaction networks are estimated by CTRHMM. CTRHMM can estimate time-varying interaction networks using interaction states, which are defined as hidden variables. Umibato outperformed the existing methods on synthetic datasets. In addition, it yielded reasonable estimations in experiments on a mouse gut microbiota dataset, thus providing novel insights into the relationship between consumed diets and the gut microbiota.

AvailabilityThe C++ and python source codes of the Umibato software are available at https://github.com/shion-h/Umibato

Contactshion_hosoda@asagi.waseda.jp, mhamada@waseda.jp

sentences: The effect of resource depletion on the thermal response of mosquito population fitness The temperature-dependencies of life history traits are increasingly being used to predict how climatic warming will affect vector-borne disease dynamics, partially by affecting the abundance dynamics of the vector population. Such predictions generally arise from mathematical models that incorporate the temperature dependence of traits measured under laboratory conditions. These temperature-trait relationships are typically estimated from juvenile populations reared under optimal resource conditions, even though natural populations experience intermittent resource depletion. Using laboratory experiments on the mosquito Aedes aegypti, combined with a stage-structured population model, we show that resource depletion in the juvenile habitat can significantly depress the vectors maximal population growth rate (rm) across the entire temperature range, cause it to peak at a lower temperature, and narrow its thermal niche width. Our results provide compelling evidence for future studies to consider resource depletion when predicting the effects of global change on vector-borne disease transmission, disease vectors and other arthropods.

sentences: Evolutionary dynamics of piRNA clusters in Drosophila Small RNAs produced from transposable element (TE) rich sections of the genome, termed piRNA clusters, are a crucial component in the genomic defense against selfish DNA. In animals it is thought the invasion of a TE is stopped when a copy of the TE inserts into a piRNA cluster, triggering the production of cognate small RNAs that silence the TE. Despite this importance for TE control, little is known about the evolutionary dynamics of piRNA clusters, mostly because these repeat rich regions are difficult to assemble and compare. Here we establish a framework for studying the evolution of piRNA clusters quantitatively. Previously introduced quality metrics and a newly developed software for multiple alignments of repeat annotations (Manna) allow us to estimate the level of polymorphism segregating in piRNA clusters and the divergence among homologous piRNA clusters. By studying 20 conserved piRNA clusters in multiple assemblies of four Drosophila species we show that piRNA clusters are evolving rapidly. While 70-80% of the clusters are conserved within species, the clusters share almost no similarity between species as closely related as D. melanogaster and D. simulans. Furthermore, abundant insertions and deletions are segregating within the Drosophila species. We show that the evolution of clusters is mainly driven by large insertions of recently active TEs, and smaller deletions mostly in older TEs. The effect of these forces is so rapid that homologous clusters often do not contain insertions from the same TE families.x

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An open source platform for presenting dynamic visual stimuli Operant behavior procedures often rely on visual stimuli to cue the initiation or secession of a response, and to provide a means for discriminating between two or more simultaneously available responses. While primate and human studies typically use LCD or OLED monitors and touch screens, rodent studies use a variety of methods to present visual cues ranging from traditional incandescent light bulbs, single LEDs, and, more recently, touch screen monitors. Commercially available systems for visual stimulus presentation are costly, challenging to customize, and are typically closed source. We developed an open-source, highly-modifiable visual stimulus presentation platform that can be combined with a 3D-printed operant response device. The device uses an eight by eight matrix of LEDs, and can be expanded to control much larger LED matrices. Implementing the platform is low-cost (<$70 USD per device in the year 2020). Using the platform, we trained rats to make nosepoke responses and discriminate between two distinct visual cues in a location-independent manner. This visual stimulus presentation platform is a cost-effective way to implement complex visually-guided operant behavior, including the use of moving or dynamically changing visual stimuli.

Significance StatementThe design of an open source and low cost device for presenting visual stimuli is described. It is capable of presenting complex visual patterns and dynamically changing stimuli. A practical demonstration of the device is also reported, from an experiment in which rats performed a luminance based visual discrimination. The device has utility for studying visual processing, psychophysics, and decision making in a variety of species.

sentences: Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants The causative agent of COVID-19 pandemic, the SARS-CoV-2 coronavirus, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 100 modified bases that are essential for proper function. Among internal modified bases, the N6-methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced so far are in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because incorporation of dNTPs hides RNA base modifications. Here, in this perspective paper, we present an initial exploration of the Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3-untranslated region. We identified 15 m6A methylated positions, of which, 6 are in ORF N. Also, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants we show that variants Beta and Eta have a fourth position C>U change in DRACH at 28,884b that could affect methylation. The Nanopore technology offers a unique opportunity for the study of viral epitranscriptomics. This technique is PCR-free and is not sequencing-by-synthesis, therefore, no PCR bias and synthesis errors are introduced. The modified bases are preserved and assessed directly with no need for chemical treatments or antibodies. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.

sentences: Niche-specific genome degradation and convergent evolution shaping Staphylococcus aureus adaptation during severe infections During severe infections, Staphylococcus aureus moves from its colonising sites to blood and tissues, and is exposed to new selective pressures, thus potentially driving adaptive evolution. Previous studies have shown the key role of the agr locus in S. aureus pathoadaptation, however a more comprehensive characterisation of genetic signatures of bacterial adaptation may enable prediction of clinical outcomes and reveal new targets for treatment and prevention of these infections. Here, we measured adaptation using within-host evolution analysis of 2,590 S. aureus genomes from 396 independent episodes of infection. By capturing a comprehensive repertoire of single-nucleotide and structural genome variations, we found evidence of a distinctive evolutionary pattern within the infecting populations compared to colonising bacteria. These invasive strains had up to 20-fold enrichments for genome degradation signatures and displayed significantly convergent mutations in a distinctive set of genes, linked to antibiotic response and pathogenesis. In addition to agr-mediated adaptation we identified non-canonical, genome-wide significant loci including sucA-sucB and stp1. The prevalence of adaptive changes increased with infection extent, emphasising the clinical significance of these signatures. These findings provide a high-resolution picture of the molecular changes when S. aureus transitions from colonisation to severe infection and may inform correlation of infection outcomes with adaptation signatures.

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Deep neural network analysis employing diffusion basis spectrum imaging metrics as classifiers improves prostate cancer detection and grading Structural and cellular complexity of prostatic histopathology limits the accuracy of noninvasive detection and grading of prostate cancer (PCa). We addressed this limitation by employing a novel diffusion basis spectrum imaging (DBSI) to derive structurally-specific diffusion fingerprints reflecting various underlying prostatic structural and cellular components. We further developed diffusion histology imaging (DHI) by combining DBSI-derived structural fingerprints with a deep neural network (DNN) algorithm to more accurately classify different histopathological features and predict tumor grade in PCa. We examined 243 patients suspected with PCa using in vivo DBSI. The in vivo DBSI-derived diffusion metrics detected coexisting prostatic pathologies distinguishing inflammation, PCa, and benign prostatic hyperplasia. DHI distinguished PCa from benign peripheral and transition zone tissues with over 95% sensitivity and specificity. DHI also demonstrated over 90% sensitivity and specificity for Gleason score noninvasively. We present DHI as a novel diagnostic tool capable of noninvasive detection and grading of PCa.

One sentence summaryDiffusion histology imaging noninvasively and accurately detects and grades prostate cancer.

sentences: Proteome of actin stress fibers Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from huma fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 263 proteins are associated with SFs, and 101 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

sentences: Unified classification of mouse retinal ganglion cells using function, morphology, and gene expression Classification and characterization of neuronal types are critical for understanding their function and dysfunction. Neuronal classification schemes typically rely on measurements of electrophysiological, morphological, and molecular features, but aligning such datasets has been challenging. Here, we present a unified classification of mouse retinal ganglion cells (RGCs), the sole retinal output neurons. We used visually-evoked responses to classify 1859 mouse RGCs into 42 types. We also obtained morphological or transcriptomic data from subsets and used these measurements to align the functional classification to publicly available morphological and transcriptomic data sets. We created an online database that allows users to browse or download the data and to classify RGCs from their light responses using a machine learning algorithm. This work provides a resource for studies of RGCs, their upstream circuits in the retina, and their projections in the brain, and establishes a framework for future efforts in neuronal classification and open data distribution.

sentences: Substrate rigidity modulates traction forces and stoichiometry of cell matrix adhesions. In cell matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative superresolution microscopy to cell matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application whereas a ~60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate of effective stiffness of 47 kPa. On a substrate with a four-fold lower effective stiffness the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ~60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relation between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

sentences: Modulation of tactile feedback for the execution of dexterous movement While dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive to behavior. Yet how somatosensory feedback from the hands is regulated as it first enters the brain, and whether this modulation exerts any influence on movement, remain unclear. Leveraging molecular-genetic access in mice, we find that tactile afferents from the hand recruit neurons in the brainstem cuneate nucleus whose activity is modulated by distinct classes of local inhibitory neurons. Selective manipulation of these inhibitory circuits can suppress or enhance the transmission of tactile information, affecting behaviors that rely on movement of the hands. Investigating whether these local circuits are subject to top-down control, we identify distinct descending cortical pathways that innervate cuneate in a complementary pattern. Somatosensory cortical neurons target the core tactile region of cuneate, while a large rostral cortical population drives feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation, enabling the effective execution of dexterous movement.

sentences: Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigate the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

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Lower limb in sprinters with larger relative mass but not larger normalized moment of inertia Inhomogeneous muscle hypertrophy in response to daily motor executions is a morphological adaptation in humans. Inhomogeneous morphological adaptation would change the mass distribution, which in turn may affect the mechanical difficulty in moving from an inertia perspective; however, the morphological adaptation from the inertia perspective has not been examined. Here we show no corresponding differences in the normalized mass and normalized moment of inertia between the sprinters and the untrained non-sprinters. We analyzed the fat- and water-separated magnetic resonance images from the lower limbs of 11 male sprinters (100 m best time of 10.44-10.83 s) and 12 untrained non-sprinters. The lower-limb relative mass was significantly larger in sprinters (18.7{+/-}0.7% body mass) than in non-sprinters (17.6{+/-}0.6% body mass), while the normalized moment of inertia of the lower limb around the hip in the anatomical position was similar between them (0.044{+/-}0.002 vs. 0.042{+/-}0.002 [a. u.]). The thigh relative mass in sprinters (12.9{+/-}0.4% body mass) was significantly larger than that in non-sprinters (11.9{+/-}0.4% body mass), whereas the shank and foot relative masses were similar. We revealed that the adaptation to sprinting in humans does not relatively increase the mechanical difficulty to swing the lower limb in terms of inertia, even though the lower-limb mass increases, reflecting muscular hypertrophy. This is suggested to be one of the significant advantages of human morphology for adaptability to explosive motor tasks.

NEW & NOTEWORTHYWe compared the lower-limb inertia properties between male sprinters and untrained non-sprinters by using the fat- and water-separated magnetic resonance images. Sprinters had lower limbs with a larger relative mass but not a larger normalized moment of inertia than non-sprinters. We suggest one of the practical advantages of human morphological characteristics for their adaptation to explosive motor tasks, which provides a novel perspective to the literature for understanding human morphological adaptability.

sentences: Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2x259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2x259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2x259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2x259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

sentences: The Hox gene Antennapedia regulates wing development through 20-hydroxyecdysone in insect A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species, Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila, and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20E. In turn, 20E signaling also up-regulates Antp. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28, and CPG9, essential for wing development. We propose, thus, that insect wing development occurs in an Antp-dependent manner.

sentences: Redefining the architecture of ferlin proteins: insights into multi-domain protein structure and function Ferlins are complex, multidomain proteins whose functions involve membrane fusion, membrane repair, and exocytosis. Progress in studying the domain composition of ferlins has been problematic due to the large size of the ferlin proteins and the difficulty in computing accurate residue ranges for each respective ferlin protein domain. However, recent advances in in silico protein folding methods have significantly enhanced the understanding of the domain structure of these complex proteins. To compute unbiased domain boundaries, we used RoseTTAFold to assemble full-length models for each of the six human ferlin proteins (dysferlin, myoferlin, otoferlin, Fer1L4, Fer1L5, and Fer1L6). Despite the differences in amino acid sequence between the ferlin proteins, the domain ranges and the distinct subdomains in some of the ferlin domains were remarkably consistent. Further, the RoseTTAFold/AlphaFold2 in silico boundary prediction methods allowed us to describe and characterize a previously unknown C2 domain, ubiquitous in all human ferlins, which we refer to as C2-FerA. At present, the ferlin domain-domain interactions implied by the full-length in silico models are predicted to have a low accuracy; however, the use of RoseTTAFold and AlphaFold2 as a domain finder has proven to be a powerful research tool in understanding ferlin structure.

sentences: A systematic comparison of fibroblasts derived from postmortem human dura mater versus dermal epithelium for neurodegenerative disease modeling Patient-derived cells hold great promise for precision medicine approaches in human health. Fibroblast cells have been a major source of human cells for reprogramming and differentiating into specific cell types for disease modeling. Such cells can be isolated at various stages during life (presymptomatic, symptomatic, and postmortem) and thus can potentially be used to model different phases of disease progression. In certain circumstances, however, tissues are not collected during life and only postmortem tissues are the only available source of fibroblasts. Fibroblasts cultured from postmortem human dura mater of individuals with neurodegenerative diseases have been suggested as a primary source of cells for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols requires further characterization. In this study, cells derived from dermal biopsies performed in living subjects were compared to cells derived from postmortem dura mater. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences between the dermis and dura mater-derived cell lines were found. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, exhibited slower growth rates, failed to express fibroblast protein markers, and exhibited significant differences in gene expression profiles. In addition, dura mater-derived cells were found to exhibit a high rate of chromosomal abnormalities, particularly in the loss of the Y chromosome. Our study highlights potential limitations of postmortem human dura mater-derived cells for disease modeling, argues for rigorous karyotyping prior to reprograming, and brings into question the identity of dura mater-derived cells as belonging to a fibroblast lineage.

sentences: Field Potential Image Classification of Pacemaker Micro-coordination in the Intestine of the Mouse The flexible and sophisticated movement of the gastrointestinal (GI) tract implies the involvement of mechanisms other than enteric neural circuits, to coordinate excitation in microregions. We thus performed microimaging of pacemaker dynamics in the small intestine of mice since it contains typical network-forming pacemaker cells. A dialysis membrane-reinforced low-impedance microelectrode array (MEA) enabled field potentials over a wide frequency range to be stably measured in microregions. The pacemaker dynamics were classified into basic patterns despite large variations. In the developmental process, pacemaker activity was categorized as either an  expanding or a  migrating pattern that was initiated in or propagated to the MEA sensing area, respectively. The intercellular current of the volume conductor complicated the waveform of both activities. The existence of  expanding and  migrating patterns was attributable to duplicated pacemaker systems such as intracellular Ca2+ oscillation-activated and voltage-gated mechanisms. Additionally, from the spatio-temporal feature during the period of pacemaker events, the  bumpy/aberrant pattern was defined by aberrant, incoherent propagation, and associated with local impairment of excitability, while the  colliding/converging pattern involved the interaction of multiple activities in the MEA area. Interconversion between the four micro-coordination patterns occurred in the same microregion. 5-Hydroxytryptamine (5-HT) promoted  migrating activity, implying an improvement or restoration of spatial conductivity. These results agree well with the action of 5-HT to change GI movement toward propulsion. In conclusion, our MEA method of microimaging classification enables the quantitative assessment of spatio-temporal electric coordination underlying GI motility, suggesting its application to small model animals.

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Regulation of ammonium acquisition and use in Oryza longistaminata ramets under nitrogen source heterogeneity Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability by using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin, a cytokinin, was up-regulated in response to the nitrogen supply, but trans-zeatin appears not to target the compensatory regulation. Our results also implied that the O. longistaminata ortholog of OsCEP1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory up-regulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

One sentence summaryOryza longistaminata, a rhizomatous wild rice, systemically regulates ammonium acquisition and use in response to spatially heterogeneous nitrogen availability via inter-ramet communication.

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A chromosome-level assembly of the "Cascade" hop (Humulus lupulus) genome uncovers signatures of molecular evolution and improves time of divergence estimates for the Cannabaceae family O_LIWe present a chromosome-level assembly of the Cascade hop (Humulus lupulus L. var. lupulus) genome. The hop genome is large (2.8 Gb) and complex, and early attempts at assembly resulted in fragmented assemblies. Recent advances have made assembly of the hop genome more tractable, transforming the extent of investigation that can occur.
C_LIO_LIThe chromosome-level assembly of Cascade was developed by scaffolding the previously-reported Cascade assembly generated with PacBio long-read sequencing, and polishing with Illumina short-read DNA sequencing. We developed gene models and repeat annotations, and used a controlled bi-parental mapping population to identify significant sex-associated markers. We assess molecular evolution in gene sequences, gene family expansion and contraction, and time divergence using Bayesian inference.
C_LIO_LIWe identified the putative sex chromosome in the female genome based on significant sex-associated markers from the bi-parental mapping population. While the estimate of repeat content (~64%) is similar to the hemp genome, syntenic blocks in hop contain a greater percentage of LTRs. Hop is enriched for disease resistance-associated genes in syntenic gene blocks and expanded gene families.
C_LIO_LIThe Cascade chromosome-level assembly will inform cultivation strategies and serve to deepen our understanding of the hop genomic landscape, benefiting hop researchers and the Cannabaceae genomics community.
C_LI

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6',6'-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6',6'-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5 end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a [~]100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication.

ImportanceCurrently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.

sentences: TissUUmaps 3: Interactive visualization and quality assessment of large-scale spatial omics data TissUUmaps is a browser-based tool for GPU-accelerated visualization and interactive exploration of 107+ datapoints overlaying tissue samples. Users can visualize markers and regions, explore spatial statistics and quantitative analyses of tissue morphology, and assess the quality of decoding in situ transcriptomics data. TissUUmaps provides instant multi-resolution image viewing, can be customized, shared, and also integrated in Jupyter Notebooks. We envision TissUUmaps to contribute to broader dissemination and flexible sharing of large-scale spatial omics data.

sentences: What are housekeeping genes? The concept of "housekeeping genes" has been used for four decades but remains loosely defined. Housekeeping genes are commonly described as "essential for cellular existence regardless of their specific function in the tissue or organism", and "stably expressed irrespective of tissue type, developmental stage, cell cycle state, or external signal". However, experimental support for the tenet that gene essentiality is linked to stable expression across cell types, conditions, and organisms has been limited. Here we use genome-scale functional genomic screens, bulk and single-cell sequencing technologies to test this link and optimize a quantitative and experimentally validated definition of housekeeping gene. Using the optimized definition, we identify, characterize, and provide as resources, housekeeping gene lists extracted from several human datasets, and 12 other animal species that include primates, chicken, and C. elegans. We find that stably expressed genes are not necessarily essential, and that the individual genes that are essential and stably expressed can considerably differ across organisms; yet the pathways enriched among these genes are conserved. Further, the level of conservation of housekeeping genes across the analyzed organisms captures their taxonomic groups, showing evolutionary relevance for our definition. Therefore, we here present a quantitative and experimentally validated definition of housekeeping genes that can contribute to better understanding of their unique biological and evolutionary characteristics.

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Ariadne: Barcoded Linked-Read Deconvolution Using de Bruijn Graphs De novo assemblies are critical for capturing the genetic composition of complex samples. Linked-read sequencing techniques such as 10x Genomics Linked-Reads, USTs TELL-Seq, Loop Genomics LoopSeq, and BGIs Long Fragment Read combines 3' barcoding with standard short-read sequencing to expand the range of linkage resolution from hundreds to tens of thousands of base-pairs. The application of linked-read sequencing to genome assembly has demonstrated that barcoding-based technologies balance the tradeoffs between long-range linkage, per-base coverage, and costs. Linked-reads come with their own challenges, chief among them the association of multiple long fragments with the same 3' barcode. The lack of a unique correspondence between a long fragment and a barcode, in conjunction with low sequencing depth, confounds the assignment of linkage between short-reads.

ResultsWe introduce Ariadne, a novel linked-read deconvolution algorithm based on assembly graphs, that can be used to extract single-species read-sets from a large linked-read dataset. Ariadne deconvolution of linked-read clouds increases the proportion of read clouds containing only reads from a single fragment by up to 37.5-fold. Using these enhanced read clouds in de novo assembly significantly improves assembly contiguity and the size of the largest aligned blocks in comparison to the non-deconvolved read clouds. Integrating barcode deconvolution tools, such as Ariadne, into the postprocessing pipeline for linked-read technologies increases the quality of de novo assembly for complex populations, such as microbiomes. Ariadne is intuitive, computationally efficient, and scalable to other large-scale linked-read problems, such as human genome phasing.

AvailabilityThe source code is available on GitHub: https://github.com/lauren-mak/Ariadne

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CHIQUITA1 maintains temporal transition between proliferation and differentiation in Arabidopsis thaliana Body size varies widely among species, populations, and individuals depending on the environment. Transitioning between proliferation and differentiation is a crucial determinant of final organ size, but how the timing of this transition is established and maintained remains unknown. Using cell proliferation markers and genetic analysis, we show that CHIQUITA1 (CHIQ1) is required to maintain the timing of the transition from proliferation to differentiation in Arabidopsis thaliana. Combining kinematic and cell lineage tracking studies, we found that the number of actively dividing cells in chiquita1-1 plants decreases prematurely compared to wild type plants, suggesting CHIQ1 maintains the proliferative capacity in dividing cells and ensures that cells divide a certain number of times. CHIQ1 belongs to a plant-specific gene family of unknown molecular function and physically and genetically interacts with three close members of its family to control the timing of proliferation exit. Our work reveals the interdependency between cellular and organ-level processes underlying final organ size determination.

SignificanceTiming of the transition between proliferation and differentiation is fundamental for determining the final size of organs and organisms. In agriculture, controlling organ and organism size can influence key agronomic traits such as yield and biomass. Dwarfism prevents lodging and was the trait responsible for the Green Revolution. Today, more sophisticated traits are needed for generating crops that are both resilient and sustainable. Revealing the molecular mechanisms that control the temporal transition between proliferation and differentiation will help unlock the potential of next-generation crops. Here, we report that CHIQUITA 1 in Arabidopsis thaliana is needed to maintain the proper timing of the transition between proliferation and differentiation in leaves and roots.

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Architecture of the Neuro-Glia-Vascular System Astrocytes connect the vasculature to neurons and mediate the supply of nutrients and biochemicals. They also remove metabolites from the neurons and extracellular environment. They are involved in a growing number of physiological and pathophysiological processes. Understanding the biophysical, physiological, and molecular interactions in this neuro-glia-vascular ensemble (NGV) and how they support brain function is severely restricted by the lack of detailed cytoarchitecture. To address this problem, we used data from multiple sources to create a data-driven digital reconstruction of the NGV at micrometer anatomical resolution. We reconstructed 0.2 mm3 of rat somatosensory cortical tissue with approximately 16000 morphologically detailed neurons, its microvasculature, and approximately 2500 morphologically detailed protoplasmic astrocytes. The consistency of the reconstruction with a wide array of experimental measurements allows novel predictions of the numbers and locations of astrocytes and astrocytic processes that support different types of neurons. This allows anatomical reconstruction of the spatial microdomains of astrocytes and their overlapping regions. The number and locations of end-feet connecting each astrocyte to the vasculature can be determined as well as the extent to which they cover the microvasculature. The structural analysis of the NGV circuit showed that astrocytic shape and numbers are constrained by vasculatures spatial occupancy and their functional role to form NGV connections. The digital reconstruction of the NGV is a resource that will enable a better understanding of the anatomical principles and geometric constraints which govern how astrocytes support brain function.

Table of contentsO_ST_ABSMain pointsC_ST_ABSO_LIThe Blue Brain Project digitally reconstructs a part of neocortical Neuro-Glia-Vascular organization
C_LIO_LIInterdependencies and topological methods allow dense in silico reconstruction from sparse experimental data
C_LIO_LIThe polarized role of protoplasmic astrocytes constrains their shapes and numbers
C_LI

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sentences: A quantitative review of abundance-based species distribution models The contributions of species to ecosystem functions or services depend not only on their presence in a given community, but also on their local abundance. Progress in predictive spatial modelling has largely focused on species occurrence, rather than abundance. As such, limited guidance exists on the most reliable methods to explain and predict spatial variation in abundance. We analysed the performance of 68 abundance-based species distribution models fitted to 800,000 standardised abundance records for more than 800 terrestrial bird and reef fish species. We found high heterogeneity in performance of abundance-based models. While many models performed poorly, a subset of models consistently reconstructed range-wide abundance patterns. The best predictions were obtained using random forests for frequently encountered and abundant species, and for predictions within the same environmental domain as model calibration. Extending predictions of species abundance outside of the environmental conditions used in model training generated poor predictions. Thus, interpolation of abundances between observations can help improve understanding of spatial abundance patterns, but extrapolated predictions of abundance, e.g. under climate change, have a much greater uncertainty. Our synthesis provides a roadmap for modelling abundance patterns, a key property of species distributions that underpins theoretical and applied questions in ecology and conservation.

sentences: A Connectivity-based Psychometric Prediction Framework for Brain-behavior Relationship Studies The recent availability of population-based studies with neuroimaging and behavioral measurements opens promising perspectives to investigate the relationships between interindividual variability in brain regions connectivity and behavioral phenotypes. However, the multivariate nature of connectivity-based prediction model severely limits the insight into brain-behavior patterns for neuroscience. To address this issue, we propose a connectivity-based psychometric prediction framework based on individual regions connectivity profiles. We first illustrate two main applications: 1) single brain regions predictive power for a range of psychometric variables, and 2) single psychometric variables predictive power variation across brain region. We compare the patterns of brain-behavior provided by these approaches to the brain-behavior relationships from activation approaches. Then, capitalizing on the increased transparency of our approach, we demonstrate how the influence of various data processing and analyses can directly influence the patterns of brain-behavior relationships, as well as the unique insight into brain-behavior relationships offered by this approach.

sentences: Nanoscale prognosis of colorectal cancer metastasis from AFM image processing of histological sections Early ascertainment of metastatic tumour phases is crucial to improve cancer survival, formulate an accurate prognostic report of disease advancement and, most important, quantify the metastatic progression and malignancy state of primary cancer cells with a universal numerical indexing system. This work proposes an early improvement of cancer detection with 97 nm spatial resolution by indexing the metastatic cancer phases from the analysis of atomic force microscopy images of human colorectal cancer histological sections. The procedure applies variograms of residuals of Gaussian filtering and theta statistics of colorectal cancer tissue image settings. The methodology elucidates the early metastatic progression at the nanoscale level by setting metastatic indexes and critical thresholds from relatively large histological sections and categorising the malignancy state of a few suspicious cells not identified with optical image analysis. In addition, we sought to detect early tiny morphological differentiations indicating potential cell transition from epithelial cell phenotypes of low to high metastatic potential. The metastatic differentiation, also identified by higher moments of variograms, sets different hierarchical levels for the metastatic progression dynamic, potentially impacting therapeutic cancer protocols.

sentences: Hyphal compartmentalization and sporulation in Streptomyces require the conserved cell division protein SepX Filamentous actinobacteria such as Streptomyces undergo two distinct modes of cell division, leading to partitioning of growing hyphae into multicellular compartments via cross-walls, and to septation and release of unicellular spores. Specific determinants for cross-wall formation and the importance of hyphal compartmentalization for Streptomyces development are largely unknown. Here we show that SepX, an actinobacterial-specific protein, is crucial for both cell division modes in Streptomyces venezuelae. Importantly, we find that sepX-deficient mutants grow without cross-walls and that this substantially impairs the fitness of colonies and the coordinated progression through the developmental life cycle. Protein interaction studies and live-cell imaging suggest that SepX contributes to the stabilization of the divisome, a mechanism that also requires the dynamin-like protein DynB. Thus, our work identifies an important determinant for cell division in Streptomyces that is required for cellular development and sporulation.

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Differential metabolism of arsenicals regulates Fps1-mediated arsenite transport Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1, but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins; whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1, but also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.

Summary (for JCB table of contents)

Jongmin Lee, David E. Levin

Lee and Levin investigate the question of how different stressors can drive divergent outputs from an active SAPK. The work describes the mechanism by which two forms of arsenic that both activate the yeast SAPK Hog1 differentially regulate the glycerol channel Fps1.

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Nitric oxide-induced tyrosine nitration of TrkB impairs BDNF signaling and restrains neuronal plasticity Nitric oxide has been long recognized as an important modulator of neural plasticity, but characterization of the molecular mechanisms involved - specially the guanylyl cyclase-independent ones - has been challenging. There is evidence that NO could modify BDNF-TRKB signaling, a key mediator of neuronal plasticity. However, the mechanism underlying the interplay of NO and TRKB remains unclear. Here we show that nitric oxide induces nitration of the tyrosine 816 in the TRKB receptor in vivo and in vitro, and that post-translational modification inhibits TRKB phosphorylation and binding of phospholipase C{gamma}1 (PLC{gamma}1) to this same tyrosine residue. Additionally, nitration triggers clathrin-dependent endocytosis of TRKB through the adaptor protein AP2M and ubiquitination, thereby increasing translocation of TRKB away from the neuronal surface and directing it towards lysosomal degradation. Accordingly, inhibition of nitric oxide increases TRKB phosphorylation and TRKB-dependent neurite branching in neuronal cultures. In vivo, chronic inhibition of neuronal nitric oxide synthase (nNOS) dramatically reduced TRKB nitration and facilitated TRKB signaling in the primary visual cortex, and promoted a shift in ocular dominance upon monocular deprivation in the visual cortex - an indicator of increased plasticity. Altogether, our data describe and characterize a new molecular brake on plasticity, namely nitration of TRKB receptors.

Significance statementWe described the nitration of TRKB receptors at the tyrosine residue 816 as a new post-translational modification (PTM) that restrains the signaling of the neurotrophic factor BDNF in neurons. This new PTM leads to endocytosis and degradation of the TRKB receptors. Intriguingly, this mechanism is tonically active under physiological conditions in vivo, and it is important for restricting ocular dominance plasticity in the visual cortex. This mechanism directly links two major systems involved in brain plasticity, BDNF/TRKB and nitric oxide. Our data provides a model for how NO production from nNOS can compromise TRKB function, and for the effects of nNOS inhibitors promoting plasticity.

sentences: Serial Dependence in Temporal Perception Reveals the Dynamics of Constructing an Internal Reference Frame Temporal perception is crucial to cognitive functions. To better estimate temporal durations, the observers need to construct an internal reference frame based on past experience and apply it to guide future perception. However, how this internal reference frame is constructed remains largely unclear. Here we showed the dynamics of the internal reference construction from the perspective of serial dependence in temporal reproduction tasks. We found the current duration estimation is biased towards both perceived and reproduced durations in previous trials. Moreover, this effect is regulated by the variability of sample durations. The influence of previous trials was stronger when the observers were exposed to context with more variable durations, which is inconsistent with previous theories that the similarity between successive stimuli induces serial dependence. We proposed a Bayesian model with an adaptive reference updated continuously after each observation, which can better explain the serial dependence observed in temporal perception.

sentences: Evolving the Olfactory System with Machine Learning The convergent evolution of the fly and mouse olfactory system led us to ask whether the anatomic connectivity and functional logic of olfactory circuits would evolve in artificial neural networks trained to perform olfactory tasks. Artificial networks trained to classify odor identity recapitulate the connectivity inherent in the olfactory system. Input units are driven by a single receptor type, and units driven by the same receptor converge to form a glomerulus. Glomeruli exhibit sparse, unstructured connectivity to a larger, expansion layer of Kenyon cells. When trained to both classify odor identity and to impart innate valence onto odors, the network develops independent pathways for identity and valence classification. Thus, the defining features of fly and mouse olfactory systems also evolved in artificial neural networks trained to perform olfactory tasks. This implies that convergent evolution reflects an underlying logic rather than shared developmental principles.

sentences: Genome-wide characterization of the R2R3-MYB transcription factors in pepper (Capsicum spp.) unveils the role of CaMYB101 as repressor in anthocyanin biosynthesis Fruit colour is one of the most important commercial traits of pepper (Capsicum spp.), a major horticultural crop worldwide. Some pepper accessions temporarily accumulate anthocyanins during fruit development and gradually lose them upon fruit ripening. Meanwhile, anthocyanin biosynthesis gradually stops. However, how this process is exactly regulated is still largely unknown. R2R3-MYB is one of the largest plant transcription factor families, and it is considered the most important regulator for the biosynthesis of anthocyanins and other flavonoids. Although R2R3-MYBs are widely studied in many plants, research in pepper has been limited. In this study, we performed a genome-wide analysis of R2R3-MYBs across three cultivated pepper species (C. annuum, C. baccatum, and C. chinense ) involving identification, chromosome localization, gene structure analysis, phylogenetic analysis and collinearity analysis. Candidate R2R3-MYB repressors were further identified based on repression motifs. An R2R3-MYB gene, CaMYB101, was selected based on its high homology with anthocyanin biosynthesis repressors in tomato and petunia as well as its high expression level in fruit when purple pigmentation started to discolour. By using virus-induced gene silencing, CaMYB101 was characterized as an anthocyanin biosynthesis repressor. To our knowledge, CaMYB101 is the first transcriptional repressor associated with anthocyanin biosynthesis identified in pepper.

sentences: Frequency-specific neural signatures of perceptual content and perceptual stability In the natural environment, we often form stable perceptual experiences from ambiguous and fleeting sensory inputs. Which neural activity underlies the content of perception and which neural activity supports perceptual stability remains an open question. We used a bistable perception paradigm involving ambiguous images to behaviorally dissociate perceptual content from perceptual stability, and magnetoencephalography (MEG) to measure whole-brain neural dynamics in humans. Combining multivariate decoding and neural state-space analyses, we found frequency band-specific neural signatures that underlie the content of perception and promote perceptual stability, respectively. Across different types of images, non-oscillatory neural activity in the slow cortical potential (SCP, <5 Hz) range supported the content of perception. By contrast, perceptual stability is influenced by the amplitude of alpha and beta oscillations. In addition, neural activity underlying perceptual memory, which supports perceptual stability when sensory input is temporally removed from view, also encodes elapsed time. Together, these results reveal distinct neural mechanisms that support the content vs. stability of visual perception.

sentences: Mistic: an open-source multiplexed image t-SNE viewer Understanding the complex ecology of a tumor tissue and the spatio-temporal relationships between its cellular and microenvironment components is becoming a key component of translational research, especially in immune-oncology. The generation and analysis of multiplexed images from patient samples is of paramount importance to facilitate this understanding. In this work, we present Mistic, an open-source multiplexed image t-SNE viewer that enables the simultaneous viewing of multiple 2D images rendered using multiple layout options to provide an overall visual preview of the entire dataset. In particular, the positions of the images can be taken from t-SNE or UMAP coordinates. This grouped view of all the images further aids an exploratory understanding of the specific expression pattern of a given biomarker or collection of biomarkers across all images, helps to identify images expressing a particular phenotype or to select images for subsequent downstream analysis. Currently there is no freely available tool to generate such image t-SNEs. Mistic is open-source and can be downloaded at: https://github.com/MathOnco/Mistic.

sentences: Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination of Castanea henryi Seed germination is the beginning of the plants life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however, Castanea henryi as an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes in C. henryi at different germination stages with RT-qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT-qPCR, the results of RT-qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination of C. henryi seeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT- qPCR data for germination of seed plants.

sentences: Effects of short-term salinity exposure on haemolymph osmolality, gill morphology and Na+/K+ - ATPase activity in Solenaia oleivora In order to explore the physiological reaction to hyperosmotic environment, Solenaia oleivora were exposed to 2.23{per thousand} salinity. In 48h, the hemolymph osmolality kept increasing, and the hemolymph protein concentration increased in the first 6h and then decreased significantly, while the free amino acid content increased in the first 24h and then kept stable (P < 0.05). The activity of Na+/K+-ATPase at 0h was significantly higher than other times in most organs except intestine, which was highest at 3h (P < 0.05). The ions concentration were also influenced. The concentration of Na+ rose in haemolymph, axe foot and intestine, but decreased in gill and hepatopancreas. In hemolymph, gill, hepatopancreases and adductor muscle, the K+ concentration was the highest at 0h, while in axe foot and intestine, it showed a positive tendency. The concentration of Cl- in haemolymph, adductor muscle, intestine and axe foot were positively correlated with treatment time, while hepatopancreas showed opposite tendency. High salinity stress caused a difference in the gill histological structure, the gill structure shrunk, the gill lamellas space and shrinking degree showed an enlarging trend with salinity treatment time.

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GuideMaker: Software to design CRISPR-Cas guide RNA pools in non-model genomes BackgroundCRISPR-Cas systems have expanded the possibilities for gene editing in bacteria and eukaryotes. There are many excellent tools for designing the CRISPR-Cas guide RNAs for model organisms with standard Cas enzymes. GuideMaker is intended as a fast and easy-to-use design tool for atypical projects with 1) non-standard Cas enzymes, 2) non-model organisms, or 3) projects that need to design a panel of guide RNAs (gRNA) for genome-wide screens.

FindingsGuideMaker can rapidly design gRNAs for gene targets across the genome from a degenerate protospacer adjacent motif (PAM) and a GenBank file. The tool applies Hierarchical Navigable Small World (HNSW) graphs to speed up the comparison of guide RNAs. This allows the user to design gRNAs targeting all genes in a typical bacterial genome in about 1-2 minutes.

ConclusionsGuidemaker enables the rapid design of genome-wide gRNA for any CRISPR-Cas enzyme in non-model organisms. While GuideMaker is designed with prokaryotic genomes in mind, it can efficiently process smaller eukaryotic genomes as well. GuideMaker is available as command-line software, a stand-alone web application, and a tool in the CyCverse Discovery Environment. All versions are available under a Creative Commons CC0 1.0 Universal Public Domain Dedication.

sentences: Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.

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Saccades and pre-saccadic stimulus repetition alter cortical network topology and dynamics: evidence from EEG and graph theoretical analysis Parietal and frontal cortex are involved in saccade generation, but their output signals also modify visual signals throughout cortex. These signals produce well-documented behavioral phenomena (saccades, saccadic suppression, various perisaccadic perceptual distortions) but their underlying influence on cortical network dynamics is not known. Here, we combined electroencephalography (EEG) with frequency-dependent source localization and graph theory analysis (GTA) to understand how saccades and pre-saccadic visual stimuli interactively alter cortical network dynamics in humans. 21 participants viewed series of 1-3 vertical or horizontal grids, followed by grid with the opposite orientation just before a horizontal saccade or continued fixation. EEG signals from the presaccadic interval (cue + 200ms, or equivalent fixation period) were used for source localization. Source localization (saccade - fixation) identified bilateral dorsomedial frontoparietal activity across frequency bands, whereas stimulus repetition produced band-specific modulations in left prefrontal, posterior parietal, and central-superior frontal and/or parietal cortex, with significant saccade-repetition interactions in frontal and parietal regions. GTA analysis revealed a saccade-specific functional network with major hubs in inferior parietal cortex (alpha) and the frontal eye fields (beta), and major saccade-repetition interactions in left prefrontal (theta) and supramarginal gyrus (gamma). Overall, quantitative measures of whole-brain network topology and dynamics (segregation, integration, synchronization, complexity) were enhanced during the presaccadic interval, but repetition interactions reduced synchronization and complexity. These results show that presaccadic signals have widespread, coherent influence on cortical network dynamics, likely responsible for both saccade production and the perceptual phenomena associated with saccades.

HighlightsO_LISource localization & graph theory were used to analyze presaccadic EEG signals
C_LIO_LIPresaccadic signals produced band-specific modulations/hubs in parietofrontal cortex
C_LIO_LIFrontal/parietal eye fields showed extensive functional connectivity across all lobes
C_LIO_LIPresaccadic stimulus repetition further modulated functional network connectivity
C_LIO_LISaccades and repetition both influenced network clustering, integration, & complexity
C_LI

sentences: Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

sentences: Xanthomonas indica sp. nov., a non-pathogenic bacterium isolated from healthy rice (Oryza sativa) seeds from India Two yellow pigmented bacterial strains were isolated from healthy rice seeds. The strains designated as PPL560T and PPL568 were identified as members of genus Xanthomonas based on analysis of biochemical and 16S rRNA gene sequence retrieved from whole genome sequence. Isolates formed a distinct monophyletic lineage with X. sontii and X. sacchari as the closest relatives in the phylogenetic tree based on core gene content shared by reported species in genus Xanthomonas. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridisation values calculated against other species of Xanthomonas were below their respective cut-offs. In planta studies revealed that PPL560T and PPL568 are non-pathogenic to rice plants upon leaf clip inoculation. Absence of type III secretion system related genes and effectors further supported their non-pathogenic status. Herein, we propose Xanthomonas indica sp. nov. as novel species of genus Xanthomonas with PPL560T=MTCC13185 as its type strain and PPL568 as another constituent member.

sentences: Marine heatwaves depress metabolic activity and impair cellular acid-base homeostasis in reef-building corals regardless of bleaching susceptibility Ocean warming is causing global coral bleaching events to increase in frequency, resulting in widespread coral mortality and disrupting the function of coral reef ecosystems. However, even during mass bleaching events, many corals resist bleaching despite exposure to abnormally high temperatures. While the physiological effects of bleaching have been well documented, the consequences of heat stress for bleaching resistant individuals are not well understood. In addition, much remains to be learned about how heat stress affects cellular level processes that may be overlooked at the organismal level, yet are crucial for coral performance in the short term and ecological success over the long term. Here we compared the physiological and cellular responses of bleaching resistant and bleaching susceptible corals throughout the 2019 marine heatwave in Hawai i, a repeat bleaching event that occurred four years after the previous regional event. Relative bleaching susceptibility within species was consistent between the two bleaching events, yet corals of both resistant and susceptible phenotypes exhibited pronounced metabolic depression during the heatwave. At the cellular level, bleaching susceptible corals had lower intracellular pH than bleaching resistant corals at the peak of bleaching for both symbiont-hosting and symbiont-free cells, indicating greater disruption of acid-base homeostasis in bleaching susceptible individuals. Notably, cells from both phenotypes were unable to compensate for experimentally induced cellular acidosis, indicating that acid-base regulation was significantly impaired at the cellular level even in bleaching resistant corals and in cells containing symbionts. Thermal disturbances may thus have substantial ecological consequences, as even small reallocations in energy budgets to maintain homeostasis during stress can negatively affect fitness. These results suggest concern is warranted for corals coping with ocean acidification alongside ocean warming, as the feedback between temperature stress and acid-base regulation may further exacerbate the physiological effects of climate change.

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Polar growth protein Wag31 undergoes changes in homo-oligomeric network topology, and has distinct functions at both cell poles and the septum Mycobacterial cell elongation occurs at the cell poles; however, it is not clear how cell wall insertion is restricted to the pole and organized. Wag31 is a pole-localized cytoplasmic protein that is essential for polar growth, but its molecular function has not been described. Wag31 homo-oligomerizes in a network at the poles, but it is not known how the structure of this network affects Wag31 function. In this study we used a protein fragment complementation assay to identify Wag31 residues involved in homo-oligomeric interactions, and found that amino acids all along the length of the protein mediate these interactions. We then used both N-terminal and C-terminal splitGFP fusions to probe Wag31 network topology at different sites in the cell, and found that Wag31 C-terminal-C-terminal interactions predominate at the septa, while C-terminal-C-terminal and C-terminal-N-terminal interactions are found equally at the poles. This suggests the Wag31 network is formed through an ordered series of associations. We then dissected Wag31s functional roles by phenotyping a series of wag31 alanine mutants; these data show that Wag31 has separate functions in not only new and old pole elongation, but also inhibition of both septation and new pole elongation. This work establishes new functions for Wag31, and indicates that changes in Wag31 homo-oligomeric network topology may contribute to cell wall regulation in mycobacteria.

ImportanceMany bacteria restrict cell wall elongation to their cell poles, but it is not known how polar growth is affected on the molecular level. Wag31 is a protein that is required for this polar elongation. In this work, we show that Wag31 actually has at least four distinct functions in regulating the cell wall: it promotes elongation at both poles in different ways, and it can also inhibit cell wall metabolism at the new pole and the septum. In addition, we propose a new model for how Wag31 self-associates into a protein network. This work is important because it shows that a DivIVA homolog can have distinct functions depending on cell context. And, this work clarifies that Wag31 is doing several different things in the cell, and gives us genetic tools to disentangle its functions.

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Identification of the key role of white matter alteration in the pathogenesis of Huntington's Disease Pathogenesis of the inherited neurodegenerative disorder Huntingtons Disease (HD) is complex and progressive, with a long presymptomatic phase in which subtle changes occur in the brain of gene carriers up to 15 years before the onset of symptoms. Thus, there is a need of early, functional biomarker to better understand disease progression and to evaluate treatment efficacy far from onset. In particular, recent studies have shown that white matter may be affected early in HD. In this study, we scanned longitudinally Ki140CAG mice with structural MRI, Diffusion Tensor Imaging (DTI), Chemical Exchange Saturation Transfer of glutamate (gluCEST) and Magnetization Transfer (MT) imaging, in order to assess white matter integrity over the life of this very progressive mouse model. Our results show early defects of diffusion properties in the anterior part of the corpus callosum, preceding gluCEST defects in the same region (-10.8% at 8 months, -19% at 12 months) that spread to adjacent regions. At 12 months, frontal (-7.3%) and piriform (-16.7%) cortices showed reduced gluCEST, as well as the pallidum (-21.0%). MT imaging showed reduced signal in the septum (-21.7%) at 12 months. Cortical and striatal atrophy then appear at 18 months. Vulnerability of the striatum and motor cortex, combined with alterations of anterior corpus callosum, seems to point out the pivotal role of white matter, in the pathogenesis of HD and the pertinence of gluCEST and DTI as biomarkers in HD.

Highlights- A knock-in mouse model of Huntingtons disease is longitudinally characterized
- A multimodal MRI protocol is performed to identify biomarkers of the disease
- The white matter plays a pivotal role in the pathogenesis of the disease
- The cortico-striatal pathway seems particularly vulnerable in Huntingtons disease

sentences: Deceleration of cell cycle underpins a switch from proliferative- to terminal division in plant stomatal lineage Differentiation of specialized cell types from self-renewing progenitors requires precise cell cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor meristemoid followed by the single symmetric division that creates an adjustable pore surrounded by paired guard cells. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and triggers differentiation. However, the role of cell cycle machinery in this transition remains unknown. Through time-lapse imaging, we discover that the symmetric division is slower than the asymmetric division. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a molecular brake that decelerates cell cycle during this transition. SMR4 is directly induced by MUTE and transiently accumulates in differentiating meristemoids. SMR4 physically and functionally associates with CYCD3;1 and extends G1-phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for the stomatal fate specification.

sentences: GNPS Dashboard: Collaborative Analysis of Mass Spectrometry Data in the Web Browser Access to web-based platforms has enabled scientists to perform research remotely. A critical aspect of mass spectrometry data analysis is the inspection, analysis, and visualization of the raw data to validate data quality and confirm statistical observations. We developed the GNPS Dashboard, a web-based data visualization tool, to facilitate synchronous collaborative inspection, visualization, and analysis of private and public mass spectrometry data remotely.

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Analysis of gene expression and mutation data points on contribution of transcription to the mutagenesis by APOBEC enzymes Since the discovery of the role of the APOBEC enzymes in human cancers, the mechanisms of this type of mutagenesis remain little understood. Theoretically, targeting of single-stranded DNA by the APOBEC enzymes could occur during cellular processes leading to the unwinding of DNA double-stranded structure. Some evidence points to the importance of replication in the APOBEC mutagenesis, while the role of transcription is still underexplored. Here, we analyzed gene expression and whole genome sequencing data from five types of human cancers with substantial APOBEC activity to estimate the involvement of transcription in the APOBEC mutagenesis and compare its impact with that of replication. Using the TCN motif as the mutation signature of the APOBEC enzymes, we observed a correlation of active APOBEC mutagenesis with gene expression, confirmed the increase of APOBEC-induced mutations in early-replicating regions, and estimated the relative impact of transcription and replication on the APOBEC mutagenesis, which turned out to be approximately equal in transcribed regions. We also found that the known effect of higher density of APOBEC-induced mutations on the lagging strand was highest in middle-replicating regions, and observed higher APOBEC mutation density on the sense strand, the latter bias positively correlated with the gene expression level.

Bullet points- The APOBEC mutagenesis rate is higher in actively expressed genes
- The APOBEC mutation density is higher on the sense strand
- The lagging/leading strand ratio of the APOBEC mutational density is highest in middle-replicating regions

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Tsc1 haploinsufficiency leads to Pax2 dysregulation in the developing murine cerebellum Tuberous sclerosis complex 1 (TSC1) is a tumour suppressor gene that inhibits the mechanistic target of rapamycin (mTOR) pathway. Mutations in TSC1 lead to a rare complex disorder of the same name, in which up to 50% of patients present with autism spectrum disorder (ASD). ASD is a highly prevalent, early-onset neurodevelopmental disorder, characterized by social deficits and repetitive behaviours, although the type and severity of symptoms show wide variability across individuals. Amongst different brain areas proposed to play a role in the development of ASD, the cerebellum is commonly reported to be altered, and cerebellar-specific deletion of Tsc1 in mice is sufficient to induce an ASD-like phenotype. Given that the mTOR pathway is crucial for proper cell replication and migration, this suggests that dysregulation of this pathway, particularly during critical phases of cerebellar development, could contribute to the establishment of ASD.

Here, we used a mouse model of TSC to investigate gene and protein expression during embryonic and early postnatal periods of cerebellar development. We found that, at E18 and P7, mRNA levels of the cerebellar inhibitory interneuron marker Pax2 were dysregulated. This was accompanied by changes in the expression of mTOR pathway-related genes and downstream phosphorylation of S6. Differential gene correlation analysis revealed dynamic changes in correlated gene pairs across development, with an overall loss of correlation between mTOR- and cerebellar-related genes in Tsc1 mutants compared to controls. We corroborated the genetic findings by characterizing the mTOR pathway and cerebellar development on protein and cellular levels with Western blot and immunohistochemistry. We found that Pax2-expressing cells were hypertrophic at E18 while, at P7, their number was increased and maturation into parvalbumin-expressing cells delayed. Our findings indicate that E18 and P7 are crucial time points in cerebellar development in mice that are particularly susceptible to mTOR pathway dysregulation.

Manuscript contribution to the fieldASD is one of the most prevalent neurodevelopmental disorders, however little is known about the shared mechanisms underlying its aetiology. At the anatomical level, the cerebellum has been identified as one of the key structures involved in the development of ASD, whereas at the molecular level, mutations in the mTOR signalling pathway, essential for cell growth and proliferation, carry a high genetic risk for this disorder. We used a haploinsufficient tuberous sclerosis complex 1 (Tsc1) mouse model to investigate the effects of mTOR overactivation in the developing cerebellum. Tsc1 inhibits the mTOR pathway, and mice with cerebellar-specific deletion of Tsc1 have been shown to harbour an ASD-like phenotype. We found that Pax2 expression in the cerebellum is dysregulated at prenatal and early postnatal time points, leading to a delayed maturation of inhibitory interneurons. Our findings indicate that mTOR overactivity in the cerebellum selectively affects the development of cerebellar interneurons. This finding is in line with other studies, which found decreased numbers of inhibitory interneurons in other models of ASD. Therefore, deficits in the maturation of the inhibitory signalling could be one of the mechanisms integrating high-risk mutations that underlie ASD aetiology.

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Light/dark and temperature cycling modulate metabolic electron flow in Pseudomonas aeruginosa biofilms Sunlight drives phototrophic metabolism, which affects redox conditions and produces substrates for non-phototrophs. These environmental parameters fluctuate daily due to Earths rotation, and non-phototrophic organisms can therefore benefit from the ability to respond to, or even anticipate, such changes. Circadian rhythms, such as diurnal changes in body temperature, in host organisms can also affect local conditions for colonizing bacteria. Here, we investigated the effects of light/dark and temperature cycling on biofilms of the opportunistic pathogen Pseudomonas aeruginosa PA14. We grew biofilms in the presence of a respiratory indicator dye and found that greater dye reduction occurred in biofilm zones that formed during dark intervals and at lower temperatures. This pattern formation occurred with cycling of blue, red, or far-red light, and a screen of mutants representing potential sensory proteins identified two with defects in pattern formation specifically under red light cycling. We also found that the physiological states of biofilm subzones formed under specific light and temperature conditions were retained during subsequent condition cycling. Light/dark and temperature cycling affected expression of genes involved in primary metabolic pathways and redox homeostasis, including those encoding electron transport chain components. Consistent with this, we found that cbb3-type oxidases contribute to dye reduction under light/dark cycling conditions. Together, our results indicate that cyclic changes in light exposure and temperature have lasting effects on redox metabolism in biofilms formed by a non-phototrophic, pathogenic bacterium.

IMPORTANCEOrganisms that do not obtain energy from light can nevertheless be affected by diurnal changes in light exposure. Many aspects of animal and fungal physiology fluctuate in response to these changes, including body temperature and the activities of antioxidant and other redox enzymes that play roles in metabolism. Whether redox metabolism is affected by light/dark and temperature cycling in bacteria that colonize such circadian organisms has not been studied in detail. Here we show that growth under light/dark and temperature cycling leads to rhythmic changes in redox metabolism in Pseudomonas aeruginosa and identify proteins involved in this response. P. aeruginosa is a major cause of healthcare-associated infections and designated as a serious threat by the CDC due to its recalcitrance during treatments. Our findings have the potential to inform therapeutic strategies that incorporate controlled light exposure or consider P. aeruginosas responses to conditions in the host.

sentences: EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or highly related fusions. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. EWS/FLI binding is primarily associated with compartment activation, establishment of topologically-associated domain (TAD) boundaries, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators through three-dimensional reprogramming of chromatin.

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

sentences: Diurnal stomatal apertures and density ratios affect whole-canopy stomatal conductance, water-use efficiency and yield Key physiological traits of plants, such as transpiration and stomatal conductance, are usually studied under steady-state conditions or modeled using only a few measured data points. Those measurements do not reflect the dynamic behavior of the plant in response to field conditions. To overcome this bottleneck, we used a gravimetric functional-phenotyping platform and a reverse-phenotyping method to examine the dynamic whole-plant water-regulation responses of tomato introgression lines and compared those responses with several years of yield performance in commercial fields. Ideotype lines had highly plastic stomatal conductance and high abaxial-adaxial stomatal density ratios and the size of their stomatal apertures peaked early in the day under water-deficit conditions. These traits resulted in dynamic daily water-use efficiency, which allowed for the rapid recovery of transpiration when irrigation was resumed after a period of imposed drought. We found that stomatal density, the abaxial-adaxial stomatal density ratio and the time of maximum stomatal apertures are crucial for plant adaptation and productivity under drought-stress conditions. Abaxial stomatal density was also found to be strongly correlated with the expression of the stomatal-development genes SPCH and ZEP. This study demonstrates how a reverse functional phenotyping approach based on field yield data, continuous and simultaneous whole-plant water-balance measurements and anatomical examination of individual leaves can help us to understand and identify dynamic and complex yield-related physiological traits.

sentences: Reverse engineering environmental metatranscriptomes clarifies best practices for eukaryotic assembly Diverse communities of microbial eukaryotes in the global ocean provide a variety of essential ecosystem services, from primary production and carbon flow through trophic transfer to cooperation via symbioses. Increasingly, these communities are being understood through the lens of omics tools, which enable high-throughput processing of communities of multiple species. Metatranscriptomics in particular offers an understanding of real-time gene expression in microbial eukaryotic communities, providing a window into community metabolic activity. However, these approaches are rarely validated. A systematic validation of metatranscriptome assembly and annotation methods is necessary to appropriately assess (1) the fidelity of our community composition measurements and (2) the confidence of novel taxonomic and functional content discovered with metatranscriptomics. Here, we present euk rhythmic, an automated and customizable multi-assembler workflow for eukaryotic metatranscriptome assembly, annotation, and analysis, and validate the ability of the pipeline to recapitulate simulated eukaryotic community-level expression data treated as a gold standard. Finally, we apply this pipeline to two previously published metatranscriptomic datasets, showing both recapitulation of previous results as well as enhanced taxonomic and functional gene discovery.

sentences: Post-interval EEG activity is related to task-goals in temporal discrimination Studies investigating the neural mechanisms of time perception often measure brain activity while participants perform a temporal task. However, several of these studies are based exclusively on tasks in which time is relevant, making it hard to dissociate activity related to decisions about time from other task-related patterns. In the present study, human participants performed a temporal or color discrimination task of visual stimuli. Participants were informed which magnitude they would have to judge before or after presenting the two stimuli (S1 and S2) in different blocks. Our behavioral results showed, as expected, that performance was better when participants knew beforehand which magnitude they would judge. Electrophysiological data (EEG) was analyzed using Linear Discriminant Contrasts (LDC) and a Representational Similarity Analysis (RSA) approach to investigate whether and when information about time and color was encoded. During the presentation of S1, we did not find consistent differences in EEG activity as a function of the task. On the other hand, during S2, we found that temporal and color information was encoded in a task-relevant manner. Taken together, our results suggest that task goals strongly modulate decision-related information in EEG activity.

sentences: Exploring the conformational landscape and stability of Aurora A using ion-mobility mass spectrometry and molecular modelling Protein kinase inhibitors are proving highly effective in helping treat a number of non-communicable diseases driven by aberrant kinase signaling. They are also extremely valuable as chemical tools to help delineate cellular roles of kinase signaling complexes. The binding of small molecule inhibitors induces conformational effects on kinase dynamics; evaluating the effect of such interactions can assist in developing specific inhibitors and is deemed imperative to understand both inhibition and resistance mechanisms. Using gas-phase ion mobility-mass spectrometry (IM-MS) we characterized changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations: one highly-populated compact conformer similar to that observed in most crystal structures, a second highly-populated conformer possessing a more open structure that is infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Comparison of active (phosphorylated) and inactive (non-phosphorylated) forms of Aur A revealed that the active enzyme has different conformer weightings and is less stable than the inactive enzyme. Notably, inhibitor binding shifts conformer balance towards the more compact configurations adopted by the unbound enzyme, with both IM-MS and modelling revealing inhibitor-mediated stabilisation of active Aur A. These data highlight the power of IM-MS in combination with molecular dynamics simulations to probe and compare protein kinase structural dynamics that arise due to differences in activity and as a result of compound binding.

sentences: Stable coexistence in plant-pollinator-herbivore communities requires balanced mutualistic vs antagonistic interactions Ecological communities consist of multiple species interacting in diverse ways. Understanding the mechanisms supporting coexistence requires accounting for such a diversity. Because most works focus either on mutualism or predation, how pollination and herbivory interactively determine the stable coexistence in plant-pollinator-herbivore communities is still poorly understood. Studying the typical three-species module of such communities, we determine the conditions allowing stable coexistence then investigate how its maintenance constrains the relative interaction strengths. Our results show that coexistence is possible if pollination is sufficiently strong relative to herbivory, while its stability is possible if herbivory is sufficiently strong relative to pollination. A balance between pollination and herbivory is therefore required. Interestingly, shared preferences for plant phenotypes, that would favor such balance, have been frequently reported in the empirical literature. The identified ecological trade-off between attracting pollinators and deterring herbivores therefore also appears as an emergent property of stable plant-pollinator-herbivore communities.

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STRipy: a graphical application for enhanced genotyping of pathogenic short tandem repeats in sequencing data Short tandem repeats (STRs) are highly polymorphic with high mutation rates and expansions of STRs have been implicated as the causal variant in diseases. The application of genome sequencing in patients has recently allowed many new discoveries with over 50 disease causing loci known to date. There are several tools which allow genotyping of STRs from high-throughput sequencing (HTS) data. However, running these tools out of the box only allow around half of the known disease-causing loci to be genotyped, with lengths often limited to either read or fragment length which is less than the pathogenic cut-off for some diseases. While analysis tools can be customised to genotype extra loci, this requires proficiency in bioinformatics to set up, use, and analyse the resulting data, limiting their widespread usage by other researchers and clinicians.

To address these issues, we have created a new software called STRipy that has an intuitive graphical interface and requires no specific skills for usage, thus significantly simplifying detection of STRs expansions from human HTS data. STRipy is able to target all known disease-causing STRs with genotyping performed with an established tool, ExpansionHunter, that is incorporated into the software. We have created additional functionality into STRipy to work with long alleles exceeding the fragment length.

STRipy was validated using over 60 thousand simulated samples and was shown to work on whole genome sequencing of biological samples with pathogenic variants. Finally, we have used STRipy to acquire genotypes of pathogenic loci for thousands of samples from various populations which are provided to the user along with the data from the literature to assist with results interpretation. We believe the simplicity and breadth of STRipy will increase the testing of STR diseases in current datasets resulting in further diagnoses of rare diseases caused by STRs expansions.

sentences: Genome-wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification Current preimplantation genetic testing (PGT) enables the selection of embryos based on fetal aneuploidy or the presence a small number of preselected disease-associated variants. Here we present a new approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call most early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. With this approach, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide information to compute polygenic risk scores for common diseases. Although numerous computational, interpretive, and ethical challenges remain, this approach establishes the technical feasibility of screening for and preventing numerous debilitating inherited diseases.

sentences: Musical expertise shapes visual-melodic memory integration Music can act as a powerful mnemonic device that can elicit vivid episodic memories. However, how musical information is integrated with non-musical information is largely unknown. Here, we investigated whether and how musical expertise modulates binding of melodies and visual information into integrated memory representations. We reasoned that the significant mnemonic demands of musicianship might alter the underlying integration process and reveal mechanisms by which music promotes retrieval of non-musical memories. Professional musicians and musical laypersons learned overlapping pairs of everyday objects and melodies (AB- and BC-pairs, object-melody and melody-object pairs). Participants were then tested for memory of studied pairs (direct trials) and for inferential AC-decisions (indirect trials). Although musicians showed a higher overall performance than non-musicians, both groups performed well above chance level in both trial types. Non-musicians reacted faster in indirect compared to direct trials, whereas the reverse pattern was found in musicians. Differential correlations of trial type performance between groups further suggested that non-musicians efficiently formed integrated ABC-triplets already during the encoding phase of the task, while musicians separately memorized AB- and BC-pairs and recombined them at retrieval for AC-decisions. Our results suggest that integrative encoding is a default mechanism for integration of musical and non-musical stimuli that works with great efficacy even in musically untrained subjects and may contribute to the everyday experience of music-evoked episodic memories. By contrast, recombination at retrieval seems to be an advanced strategy for memory integration that critically depends on an expert ability to maintain and discriminate musical stimuli across extended memory delays.

sentences: Passive plasma membrane transporters play a critical role in perception of carbon availability in yeast Recently, our lab found that the canonical glucose/galactose regulation pathway in yeast makes the decision to metabolize galactose based on the ratio of glucose to galactose concentrations in the external medium. This led to the question of where and how the ratio-sensing is achieved. Here, we consider the possibilities of an intracellular, extracellular, or membrane bound ratio sensing mechanisms. We show that hexose transporters in the plasma membrane are mainly responsible for glucose/galactose ratio-sensing in yeast. Further, while the glucose sensors Gpr1, Snf3, and Rgt2 are not required for ratio sensing, they help modulate the ratio sensing phenotype by regulating the expression of individual transporters in different environments. Our study provides an example of an unexpected, but potentially widespread, mechanism for making essential decisions.

sentences: Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

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NAD metabolism modulates mitochondrial function and inflammation and prevents progression of diabetic kidney disease BackgroundDiabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. In spite of all of the beneficial interventions implemented in patients with diabetes, there remains a need for additional therapeutic targets in diabetic kidney disease (DKD). Mitochondrial dysfunction and inflammation are increasingly recognized as important causes of the development and progression of DKD. However, the molecular connection between mitochondrial function, inflammation, and fibrosis remains to be elucidated.

MethodsIn the present studies we tested the hypothesis that enhancing NAD metabolism could increase mitochondrial sirtuin 3 (SIRT3) activity, improve mitochondrial function, decrease mitochondrial DNA damage, and prevent inflammation and progression of DKD.

ResultsWe found that treatment of db-db mice with type 2 diabetes with nicotinamide riboside (NR) prevented albuminuria, increased urinary KIM1 excretion, and several parameters of DKD. These effects were associated with increased SIRT3 activity, improved mitochondrial function, and decreased inflammation at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway.

ConclusionsNR supplementation boosted the NAD metabolism to modulate mitochondrial function and inflammation and prevent progression of diabetic kidney disease.

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Gut microbiome and its cofactors are linked tolipoprotein distribution profiles Increasing evidence indicates that the gut microbiome (GM) plays an important role in the etiology of dyslipidemia. To date, however, no in-depth characterization of the associations between GM and its metabolic attributes with deep profiling of lipoproteins distributions (LPD) among healthy individuals has been conducted. To determine associations and contributions of GM composition and its cofactors with distribution profiles of lipoprotein subfractions, we studied blood plasma LPD, fecal short-chain fatty acids (SCFA) and GM of 262 healthy Danish subjects aged 19-89 years.

Stratification of LPD segregated subjects into three clusters of profiles that reflected differences in the lipoprotein subclasses, corresponded well with limits of recommended levels of main lipoprotein fractions and were largely explained by host characteristics such as age and body mass index. Higher levels of HDL, particularly driven by large subfractions (HDL2a and HDL2b), were associated with a higher relative abundance of Ruminococcaceae and Christensenellaceae. Increasing levels of total cholesterol and LDL, which were primarily associated with large 1 and 2 subclasses, were positively associated with Lachnospiraceae and Coriobacteriaceae, and negatively with Bacteroidaceae and Bifidobacteriaceae. Metagenome sequencing showed a higher abundance of genes involved in the biosynthesis of multiple B-vitamins and SCFA metabolism among subjects with healthier LPD profiles. Metagenomic assembled genomes (MAGs) affiliated mainly to Eggerthellaceae and Clostridiales were identified as the contributors of these genes and whose relative abundance correlated positively with larger subfractions of HDL.

The results of this study demonstrate that remarkable differences in composition and metabolic traits of the GM are associated with variations in LPD among healthy subjects. Findings from this study provide evidence for GM considerations in future research aiming to shade light on mechanisms of the GM - dyslipidemia axis.

sentences: DEVELOPMENT OF A SOYBEAN MATURITY PREDICTION MODEL FOR SOYBEAN GROWN IN AFRICAN ENVIRONMENTS AO_SCPLOWBSTRACTC_SCPLOWTime-to-maturity (TTM) is an important trait in soybean breeding programs. However, soybean is a relatively new crop in Africa. As such, TTM information is not yet well defined as in other major producing areas. Multi Environment trials (MET) allow breeders to analyze crop performance across diverse conditions but also pose statistical challenges (e.g. unbalanced data). Modern statistical methods, e.g.. Generalized Additive Models (GAM), can flexibly smooth a range of responses while retaining observations that could be lost under other approaches. We leveraged 5 years of data from a MET breeding program in Africa to identify the best geographical and seasonal variables to explain site and genotypic differences in soybean TTM. Using soybean-cycle features (minimum temperature, daylength) along with trial geolocation (longitude, latitude), a GAM model predicted soybean TTM within {+/-} 10 days of the average observed TTM [[Formula] days post-planting]. Further, we found significant differences between cultivars (p<0.05) in TTM sensitivity to minimum temperature and daylength. Our results show promise to advance the design of maturity systems that enhance soybean planting and breeding decisions in Africa.

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Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach AO_SCPLOWBSTRACTC_SCPLOWBacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35-Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35-B. thuringiensis interaction at the molecular level.

AO_SCPLOWUTHORC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWSUMMARYC_SCPLOWMembers of the family Tectiviridae, composed of non-tailed icosahedral, membrane-containing bacteriophages, have been increasingly scrutinized in recent years for their possible role in the origin of dsDNA viruses. In particular, the genus Betatectivirus receives increased attention as these phages can infect clinical strains as well as industrially relevant members of the B. cereus group. However, little is known about the interactions between these temperate viruses and their hosts. Here, we present the first high-throughput study of tectivirus-host protein-protein interactions focusing on Bam35, model virus of betatectiviruses, and its host B. thuringiensis, an important entomopathogenic bacterium. We adapted the well-known technique yeast-two-hybrid and integrated high-throughput sequencing and bioinformatics for the downstream analysis of the results which enables large-scale analysis of protein-protein interactions. In total, 182 detected interactions show an enrichment in host metabolic proteins and peptidases, in contrast with the current knowledge on host-phage PPIs. Specific host-viral protein-protein interactions were also detected enabling us to propose functions for uncharacterized proteins.

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OTUs clustering should be avoided for defining oral microbiome This in silico investigation aimed to: 1) evaluate a set of primer pairs with high coverage, including those most commonly used in the literature, to find the different oral species with 16S rRNA gene amplicon similarity/identity (ASI) values [≥]97%; and 2) identify oral species that may be erroneously clustered in the same operational taxonomic unit (OTU) and ascertain whether they belong to distinct genera or other higher taxonomic ranks.

Thirty-nine primer pairs were employed to obtain amplicon sequence variants (ASVs) from the complete genomes of 186 bacterial and 135 archaeal species. For each primer, ASVs without mismatches were aligned using BLASTN and their similarity values were obtained. Finally, we selected ASVs from different species with an ASI value [≥]97% that were covered 100% by the query sequences. For each primer, the percentage of species-level coverage with no ASI[≥]97% (SC-NASI[≥]97%) was calculated.

Based on the SC-NASI[≥]97% values, the best primer pairs were OP_F053-KP_R020 for bacteria (65.05%), KP_F018-KP_R002 for archaea (51.11%), and OP_F114-KP_R031 for bacteria and archaea together (52.02%). Eighty percent of the oral-bacteria and oralarchaea species shared an ASI[≥]97% with at least one other taxa, including Campylobacter, Rothia, Streptococcus, and Tannerella, which played conflicting roles in the oral microbiota. Moreover, around a quarter and a third of these two-by-two similarity relationships were between species from different bacteria and archaea genera, respectively. Furthermore, even taxa from distinct families, orders, and classes could be grouped in the same cluster.

Consequently, irrespective of the primer pair used, OTUs constructed with a 97% similarity provide an inaccurate description of oral-bacterial and oral-archaeal species, greatly affecting microbial diversity parameters. As a result, clustering by OTUs impacts the credibility of the associations between some oral species and certain health and disease conditions. This limits significantly the comparability of the microbial diversity findings reported in oral microbiome literature.

sentences: Pneumococcal within-host diversity during colonisation, transmission and treatment Characterising the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition, and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples. We demonstrate deep sequencing has unsurpassed sensitivity for detecting multiple colonisation, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared to gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the childs life. Comprehensive treatment data demonstrated infants were at an elevated risk of both the acquisition, and persistent colonisation, of a multidrug resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.

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Post-embryonic development and aging of the appendicular skeleton in Ambystoma mexicanum BackgroundThe axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop post-embryonically. In this work, we evaluated the post-embryonic development of the appendicular skeleton and its changes with aging.

ResultsThe juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age towards the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to L-thyroxine, increasing the rate of bone formation.

ConclusionsIn axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

sentences: Development of PRPK Directed Phthalimides Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies.

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rescomp: An R package for defining, simulating and visualizing ODE models of consumer-resource interactions O_LIMechanistic models of resource competition underpin numerous foundational concepts and theories in ecology, and continue to be employed widely to address diverse research questions. Nevertheless, current software tools present a comparatively steep barrier to entry.
C_LIO_LII introduce the R package rescomp to support the specification, simulation and visualisaton of a broad spectrum of consumer-resource interactions. rescomp is compatible with diverse model specifications, including an unlimited number of consumers and resources, different consumer functional responses (type I, II and III), different resource types (essential or substitutable) and supply dynamics (chemostats, logistic and/or pulsed), delayed consumer introductions, time dependent growth and consumption parameters, and instantaneous changes to consumer and/or resource densities.
C_LIO_LISeveral examples on implementing rescomp are provided. In addition, a wide variety of additional examples can be found in the package vignettes, including using rescomp to reproduce the results of several well known studies from the literature.
C_LIO_LIrescomp provides users with an accessible tool to reproduce classic models in ecology, to specify models resembling a wide range of experimental designs, and to explore diverse novel model formulations.
C_LI

sentences: A multi-measure approach for assessing the performance of fMRI preprocessing strategies in resting-state functional connectivity It is well established that head motion and physiological processes (e.g. cardiac and breathing activity) should be taken into consideration when analyzing and interpreting results in fMRI studies. However, even though recent studies aimed to evaluate the performance of different preprocessing pipelines there is still no consensus on the optimal strategy. This is partly due to the fact that the quality control (QC) metrics used to evaluate differences in performance across pipelines have often yielded contradictory results. Furthermore, preprocessing techniques based on physiological recordings or data decomposition techniques (e.g. aCompCor) have not been comprehensively examined. Here, to address the aforementioned issues, we propose a framework that summarizes the scores from eight previously proposed and novel QC metrics to a reduced set of two QC metrics that reflect the signal-to-noise ratio and the reduction in motion artifacts and biases in the preprocessed fMRI data. Using this framework, we evaluate the performance of three commonly used practices on the quality of data: 1) Removal of nuisance regressors from fMRI data, 2) discarding motion-contaminated volumes (i.e., scrubbing) before regression, and 3) low-pass filtering the data and the nuisance regressors before their removal. Using resting-state fMRI data from the Human Connectome Project, we show that the scores of the examined QC metrics improve the most when the global signal (GS) and about 17% of principal components from white matter (WM) are removed from the data. Finally, we observe a small further improvement with low-pass filtering at 0.20 Hz and milder variants of WM denoising, but not with scrubbing.

sentences: Comparative study between radiofrequency- and muscimol-induced inhibition of cultured networks of cortical neuron Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode array are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and dissimilarities between the inhibitory effect of RF fields (continuous wave, 1800 MHz) to the {gamma}-Aminobutyric acid type A (GABAA) receptor agonist muscimol (MU). Inhibition of network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of [~]1 {degrees}C. Differently to a pharmacological inhibition with MU, exposure to RF fields preferentially inhibits bursting over spiking activity and exerts less constrains on neural network bursting synchrony. Network rebound excitation, a phenomenon relying on intrinsic properties of cortical neurons, was observed consecutively to the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure which implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As change in AP waveform tightly influence efficiency of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reduce network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of action of RF fields differ from the ones mediated by the activation of GABAA receptor. The rapid onset of the inhibitory effect of RF fields and its reversibility strikingly similar to MU are in favour of a mechanism interacting with fast operating targets at the membrane such as ion channels.

sentences: Structural visualization of de novo initiation of RNA polymerase II transcription Structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription were previously facilitated by the use of synthetic oligonucleotides. Here we report structures of initiation complexes de novo converted from pre-initiation complex (PIC) through catalytic activities and stalled at different template positions. Contrary to previous models, the closed-to-open promoter transition was accompanied by a large positional change of the general transcription factor TFIIH which became in closer proximity to TFIIE for the active delivery of the downstream DNA to the pol II active center. The initially-transcribing complex (ITC) reeled over 80 base pairs of the downstream DNA by scrunching, while retaining the fixed upstream contact, and underwent the transition to elongation when it encountered promoter-proximal pol II from a preceding round of transcription. TFIIH is therefore conducive to promoter melting, TSS scanning, and promoter escape, extending far beyond synthesis of a short transcript.

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Antigenic characterization and pandemic risk assessment of North American H1 influenza A viruses circulating in swine The first pandemic of the 21st century was caused by an H1N1 influenza A virus (IAV) introduced from pigs into humans, highlighting the importance of swine as reservoirs for pandemic viruses. Two major lineages of swine H1 circulate in North America: the 1A classical swine lineage (including the 2009 pandemic H1N1) and 1B human seasonal-like lineage. Here, we investigated the evolution of these H1 IAV lineages in North American swine and their potential pandemic risk. We assessed the antigenic distance between the HA of representative swine H1 and human seasonal vaccine strains (1978-2015) in hemagglutination inhibition (HI) assays using a panel of monovalent anti-sera raised in pigs. Antigenic cross-reactivity varied by strain but was associated with genetic distance. Generally, swine 1A lineage viruses that seeded the 2009 H1 pandemic were antigenically most similar to H1 pandemic vaccine strains, with the exception of viruses in the genetic clade 1A.1.1.3 that had a two-amino acid deletion mutation near the receptor-binding site, dramatically reducing antibody recognition. The swine 1B lineage strains, which arose from previously circulating (pre-2009 pandemic) human seasonal viruses, were more antigenically similar to pre-2009 human seasonal H1 vaccine viruses than post-2009 strains. Human population immunity was measured by cross-reactivity in HI assays to representative swine H1 strains. There was a broad range of titers against each swine strain that was not associated with age, sex, or location. However, there was almost no cross-reactivity in human sera to the 1A.1.1.3 and 1B.2.1 genetic clades of swine viruses, and the 1A.1.1.3 and 1B.2.1 clades were also the most antigenically distant from all human vaccine strains. Our data demonstrate that antigenic distances of representative swine strains from human vaccine strains represent a rational assessment of swine IAV for zoonotic risk research and pandemic preparedness prioritization.

ImportanceHuman H1 influenza A viruses (IAV) spread to pigs in North America, resulting in sustained circulation of two major groups of H1 viruses in swine. We quantified the genetic diversity of H1 in swine and measured antigenic phenotypes. We demonstrated that swine H1 lineages were significantly different from human vaccine strains and this antigenic dissimilarity increased over time as the viruses evolved in swine. Pandemic preparedness vaccine strains for human vaccines also demonstrated a loss in similarity with contemporary swine strains. Human sera revealed a range of responses to swine IAV, including two groups of viruses with little to no immunity. Surveillance and risk assessment of IAV diversity in pig populations are essential to detect strains with reduced immunity in humans, providing critical information for pandemic preparedness.

sentences: RTEL1 and MCM10 overcome topological stress during vertebrate replication termination Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.

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Aliquoting of isobaric labeling reagents for low concentration and single cell proteomics samples The introduction of isobaric tagging reagents enabled more accurate, high-throughput quantitative proteomics by enabling multiple samples to be multiplexed. One drawback of these workflows is the relative expense of the proprietary isobaric reagents, which is often only second to the expense of the instruments themselves. These highly reactive chemical tags are only commercially available in relatively large aliquots compared to the typical amounts of peptides analyzed in proteomic workflows today. Excess reagents are typically disposed of following a single labeling experiment or those performed within a few days of opening a new kit. We present a simple procedure to aliquot commercial isobaric tagging reagents and demonstrate the successful and high efficiency labeling of multiple samples over a period of six months. The samples presented herein were selected as the most diverse ones labeled by prepared aliquots from a single labeling reagent kit over this period. We observe comparable labeling efficiency from 100 microgram to 100 picograms of peptide when labeling samples from both human digest standards, cancer cell lines prepared in-house and from cells directly obtained from human organ donors, despite differences in cell type, lysis, and digestion procedures. No labeling experiment of whole human proteomics samples achieved less than 92% labeling efficiency over this period. When preparing phosphoproteomic samples 6 months after the date of the aliquoting procedure, we observed a decrease in labeling efficiency to approximately 86%, indicating the end of the useful lifetime of aliquots prepared in this manner. Over this period, we have effectively reduced the reagent costs of each experiment to less than 10% of the predicted costs when following the manufacturer instructions for use and disposal. While aliquoting of reagents can be performed by hand, we provide a complete template for automatic aliquoting using an affordable liquid handling robot, including plans for 3D printing of two parts we have found useful for streamlining this procedure.

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sentences: ETV2 regulates PARP-1 binding protein to induce ER stress-mediated cell death in tuberin-deficient cells Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (Tsc1 or Tsc2), and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). The effectiveness of mTORC1 inhibitors is limited by their lack of cytotoxic effects. Here, we report that E26 transformation specific (ETS) Variant Transcription Factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. Nuclear localization of ETV2 in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP), a coregulator of transcription, mRNA and protein expression. Silencing of ETV2 or PARPBP in Tsc2-deficient cells induced ER-stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2 lending support to the translational relevance of our findings. In conclusion, we report a novel signaling axis unique to Syk-inhibition is mTORC1-independent and promotes a cytocidal response in Tsc2-deficient cells, and therefore, maybe a potential alternative therapeutic target in LAM.

sentences: Pervasive transcription enhances the accessibility of H-NS-silenced promoters and generates bistability in Salmonella virulence gene expression In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analysed the basis for the upregulation of H-NS-silenced Salmonella Pathogenicity Island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semi-quantitative 5 RACE-Seq and ChiP-Seq shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilDs ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that, although genetically identical, either express or dont express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.

sentences: Can we improve slow learning in cerebellar patients? We tested whether training paradigms targeting slow learning alleviate motor learning deficits of cerebellar patients compared to matched controls using four visuomotor tasks: standard, gradual, overlearning and long intertrial interval. We measured slow learning using spontaneous recovery, the return to learned adaptation after brief wash out of fast adaptation. Spontaneous recovery increased in both groups only in overlearning. A model of adaptation suggested that in controls the increase reflects changes in slow system dynamics. In contrast, because cerebellar participants plateaued more slowly than controls, added trials in models of cerebellar participants increased the level of slow learning. Whereas, under the model, slow learning became more resilient in controls, there was no change in its dynamics for cerebellar participants. Our results and modeling suggest that while residual slow learning does exist in cerebellar patients and can be expressed through increased training trials, the primary cerebellar deficit cannot be improved by overlearning paradigms.

sentences: Hypertrophied human adipocyte spheroids as in vitro model of weight gain and adipose tissue dysfunction The rise in obesity prevalence has created an urgent need for new and improved methods to study human adipocytes and the pathogenic effects of weight gain in vitro. Despite numerous studies showing the advantages of culturing adipocyte progenitors as 3D structures, the majority continue using traditional 2D cultures which result in small, multilocular adipocytes with poor representability. We hypothesized that providing differentiating pre-adipocytes with a vascular growth niche would mimic in vivo adipogenesis and improve the differentiation process. Here we present a simple, easily applicable culture protocol that allows for the differentiation and culturing of human adipocytes with a more unilocular morphology and larger lipid droplets than previous protocols. We moreover offer a protocol for inducing adipocyte enlargement in vitro, resulting in larger lipid droplets and development of several key features of adipocyte dysfunction, including altered adipokine secretions and impaired lipolysis. Taken together, our hypertrophied human adipocyte spheroids offer an improved culture system for studying the cellular and molecular mechanisms causing metabolic dysfunction and inflammation during weight gain.

sentences: Perception of RNA Nanotechnology among students and recommendations towards improved educational outreach Translating new technologies to industrial and biomedical applications requires a highly skilled workforce. In the past, colleges and graduate schools played a primary role in preparing students for various areas of industry and medicine. The learning process and introduction of new concepts have recently extended beyond college education. High schools saw the rise of specialized career programs, while both high and middle school curricula got infused with challenging concepts. Nucleic acids, such as DNA and RNA, are broadly known for their role in the foundation of life. However, nucleic acid nanotechnology, an area of material science manipulating DNA and RNA to create complex structures with controlled properties, is less known. Herein, I report the results of a study investigating the perception of RNA nanotechnology among school students and suggest educational resources to improve understanding of RNA nanotechnology.

sentences: In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

sentences: Temporal Gene Expression in Apical Culms Shows Early Changes in Cell Wall Biosynthesis Genes in Sugarcane Multiple genes in sugarcane control sucrose accumulation and the biosynthesis of cell wall components; however, it is unclear how these genes are expressed in its apical culms. To better understand this process, we sequenced mRNA from +1 stem internodes collected from four genotypes with different concentrations of soluble solids. Culms were collected at four different time points, ranging from six to 12-month-old plants. Here we show differentially expressed genes related to sucrose metabolism and cell wall biosynthesis, including genes encoding invertases, sucrose synthase and cellulose synthase. Our results showed increased expression of invertases in IN84-58, the genotype with lower sugar and higher fiber content, as well as delayed expression of secondary cell wall-related cellulose synthase for the other genotypes. Interestingly, genes involved with hormone metabolism were differentially expressed across time points in the three genotypes with higher soluble solids content. A similar result was observed for genes controlling maturation and transition to reproductive stages, possibly a result of selection against flowering in sugarcane breeding programs. These results indicate that carbon partitioning in apical culms of contrasting genotypes is mainly associated with differential cell wall biosynthesis, and may include early modifications for subsequent sucrose accumulation. Co-expression network analysis identified transcription factors related to growth and development, showing a probable time shift for carbon partitioning occurred in 10-month-old plants.

sentences: On the stability and layered organization of protein-DNA condensates Multi-component phase separation is emerging as a key mechanism for the formation of biological condensates that play essential roles in signal sensing and transcriptional regulation. The molecular factors that dictate these condensates stability and spatial organization are not fully understood, and it remains challenging to predict their microstructures. Using a near-atomistic, chemically accurate force field, we studied the phase behavior of chromatin regulators that are crucial for heterochromatin organization and their interactions with DNA. Our computed phase diagrams recapitulated previous experimental findings on different proteins. They revealed a strong dependence of condensate stability on the protein-DNA mixing ratio as a result of balancing protein-protein interactions and charge neutralization. Notably, a layered organization was observed in condensates formed by mixing HP1, histone H1, and DNA. This layered organization may be of biological relevance as it enables cooperative DNA packaging between the two chromatin regulators: histone H1 softens the DNA to facilitate the compaction induced by HP1 droplets. Our study supports near atomistic models as a valuable tool for characterizing the structure and stability of biological condensates.

sentences: Circular RNA Vaccines against SARS-CoV-2 and Emerging Variants Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants of concern (VOC), such as Delta (B.1.617.2) and Omicron (B.1.1.529), has continued to drive the worldwide pandemic. Therefore, there is a high demand for vaccines with enhanced efficacy, high thermostability, superior design flexibility, and fast manufacturing speed. Here, we report a circular RNA (circRNA) vaccine that encodes the trimeric RBD of SARS-CoV-2 Spike protein. Without the need of nucleotide modification, 5-capping or 3-polyadenylation, circRNA could be rapidly produced via in vitro transcription and is highly thermostable whether stored in naked or lipid-nanoparticle (LNP)-encapsulated format. LNP-encapsulated circRNARBD elicited potent neutralizing antibodies and T cell responses, providing robust protection against Beta (B.1.351) and native viruses in mice and rhesus macaques, respectively. Notably, circRNA vaccine enabled higher and more durable antigen production than 1m{Psi}-modified mRNA vaccine, eliciting a higher proportion of neutralizing antibodies and stronger Th1-biased immune responses. Importantly, we found that circRNARBD-Omicron vaccine induced effective neutralizing antibodies against only Omicron but not Delta variant. By contrast, circRNARBD-Delta could elicit high level of neutralizing antibodies against both Delta and Omicron. Following two doses of either native- or Delta-specific vaccination, circRNARBD-Delta, but not Omicron or Beta vaccines, could effectively boost the neutralizing antibodies against both Delta and Omicron variants. These results suggest that circRNARBD-Delta is a favorable choice for vaccination to provide a broad-spectrum protection against the current variants of concern of SARS-CoV-2.

sentences: Reaction-diffusion patterning of DNA-based artificial cells Biological cells display complex internal architectures, with distinct micro environments that establish the chemical heterogeneity needed to sustain cellular functions. The continued efforts to create advanced cell mimics - artificial cells - demands strategies to robustly engineer micro-compartmentalised architectures, where the molecular makeup of distinct regions is coupled with localised functionalities. Here, we introduce a platform for constructing membrane-less artificial cells from the self-assembly of synthetic DNA nanostructures, in which internal domains can be established thanks to a rationally designed reactiondiffusion process. The method, rationalised through numerical modelling, enables the formation of up to five distinct and addressable environments, in which functional moieties can be localised. As a proof-of-concept, we apply this platform to build artificial cells in which a prototypical nucleus synthesises fluorescent RNA aptamers, which then accumulate in a surrounding storage shell, thus demonstrating spatial segregation of functionalities reminiscent of that observed in biological cells.

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Multi-objective prioritisation of candidate epitopes for diagnostic test development BackgroundThe development of peptide-based diagnostic tests requires the identification of epitopes that are at the same time highly immunogenic and, ideally, unique to the pathogen of interest, to minimise the chances of cross-reactivity. Existing computational pipelines for the prediction of linear B-cell epitopes tend to focus exclusively on the first objective, leaving considerations of cross-reactivity to later stages of test development.

ResultsWe present a multi-objective approach to the prioritisation of candidate epitopes for experimental validation, in the context of diagnostic test development. The dual objectives of uniqueness (measured as dissimilarity from known epitope sequences from other pathogens) and predicted immunogenicity (measured as the probability score returned by the prediction model) are considered simultaneously. Validation was performed using data from three distinct pathogens (namely the nematode Onchocerca volvulus, the Epstein-Barr Virus and the Hepatitis C Virus), with predictions derived using an organism-specific prediction approach. The multi-objective rankings returned sets of non-dominated solutions as potential targets for the development of diagnostic tests with lower probability of false positives due to cross-reactivity.

ConclusionsThe application of the proposed approach to three test pathogens led to the identification of 20 new potential epitopes, with both high probability and a high degree of exclusivity to the target organisms. The results indicate the potential of the proposed approach to provide enhanced filtering and ranking of potential candidates, highlighting potential cross-reactivities and including this information into the test development process right from the target identification and prioritisation step.

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

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Mitochondrial pyruvate supports lymphoma proliferation by fueling a non-canonical glutamine metabolism pathway The fate of pyruvate, which is modulated by the activity of the mitochondrial pyruvate carrier (MPC), is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogeneous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate was uniformly consumed by glutamate pyruvate transaminase 2 (GPT2) to support -ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid (TCA) cycle in DLBCLs. Furthermore, we found that MPC inhibition unexpectedly leads to decreased glutaminolysis, which is contrary to previous observations in other cell types. We also discovered that MPC inhibition and depletion only decreased DLBCL proliferation in an extracellular matrix (ECM) environment and in vivo xenografts, but not in the typical DLBCL suspension environment. We also have found that the metabolic profile of DLBCL cells in ECM is markedly different from cells in suspension environment. Thus, we report that besides the canonical glutamate dehydrogenase (GDH)-mediated glutaminolysis, the non-canonical GPT2 mediated consumption and assimilation of glutamine and pyruvate in DLBCLs enables their proliferation in an extracellular environment-dependent manner.

HIGHLIGHTSO_LIGlutamine, but not glucose, is a major carbon source for the tricarboxylic acid cycle in diffuse large B-cell lymphomas.
C_LIO_LIMitochondrial pyruvate supports glutaminolysis in diffuse large B-cell lymphomas by supplying pyruvate for glutamate pyruvate transaminase 2-mediated  -ketoglutarate production.
C_LIO_LIMitochondrial pyruvate carrier inhibition leads to decreased glutaminolysis in diffuse large B-cell lymphomas.
C_LIO_LI -ketoglutarate production is important for diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment.
C_LIO_LIMitochondrial pyruvate carrier activity supports diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment and in mouse xenografts.
C_LI

sentences: Perceptual decisions and oculomotor responses rely on temporally distinct streams of evidence Perceptual decisions often require the integration of noisy sensory evidence over time. This process is formalized with sequential sampling models, where evidence is accumulated up to a decision threshold before a choice is made. Although intuition suggests that decision formation must precede the preparation of a motor response (i.e., the action used to communicate the choice), neurophysiological findings have suggested that these two processes might be one and the same. To test this idea, we developed a reverse-correlation protocol in which the visual stimuli that influence decisions can be distinguished from those guiding motor responses. In three experiments, we found that the temporal weighting function of oculomotor responses did not overlap with the relatively early weighting function of stimulus properties having an impact on decision formation. These results support a timeline in which perceptual decisions are formed, at least in part, prior to the preparation of a motor response.

sentences: Droplet array-based platform for parallel optical analysis of dynamic extracellular vesicle secretion from single cells Extracellular vesicles (EVs) are essential intercellular communication tools, but the regulatory mechanisms governing heterogeneous EV secretion are still unclear due to the lack of methods for precise analysis. Monitoring the dynamics of secretion from individually isolated cells is crucial because, in bulk analysis, secretion activity can be perturbed by cell-cell interactions, and a cell population rarely performs secretion in a magnitude- or duration-synchronized manner. Although various microfluidic techniques have been adopted to evaluate the abundance of single-cell-derived EVs, none can track their secretion dynamics continually for extended periods. Here, we have developed a droplet array-based method that allowed us to optically quantify the EV secretion dynamics of >300 single cells every 2 hours for 36 hours, which covers the cell doubling time of many cell types. The experimental results clearly show the highly heterogeneous nature of single-cell EV secretion and suggest that cell division facilitates EV secretion, showing the usefulness of this platform for discovering EV regulation machinery.

sentences: The accuracy of absolute differential abundance analysis from relative count data Concerns have been raised about the use of relative abundance data derived from next generation sequencing as a proxy for absolute abundances. For example, in the differential abundance setting, compositional effects in relative abundance data may give rise to spurious differences (false positives) when considered from the absolute perspective. In practice however, relative abundances are often transformed by renormalization strategies intended to compensate for these effects and the scope of the practical problem remains unclear. We used simulated data to explore the consistency of differential abundance calling on renormalized relative abundances versus absolute abundances and find that, while overall consistency is high, with median sensitivities (true positive rates) and specificities (1 - false positive rates) each of around 0.90, consistency can be much lower where there is widespread change in the abundance of features across conditions. We confirm these findings on a large number of real data sets drawn from 16S metabarcoding, expression array, bulk RNA-seq, and single-cell RNA-seq experiments, where data sets with the greatest change between experimental conditions are also those with the highest false positive rates. Finally, we evaluate the predictive utility of summary features of relative abundance data themselves. Estimates of sparsity and the prevalence of feature-level change in relative abundance data give accurate predictions of discrepancy in differential abundance calling in simulated data and provide useful bounds for worst-case outcomes in real data.

sentences: Hypoxia-inducible factor 1 signaling drives placental aging and can elicit inflammatory changes in uterine myocytes Most cases of pre-term labor have unknown cause, and the burden of preterm birth is immense. Placental aging has been proposed to promote labor onset, but specific mechanisms remain elusive. We report findings stemming from unbiased transcriptomic analysis of mouse placenta, which revealed that hypoxia-inducible factor 1 (HIF-1) stabilization is a hallmark of advanced gestational timepoints, accompanied by mitochondrial dysfunction and cellular senescence; we validated these findings in human placenta. In parallel in primary mouse trophoblasts and human choriocarcinoma cells, we modeled HIF-1 induction using prolyl hydroxylase inhibitors, and demonstrated resultant mitochondrial dysfunction and cellular senescence. Transcriptomic analysis revealed that HIF-1 stabilization recapitulated gene signatures observed in aged placenta. Further, conditioned media from trophoblasts following HIF-1 induction was sufficient to induce a contractile phenotype in immortalized uterine myocytes, suggesting a mechanism by which the aging placenta may drive the transition from uterine quiescence to contractility at the onset of labor. These results provide clear evidence for placental aging in normal pregnancy. Mechanistic dissection of this phenomenon demonstrates how hypoxia in late gestation may be a causal determinant of the mitochondrial dysfunction and senescence observed within the trophoblast as well as a trigger for uterine contraction.

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skater: An R package for SNP-based Kinship Analysis, Testing, and Evaluation MotivationSNP-based kinship analysis with genome-wide relationship estimation and IBD segment analysis methods produces results that often require further downstream processing and manipulation. A dedicated software package that consistently and intuitively implements this analysis functionality is needed.

ResultsHere we present the skater R package for SNP-based kinship analysis, testing, and evaluation with R. The skater package contains a suite of well-documented tools for importing, parsing, and analyzing pedigree data, performing relationship degree inference, benchmarking relationship degree classification, and summarizing IBD segment data.

AvailabilityThe skater package is implemented as an R package and is released under the MIT license at https://github.com/signaturescience/skater. Documentation is available at https://signaturescience.github.io/skater.

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

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GBS-SBG - GBS Serotyping by Genome Sequencing Group B Streptococcus agalactiae (GBS; Streptococcus agalactiae) is the most common cause of neonatal meningitis and a rising cause of sepsis in adults. Recently, it has also been shown to cause foodborne disease. As with many other bacteria, the polysaccharide capsule of GBS is antigenic, enabling its use for strain serotyping. Recent advances in DNA sequencing have made sequence-based typing attractive (as has been implemented for several other bacteria, including Escherichia coli, Klebsiella pneumoniae species complex, Streptococcus pyogenes, and others). For GBS, existing WGS-based serotyping systems do not provide complete coverage of all known GBS serotypes (specifically including subtypes of serotype III), and none are simultaneously compatible with the two most common data types, raw short reads and assembled sequences. Here, we create a serotyping database (GBS-SBG, GBS Serotyping by Genome Sequencing), with associated scripts and running instructions, that can be used to call all currently described GBS serotypes, including subtypes of serotype III, using both direct short-read- and assembly-based typing. We achieved higher concordance using GBS-SBG on a previously reported data set of 790 strains. We further validated GBS-SBG on a new set of 572 strains, achieving 99.8% concordance with PCR-based molecular serotyping using either short-read- or assembly-based typing. The GBS-SBG package is publicly available and will accelerate and simplify serotyping by sequencing for GBS.

DATA SUMMARYO_LIThe GBS-SBG package is open source and available for at Github under the MIT license (URL - https://github.com/swainechen/GBS-SBG)
C_LIO_LIAccession numbers of the sequencing reads and reference sequences used in the study from earlier reports have been provided within the article and the supplementary data
C_LIO_LIThe WGS data for the 572 isolates used in the study is available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA293392
C_LI

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A real-time cellular thermal shift assay (RT-CETSA) to monitor target engagement Determining a molecules mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a proteins unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered that withstood unfolding at temperatures greater than 90 degrees Celsius and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. The RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule.

SignificanceValidating target engagement is a critical step when characterizing a small molecule modulator. The cellular thermal shift assay (CETSA) is a common approach to examine target engagement, as alterations in the thermal stability of a protein can be conferred by ligand binding. An advantage of CETSA is that it does not require modification of the protein target or small molecule. Major limitations are the throughput and ease-of-use, as the traditional detection method uses western blots, which limits the number of samples that can be processed. Higher-throughput CETSA methods have been developed but are performed at a single temperature and require target-specific optimization. We developed a high-throughput real-time CETSA to circumvent these challenges, providing a rapid and cost-effective strategy to assess on-target activity of a small molecule in living cells.

sentences: Laminar Mechanisms of Saccadic Suppression in Primate Visual Cortex Saccades are a ubiquitous and crucial component of our visual system, allowing for the efficient deployment of the fovea and its accompanying neural resources. Initiation of a saccade is known to cause saccadic suppression, a temporary reduction in visual sensitivity1, 2 and visual cortical firing rates3-6. While saccadic suppression has been well characterized at the level of perception and single neurons, relatively little is known about the visual cortical networks governing this phenomenon. Here we examine the effects of saccadic suppression on distinct neural subpopulations within visual area V4. We find cortical layer- and cell type-specific differences in the magnitude and timing of peri-saccadic modulation. Neurons in the input layer show changes in firing rate and inter-neuronal correlations prior to saccade onset, indicating that this layer receives information about impending saccades. Putative inhibitory interneurons in the input layer elevate their firing rate during saccades, suggesting they play a role in suppressing the activity of other cortical subpopulations. A computational model of this circuit recapitulates our empirical observations and demonstrates that an input layer-targeting pathway can initiate saccadic suppression by enhancing local inhibitory activity. Collectively, our results provide a mechanistic understanding of how eye movement signaling interacts with cortical circuitry to enforce visual stability.

sentences: Genetic variations within human gained enhancer elements affect human brain sulcal morphology. The expansion of the cerebral cortex is one of the most distinctive changes in the evolution of the human brain. Cortical expansion and related increases in cortical folding may have contributed to emergence of our capacities for high-order cognitive abilities. Molecular analysis of humans, archaic hominins, and non-human primates has allowed identification of chromosomal regions showing evolutionary changes at different points of our phylogenetic history. In this study, we assessed the contributions of genomic annotations spanning 30 million years to human sulcal morphology measured via MRI in more than 18,000 participants from the UK Biobank. We found that variation within brain-expressed human gained enhancers, regulatory genetic elements that emerged since our last common ancestor with Old World monkeys, explained more trait heritability than expected for the left and right calloso-marginal posterior fissures and the right central sulcus. Intriguingly, these are sulci that have been previously linked to the evolution of locomotion in primates and later on bipedalism in our hominin ancestors.

sentences: The oscillation of mitotic kinase governs cell cycle latches In order to transmit a eukaryotic cells genome accurately from mother cell to daughter cells, it is essential that the basic events of the cell division cycle (DNA synthesis and mitosis) occur once and only once per cycle, i.e., that a cell progresses irreversibly from G1 to S to G2 to M and back to G1. Irreversible progression through the cell cycle is assured by a sequence of  latching molecular switches, based on molecular interactions among cyclin-dependent kinases and their auxiliary partners. Positive feedback loops (++ or --) create bistable switches with latching properties, and negative feedback loops drive progression from one stage to the next. In budding yeast (Saccharomyces cerevisiae) these events are coordinated by double-negative feedback loops between Clb-dependent kinases (Clb1-6) and their antagonists (APC:Cdh1 and Sic1). If the coordinating signal is compromised, either by deletion of Clb1-5 proteins or expression of non-degradable Clb2, then irreversibility is lost and yeast cells exhibit multiple rounds of DNA replication or mitotic exit events (Cdc14 endocycles). Using mathematical modelling of a stripped-down control network, we show how endocycles arise because the switches fail to latch, and the gates swing back and forth by the action of the negative feedback loops.

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

sentences: Distinct Patterns of Emergence of SARS-CoV-2 Spike Variants including N501Y in Clinical Samples in Columbus Ohio Following the worldwide emergence of the p.Asp614Gly shift in the Spike (S) gene of SARS-CoV-2, there have been few recurring pathogenic shifts occurring during 2020, as assessed by genomic sequencing. This situation has evolved in the last several months with the emergence of several distinct variants (first identified in the United Kingdom and South Africa) that manifest multiple changes in the S gene, particularly p.Asn501Tyr (N501Y), that likely have clinical impact. We report here the emergence in Columbus, Ohio in December 2020 of two novel SARS-CoV-2 clade 20G variants. One variant, that has become the predominant virus found in nasopharyngeal swabs in the December 2020-January 2021 period, harbors S p.Gln677His (Q677H), affecting a consensus QTQTN domain near the S1/S2 furin cleavage site, nucleocapsid (N) p.Asp377Tyr (D377Y) and membrane glycoprotein (M) p.Ala85Ser (A85S) mutations, with additional S mutations in subsets. The other variant present in two samples, contains S N501Y, which is a marker of the UK-B.1.1.7 (clade 20I/501Y.V1) strain, but lacks all other mutations from that virus. The Ohio variant is from a different clade and shares multiple mutations with the clade 20G viruses circulating in the area prior to December 2020. These two SARS-CoV-2 viruses, which we show are also present and evolving currently in several other parts of North America, add to the diversity of S gene shifts occurring worldwide. These and other shifts in this period of the pandemic support multiple independent acquisition of functionally significant and potentially complementing mutations affecting the S QTQTN site (Q675H or Q677H) and certain receptor binding domain mutations (e.g., E484K and N501Y).

sentences: Loss of PRMT2 in myeloid cells in normoglycemic mice phenocopies impaired regression of atherosclerosis in diabetic mice Regression of atherosclerosis is impaired in diabetes. However, the factors mediating this effect remain incomplete. We identified Protein Arginine Methyltransferase 2 (PRMT2) as a protein whose expression in macrophages is reduced in hyperglycemia and diabetes. PRMT2 catalyzes arginine methylation to target proteins to modulate gene expression. Because PRMT2 expression is reduced in cells in hyperglycemia, we wanted to determine whether PRMT2 plays a causal role in the impairment of atherosclerosis regression in diabetes. We therefore examined the consequence of deleting PRMT2 in myeloid cells during regression of atherosclerosis in normal and diabetic mice. Remarkably, we found a significant impairment of atherosclerosis regression under normoglycemic conditions in mice lacking PRMT2 (Prmt2-/-) in myeloid cells that mimics the decrease in regression of atherosclerosis in WT mice under diabetic conditions. This was associated with increased plaque macrophage retention, as well as increased apoptosis and necrosis. PRMT2-deficient plaque CD68+ cells under normoglycemic conditions showed increased expression of genes involved in cytokine signaling and inflammation compared to WT. Consistently Prmt2-/- bone marrow derived macrophages (BMDMs) showed an increased response of pro-inflammatory genes to LPS, and a decreased response of inflammation resolving genes to IL-4. This increased response to LPS in Prmt2-/- BMDMs is via enhanced NF-kappa B activity. Thus, the loss of PRMT2 is causally linked to impaired atherosclerosis regression via a heightened inflammatory response in macrophages. That PRMT2 expression was lower in myeloid cells in plaques from human subjects with diabetes supports the relevance of our findings to human atherosclerosis.

sentences: Plasma ST6Gal1 is Dispensable for IgG Sialylation The glycosylation of IgG has attracted increased attention due to the impact of N-glycan modifications at N297 on IgG function, acting primarily through modulation of Fc domain conformation and Fc{gamma} receptor binding affinities and signaling. However, the mechanisms regulating IgG glycosylation and especially 2,6-sialylation of its N-glycan remain poorly understood. We observed previously that IgG is normally sialylated in mice with B cells lacking the sialyltransferase ST6Gal1. This supported the hypothesis that IgG may be sialylated outside of B cells, perhaps through the action of hepatocyte-released plasma ST6Gal1. Here we demonstrate that this model is incorrect. Animals lacking hepatocyte expressed ST6Gal1 retain normal IgG 2,6-sialylation, despite the lack of detectable ST6Gal1 in plasma. Moreover, we confirmed that B cells were not a redundant source of IgG sialylation. Thus, while 2,6-sialylation is lacking in IgG from mice with germline ablation of ST6Gal1, IgG 2,6-sialylation is normal in mice lacking ST6Gal1 in either hepatocytes or B cells. These results indicate that IgG 2,6-sialylation arises after release from a B cell, but is not dependent on plasma-localized ST6Gal1 activity.

sentences: Developmental and Intergenerational Landscape of Human Circulatory Lipidome and its Association with Obesity Risk Lipids play a vital role in human health and development, but changes to their circulatory levels during gestation and in early life are poorly understood. Here we present the first developmental and intergenerational landscape of the human circulatory lipidome, derived by profiling of 480 lipid species representing 25 lipid classes, in mothers and their offspring (n=2491). Levels of 66% of the profiled lipids increased in maternal circulation during gestation, while cord blood had higher concentrations of acylcarnitines and lysophospholipids. The offspring lipidome at age six years revealed striking similarities with postnatal maternal lipidome (adult) in its lipid composition and concentrations. Comparison of lipids associated with child and maternal adiposity identified a 92% overlap, implying intergenerational similarities in the lipid signatures of obesity risk. We also catalogued lipid signatures linked with maternal adiposity during gestation and offspring birthweight, and validated (>70% overlap) the findings in an independent birth-cohort (n=1935).

sentences: Emergent dynamics of a three-node regulatory network explain phenotypic switching and heterogeneity: a case study of Th1/Th2/Th17 cell differentiation Naive helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a  master regulator - T-bet (Th1), GATA3 (Th2) and ROR{gamma}T (Th17) - that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotypes - Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17 and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remains unclear. Here, through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any  master regulator can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/ROR{gamma}T.

sentences: An interpretable and versatile machine learning approach for oocyte phenotyping Meiotic maturation is a crucial step of oocyte development allowing its potential fertilization and embryo development. Elucidating this process is important both for fundamental research and assisted reproductive technology. However, only few computational tools, based on non-invasive measurements, are currently available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images or movies acquired exclusively in transmitted light. We first trained neural networks to segment the contours of oocytes and their zona pellucida using a diverse cohort of both mouse and human oocytes. We then defined a comprehensive set of morphological features to describe a single oocyte. We have implemented these steps in a versatile and user-friendly open source Fiji plugin available to the mouse and human oocyte community. Then, we present a machine learning pipeline based on selected features to automatically recognize oocyte populations and determine their morphological differences. Its first application is a novel approach to screen oocyte strains and automatically identify their morphological characteristics. We demonstrate its potential by phenotyping a well characterized genetically modified mouse oocyte strain. Its second application is to predict and characterize the maturation potential of oocytes. Importantly, we identify two new features to assess mouse oocyte maturation potential, consisting in the texture of the zona pellucida and the cytoplasmic particles size. Eventually, we tested whether these mouse oocyte quality features were applicable to human oocytes developmental potential.

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The landscape of human brain immune response in patients with severe COVID-19 In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between brain tropism, neuroinflammation and host immune response has not been well characterized. We analyzed 68,557 single-nucleus transcriptomes from three brain regions (dorsolateral prefrontal cortex, medulla oblongata and choroid plexus) and identified an increased proportion of stromal cells and monocytes in the choroid plexus of COVID-19 patients. Differential gene expression, pseudo-temporal trajectory and gene regulatory network analyses revealed microglial transcriptome perturbations, mediating a range of biological processes, including cellular activation, mobility and phagocytosis. Quantification of viral spike S1 protein and SARS-CoV-2 transcripts did not support the notion of brain tropism. Overall, our findings suggest extensive neuroinflammation in patients with acute COVID-19.

One Sentence SummarySingle-nucleus transcriptome analysis suggests extensive neuroinflammation in human brain tissue of patients with acute coronavirus disease 2019.

sentences: Transcriptome innovations in primates revealed by single-molecule long-read sequencing Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Our transcriptomes reveal thousands of novel transcripts, some of them under active translation, expanding and completing the repertoire of primate gene models. Our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

sentences: Vertical transmission of tissue microbiota in 	Caenorhabditis elegans The past forty-five years has witnessed Caenorhabditis elegans as the most significant model animal in life science since its discovery seventy years ago1,2, as it introduced principles of gene regulated organ development, and RNA interference into biology3-5. Meanwhile, it has become one of the lab animals in gut microbiota studies as these symbionts contribute significantly to many aspects in host biology6,7. Meanwhile, the origin of gut microbiota remains debatable in human8- 11, and has not been investigated in other model animals. Here we show that the symbiont bacteria in C. elegans not only vertically transmit from the parent generation to the next, but also distributes in the worm tissues parallel with its development. We found that bacteria can enter into the embryos of C. elegans, a step associated with vitellogenin, and passed to the next generation. These vertically transmitted bacteria share global similarity, and bacterial distribution in worm tissues changes as they grow at different life stages. Antibiotic treatment of worms increased their vulnerability against pathogenic bacteria, and replenishment of tissue microbiota restored their immunity. These results not only offered a molecular basis of vertical transmission of bacteria in C. elegans, but also signal a new era for the mixed tissue cell-bacteria multi-species organism study.

sentences: Primer biases in the molecular assessment of diet in multiple insectivorous mammals Our understanding of trophic interactions of small insectivorous mammals has been drastically improved with the advent of DNA metabarcoding. The technique has continued to be optimised over the years, with primer choice repeatedly being a vital factor for dietary inferences. However, the majority of dietary studies examining the effect of primer choice often rely on in silico analyses or comparing single-niche species. Here we apply DNA metabarcoding to empirically compare the prey detection capabilities of two widely used primer sets when assessing the diets of a flying (lesser horseshoe bat; Rhinolophus hipposideros) and two ground dwelling insectivores (greater white-toothed shrew; Crocidura russula and pygmy shrew; Sorex minutus). Although R. hipposideros primarily rely on two prey orders (Lepidoptera and Diptera), the unique taxa detected by each primer shows that a combination of primers may be the best approach to fully describe bat trophic ecology. However, random forest classifier analysis suggest that one highly degenerate primer set detected the majority of both shrews diet despite higher levels of host amplification. The wide range of prey consumed by ground-dwelling insectivores can therefore be accurately documented from using a single broad-range primer set, which can decrease cost and labour. The results presented here show that dietary inferences will differ depending on the primer or primer combination used for insectivores occupying different niches (i.e. hunting in the air or ground) and demonstrate the importance of performing empirical pilot studies for novel study systems.

sentences: AMPing up the search: An in silico approach to identifying Antimicrobial Peptides (AMPs) with potential anti-biofilm activity Antibiotic resistance is a public health threat, and the rise of multidrug-resistant bacteria, including those that form protective biofilms, further compounds this challenge. Antimicrobial peptides (AMPs) have been recognized for their anti-infective properties, including their ability to target processes important for biofilm formation. However, given the vast array of natural and synthetic AMPs, determining potential candidates for anti-biofilm testing is a significant challenge. In this study, we present an in silico approach, based on open-source tools, to identify AMPs with potential anti-biofilm activity. This approach is developed using the sortase-pilin machinery, important for adhesion and biofilm formation, of the multidrug-resistant, biofilm-forming pathogen C. striatum as the target. Using homology modeling, we modeled the structure of the C. striatum sortase C protein, resembling the semi-open lid conformation adopted during pilus biogenesis. Next, we developed a structural library of 5544 natural and synthetic AMPs from sequences in the DRAMP database. From this library, AMPs with known anti-Gram positive activity were filtered, and 100 select AMPs were evaluated for their ability to interact with the sortase C protein using in-silico molecular docking. Based on interacting residues and docking scores, we built a preference scale to categorize candidate AMPs in order of priority for future in vitro and in vivo biofilm studies. The considerations and challenges of our approach, and the resources developed, which includes a search-enabled repository of predicted AMP structures and protein-peptide interaction models relevant to biofilm studies (B-AMP), can be leveraged for similar investigations across other biofilm targets and biofilm-forming pathogens.

sentences: Chronic Pb2+ exposure causes theta-band hypersynchrony disrupting sensory motor gating and exacerbating absence seizures Chronic lead (Pb2+) exposure from childhood contributes to an array of cognitive and behavioral dysfunctions including impaired attention and intellectual ability, learning and memory deficits, and delinquency. It is also an environmental risk factor for adult psychopathologies, most notably schizophrenia and epilepsy. Pb2+ is a potent N-methyl-D-aspartate receptor (NMDAR) antagonist and exposure during early life elicits a cascade of cellular neurotoxic effects that alter the developmental trajectory leading to a loss of parvalbumin-expressing interneurons in hippocampus and altered synaptic transmission. Little is known, however, about the impact of chronic Pb2+ exposure on hippocampal network dynamics which serve as a link between cellular-molecular effects and cognitive-behavioral consequences of Pb2+ neurotoxicity. Here, we tested the effects of chronic Pb2+ exposure on the hippocampal local field potential (LFP) of freely-behaving rats. Pb2+ exposure caused striking theta rhythmic hypersynchrony and heightened behavioral modulation of theta during locomotor behavior. Pb2+ exposure also markedly exacerbated absence seizures appearing in the LFP as spike-wave discharges with a theta-band fundamental frequency and strong theta-harmonic synchronization. Mechanisms of theta rhythmogenesis have been implicated in impairments of prepulse inhibition of the acoustic startle reflex (PPI), so we tested the effect of Pb2+ exposure on PPI in male and female rats at different developmental timepoints. We found that adult males (PN50 and 120), but neither females nor juvenile males showed reduced PPI independent of changes in the startle reflex. This pattern recapitulates sex- and age-dependencies of PPI disruption in schizophrenic patients. Overall, these results are consistent with the hypothesis that Pb2+ is an environmental risk factor for psychopathology in adulthood, especially those symptoms related to cognitive and sensory-motor gating processes that depend on rhythmic coordination of network activity in the hippocampus.

sentences: The Population Genetics of Collateral Resistance and Sensitivity Resistance mutations against one drug can elicit collateral sensitivity against other drugs. Multi-drug treatments exploiting such trade-offs can help slow down the evolution of resistance. However, if mutations with diverse collateral effects are available, a treated population may evolve either collateral sensitivity or collateral resistance. How to design treatments robust to such uncertainty is unclear. We show that many resistance mutations in Escherichia coli against various antibiotics indeed have diverse collateral effects. We propose to characterize such diversity with a joint distribution of fitness effects (JDFE) and develop a theory for describing and predicting collateral evolution based on simple statistics of the JDFE. We show how to robustly rank drug pairs to minimize the risk of collateral resistance and how to estimate JDFEs. In addition to practical applications, these results have implications for our understanding of evolution in variable environments.

sentences: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by the N-terminal peptide of lactoferrin In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.

sentences: A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity The Ras-like GTPase MglA is a key regulator of front-rear polarity in rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two motility machineries. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY as a co-GAP that stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting complex formation to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that MglB GAP activity is spatially precisely confined to the lagging pole, thereby constraining MglA-GTP to the leading pole establishing front-rear polarity.

sentences: A comparison of mtDNA deletion mutant proliferation mechanisms In this paper we use simulation methods to investigate the proliferation of deletion mutations of mitochondrial DNA in neurons. We simulate three mtDNA proliferation mechanisms, namely, random drift, replicative advantage and vicious cycle. For each mechanism, we investigated the effect mutation rates have on neuron loss within a human host. We also compare heteroplasmy of each mechanism at mutation rates that yield the levels neuron loss that would be associated with dementia. Both random drift and vicious cycle predicted high levels of heteroplasmy, while replicative advantage showed a small number of dominant clones with a low background of heteroplasmy.

sentences: Thermophoresis of Molecules and Structures of different Sizes in Self-assembled Biomatrices Upon subjecting biomolecules to non-equilibrium conditions, many biochemical and biophysical features such as biomolecular diffusion, protein folding, interaction kinetics, as well as enzyme-catalyzed reactions can be characterized in an aqueous solution. However, most assays under non-equilibrium conditions cannot be performed in complex self-assembled biomatrices (e.g. extracellular matrices) due to the limitations associated with sample handling, reaction design, and optical detection. Herein, we report the study of biomolecular thermodiffusion in non-covalently assembled synthetic or naturally derived hydrogels. This approach has been demonstrated with a large variety of analytes, including small molecules, polysaccharides, DNAs, DNA origami, and proteins in various polymer networks. The in-biomatrix method has also shown advantages over in-solution measurements: First, it allows us to analyze biomolecules in 3D matrices in a high-throughput fashion. Second, the aggregation of analytes can be remarkably prevented. Although the underlying physics of thermodiffusion is still not well-understood, we demonstrated that the thermodiffusion of surrounding networks will enhance the thermodiffusion of the analyte, an effect counteracting the hindered movement by the polymer network.

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Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis ObjectiveInclusion body myositis (IBM) has an unclear molecular etiology due to the co-existence of characteristic cytotoxic T-cell activity and degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM.

MethodsWe performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n=24), tibial muscular dystrophy (n=6), and histopathologically normal group (n=9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs).

ResultsWe observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+ induced apoptotic pathway of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows altered expression and splicing usage in IBM muscles

InterpretationOur analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.

sentences: Fully-automated and ultra-fast cell-type identification using specific marker combinations from single-cell transcriptomic data Single-cell transcriptomics enables systematic charting of cellular composition of complex tissues. Identification of cell populations often relies on unsupervised clustering of cells based on the similarity of their scRNA-seq profiles, followed by manual annotation of cell clusters using established marker genes. However, manual selection of marker genes is a time-consuming process that may lead to sub-optimal annotation results as the selected markers must be informative of both the individual cell clusters and various cell types present in the complex samples. Here, we developed a computational platform, termed ScType, which enables data-driven, fully-automated and ultra-fast cell-type identification based solely on given scRNA-seq data, combined with our comprehensive cell marker database as background information. Using a compendium of six scRNA-seq datasets from various human and mouse tissues, we show how ScType provides an unbiased and accurate cell-type annotation by guaranteeing the specificity of positive and negative marker genes both across cell clusters and cell types. We also demonstrate how ScType enables distinguishing between healthy and malignant cell populations, based on single-cell calling of single-nucleotide variants, making it a versatile tool for exploration and use of single-cell transcriptomic data for anticancer applications. The widely-applicable method is deployed both as an interactive web-tool (https://sctype.app), and as an open-source R-package, connected with a comprehensive ScType database of specific markers.

sentences: Immune competence and spleen size scale with colony status in the naked mole-rat Naked mole-rats (NM-R; Heterocephalus glaber) live in multi-generational colonies with a social hierarchy, show low cancer incidence and long life-spans. Here we asked if such extreme physiology might have an immune component. The spleen is the largest lymphoid organ and plays an essential role in response to immunological insults and may participate in combating cancer and slowing ageing. We investigated the anatomy, molecular composition and function of the NM-R spleen using RNA-sequencing and histological analysis in healthy animals. We found that spleen size in healthy NM-Rs varies considerably. We therefore classified NM-Rs according to spleen size as NM-Rs with small spleens or enlarged spleens. Animals with enlarged spleens showed potentially better anti-microbial profiles and were much more likely to have a high rank within the colony. Splenomegaly was associated with infection in sick NM-Rs, but not in NM-Rs with enlarged spleens. In all healthy NM-Rs splenic erythropoiesis, megakaryopoiesis and myelopoiesis were increased, but B lymphopoiesis was reduced and splenic marginal zone showed markedly altered morphology when compared to other rodents. However, in NM-Rs lymphocytes were found in secondary sites such as lymph nodes, gut lymphoid nodules and thymus. Thus, the NM-R spleen is a major site of adult hematopoiesis under normal physiological conditions. Overall, the NM-R immune system seems to rely mainly on innate immune responses with a more restricted adaptive immune response. We propose that the anatomical plasticity of the spleen might be regulated by social interaction and gives immunological advantage to increase the life-span of higher ranked animals.

sentences: Effective connectivity reveals distinctive patterns in response to others' genuine affective experience of disgust as compared to pain Empathy is significantly influenced by the identification of others emotions. In a recent study, we have found increased activation in the anterior insular cortex (aIns) that could be attributed to affect sharing rather than perceptual saliency, when seeing another person genuinely experiencing pain as opposed to merely acting to be in pain. In that prior study, effective connectivity between aIns and the right supramarginal gyrus (rSMG) was revealed to track what another person really feels. In the present study, we used a similar paradigm to investigate the corresponding neural signatures in the domain of empathy for disgust - with participants seeing others genuinely sniffing unpleasant odors as compared to pretending to smell something disgusting. Consistent with the previous findings on pain, we found stronger activations in aIns associated with affect sharing for genuine disgust compared with pretended disgust. However, instead of rSMG we found engagement of the olfactory cortex. Using dynamic causal modeling (DCM), we estimated the neural dynamics of aIns and the olfactory cortex between the genuine and pretended conditions. This revealed an increased excitatory modulatory effect for genuine disgust compared to pretended disgust. For genuine disgust only, brain-to-behavior regression analyses highlighted a link between the observed modulatory effect and the perspective-taking empathic trait. Altogether, the current findings complement and expand our previous work, by showing that perceptual saliency alone does not explain responses in the insular cortex. Moreover, it reveals that different brain networks are implicated in a modality-specific way when sharing the affective experiences associated with pain vs. disgust.

sentences: An internal expectation guides Drosophila egg-laying decisions When presented with two egg-laying substrates, Drosophila lay most of their eggs on the option with higher relative value. How do flies make this relative-value decision? Might the strategy they use allow them to choose the best option even when they experience substrates with a more complex spatiotemporal profile than in canonical two-choice paradigms? We measured Drosophila egg-laying behavior in diverse substrate environments. In all cases, we found that flies dynamically increase or decrease their egg-laying rates as they explore substrates for a deposition site so as to target eggs to the best, recently visited option. Visiting the best option typically led to a strong reduction in egg laying on other substrates for several minutes, with this timescale varying across fly strains. Our data support a model in which flies compare the value of the current substrate with an internally constructed expectation on the value of available options to regulate the likelihood of laying an egg. We show that dopamine-neuron activity is critical for learning and/or expressing this expectation, similar to its role in certain tasks in vertebrates. Integrating sensory experiences over minutes to generate an internal sense of the quality of available options, i.e., forming an expectation, allows flies to use a dynamic reference point for judging the current substrate and might be a general way in which decisions are made, even beyond flies and egg laying.

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Quality control strategies for brain MRI segmentation and parcellation: practical approaches and recommendations - insights from The Maastricht Study BackgroundQuality control of brain segmentation is a fundamental step to ensure data quality. Manual quality control is the current gold standard, despite unfeasible in large neuroimaging samples. Several options for automated quality control have been proposed, providing potential time efficient and reproducible alternatives. However, those have never been compared side to side, which prevents to reach consensus in the appropriate QC strategy to use. This study aims to elucidate the changes manual editing of brain segmentations produce in morphological estimates, and to analyze and compare the effects of different quality control strategies in the reduction of the measurement error.

MethodsWe used structural MR images from 259 participants of The Maastricht Study. Morphological estimates were automatically extracted using FreeSurfer 6.0. A subsample of the brain segmentations with inaccuracies was manually edited, and morphological estimates were compared before and after editing. In parallel, 11 quality control strategies were applied to the full sample. Those included: a manual strategy, manual-QC, in which images were visually inspected and manually edited; five automated strategies where outliers were excluded based on the tools MRIQC and Qoala-T, and the metrics morphological global measures, Euler numbers and Contrast-to-Noise ratio; and five semi-automated strategies, were the outliers detected through the mentioned tools and metrics were not excluded, but visually inspected and manually edited. We used a regression of morphological brain measures against age as a test case to compare the changes in relative unexplained variance that each quality control strategy produces, using the reduction of relative unexplained variance as a measure of increase in quality.

ResultsManually editing brain surfaces produced changes particularly high in subcortical brain volumes and moderate in cortical surface area, thickness and hippocampal volumes. The exclusion of outliers based on Euler numbers yielded a larger reduction of relative unexplained variance for measurements of cortical area, subcortical volumes and hippocampal subfields, while manual editing of brain segmentations performed best for cortical thickness. MRIQC produced a lower, but consistent for all types of measures, reduction in relative unexplained variance. Unexpectedly, the exclusion of outliers based on global morphological measures produced an increase of relative unexplained variance, potentially removing more morphological information than noise from the sample.

ConclusionOverall, the automatic exclusion of outliers based on Euler numbers or MRIQC are reliable and time efficient quality control strategies that can be applied in large neuroimaging cohorts.

sentences: Legacy effect of constant and diurnally oscillating temperatures on soil respiration and microbial community structure 1.0Laboratory incubation studies evaluating the temperature sensitivity of soil respiration often use measurements of respiration taken at a constant incubation temperature from soil that has been pre-incubated at the same constant temperature. However, such constant temperature incubations do not represent the field situation where soils undergo diurnal temperature oscillations. We investigated the effects of constant and diurnally oscillating temperatures on soil respiration and soil microbial community composition. A grassland soil from the UK was either incubated at a constant temperature of 5 {degrees}C, 10 {degrees}C, or 15 {degrees}C, or diurnally oscillated between 5 {degrees}C and 15 {degrees}C. Soil CO2 flux was measured by temporarily moving incubated soils from each of the abovementioned treatments to 5 {degrees}C, 10 {degrees}C or 15 {degrees}C, such that soils incubated at each temperature had CO2 flux measured at every temperature. We hypothesised that, irrespective of measurement temperature, CO2 emitted from the 5 {degrees}C to 15 {degrees}C oscillating incubation would be most similar to the soil incubated at 10 {degrees}C. The results showed that both incubation and measurement temperatures influence soil respiration. Incubating soil at a temperature oscillating between 5 {degrees}C and 15 {degrees}C resulted in significantly greater CO2 flux than constant incubations at 10 {degrees}C or 5 {degrees}C, but was not significantly different to the 15 {degrees}C incubation. The greater CO2 flux from soils incubated at 15 {degrees}C, or oscillating between 5 {degrees}C and 15 {degrees}C, coincided with a depletion of dissolved organic carbon and a shift in the phospholipid fatty acid profile of the soil microbial community, consistent with the thermal adaptation of microbial communities to higher temperatures. However, diurnal temperature oscillation did not significantly alter Q10. Our results suggest that daily maximum temperatures are more important than daily minimum or daily average temperatures when considering the response of soil respiration to warming.

sentences: Defining the dynamics of naive CD4 and CD8 T cells across the mouse lifespan Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we employ multiple experimental systems to identify a unified model of naive CD4 and CD8 T cell population dynamics across the mouse lifespan. We infer that both subsets divide rarely and progressively increase their survival capacity with cell age. Strikingly, this simple model captures naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics from birth into old age.

sentences: CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production Cassava (Manihot esculenta Crantz) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces toxic cyanogenic compounds and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. Reducing the cyanogen content by conventional breeding is problematic due to the heterozygous nature of the crop; recombination will generally disrupt a clonally propagated cultivars suite of desirable traits. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444 and the West African, farmer-preferred cultivar TME 419. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. Our work demonstrates cassava genome editing for food safety, reduced processing requirements, and environmental benefits that could be readily extended to other farmer-preferred cultivars.

sentences: Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-Cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). Inhibition of E- and N-Cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.

sentences: The Campylobacter jejuni CadF and FlpA fibronectin binding proteins facilitate signaling via host cell focal adhesions to inhibit cell motility and impede wound repair Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblots, and super-resolution iPALM. We found that C. jejuni infection of epithelial cells results in an increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, as evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction of collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially re-establishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions.

sentences: Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 sixteen-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of E. coli resistant to fluoroquinolones, tetracycline, amoxicillin, and streptomycin, but not to cefalexin or cefotaxime. Whole genome sequencing of 30 fluoroquinolone-resistant (FQ-R), 22 cefotaxime-resistant (CTX-R) and seven dual FQ-R/CTX-R E. coli isolates showed a wide range of sequence types (STs), an approximately 50:50 split of CTX-M:AmpC-mediated CTX-R, and almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 x 50 km region with those isolated from human urinary tract and bloodstream infections (isolated in parallel in the same region) identified a clone of ST963 E. coli carrying chromosomal blaCMY.2 in two puppies and causing two urinary tract infections and one bloodstream infection. Furthermore, an ST744 FQ-R clone was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at sixteen weeks of age and that bacteria carried by dogs are shared with humans.

sentences: Prediction and validation of host cleavage targets of SARS-CoV-2 3C like protease How SARS-CoV-2 causes the observed range of clinical manifestations and disease severity remains poorly understood. SARS-CoV-2 encodes for two proteases (3CLPro and PLPro), vital for viral production, but also promiscuous with respect to host protein targets, likely contributing to the range of disease. Pharmacological inhibition of the 3C-like3 protease has revealed remarkable reduction in hospitalization and death in phase 2/3 clinical studies. However, the mechanisms responsible for the pathology mediated by those proteases are still unclear. In this study, we develop a bioinformatic algorithm, leveraging experimental data from SARS-CoV, to predict host cleavage targets of the SARS-CoV-2 3C-like protease, or 3CLPro. We capture targets of the 3CL protease described previously for SARS-CoV, and we identify hundreds of new putative targets. We experimentally validate a number of these predicted targets, including the giant sarcomeric protein Obscurin, and show that expression of 3CL protease alone recapitulates the sarcomeric disorganization seen by SARS-CoV-2 infection of hiPSC-derived cardiomyocytes. Our data provide a resource to identify putative host cleavage targets of 3CL protease that contribute to mechanisms and heterogeneity of disease in COVID-19 and future coronavirus outbreaks.

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On co-activation pattern analysis and non- stationarity of resting brain activity The non-stationarity of resting-state brain activity has received increasing attention in recent years. Functional connectivity (FC) analysis with short sliding windows and coactivation pattern (CAP) analysis are two widely used methods for assessing the dynamic characteristics of brain activity observed with functional magnetic resonance imaging (fMRI). However, the statistical nature of the dynamics captured by these techniques needs to be verified. In this study, we found that the results of CAP analysis were similar for real fMRI data and simulated stationary data with matching covariance structures and spectral contents. We also found that, for both the real and simulated data, CAPs were clustered into spatially heterogeneous modules. Moreover, for each of the modules in the real data, a spatially similar module was found in the simulated data. The present results suggest that care needs to be taken when interpreting observations drawn from CAP analysis as it does not necessarily reflect non-stationarity or a mixture of states in resting brain activity.

HighlightsO_LICAP was conducted for real fMRI data and a stationary null model.
C_LIO_LIThe results of CAP analysis were similar for the real and simulated data.
C_LIO_LISimilar results for real and simulated data in both ROI- and voxel-based analyses.
C_LIO_LIResults of CAP analysis may not reflect non-stationarity of resting brain activity.
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High-resolution epigenetic profiling identifies novel regulators of COPD in human lung fibroblasts Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering the environmental cause of COPD (e.g., cigarette smoke) and disease phenotypes, including stem-cell senescence and impaired differentiation, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) isolated from distal parenchyma of ex-smoker controls and COPD patients, with both mild and severe disease. The epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD patients, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes, including epigenetic factors. Using phenotypic screens, we verified the regulator capacity of multiple candidates and linked them to repair processes in the human lung.

Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across stages of COPD. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic respiratory diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

sentences: Somatic HCN channels augment and speed up GABAergic basket cell input-output function in human neocortex Neurons in the mammalian brain exhibit evolution-driven species-specific differences in their functional properties. Therefore, understanding the human brain requires unraveling the human neuron "uniqueness" and how it contributes to the operation of specific neuronal circuits. We show here that a highly abundant type of inhibitory neurons in the neocortex, GABAergic parvalbumin-expressing basket cell (pv+BC), exhibits in the human brain a specific somatic leak current mechanism, which is absent in their rodent neuronal counterparts. Human pv+BC soma shows electric leak conductance mediated by hyperpolarization-activated cyclic nucleotide-gated channels. This leak conductance has depolarizing effects on the resting membrane potential and it accelerates the rise of synaptic potentials in the cell soma. The leak facilitates the human pv+BC input-to-output fidelity and shortens the action potential generation to excitatory inputs. This mechanism constitutes an adaptation that enhances signal transmission fidelity and speed in the common inhibitory circuit in the human but not in the rodent neocortex.

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Characterization of plasma circulating small extracellular vesicles in patients with metastatic solid tumors and newly diagnosed brain metastasis. PurposeNearly 40% of the advanced cancer patients will present brain metastases during the course of their disease, with a 2-year life expectancy of less than 10%. Immune system impairment, including the modulation of both STAT3 and PD-L1, is one of the hallmarks of brain metastases. Liquid biopsy could offer several advantages in brain metastases management, such as the possibility of non-invasive dynamic monitoring. Extracellular vesicles (EVs) have been recently proposed as novel biomarkers especially useful in liquid biopsy due to their secretion in biofluids and their role in cell communication during tumor progression.

Materials and MethodsThe main aim of this work was to characterize the size and protein cargo of plasma circulating EVs in patients with solid tumors and their correlation with newly diagnosed brain metastases, in addition to their association with other relevant clinical variables.

ResultsWe analyzed circulating EVs in the plasma of 123 patients: 42 patients with brain metastases, 50 without brain metastases and 31 healthy controls. Patients with newly diagnosed brain metastases had a lower number of circulating EVs in the plasma and a higher protein concentration in small EVs (sEVs) compared to patients without brain metastases and healthy controls. Interestingly, melanoma patients with brain metastases presented decreased STAT3 activation and increased PD-L1 levels in circulating sEVs compared to patients without central nervous system metastases.

ConclusionsDecreased STAT3 activation and increased PD-L1 in plasma circulating sEVs identify melanoma patients with brain metastasis.

Statement of translational relevanceBrain metastases are critical for outcomes and quality of life in almost 50% of oncological patients, generally associated with a poor short-term prognosis. Early or preventive diagnosis of this complication represents an unmet need. There is a necessity of discovering new biomarkers that could aid to predict disease outcome.

In this study, we analyzed plasma circulating extracellular vesicles (EVs) from a cohort of 92 patients with different solid tumors (lung, breast, kidney cancer and melanoma) and found that newly diagnosed patients with brain metastases presented lower number of circulating particles and a higher protein concentration in small extracellular vesicles (sEVs) compared to patients without brain metastases and healthy controls. Out of all groups analyzed, melanoma patients with brain metastases presented decreased STAT3 activation and increased PD-L1 levels in circulating sEVs compared to patients without central nervous system metastases.

The data presented in this work suggest that circulating sEVs may represent the immunosuppressive status of newly diagnosed brain metastases characterized by the reduced phospho-STAT3 (pSTAT3) and increased PD-L1, although the origin of these molecules found in circulating sEVs remains to be uncovered.

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Seizures initiate in zones of relative hyperexcitation in a zebrafish epilepsy model Seizures are thought to arise from an imbalance of excitatory and inhibitory neuronal activity. While most classical studies suggest excessive excitatory neural activity plays a generative role, some recent findings challenge this view and instead argue that excessive activity in inhibitory neurons initiates seizures. We investigated this question of imbalance in a zebrafish seizure model with multi-regional two-photon imaging of excitatory and inhibitory neuronal activity using a nuclear-localized calcium sensor. We found that seizures consistently initiated in circumscribed zones of the midbrain before propagating to other brain regions. Excitatory neurons were both more prevalent and more likely to be recruited than inhibitory neurons in initiation as compared with propagation zones. These findings support a mechanistic picture whereby seizures initiate in a region of hyper-excitation, then propagate more broadly once inhibitory restraint in the surround is overcome.

TeaserWe uncover the roles of excitation and inhibition during seizures, thus opening a path to more targeted therapy of epilepsy.

sentences: CRISPR-Cas9 bends and twists DNA to read its sequence In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 "reads" snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

sentences: A two-year study on the phenology, host and habitat associations, and pathogens of Haemaphysalis longicornis in Virginia, U.S.A. Understanding the abiotic and biotic variables affecting tick populations is essential for studying the biology and health risks associated with vector species. We conducted a study on the phenology of exotic Haemaphysalis longicornis (Asian longhorned tick) at a site in Albemarle County, Virginia. We assessed the importance of available wildlife hosts, habitats, and microclimate variables such as temperature, relative humidity, and wind speed on this exotic ticks presence and abundance. In addition, we determined the prevalence of selected tick-borne pathogens potentially transmitted by H. longicornis. We determined that the seasonal activity of H. longicornis was slightly different from previous studies in the northeastern United States. We observed nymphal ticks persist year-round but were most active in the spring, followed by a peak in adult activity in the summer and larval activity in the fall seasons. We also observed a lower probability of detecting H. longicornis in field habitats and the summer months. In addition, we detected H. longicornis on several wildlife hosts, including coyote (Canis latrans), eastern cottontail (Sylvilagus floridanus), raccoon (Procyon lotor), Virginia opossum (Didelphis virginiana), white-tailed deer (Odocoileus virginianus), woodchuck (Marmota monax), and a Peromyscus sp. This is the first detection of this tick on a rodent host important to the epidemiology of tick-borne pathogens of humans and animals. Finally, we continued to detect the exotic piroplasm parasite, Theileria orientalis Ikeda, in H. longicornis as well as other pathogens, including Rickettsia felis, Anaplasma phagocytophilum (AP-1), and a Hepatozoon sp. previously characterized in Amblyomma americanum. These represent some of the first detections of arthropod-borne pathogens native to the United States in host-seeking H. longicornis. These data increase our understanding of H. longicornis biology in the United States and provide valuable information into the future health risks associated with this tick and pathogens.

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Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model 1 -Digital light microscopy provides powerful tools for quantitatively probing the real-time dynamics of subcellular structures. While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). Keeping notes on microscopy experiments and quality control procedures ought to be straightforward, as the microscope is a machine whose components are defined and the performance measurable. Nevertheless, to this date, no universally adopted community-driven specifications exist that delineate the required information about the microscope hardware and acquisition settings (i.e., microscopy "data provenance" metadata) and the minimally accepted calibration metrics (i.e., microscopy quality control metadata) that should be automatically recorded by both commercial microscope manufacturers and customized microscope developers. In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To add to the confusion, microscopy experiments vary greatly in aim and complexity, ranging from purely descriptive work to complex, quantitative and even sub-resolution studies that require more detailed reporting and quality control measures.

To solve this problem, the 4D Nucleome Initiative (4DN) (1, 2) Imaging Standards Working Group (IWG), working in conjunction with the BioImaging North America (BINA) Quality Control and Data Management Working Group (QC-DM-WG) (3), here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a revision of the Core of the Open Microscopy Environment (OME) Data Model, which forms the basis for the widely adopted Bio-Formats library (4-6), accompanied by a suite of three extensions, each with three tiers, allowing the classification of imaging experiments into levels of increasing imaging and analytical complexity (7, 8). Hence these specifications not only provide an OME-based comprehensive set of metadata elements that should be recorded, but they also specify which subset of the full list should be recorded for a given experimental tier. In order to evaluate the extent of community interest, an extensive outreach effort was conducted to present the proposed metadata specifications to members of several core-facilities and international bioimaging initiatives including the European Light Microscopy Initiative (ELMI), Global BioImaging (GBI), and European Molecular Biology Laboratory (EMBL) - European Bioinformatics Institute (EBI). Consequently, close ties were established between our endeavour and the undertakings of the recently established QUAlity Assessment and REProducibility for Instruments and Images in Light Microscopy global community initiative (9). As a result this flexible 4DN-BINA-OME (NBO namespace) framework (7, 8) represents a turning point towards achieving community-driven Microscopy Metadata standards that will increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of different datasets, experimental setups, and assays, and it demonstrates the method for future extensions. Such universally accepted microscopy standards would serve a similar purpose as the Encode guidelines successfully adopted by the genomic community (10, 11). The intention of this proposal is therefore to encourage participation, critiques and contributions from the entire imaging community and all stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders.

sentences: Sex differences in allometry for phenotypic traits indicate that females are not scaled males Sex differences in the lifetime risk and expression of disease are well-known. Paradoxically, preclinical research targeted at improving treatment, increasing health span and reducing the financial burden of health care, has mostly been conducted on male animals and cells. Females are assumed to be the same or scaled versions of males, yet sex differences in the allometric relationship between phenotypic traits and body size, needed to evaluate the validity of this assumption, have not been established. We quantify allometry for 297 phenotypic traits in male and female mice, recorded in >2.1 million measurements from the International Mouse Phenotyping Consortium. We find sex differences in allometric parameters (slope, intercept, residual SD) are common. Thus, the allometric relationship varies between the sexes: females are not scaled males. Our results support a complex, trait-specific patterning of sex differences in phenotypic traits, promoting case-specific approaches to therapeutic intervention and drug dosage scaled by body weight.

sentences: Transgenic resources for functional studies on the hsrω gene in Drosophila The hsr{omega} gene of Drosophila melanogaster, an early discovered non-coding developmentally active and stress-inducible gene, has pleiotropic actions through its multiple nuclear and cytoplasmic non-coding transcripts. With a view to understand this diverse functions and mechanisms of actions of this gene, we generated a series of transgenic lines for over-expression or RNAi of different transcripts and CRISPR-Cas9 mediated deletion alleles. These are described here.

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Metabolic modeling of the International Space Station microbiome reveals key microbial interactions BackgroundRecent studies have provided insights into the persistence and succession of microbes aboard the International Space Station (ISS), notably the dominance of Klebsiella pneumoniae. However, the interactions between the various microbes aboard the ISS, and how it shapes the microbiome remain to be clearly understood. In this study, we apply a computational approach to predict possible metabolic interactions in the ISS microbiome and shed further light on its organization.

ResultsThrough a combination of a systems-based graph-theoretical approach, and a constraint-based community metabolic modelling approach, we demonstrated several key interactions in the ISS microbiome. These complementary approaches provided insights into the metabolic interactions and dependencies present amongst various microbes in a community, highlighting key interactions and keystone species. Our results showed that the presence of K. pneumoniae is beneficial to many other microorganisms it coexists with, notably those from the Pantoea genus. Species belonging to the Enterobacteriaceae family were often found to be the most beneficial for the survival of other microorganisms in the ISS microbiome. However, K. pneumoniae was found to exhibit parasitic and amensalistic interactions with Aspergillus and Penicillium species, respectively. To prove this metabolic prediction, K. pneumoniae and Aspergillus fumigatus were co-cultured under normal and simulated microgravity, where K. pneumoniae cells showed parasitic characteristics to the fungus. The electron micrography revealed that the presence of K. pneumoniae compromised the morphology of fungal conidia and its biofilm biofilm-forming structures.

ConclusionsOur study underscores the importance of K. pneumoniae in the ISS, and its potential contribution to the survival (mutualism) and eradication (parasitism) of other microbes, including potential pathogens. This integrated modelling approach, combined with experiments, demonstrates immense potential for understanding the organization of other such microbiomes, unravelling key organisms and their interdependencies.

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Heterotrimeric G protein α subunit (GPA1) regulates the response to low-nitrogen stress in Arabidopsis by interacting with AtNRT1.4 and AtATG8a Efficient nitrogen absorption and utilization are important factors for higher plants to increase yield and reduce eutrophication (caused by excessive use of nitrogen fertilizers). Heterotrimeric G proteins, including three subunits of , {beta}, and {gamma}, participate in the pathway regulating nitrogen absorption and utilization in plants. However, the regulatory mechanism remains largely obscured. In this study, our results revealed that the G protein  subunit (AtGPA1) mutant gpa1-4 was tolerant to low-nitrogen stress in Arabidopsis. AtGPA1 was shown to directly interact with a nitrate transporter (AtNRT1.4) and a key autophagy-related protein (AtATG8a) on the plasma membrane using the yeast hybrid system and pull-down assay (in vitro) and BiFC assay (in vivo). GUS staining and subcellular localization showed that AtGPA1 and AtNRT1.4 were co-expressed in roots and leaf veins and on the plasma membrane. Under low-nitrate conditions, the single mutant gpa1-4 and NRT1.4RNAi plants (AtNRT1.4RNA interference plants) and the double mutant NRT1.4RNAi/gpa1-4 plants (AtNRT1.4RNA interference plants on a gpa1-4 background) were healthier than the wild type plants. Moreover, the phenotype of the double mutant NRT1.4RNAi/gpa1-4 plants was closer to that of the NRT1.4RNAi plants compared to that of the gpa1-4 mutants. The results of the nitrate efflux rate assay in roots were consistent with the phenotypic changes under low-nitrogen conditions. These results indicated that AtGPA1 is an upstream factor that regulated the response to low-nitrogen stress through interaction with AtNRT1.4. In addition, we found that transgenic plants overexpressing AtATG8a were more tolerant to low-nitrogen stress, and their phenotype was similar to that of gpa1-4 mutants and double mutant ATG8aOX/gpa1-4 plants (AtATG8a overexpressing plants on a gpa1-4 mutant background). Further, autophagosome observations were consistent with the phenotypes in mutant plants, indicating that AtGPA1 regulated the response to low-nitrogen stress in Arabidopsis plants by affecting the autophagosome assembly. Our findings may provide a new model for improving nitrogen-use efficiency through genetical modification to boost crop yields.

One sentence summaryAtGPA1 negative regulates low nitrogen stress response by interaction with a nitrate transporter, AtNRT1.4 and an autophagy-related protein, AtATG8a in Arabidopsis.

sentences: Phenolic profiles and antioxidant activities of Saskatchewan (Canada) bred haskap (Lonicera caerulea) berries Phenolic extracts from five Saskatoon, Saskatchewan, bred and grown haskap berry varieties (Aurora, Blizzard, Honey Bee, Indigo Gem, and Tundra) were characterized via liquid chromatography with photodiode array detection (HPLC-PDA) and mass spectrometry (HPLC-MS/MS). Tundra had the highest phenolic content (727.0 mg/100 g FW) while Indigo Gem had the highest anthocyanin content (447.8 mg/100 g FW). HPLC-MS/MS identified two previously unreported anthocyanins (Tundra variety): delphinidin-sambubioside and a peonidin-pentoside. Fruit extracts were fractionated to produce an anthocyanin rich (40% ethanol) and flavanol/flavonol rich (100% ethanol) fraction. This process affords the ability to isolate/concentrate specific subclasses for nutraceutical applications. High in vitro radical scavenging was observed for all haskap phenolic extracts. An extract from the Tundra variety delayed borage oil oxidation more effectively than commercial antioxidants (BHT and Rosamox). These results show the high phenolic content of these haskaps along with their capacity for radical scavenging/delaying lipid oxidation, indicating potential commercial value.

sentences: Insight into the ecology of vaginal bacteria through integrative analyses of metagenomic and metatranscriptomic data Vaginal bacterial communities dominated by Lactobacillus species are associated with a reduced risk to various adverse health outcomes. However, somewhat unexpectedly many healthy women have microbiota that are not dominated by lactobacilli. To determine the factors that drive vaginal community composition we characterized the genetic composition and transcriptional activities of vaginal microbiota in healthy women. We demonstrated that the abundance of a species is not always indicative of its transcriptional activity and that impending changes in community composition can be predicted from metatranscriptomic data. Functional comparisons highlight differences in the metabolic activities of these communities, notably in their degradation of host-produced mucin but not glycogen. Degradation of mucin by communities not dominated by Lactobacillus may play a role in their association with adverse health outcomes. Finally, we show that the transcriptional activities of L. crispatus, L. iners, and G. vaginalis vary with the taxonomic composition of the communities in which they reside. Notably, L. iners and G. vaginalis both demonstrated lower expression of their cholesterol-dependent cytolysins when co-resident with Lactobacillus spp. and higher expression when co-resident with other facultative and obligate anaerobes. The pathogenic potential of these species may depend on the communities in which they reside and thus could be modulated by interventional strategies. Our results provide insight to the functional ecology of vaginal microbiota and reveal strategies for management of these ecosystems.

sentences: A role for the immune system-released activating agent (ISRAA) in the ontogenetic development of brain astrocytes The Immune System-Released Activating Agent (ISRAA) was discovered as a novel molecule that functions as a mediator between the nervous and immune systems in response to a nervous stimulus following an immune challenge. This research investigated the role of ISRAA) in promoting the ontogeny of the mouse brain astrocytes. Astrocyte cultures were prepared from two-month-old BALB/c mice. Recombinant ISRAA protein was used to stimulate astrocyte cultures. Immunohistochemistry and ELISA were utilized to measure ISRAA and IFN-{gamma} levels, IFN-{gamma}R expression and STAT1 nuclear translocation. MTT-assay was used to evaluate cellular survival and proliferation. To assess astrocyte cell lysates and tyrosine-phosphorylated proteins, SDS-PAGE and western blot were used. ISRAA was highly expressed in mouse embryonic astrocytes, depending on cell age. Astrocytes aged seven days (E7) showed increased proliferation and diminished differentiation, while 21-day-old (E21) astrocytes depicted reversed effects. ISRAA stimulated the tyrosine phosphorylation of numerous cellular proteins and the nuclear translocation of STAT1. IFN-{gamma} was involved in the ISRAA action as ISRAA induced IFN-{gamma} in both age groups, but only E21 astrocytes expressed IFN-{gamma}R. The results suggest that ISRAA is involved in mouse brain development through the cytokine network involving IFN-{gamma}.

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pH profiling of mitochondrial sub-compartments reveals that IF1 is essential to prevent ATP hydrolysis under OXPHOS conditions The classical view of oxidative phosphorylation is that a proton motive force PMF generated by the respiratory chain complexes fuels ATP synthesis. Under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected the two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions in detail. pH profiles of mitochondrial sub-compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH-sensor directly at F1 and FO of ATP synthase, complex IV and in the matrix. Our results clearly show that ATP synthase activity is substantially controlling the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible {Delta}pH.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/430746v2_ufig1.gif" ALT="Figure 1">
View larger version (50K):
org.highwire.dtl.DTLVardef@1a975eforg.highwire.dtl.DTLVardef@7ddb49org.highwire.dtl.DTLVardef@c7a128org.highwire.dtl.DTLVardef@2559e8_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIThe {Delta}pH along and across the inner mitochondrial membrane is not homogeneous
C_LIO_LIThe proton motive force at cristae tips is controlled by F1 FO ATP synthase
C_LIO_LIUnder OXPHOS conditions, the pH difference between FO and F1 of active ATP synthase is almost negligible (1.2 proton vs. 1 proton equivalent)
C_LIO_LIIF1 is required to prevent the onset of ATP hydrolysis under OXPHOS conditions
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A potential prokaryotic and microsporidian pathobiome that may cause shrimp white feces syndrome (WFS) White feces syndrome (WFS) in shrimp cultivation ponds is characterized by the occurrence of shrimp with abnormal, white intestines (midguts) combined with large floating mats of white, shrimp fecal strings. The etiology for WFS is complex, similar to diarrhea in humans. EHP-WFS is a type of WFS characterized by massive quantities of spores from the microsporidian parasite Enterocytozoon hepatopenaei (EHP) together with mixed, unidentified bacteria in the shrimp hepatopancreas, midgut and fecal strings. However, WFS does not always develop in shrimp with severe EHP infections in controlled laboratory challenges. Further, in EHP-WFS outbreak ponds, some shrimp show white midguts (WG) while others in the same pond show grossly normal midguts (NG). We hypothesized that comparison of the microbial flora between WG and NG from the same EHP-WFS pond would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this hypothesis, we selected a pond exhibiting a severe EHP-WFS outbreak in cultivated Penaeus vannamei and used a combination of microscopic and microbial profiling analyses to compare WG and NG samples. By histology, EHP plasmodia and spores were confirmed in the hepatopancreas (HP) and midgut of WG and NG shrimp, but pathological severity and spore quantity was higher in the WG shrimp. In addition, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium quantity in the HP of WG shrimp was significantly higher (P = 1.08e-5) than in NG shrimp (4,506 vs. 3 copies /100 ng DNA, respectively). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as a potential eukaryote-prokaryote pathobiome that causes EHP-WFS in P. vannamei.

HighlightsO_LIWhite feces syndrome (WFS) shrimp often harbor the microsporidian Enterocytozoon hepatopenaei (EHP)
C_LIO_LIThe hepatopancreas (HP) and midgut of EHP-WFS shrimp had more EHP copies and spores than EHP-non.-WFS shrimp
C_LIO_LIVibrio spp., Propionigenium sp. and EHP dominated in HP microbiomes of EHP-WFS shrimp
C_LIO_LIPropionigenium copy numbers were uniquely high in the HP of EHP-WFS shrimp
C_LIO_LIEHP-WFS shrimp also showed intestinal microbiomes of reduced diversity but more heterogeneity
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The Legionella Lqs-LvbR regulatory network controls temperature-dependent growth onset and bacterial cell density Legionella species are facultative intracellular pathogens, which can cause a life-threatening pneumonia termed Legionnaires disease. Legionella pneumophila employs the Legionella quorum sensing (Lqs)-LvbR network to regulate virulence and motility, but its role for growth in media is ill-defined. Compared to the parental L. pneumophila strain JR32, a {Delta}lqsR mutant showed a reduced lag phase at 30{degrees}C and reached a higher cell density at 45{degrees}C, while the {Delta}lqsA, {Delta}lqsS and {Delta}lqsT mutants exhibited a longer lag phase and reached only a lower cell density. A {Delta}lvbR mutant resumed growth like the parental strain at 30{degrees}C, but exhibited a substantially reduced cell density at 45{degrees}C. Thus, LvbR is an important cell density regulator at elevated temperatures. A quantitative analysis of temperature-dependent growth characteristics of environmental and clinical strains revealed that L. pneumophila strains grew in AYE medium after distinct lag phases with similar rates at 30{degrees}C, reached different cell densities at the optimal growth temperature of 40{degrees}C, and no longer grew at 50{degrees}C. Legionella longbeachae reached a rather low cell density at 40{degrees}C and did not grow at and beyond 45{degrees}C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and the PlqsR, PlqsA, PlqsS and PlvbR promoters from strain JR32 were active in these strains. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32, and possibly also of environmental and clinical L. pneumophila isolates.

ImportanceEnvironmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires disease, the incidence of which is worldwide on the rise. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a similar mechanism as in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical or biological means will reduce the incidence of Legionnaires disease. Here we report that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is not only determined by the temperature and nutrient availability, but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.

sentences: Beyond Turing: Far-from-equilibrium patterns and mechano-chemical feedback Turing patterns are commonly understood as specific instabilities of a spatially homogeneous steady state, resulting from activator-inhibitor interaction destabilised by diffusion. We argue that this view is restrictive and its agreement with biological observations is problematic. We present two alternative to the  classical Turing analysis of patterns. First, we employ the abstract framework of evolution equations to enable the study of far-from-equilibrium patterns. Second, we introduce a mechano-chemical model, with the surface on which the pattern forms being dynamic and playing an active role in the pattern formation, effectively replacing the inhibitor. We highlight the advantages of these two alternatives vis-a-vis the  classical Turing analysis, and give an overview of recent results and future challenges for both approaches.

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Random peptides rich in small and disorder-promoting amino acids are less likely to be harmful Proteins are the workhorses of the cell, yet they carry great potential for harm via misfolding and aggregation. Despite the dangers, proteins are sometimes born de novo from non-coding DNA. Proteins are more likely to be born from non-coding regions that produce peptides that do little to no harm when translated than from regions that produce harmful peptides. To investigate which newborn proteins are most likely to "first, do no harm", we estimate fitnesses from an experiment that competed Escherichia coli lineages that each expressed a unique random peptide. A variety of peptide metrics significantly predict lineage fitness, but this predictive power stems from simple amino acid frequencies rather than the ordering of amino acids. Amino acids that are smaller and that promote intrinsic structural disorder have more benign fitness effects. We validate that the amino acids that indicate benign effects in random peptides expressed in E. coli also do so in an independent dataset of random N-terminal tags in which it is possible to control for expression level. The same amino acids are also enriched in young animal proteins.

Significance statementProteins are sometimes born de novo. In an experiment to reproduce this process in Escherichia coli, we were able to predict 15% of the variation in random peptide fitness effects from their amino acid frequencies. In contrast, which order the amino acids are in seems to make no difference, adding no predictive power on top of simple amino acid frequencies. Amino acids that are smaller and promote intrinsic structural disorder have more benign fitness effects.

sentences: Comparable human spatial memory distortions in physical, desktop virtual and immersive virtual environments Boundaries define regions of space and are integral to episodic memories. The impact of boundaries on spatial memory and neural representations of space has been extensively studied in freely-moving rodents. But less is known in humans and many prior studies have employed desktop virtual reality (VR) which lacks the body-based self-motion cues of the physical world, diminishing the potentially strong input from path integration to spatial memory. We replicated a desktop-VR study testing the impact of boundaries on spatial memory (Hartley et al., 2004) in a physical room (2.4m x 2.4m, 2m tall) by having participants (N = 27) learn the location of a circular stool and then after a short delay replace it where they thought they had found it. During the delay, the wall boundaries were either expanded or contracted. We compared performance to groups of participants undergoing the same procedure in a laser-scanned replica in both desktop VR (N = 44) and freely-walking head mounted display (HMD) VR (N = 39) environments. Performance was measured as goodness of fit between the spatial distributions of group responses and seven modelled distributions that prioritised different metrics based on boundary geometry or walking paths to estimate the stool location. The best fitting model was a weighted linear combination of all the geometric spatial models, but an individual model derived from place cell firing in Hartley et al. 2004 also fit well. High levels of disorientation in all three environments prevented detailed analysis on the contribution of path integration. We found identical model fits across the three environments, though desktop VR and HMD-VR appeared more consistent in spatial distributions of group responses than the physical environment and displayed known variations in virtual depth perception. Thus, while human spatial representation appears differentially influenced by environmental boundaries, the influence is similar across virtual and physical environments. Despite differences in body-based cue availability, desktop and HMD-VR allow a good and interchangeable approximation for examining human spatial memory in small-scale physical environments.

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An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity.

One sentence summaryA human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

sentences: Experience-Driven Rate Modulation is Reinstated During Hippocampal Replay Replay, the sequential reactivation within a neuronal ensemble, is a central hippocampal mechanism postulated to drive memory processing. While both rate and place representations are used by hippocampal place cells to encode behavioral episodes, replay has been largely defined by only the latter - based on the fidelity of sequential activity across neighboring place fields. Here we show that dorsal CA1 place cells in rats can modulate their firing rate between replay events of two different contexts. This experience-dependent phenomenon mirrors the same pattern of rate modulation observed during behavior and can be used independently from place information within replay sequences to discriminate between contexts. Our results reveal the existence of two complementary neural representations available for memory processes.

sentences: DeepMicrobeFinder sorts metagenomes into prokaryotes, eukaryotes and viruses, with marine applications Sequence classification is valuable for reducing the complexity of metagenomes and providing a fundamental understanding of the composition of metagenomic samples. Binary metagenomic classifiers offer an insufficient solution because metagenomes of most natural environments are typically derived from multiple sequence sources including prokaryotes, eukaryotes and the viruses of both. Here we introduce a deep-learning based (not reference-based) sequence classifier, DeepMicrobeFinder, that classifies metagenomic contigs into five sequence classes, e.g., viruses infecting prokaryotic or eukaryotic hosts, eukaryotic or prokaryotic chromosomes, and prokaryotic plasmids. At different sequence lengths, DeepMicrobeFinder achieved area under the receiver operating characteristic curve (AUC) scores >0.9 for most sequence classes, the exception being distinguishing prokaryotic chromosomes from plasmids. By benchmarking on 20 test datasets with variable sequence class composition, we showed that DeepMicrobeFinder obtained average accuracy scores of 0.94, 0.87, and 0.92 for eukaryotic, plasmid and viral contig classification respectively, which were significantly higher than the other state-of-the-art individual predictors. Using a 1-300 m daily time-series metagenomic dataset sampled from coastal Southern California as a case study, we showed that metagenomic read proportions recruited by eukaryotic contigs could be doubled with DeepMicrobeFinders classification compared to the counterparts of other reference-based classifiers. In addition, a positive correlation could be observed between eukaryotic read proportions and potential prokaryotic community growth rates, suggesting an enrichment of fast-growing copiotrophs with increased eukaryotic particles. With its inclusive modeling and unprecedented performance, we expect DeepMicrobeFinder will be a useful addition to the toolbox of microbial ecologists, and will promote metagenomic studies of under-appreciated sequence types.

sentences: Striatal role in everyday life and laboratory developed habits The dorsolateral striatum plays a major role in stimulus-response habits that are learned in the experimental laboratory. Here, we use meta-analytic procedures to identify the neural circuits activated during the execution of stimulus-response behaviours acquired in everyday-life and those activated by habits acquired in the laboratory. In the case of everyday-life habits we dissociated motor and associative components. We found that motor-dominant stimulus-response associations developed outside the laboratory engaged posterior dorsal putamen, supplementary motor area (SMA) and cerebellum. Associative components were also represented in the posterior putamen. Meanwhile, newly learned habits relied more on the anterior putamen with activation expanding to caudate and nucleus accumbens. Importantly, common neural representations for both naturalistic and laboratory based habits were found in posterior left and anterior right putamen. Our findings suggest a common striatal substrate for behaviours with significant stimulus-response associations, independently of whether they were acquired in the laboratory or everyday-life.

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Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in [~]1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.

One sentence summaryCo-targeting two genes by co-electroporation of CRISPR/Cas9 RNPs and ssODN repair templates allows concomitant genome editing to create a selectable marker gene and to introduce precise modifications in another gene of interest.

sentences: Mapping cherry blossoms from geotagged street-level photos Floral phenology is useful information as an indicator on climate change and ecosystem services, however its observation is not straightforward over space and time. Satellite remote sensing and official and volunteer-based in-situ observations have been conducting, but the long-term and accurate data collection is challenging due to the insufficient quality and quantity of observations and the lack of financial and human resources to sustain. Here, we demonstrate a flower detection model from street-level photos, which can be the core function of a semi-automatic observation system to tackle those issues above. We detected cherry blossoms by this model from geotagged images with the observation date, obtained from Mapillary, which is one of social sensing data sources, and mapped dates of flowering in a study site, Aizuwakamatsu, Japan in April 2018. This approach enables us to collect floral phenology information semi-automatically as a data-driven approach. It is expected to collect a large number of observations with a certain level of quality by avoiding human-induced biases for the observations.

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Surface engineering enhances the therapeutic potential of extracellular vesicles following acute myocardial infarction Structured AbstractO_ST_ABSObjectivesC_ST_ABSThe objective of the study was to assess the therapeutic efficacy of targeting remote zone cardiomyocytes with cardiosphere-derived cell (CDC) extracellular vesicles (EVs) in acute myocardial infarction.

BackgroundCardiomyocyte (CM) cell death plays a significant role in left ventricular (LV) remodeling and cardiac dysfunction following myocardial infarction (MI). While EVs secreted by CDCs have shown efficacy in promoting cardiac repair in preclinical models of MI, their translational potential is limited by their biodistribution. We hypothesized that targeting therapeutic EVs to CMs would result in further reduction of cardiomyocyte (CM) cell death in vivo and improvement in cardiac function post-MI.

MethodsCDC-derived EVs were engineered to express a CM-specific binding peptide (CMP) on their surface and characterized for size, morphology, and protein expression. Mice with acute MI underwent delivery of human CDC EVs, CMP-EVs and placebo in a double-blind study. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of EV biodistribution and histological endpoints.

ResultsCMP-EVs demonstrated superior cardiac targeting and retention when compared with control EVs 24 hours post MI. While intramyocardial administered CDC-EVs improved LVEF compared with placebo at 4 weeks, mice treated with CMP-EVs demonstrated a significant improvement in LVEF compared with non-targeted EVs. Likely accounting for their augmented therapeutic efficacy, CMP-EVs demonstrated enhanced reduction of remote zone cardiomyocyte apoptosis.

ConclusionsTargeting CMP-EVs to CMs post-MI improved cardiac function compared with unmodified EVs demonstrating a strategy to further optimize therapeutic EV delivery to increase efficacy and decease off-target effects.

Condensed AbstractExtracellular vesicles (EVs) offer several potential advantages over small molecule therapeutics for cardiovascular disease (CVD). However, their potential is limited by their biodistribution and lack of specificity. We engineered cardiosphere-derived cells (CDCs) to express Lamp2b fused to a cardiomyocyte specific peptide (CMP), generating EVs with increased cardiomyocyte uptake and cardiac retention. CMP-EVs enhanced cardiac function following acute MI and further reduced remote zone apoptosis when compared with unmodified CDC-EVs. This work highlights a role for targeting therapeutic EVs to cardiomyocytes following injury and serves as a proof-of-concept study for the utility of EV surface engineering in the treatment of CVD.

sentences: RAMPs regulate signalling bias and internalisation of the GIPR Gastric inhibitory polypeptide (GIP) receptor is a class B1 GPCR, that responds to GIP and physiologically potentiates glucose-stimulated insulin secretion. Like most class B1 GPCRs, GIPR has been shown to interact with RAMPs, yet the effects of RAMPs on its signalling and trafficking remain poorly understood. We demonstrate that RAMPs modulate G protein activation and GIPR internalisation profiles. RAMP3 reduced GIPR Gs activation and cAMP production but retained GIPR at the cell surface, and this was associated with prolonged ERK1/2 phosphorylation and {beta}-arrestin association. By contrast, RAMP1/2 reduced Gq/11/15 activation of the GIPR. Through knockout mice studies, we show that RAMP1 is important to the normal physiological functioning of GIPR to regulate blood glucose levels. Thus, RAMPs act on G protein/{beta}-arrestin complexes, having both acute and chronic effects on GIPR function, while this study also raises the possibility of a more general role of RAMP3 to enhance GPCR plasma membrane localisation.

sentences: Temporal dynamics of base excision / single-strand break repair protein complex assembly and disassembly are modulated by the PARP1/NAD+/SIRT6 axis Assembly and disassembly of DNA repair protein complexes at sites of DNA damage is essential to maintain genomic integrity. We investigated factors coordinating assembly of the base excision repair (BER) proteins, DNA polymerase {beta} (Pol{beta}) and XRCC1, to DNA lesion sites, identifying a new role for Pol{beta} in regulating XRCC1 disassembly from DNA repair complexes and conversely, demonstrating Pol{beta}s dependence on XRCC1 for complex assembly. RealPAR, a genetically-encoded probe for live cell imaging of poly(ADP-ribose) (PAR), reveals that Pol{beta} and XRCC1 require PAR for repair complex assembly and PAR degradation for disassembly. We find that BER complex assembly is further modulated by attenuation / augmentation of NAD+ biosynthesis. Finally, SIRT6 does not regulate PARP1 activation but impairs XRCC1 recruitment, leading to diminished Pol{beta} abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for both PARP1 and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

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Standardized genome-wide function prediction enables comparative functional genomics in plants: a new application area for Gene Ontologies BackgroundGenome-wide gene function annotations are useful for hypothesis generation and for prioritizing candidate genes potentially responsible for phenotypes of interest. We functionally annotated the genes of 18 crop plant genomes across 14 species using the GOMAP pipeline.

ResultsBy comparison to existing GO annotation datasets, GOMAP-generated datasets cover more genes, contain more GO terms, and produce datasets similar in quality (based on precision and recall metrics using existing gold standards as the basis for comparison). From there, we sought to determine whether the datasets across multiple species could be used together to carry out comparative functional genomics analyses in plants. To test the idea and as a proof of concept, we created dendrograms of functional relatedness based on terms assigned for all 18 genomes. These dendrograms were compared to well-established species-level evolutionary phylogenies to determine whether trees derived were in agreement with known evolutionary relationships, which they largely are. Where discrepancies were observed, we determined branch support based on jack-knifing then removed individual annotation sets by genome to identify the annotation sets causing unexpected relationships.

ConclusionsGOMAP-derived functional annotations used together across multiple species generally retain sufficient biological signal to recover known phylogenetic relationships based on genome-wide functional similarities, indicating that comparative functional genomics across species based on GO data hold promise for generating novel hypotheses about comparative gene function and traits.

sentences: Myo1e knockout in the adult podocytes leads to proteinuria but has less severe consequences for kidney function than Myo1e loss during renal development Myosin 1e (Myo1e) is expressed in specialized epithelial cells in the kidney (podocytes) and plays an important role in renal filtration. Knockout of Myo1e in mice and mutations in the MYO1E gene in humans cause proteinuria and disrupt the ultrastructure of glomeruli, the initial segments of renal nephrons that are responsible for selective excretion of waste products without the loss of proteins from the bloodstream. Previous studies have demonstrated that the loss of Myo1e early in development results in severe defects in renal function in mice and may lead to end-stage renal disease in patients homozygous for MYO1E mutations. However, little is known about the effects of Myo1e loss later in life. In this study, we used inducible knockout of Myo1e in mouse podocytes to examine the effects of Myo1e loss from the adult kidneys. We have found that Myo1e loss after the completion of renal development causes proteinuria and podocyte defects but the effects are milder and more variable compared to the effects of Myo1e knockout in developing podocytes. These findings indicate that Myo1e plays an important role in podocyte development and differentiation but may also contribute to the maintenance of the glomerular barrier in the adult kidneys.

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Multiple hybridization events punctuate the evolutionary trajectory of Malassezia furfur Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behaviour. For example, Malassezia furfur is a common commensal of human skin, and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages, and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages, and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system, and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies.

ImportanceMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohns disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of Cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.

sentences: occAssess: An R package for assessing potential biases in species occurrence data Species occurrence records from a variety of sources are increasingly aggregated into heterogeneous databases and made available to ecologists for immediate analytical use. However, these data are typically biased, i.e. they are not a probability sample of the target population of interest, meaning that the information they provide may not be an accurate reflection of reality. It is therefore crucial that species occurrence data are properly scrutinised before they are used for research. In this article, we introduce occAssess, an R package that enables straightforward screening of species occurrence data for potential biases. The package contains a number of discrete functions, each of which returns a measure of the potential for bias in one or more of the taxonomic, temporal, spatial and environmental dimensions. Users can opt to provide a set of time periods into which the data will be split; in this case separate outputs will be provided for each period, making the package particularly useful for assessing the suitability of a dataset for estimating temporal trends in species distributions. The outputs are provided visually (as ggplot2 objects) and do not include a formal recommendation as to whether data are of sufficient quality for any given inferential use. Instead, they should be used as ancillary information and viewed in the context of the question that is being asked, and the methods that are being used to answer it. We demonstrate the utility of occAssess by applying it to data on two key pollinator taxa in South America: leaf-nosed bats (Phyllostomidae) and hoverflies (Syrphidae). In this worked example, we briefly assess the degree to which various aspects of data coverage appear to have changed over time. We then discuss additional applications of the package, highlight its limitations, and point to future development opportunities.

sentences: Cationic inhalable particles for enhanced drug delivery to M. tuberculosis infected macrophages Inhalable microparticle-based drug delivery platforms are being investigated extensively for Tuberculosis (TB) treatment as they offer efficient deposition in lungs and improved pharmacokinetics of the encapsulated cargo. However, the effect of physical parameters of microcarriers on interaction with Mycobacterium tuberculosis (Mtb) infected mammalian cells is underexplored. In this study, we report that Mtb-infected macrophages are highly phagocytic and microparticle surface charge plays a major role in particle internalization by infected cells. Microparticles of different sizes (0.5 - 2 m) were internalized in large numbers by Mtb-infected THP-1 macrophages and murine primary Bone Marrow Derived Macrophages in vitro. Drastic improvement in particle uptake was observed with cationic particles in vitro and in mice lungs. Rapid uptake of rifampicin-loaded cationic microparticles allowed high intracellular accumulation of the drug and lead to enhanced anti-bacterial function when compared to non-modified rifampicin-loaded microparticles. Cytocompatibility assay and histological analysis in vivo confirmed that the formulations were safe and did not elicit any adverse reaction. Additionally, pulmonary delivery of cationic particles in mice resulted in two-fold higher uptake in resident alveolar macrophages compared to non-modified particles. This study provides a framework for future design of drug carriers to improve delivery of anti-TB drugs inside Mtb-infected cells.

sentences: Not all voxels are created equal: reducing estimation bias in regional NODDI metrics using tissue-weighted means Neurite orientation dispersion and density imaging (NODDI) estimates microstructural properties of brain tissue relating to the organisation and processing capacity of neurites, which are essential elements for neuronal communication. Descriptive statistics of NODDI tissue metrics are commonly analysed in regions-of-interest (ROI) to identify brain-phenotype associations. Here, the conventional method to calculate the ROI mean weights all voxels equally. However, this produces biased estimates in the presence of CSF partial volume. This study introduces the tissue-weighted mean, which calculates the mean NODDI metric across the tissue within an ROI, utilising the tissue fraction estimate from NODDI to reduce estimation bias. We demonstrate the proposed mean in a study of white matter abnormalities in young onset Alzheimers disease (YOAD). Results show the conventional mean induces significant bias that correlates with CSF partial volume, primarily affecting periventricular regions and more so in YOAD subjects than in healthy controls. Due to the differential extent of bias between healthy controls and YOAD subjects, the conventional mean under- or over-estimated the effect size for group differences in many ROIs. This demonstrates the importance of using the correct estimation procedure when inferring group differences in studies where the extent of CSF partial volume differs between groups. These findings are robust across different acquisition and processing conditions. Bias persists in ROIs at higher image resolution, as demonstrated using data obtained from the third phase of the Alzheimers disease neuroimaging initiative (ADNI); and when performing ROI analysis in template space. This suggests that conventional ROI means of NODDI metrics are biased estimates under most contemporary experimental conditions, the correction of which requires the proposed tissue-weighted mean. The tissue-weighted mean produces accurate estimates of ROI means and group differences when ROIs contain voxels with CSF partial volume. In addition to NODDI, the technique can be applied to other multi-compartment models that account for CSF partial volume, such as the free water elimination method. We expect the technique to help generate new insights into normal and abnormal variation in tissue microstructure of regions typically confounded by CSF partial volume, such as those in individuals with larger ventricles due to atrophy associated with neurodegenerative disease.

sentences: More or less deadly? A mathematical model that predicts SARS-CoV-2 evolutionary direction. SARS-CoV-2 has caused tremendous deaths world wild. It is of great value to predict the evolutionary direction of SARS-CoV-2. In this paper, we proposed a novel mathematical model that could predict the evolutionary trend of SARS-CoV-2. We focus on the mutational effects on viral assembly capacity. A robust coarse-grained mathematical model is constructed to simulate the virus dynamics in the host body. Both virulence and transmissibility can be quantified in this model. The relationship between virulence and transmissibility can be simulated. A delicate equilibrium point that optimizing the transmissibility can be numerically obtained. Based on this model, we predict the virulence of SARS-CoV-2 might further decrease, accompanied by an enhancement of transmissibility. However, this trend is not continuous; its virulence will not disappear but remains at a relatively stable range. We can also explain the cross-species transmission phenomenon of certain RNA virus based on this model. A small-scale model which simulates the virus packing process is also proposed. It can be explained why a small number of mutations would lead to a significant divergence in clinical performance, both in the overall particle formation quantity and virulence. This research provides a mathematical attempt to elucidate the evolutionary driving force in RNA virus evolution.

sentences: The Great Deceiver: miR-2392's Hidden Role in Driving SARS-CoV-2 Infection MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 positive patients, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters and may potentially inhibit a COVID-19 disease state in humans.

sentences: PEPPI: Whole-proteome protein-protein interaction prediction through structure and sequence similarity, functional association, and machine learning Proteome-wide identification of protein-protein interactions is a formidable task which has yet to be sufficiently addressed by experimental methodologies. Many computational methods have been developed to predict proteome-wide interaction networks, but few leverage both the sensitivity of structural information and the wide availability of sequence data. We present PEPPI, a pipeline which integrates structural similarity, sequence similarity, functional association data, and machine learning-based classification through a naive Bayesian classifier model to accurately predict protein-protein interactions at a proteomic scale. Through benchmarking against a set of 798 ground truth interactions and an equal number of noninteractions, we have found that PEPPI attains 4.5% higher AUROC than the best of other state-of-the-art methods. As a proteomic-scale application, PEPPI was applied to model the interactions which occur between SARS-CoV-2 and human host cells during coronavirus infection, where 403 high-confidence interactions were identified with predictions covering 73% of a gold standard dataset from PSICQUIC and demonstrating significant complementarity with the most recent high-throughput experiments. PEPPI is available both as a webserver and in a standalone version and should be a powerful and generally applicable tool for computational screening of protein-protein interactions.

sentences: Stress induced TDP-43 mobility loss independent of stress granules TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting "bioreactors" for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration.  Stimulated-emission depletion microscopy (STED) and  tracking and localization microscopy (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. The data provide new insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization takes place in the cytoplasm separate from SGs.

sentences: A General Birth-Death-Sampling Model for Epidemiology and Macroevolution Birth-death stochastic processes are the foundation of many phylogenetic models and are widely used to make inferences about epidemiological and macroevolutionary dynamics. There are a large number of birth-death model variants that have been developed; these impose different assumptions about the temporal dynamics of the parameters and about the sampling process. As each of these variants was individually derived, it has been difficult to understand the relationships between them as well as their precise biological and mathematical assumptions. Without a common mathematical foundation, deriving new models is non-trivial. Here we unify these models into a single framework, prove that many previously developed epidemiological and macroevolutionary models are all special cases of a more general model, and illustrate the connections between these variants. This framework centers around a technique for deriving likelihood functions for arbitrarily complex birth-death(-sampling) models that will allow researchers to explore a wider array of scenarios than was previously possible. We then use this frame-work to derive general model likelihoods for both the "single-type" case in which all lineages diversify according to the same process and the "multi-type" case, where there is variation in the process among lineages. By re-deriving existing single-type birth-death sampling models we clarify and synthesize the range of explicit and implicit assumptions made by these models.

sentences: EnTrance: Exploration of Entropy Scaling Ball Cover Search in Protein Sequences Biological sequence alignment using computational power has received increasing attention as technology develops. It is important to predict if a novel DNA sequence is potentially dangerous by determining its taxonomic identity and functional characteristics through sequence identification. This task can be facilitated by the rapidly increasing amounts of biological data in DNA and protein databases thanks to the corresponding increase in computational and storage costs. Unfortunately, the growth in biological databases has caused difficulty in exploiting this information. EnTrance presents an approach that can expedite the analysis of this large database by employing entropy scaling. This allows scaling with the amount of entropy in the database instead of scaling with the absolute size of the database. Since DNA and protein sequences are biologically meaningful, the space of biological sequences demonstrates the structure exploited by entropy scaling. As biological sequence databases grow, taking advantage of this structure can be extremely beneficial for reducing query times. EnTrance, the entropy scaling search algorithm introduced here, accelerates the biological sequence search exemplified by tools such as BLAST. EnTrance does this by utilizing a two step search approach. In this fashion, EnTrance quickly reduces the number of potential matches before more exhaustively searching the remaining sequences. Tests of EnTrance show that this approach can lead to improved query times. However, constructing the required entropy scaling indices beforehand can be challenging. To improve performance, EnTrance investigates several ideas for accelerating index build time that supports entropy scaling searches. In particular, EnTrance makes full use of the concurrency features of Go language greatly reducing the index build time. Our results identify key tradeoffs and demonstrate that there is potential in using these techniques for sequence similarity searches. Finally, EnTrance returns more matches and higher percentage identity matches when compared with existing tools.

sentences: Maternal immune activation accelerates puberty initiation and alters mechanical allodynia in male and female C57BL6/J mice. The mechanisms that link maternal immune activation (MIA) with the onset of neurodevelopmental disorders remain largely unclear. Accelerated puberty is also associated with a heightened risk for psychopathology in later life but there is a dearth of evidence on the impacts of maternal infection on pubertal timing. We examined the effects of MIA on reproductive development, mechanical allodynia, and sensorimotor gating in juvenile, adolescent, and adult male and female mice. Moreover, we investigated hypothalamic neural markers associated with the reproductive and stress axes. Finally, we tested the mitigating effects of environmental enrichment (EE), which has clinical relevancy in human rehabilitation settings. Our results show that administration of polyinosinic-polycytidylic acid (poly(I:C)) on gestational day 12.5 led to early preputial separation, vaginal openings, and age of first estrus in offspring. MIA exposure altered pain sensitivity across development and modestly altered prepulse inhibition. The downregulation of Nr3c1 and Oprk mRNA in the hypothalamus of juvenile mice suggests that MIAs effects may be mediated through disruption of hypothalamic-pituitary-adrenal axis activity. In contrast, life-long housing with EE rescued many of these MIA-induced consequences. Overall, our findings suggest that accelerated puberty may be associated with the deleterious effects of infection during pregnancy and the onset of psychopathology.

sentences: Hippocampal sharp wave-ripples and the associatedsequence replay emerge from structured synapticinteractions in a network model of area CA3 Hippocampal place cells are activated sequentially as an animal explores its environment. These activity sequences are internally recreated ("replayed"), either in the same or reversed order, during bursts of activity (sharp wave-ripples; SWRs) that occur in sleep and awake rest. SWR-associated replay is thought to be critical for the creation and maintenance of long-term memory. In order to identify the cellular and network mechanisms of SWRs and replay, we constructed and simulated a data-driven model of area CA3 of the hippocampus. Our results show that the chain-like structure of recurrent excitatory interactions established during learning not only determines the content of replay, but is essential for the generation of the SWRs as well. We find that bidirectional replay requires the interplay of the experimentally confirmed, temporally symmetric plasticity rule, and cellular adaptation. Our model provides a unifying framework for diverse phenomena involving hippocampal plasticity, representations, and dynamics, and suggests that the structured neural codes induced by learning may have greater influence over cortical network states than previously appreciated.

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Dynamic visualisation of million-tip trees: the OneZoom project O_LIThe complete tree of life is now available, but methods to visualise it are still needed to meet needs in research, teaching and science communication. Dynamic visualisation of million-tip trees requires many challenges in data synthesis, data handling and computer graphics to be overcome.
C_LIO_LIOur approach is to automate data processing, synthesise data from a wide range of available sources, then to feed these data to a client-side visualisation engine in parts. We develop a way to store the whole tree topology locally in a highly compressed form, then dynamically populate metadata such as text and images as the user explores.
C_LIO_LIThe result is a seamless and smooth way to explore the complete tree of life, including images and metadata, even on relatively old mobile devices.
C_LIO_LIThe underlying methods developed have applications that transcend tree of life visualisation. For the whole complete tree, we describe automated ID mappings between well known resources without resorting to taxonomic name resolution, automated methods to collate sets of public domain representative images for higher taxa, and an index to measure public interest of individual species.
C_LIO_LIThe visualisation layout and the client user interface are both abstracted components of the codebase enabling other zoomable tree layouts to be swapped in, and supporting multiple applications including exhibition kiosks and digital art.
C_LIO_LIAfter ten years of work, our tree of life explorer is now broadly complete, it has attracted nearly 1.5 million online users, and is backed by a novel long-term sustainability plan. We conclude our description of the OneZoom project by suggesting the next challenges that need to be solved in this field: extinct species and guided tours around the tree.
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sentences: Aedes albopictus bionomics in Procida Island, a promising Mediterranean site for the assessment of innovative and community-based integrated pest management methods. The colonization of Mediterranean Europe and of other temperate regions by Aedes albopictus created in the last decades an unprecedented nuisance problem in highly infested areas, as well as a new public health threat due to the species competence to transmit exotic arboviruses, such as dengue, chikungunya and zika. The Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT) are insecticide-free mosquito-control methods relying on mass release of irradiated/manipulated males which are believed to have a potential in complementing existing and only partially effective control tools. Testing and implementing these approaches are challenging and selection of study sites is an instrumental and crucial step. We carried out a 4-year study in Procida Island (Gulf of Naples, Italy) in strict collaboration with local administrators and citizens to estimate: i) the temporal dynamics, spatial distribution, and population size of Ae. albopictus; and ii) the dispersal and survival of irradiated males. Overall, results provide insights on the bionomics of the mosquito in Southern Europe and draw attention to Procida Island as an ideal site to test innovative control programs against Ae. albopictus which may be used in other Mediterranean and temperate areas."

sentences: Novel Hendra virus variant detected by sentinel surveillance of Australian horses. A novel Hendra virus (HeV) variant, not detected by routine testing, was identified and isolated from a Queensland horse that suffered acute, fatal disease consistent with HeV infection. Whole genome sequencing and phylogenetic analysis demonstrated the variant to have ~83% nucleotide identity to the prototype HeV strain. An updated RT-qPCR assay was designed for routine HeV surveillance. In silico and in vitro comparison of the receptor-binding protein with prototypic HeV showed that the human monoclonal antibody m102.4 used for post-exposure prophylaxis, as well as the current equine vaccine, should be effective against this variant. Genetic similarity of this virus to sequences detected from grey-headed flying-foxes suggests the variant circulates at-least in this species. Studies determining infection kinetics, pathogenicity, reservoir-species associations, viral-host co-evolution and spillover dynamics for this virus are urgently needed. Surveillance and biosecurity practices should be updated to appreciate HeV spillover risk across all regions frequented by flying foxes.

sentences: Reporter gene assays and chromatin-level assays define substantially non-overlapping sets of enhancer sequences Transcriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence. We examine here whether these two types of definitions effectively identify the same sets of sequences and find that, concerningly, the overlap between the two groups is strikingly limited. Our results raise important questions as to the appropriateness of both old and new enhancer definitions.

sentences: Distributional reinforcement learning in prefrontal cortex Prefrontal cortex is crucial for learning and decision-making. Classic reinforcement learning (RL) theories centre on learning the expectation of potential rewarding outcomes and explain a wealth of neural data in prefrontal cortex. Distributional RL, on the other hand, learns the full distribution of rewarding outcomes and better explains dopamine responses. Here we show distributional RL also better explains prefrontal cortical responses, suggesting it is a ubiquitous mechanism for reward-guided learning.

sentences: Multiscale mechanics and temporal evolution of vimentin intermediate filament networks The cytoskeleton, an intricate network of protein filaments, motor proteins, and crosslinkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics - single filament mechanics, filament length, and interactions between filaments - including their temporal evolution. Combining particle tracking, quadruple optical trapping and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions. These results indicate that cells might need to explicitly suppress attractive interactions to re-organize the extremely stable cellular vimentin network.

sentences: Taxonomic repositioning of twelve Xanthomonas campestris, seven Xanthomonas axonopodis and one Pseudomonas cissicola reference pathovars to Xanthomonas citri Here on the basis of phylo-taxonogenomics criteria we present amended description of 20 pathovars to Xanthomonas citri majority (18/20) of which were first reported from India. 7/20 are currently classified as X. axonopodis, 12/20 as X. campestris and 1/20 as Pseudomonas cissicola. Here, we have generated genome sequence data for 4 pathovars and genomes of remaining 16 were used from the published data. Genome based investigation reveals that all these pathovars belong to X. citri and not to X. axonopodis or X. campestris as previously reported. Present proposal is to aid in resolving the taxonomic confusion of the X. ctiri pathovars and prevent future usage of invalid names.

sentences: A counter-intuitive method for slowing down an analyte in a nanopore and its application to polymer sequencing and analysis An unresolved problem in nanopore sensing is the high translocation speed ([~]10-100 monomers/s) of an analyte (nucleotide, DNA, amino acid (AA), peptide) through the pore. Here a method based on reversing the pore voltage and changing the solution pH is described. A simplified Fokker-Planck model shows mean translocation times of 1-10 ms in a nanopore of length 10 nm. Simulations show that a positive-negative voltage profile can trap an analyte for [~]1 ms. This method can be used for free nucleotides, single AAs, oligonucleotides, and oligopeptides. Its applicability to existing nanopore sensing and sequencing techniques and implementation issues are discussed.

sentences: Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces In the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a weak antibiotic-producing Streptomyces strain JB140. The seven different competition experiments utilized three serial passages (three cycles of adaptation-selection of 15 days each) of a weak antibiotic-producing Streptomyces strain (wild-type) against one (biculture) or two (triculture) or three (quadriculture) target pathogens. This resulted in the evolution of a weak antibiotic-producing strain into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against bacterial pathogens. The mutant not only effectively inhibited the growth of the tested pathogens but also observed to produce antimicrobial against multidrug-resistant (MDR) E. coli. Intriguingly, the highest antimicrobial activity was registered with the Streptomyces mutants that were adaptively evolved against the three pathogens (quadriculture competition). In contrast to the adaptively evolved mutants, a weak antimicrobial activity was detected in the un-evolved, wild-type Streptomyces. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared that revealed significant polymorphism among them. These results demonstrated that competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased production of antibiotics against targeted pathogens.

sentences: Drug combination prediction for cancer treatment using disease-specific drug response profiles and single-cell transcriptional signatures Developing novel cancer treatments is a challenging task that can benefit from computational techniques matching transcriptional signatures to large-scale drug response data. Here, we present  retriever, a tool that extracts robust disease-specific transcriptional drug response profiles based on cellular response profiles to hundreds of compounds from the LINCS-L1000 project. We used retriever to extract transcriptional drug response signatures of triple-negative breast cancer (TNBC) cell lines and combined these with a single-cell RNA-seq breast cancer atlas to predict drug combinations that antagonize TNBC-specific disease signatures. After systematically testing 152 drug response profiles and 11,476 drug combinations, we identified the combination of kinase inhibitors QL-XII-47 and GSK-690693 as the topmost promising candidate for TNBC treatment. Our new computational approach allows the identification of drugs and drug combinations targeting specific tumor cell types and subpopulations in individual patients. It is, therefore, highly suitable for the development of new personalized cancer treatment strategies.

sentences: Development of Molecular Inversion Probes for Soybean Progeny Genomic Selection Genotyping Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective, high-throughput, and provides high data quality to screen breeders germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the North Central and Mid-South regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. MIPs genotyping accuracy was 93% overall while homozoygous call accuracy was 98% with less than 10% missing data. The accuracy of MIPs combined with its low per sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs.

sentences: Risk and protective factors of feline tooth resorption in 8115 Finnish cats Tooth resorption (TR) is one of the most common dental diseases in cats. It is a painful disease characterised by progressive dental destruction, which eventually results in loss of teeth. The aetiology of the disease remains unclear, but associations with old age, breed, other oral and dental diseases, and certain environmental factors have been suspected. We collected health and environmental information of 8115 Finnish cats from 41 breeds through an online survey for cat owners to study TR in Finland with an observational study. The frequency of TR that the owner reported as veterinarian-diagnosed was 3.9% in the entire data (316/8115) and 15% in cats that the owner reported as having veterinarian-diagnosed oral or dental disease (316/2070). We determined the risk factors and breed variation of feline TR by logistic regression with the entire data and a case-control study in a more defined population drawn from the entire data. In the entire data, we defined those cats that the owners had reported as being diagnosed with TR by a veterinarian as TR-cats and compared them to all other cats. In the case-control study, we included only cats with owner-reported veterinarian-diagnosed oral and dental diseases. We defined cats with TR-diagnosis made under sedation as TR-cases. Other veterinarian-diagnosed TR-cats were excluded. Cats with other oral and dental diseases were used as controls. The model with the entire population included 308 TR-cats compared with 7508 cats, and the case-control study included 198 TR-cats and 1683 controls. Although the diagnoses were reportedly made by a veterinarian, the owner answered the survey and might have understood, remembered, or interpreted the diagnoses in a way that could affect the results. The results indicated an increasing risk of TR with age. Dental calculus, gingivitis, and periodontitis were potential risk factors for TR. These findings and the interaction between dental calculus and gingivitis suggest that inflammatory changes caused by dental calculus increase the risk of TR. Keeping food available constantly was a potential protective factor. We found that Cornish Rex, European, and Ragdoll breeds were at higher risk for TR. The observed differences between breeds highlight a genetic contribution.

sentences: Intolerance of uncertainty is associated with heightened responding in the prefrontal cortex during instructed outcome uncertainty of threat Heightened responding to uncertain threat is considered a hallmark of anxiety disorder pathology. Here, we sought to determine if individual differences in self-reported intolerance of uncertainty (IU), a key transdiagnostic dimension in anxiety-related pathology, underlies differential recruitment of neural circuitry during instructed outcome uncertainty of threat (n = 42). In an instructed threat of shock task, cues signalled uncertain threat of shock (50%) or certain safety from shock outcomes. Ratings of arousal and valence, skin conductance response (SCR) and functional magnetic resonance imaging were acquired. Overall, participants displayed greater ratings of arousal and negative valence, SCR, and amygdala activation to uncertain threat vs. safe cues. IU was not associated with greater arousal ratings, SCR or amygdala activation to uncertain threat vs. safe cues. However, we found that high IU was associated with greater ratings of negative valence and greater activity in the medial prefrontal cortex and dorsomedial rostral prefrontal cortex to uncertain threat vs safe cues. These findings suggest that during instructed outcome uncertainty of threat, individuals high in IU rate uncertain threat as aversive and engage prefrontal cortical regions known to be involved in safety-signalling and conscious threat appraisal. Taken together, these findings highlight the potential of IU in modulating safety-signalling and conscious appraisal mechanisms in situations with instructed outcome uncertainty of threat, which may be relevant to models of anxiety-related pathology.

sentences: Heterosis and Combining Ability in Pumpkin Inbreds (Cucurbita moschata Duch. ex Poir.) Twenty hybrids along with five parents evaluated in the study were mainly contemplated to find out the best cross combinations and the best general and specific combiners as well as to estimate the nature and magnitude of the gene action for different qualitative traits. Using Griffings and Haymans approach through a 5 x 5 full diallel cross fashion, an investigation on heterosis and combining ability in pumpkin was undertaken following RCBD design with three replications at the experimental field. Both positive and negative significant GCA and SCA variances were obtained from few parents and hybrids. Predominance of additive-additive gene action was noted for most of the characters except hollowness and dry matter content, where additive-dominance gene action was predominant; flesh thickness and brix (%), where dominance-dominance gene action were predominant. A single parent was not found as good combiner for more than two characters. The best specific combiners were IBD 40 X IBD 47 for beta carotene, total sugar and fruit yield; IBD23 X IBD40 for brix (%), hollowness and flesh thickness; IBD40 X IBD57 for fruit breadth; IBD47 X IBD50 for non reducing sugar; and IBD47 X IBD57 for reducing sugar. The Vr-Wr graphs exhibited complete, partial and over dominance effect of genes for different characters. Complete dominance was observed only for beta carotene whereas over dominance was noticed for hollowness and flesh thickness. Partial dominance was ensured for fruit breadth, dry matter, brix (%), reducing sugar, non-reducing sugar, total sugar and fruit yield. Significant heterosis of some crosses against mid parent and better parents were observed for some characters.

sentences: iEC7871 Quercus suber model: the first multi-tissue diel cycle genome-scale metabolic model of a woody tree In the last decade, genome-scale metabolic models have been increasingly used to study plant metabolic behavior at the tissue and multi-tissue level under different environmental conditions. Quercus suber, also known as the cork oak tree, is one of the most important forest communities of the Mediterranean/Iberian region. In this work, we present the genome-scale metabolic model of the Q. suber (iEC7871), the first of a woody plant. The metabolic model comprises 7871 genes, 6231 reactions, and 6481 metabolites across eight compartments. Transcriptomics data was integrated into the model to obtain tissue-specific models for the leaf, inner bark, and phellogen, with specific biomass compositions. The tissue-specific models were merged into a diel multi-tissue metabolic model to predict interactions among the three tissues at the light and dark phases. The metabolic models were also used to analyze the pathways associated with the synthesis of suberin monomers. Nevertheless, the models developed in this work can provide insights into other aspects of the metabolism of Q. suber, such as its secondary metabolism and cork formation.

sentences: Piezo1 ion channels inherently function as independent mechanotransducers Piezo1 is a mechanically activated ion channel involved in sensing forces in various cell types and tissues. Cryo-electron microscopy has revealed that the Piezo1 structure is bowl-shaped and capable of inducing membrane curvature via its extended footprint, which indirectly suggest that Piezo1 ion channels may bias each others spatial distribution and interact functionally. Here, we use cell-attached patch-clamp electrophysiology and pressure-clamp stimulation to functionally examine large numbers of membrane patches from cells expressing Piezo1 endogenously at low levels and cells overexpressing Piezo1 at high levels. Our data, together with stochastic simulations of Piezo1 spatial distributions, show that both at endogenous densities (1-2 channels/m2), and at non-physiological densities (10-20 channels/m2) predicted to cause substantial footprint overlap, Piezo1 density has no substantial effect on its pressure sensitivity or open probability in the nominal absence of membrane tension. The results suggest that Piezo channels, at densities likely to be physiologically relevant, inherently behave as independent mechanotransducers. We propose this property is essential for cells to transduce forces homogeneously across the entire cell membrane.

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Estimated connectivity networks outperform observed connectivity networks when classifying people with multiple sclerosis into disability groups BackgroundMultiple Sclerosis (MS), a neurodegenerative and neuroinflammatory disease, causing lesions that disrupt the brains anatomical and physiological connectivity networks, resulting in cognitive, visual and/or motor disabilities. Advanced imaging techniques like diffusion and functional MRI allow measurement of the brains structural connectivity (SC) and functional connectivity (FC) networks, and can enable a better understanding of how their disruptions cause disability in people with MS (pwMS). However, advanced MRI techniques are used mainly for research purposes as they are expensive, time-consuming and require high-level expertise to acquire and process. As an alternative, the Network Modification (NeMo) Tool can be used to estimate SC and FC using lesion masks derived from pwMS and a reference set of controls connectivity networks.

ObjectiveHere, we test the hypothesis that estimated SC and FC (eSC and eFC) from the NeMo Tool, based only on an individuals lesion masks, can be used to classify pwMS into disability categories just as well as SC and FC extracted from advanced MRI directly in pwMS. We also aim to find the connections most important for differentiating between no disability vs evidence of disability groups.

Materials and MethodsOne hundred pwMS (age:45.5 {+/-} 11.4 years, 66% female, disease duration: 12.97 8.07 years) were included in this study. Expanded Disability Status Scale (EDSS) was used to assess disability, 67 pwMS had no disability (EDSS < 2). Observed SC and FC were extracted from diffusion and functional MRI directly in pwMS, respectively. The NeMo Tool was used to estimate the remaining structural connectome (eSC), by removing streamlines in a reference set of tractograms that intersected the lesion mask. The NeMo Tools eSC was used then as input to a deep neural network to estimate the corresponding FC (eFC). Logistic regression with ridge regularization was used to classify pwMS into disability categories (no disability vs evidence of disability), based on demographics/clinical information (sex, age, race, disease duration, clinical phenotype, and spinal lesion burden) and either pairwise entries or regional summaries from one of the following matrices: SC, FC, eSC, and eFC. The area under the ROC curve (AUC) was used to assess the classification performance. Both univariate statistics and parameter coefficients from the classification models were used to identify features important to differentiating between the groups.

ResultsThe regional eSC and eFC models outperformed their observed FC and SC counterparts (p-value<0.05), while the pairwise eSC and SC performed similarly (p=0.10). Regional eSC and eFC models had higher AUC (0.66-0.68) than the pairwise models (0.60-0.65), with regional eFC having highest classification accuracy across all models. Ridge regression coefficients for the regional eFC and regional observed FC models were significantly correlated (Pearsons r = 0.52, p-value < 10e-7). Decreased estimated SC node strength in default mode and ventral attention networks and increased eFC node strength in visual networks was associated with evidence of disability.

DiscussionHere, for the first time, we use clinically-acquired lesion masks to estimate both structural and functional connectomes in patient populations to better understand brain lesion-dysfunction mapping in pwMS. Models based on the NeMo Tools estimates of SC and FC better classified pwMS by disability level than SC and FC observed directly in the individual using advanced MRI. This work provides a viable alternative to performing high-cost, advanced MRI in patient populations, bringing the connectome one step closer to the clinic.

HighlightsO_LIWe compared the accuracy of models based on observed functional connectivity (FC) and structural connectivity (SC) networks extracted from advanced MRI and estimated FC and SC networks derived using only lesion masks from conventional MRI in classifying people with multiple sclerosis (pwMS) into disability groups.
C_LIO_LIEstimated SC and FC generally outperformed observed SC and FC in classifying pwMS into no disability vs evidence of disability groups, with regional estimated SC and FC having the best performance.
C_LIO_LIIncreased estimated FC node strength of regions in the visual network was associated with disability.
C_LIO_LIDecreased estimated SC node strength of regions in the default mode and ventral attention networks was associated with disability.
C_LIO_LIDespite their varied sources of origin, feature weights for the regional estimated FC and the regional observed FC classification models was significantly correlated (Pearsons r = 0.52, p-value < 10e-7).
C_LI

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Mutagenesis-visualization: analysis of site saturation mutagenesis datasets in Python SummarySite-saturation mutagenesis experiments have been transformative in our study of protein function. Despite the rich data generated from such experiments, current tools for processing, analyzing, and visualizing the data offer only a limited set of static visualization tools that are difficult to customize. Furthermore, usage of the tools requires extensive experience and programming knowledge, slowing the research process for those in the biological field who are unfamiliar with programming. Here, we introduce mutagenesis-visualization, a Python package for creating publication-quality figures for site-saturation mutagenesis datasets without the need for prior Python or statistics experience, where each of the graphs is generated with a one-line command. The plots can be rendered as native Matplotlib objects (easy to stylize) or Plotly objects (interactive graphs). Additionally, the software offers the possibility to visualize the datasets on Pymol.

Availability and implementationThe software can be installed from PyPI or GitHub using the pip package manager and is compatible with Python [≥] 3.8. The documentation can be found at readthedocs and the source code on GitHub.

sentences: Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe  glonads, a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

sentences: The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2 Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. The canonical role of Robo receptors is to signal midline repulsion in response to their cognate Slit ligands, which bind to the N-terminal Ig1 domain in most Robo family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 cooperate to signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. Although it is clear that Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2s other activities are also Slit-dependent. To determine which of Robo2s axon guidance roles depend on its Slit-binding Ig1 domain, we have used a CRISPR/Cas9-based strategy replace the endogenous robo2 gene with a robo2 variant from which the Ig1 domain has been deleted (robo2{Delta}Ig1). We compare the expression and localization of Robo2{Delta}Ig1 protein with that of full-length Robo2 in embryonic neurons in vivo, and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that removal of the Ig1 domain from Robo2{Delta}Ig1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2{Delta}Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.

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Environmental changes dictate selection and nonspecific epistasis in an empirical phenotype-environment-fitness landscape Fitness landscapes, mappings of genotype/phenotype to their effects on fitness, are invaluable concepts in evolutionary biochemistry. Though widely discussed, measurements of phenotype-fitness landscapes in proteins remain scarce. Here, we quantify all single mutational effects on fitness and phenotype of VIM-2 {beta}-lactamase across a 64-fold range of ampicillin concentrations. We then construct a phenotype-fitness landscape that takes variations in environmental selection pressure into account. We found that a simple, empirical landscape accurately models the ~39,000 mutational data points, suggesting the evolution of VIM-2 can be predicted based on the selection environment. Our landscape provides new quantitative knowledge on the evolution of the {beta}-lactamases and proteins in general, particularly their evolutionary dynamics under sub-inhibitory antibiotic concentrations, as well as the mechanisms and environmental dependence of nonspecific epistasis.

One Sentence SummaryAn empirical fitness landscape discloses the environmental dependence of mutational effects in VIM-2 {beta}-lactamase.

sentences: Urban-adapted mammal species have more known pathogens The world is rapidly urbanising, inviting mounting concern that urban environments will experience increased zoonotic disease risk. Urban animals could have more frequent contact with humans, and therefore may transmit more zoonotic parasites; however, these animals have a specific set of underlying traits that may determine their parasite burdens while predisposing them to urban living, and they may be subject to more intense research effort, both of which could complicate our ability to reliably identify the role of urbanisation in driving zoonotic risk. Here, we test whether urban mammal species host more known zoonotic parasites, investigating the potential underlying drivers while accounting for a correlated suite of phenotypic, taxonomic, and geographic predictors. We found that urban-adapted mammals have more documented parasites, and more zoonotic parasites specifically: despite comprising only 157 of the 2792 investigated species (6%), urban mammals provided 39% of known host-parasite combinations and showed consistently higher viral discovery rates throughout the last century. However, contrary to predictions, much of the observed effect was attributable to research effort rather than to urban adaptation status itself, and urban-adapted species in fact hosted fewer zoonoses than expected given their total observed parasite richness. We conclude that extended historical contact with humans has had a limited impact on the number of observed zoonotic parasites in urban-adapted mammals; instead, their greater observed zoonotic richness likely reflects sampling bias arising from proximity to humans, which supports a near-universal underlying pattern of conflation between zoonotic risk, research effort, and synanthropy. These findings underscore the need to resolve the ecological mechanisms underlying links between anthropogenic change, sampling bias, and observed wildlife disease dynamics.

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Dimensionality reduction by sparse orthogonal projection with applications to miRNA expression analysis and cancer prediction BackgroundHigh dimensionality, i.e. p > n, is an inherent feature of machine learning. Fitting a classification model directly to p-dimensional data risks overfitting and a reduction in accuracy. Thus, dimensionality reduction is necessary to address overfitting and high dimensionality.

ResultsWe present a novel dimensionality reduction method which uses sparse, orthogonal projections to discover linear separations in reduced dimension space. The technique is applied to miRNA expression analysis and cancer prediction. We use least squares fitting and orthogonality constraints to find a set of orthogonal directions which are highly correlated to the class labels. We also enforce L1 norm sparsity penalties, to prevent overfitting and remove the uninformative features from the model. Our method is shown to offer a highly competitive classification performance on synthetic examples and real miRNA expression data when compared to similar methods from the literature which use sparsity ideas and orthogonal projections.

DiscussionA novel technique is introduced here, which uses sparse, orthogonal projections for dimensionality reduction. The approach is shown to be highly effective in reducing the dimension of miRNA expression data. The application of focus in this article is miRNA expression analysis and cancer predction. The technique may be generalizable, however, to other high dimensionality datasets.

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Can neurogenesis act as a neural regularizer? New neurons are continuously generated in the subgranular zone of the dentate gyrus throughout adulthood. These new neurons gradually integrate into hippocampal circuits, forming new naive synapses. Viewed from this perspective, these new neurons may represent a significant source of  wiring noise in hippocampal networks. In machine learning, such noise injection is commonly used as a regularization technique. Regularization techniques help prevent overfitting training data, and allow models to generalize learning to new, unseen data. Using a computational modeling approach, here we ask whether a neurogenesis-like process similarly acts as a regularizer, facilitating generalization in a category learning task. In a convolutional neural network (CNN) trained on the CIFAR-10 object recognition dataset, we modeled neurogenesis as a replacement/turnover mechanism, where weights for a randomly chosen small subset of neurons in a chosen hidden layer were re-initialized to new values as the model learned to categorize 10 different classes of objects. We found that neurogenesis enhanced generalization on unseen test data compared to networks with no neurogenesis. Moreover, neurogenic networks either outperformed or performed similarly to networks with conventional noise injection (i.e., dropout, weight decay, and neural noise). These results suggest that neurogenesis can enhance generalization in hippocampal learning through noise-injection, expanding on the roles that neurogenesis may have in cognition.

Author SummaryIn deep neural networks, various forms of noise injection are used as regularization techniques to prevent overfitting and promote generalization on unseen test data. Here, we were interested in whether adult neurogenesis- the lifelong production of new neurons in the hippocampus- might similarly function as a regularizer in the brain. We explored this question computationally, assessing whether implementing a neurogenesis-like process in a hidden layer within a convolutional neural network trained in a category learning task would prevent overfitting and promote generalization. We found that neurogenesis regularization was as least as effective as, or more effective than, conventional regularizers (i.e., dropout, weight decay and neural noise) in improving model performance. These results suggest that optimal levels of hippocampal neurogenesis may improve memory-guided decision making by preventing overfitting, thereby promoting the formation of more generalized memories that can be applied in a broader range of circumstances. We outline how these predictions may be evaluated behaviorally in rodents with altered hippocampal neurogenesis.

sentences: Nucleosome patterns in four plant pathogenic fungi with contrasted genome structures AO_SCPLOWBSTRACTC_SCPLOWFungal pathogens represent a serious threat towards agriculture, health, and environment. Control of fungal diseases on crops necessitates a global understanding of fungal pathogenicity determinants and their expression during infection. Genomes of phytopathogenic fungi are often compartmentalized: the core genome contains housekeeping genes whereas the fast-evolving genome mainly contains transposable elements and species-specific genes. In this study, we analysed nucleosome landscapes of four phytopathogenic fungi with contrasted genome organizations to describe and compare nucleosome repartition patterns in relation with genome structure and gene expression level. We combined MNase-seq and RNA-seq analyses to concomitantly map nucleosome-rich and transcriptionally active regions during fungal growth in axenic culture; we developed the MNase-seq Tool Suite (MSTS) to analyse and visualise data obtained from MNase-seq experiments in combination with other genomic data and notably RNA-seq expression data. We observed different characteristics of nucleosome profiles between species, as well as between genomic regions within the same species. We further linked nucleosome repartition and gene expression. Our findings support that nucleosome positioning and occupancies are subjected to evolution, in relation with underlying genome sequence modifications. Understanding genomic organization and its role in expression regulation is the next gear to understand complex cellular mechanisms and their evolution.

sentences: Genome-wide screening in human kidney organoids identifies novel aspects of nephrogenesis Human organoids allow studying proliferation, lineage specification, and three-dimensional tissue development. Due to the inherent multicellular complexity, interrogation by systematic genetic methodologies is challenging. Here, we present the first genome-wide CRISPR screen in iPSC-derived kidney organoids. The combination of genome editing, longitudinal sampling and sorting of specific cell populations enabled us to uncover novel biology from development to tubular morphogenesis. We validated the high quality of our screen by individual hit follow up and comparisons to kidney disease datasets. The discovery of a novel regulatory mechanism which controls epithelial proliferation via a trans-activating but cis-inhibitory effect of the Notch ligand Jagged1 proves how mosaic knockouts generated by pooled CRISPR screening in organoids can identify novel ways of communication between heterogeneous cell populations in complex tissues. Collectively, these data demonstrate the feasibility of using complex iPSC- derived organoids for genome-scale screening and serves as a benchmark for future organoid CRISPR screens.

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Dynamic Interrogation of Stochastic Transcriptome Trajectories Using Disease Associated Genes Reveals Distinct Origins of Neurological and Neuropsychiatric Disorders 1The advent of open access to genomic data offers new opportunities to revisit old clinical debates while approaching them from a different angle. We examine anew the question of whether psychiatric and neurological disorders are different from each other by assessing the pool of genes associated with disorders that are understood as psychiatric or as neurological. We do so in the context of transcriptome data tracked as human embryonic stem cells differentiate and become neurons. Building upon probabilistic layers of increasing complexity, we describe the dynamics and stochastic trajectories of the full transcriptome and the embedded genes associated with psychiatric and/or neurological disorders. From marginal distributions of a genes expression across hundreds of cells, to joint interactions taken globally to determine degree of pairwise dependency, to networks derived from probabilistic graphs along maximal spanning trees, we have discovered two fundamentally different classes of genes underlying these disorders and differentiating them. One class of genes boasts higher variability in expression and lower dependencies ("active genes"); the other has lower variability and higher dependencies ("lazy genes"). They give rise to different network architectures and different transitional states. Active genes have large hubs and a fragile topology, whereas lazy genes show more distributed code during the maturation toward neuronal state. Lazy genes boost differentiation between psychiatric and neurological disorders also at the level of tissue across the brain, spinal cord, and glands. These genes, with their low variability and asynchronous ON/OFF states that have been treated as gross data and excluded from traditional analyses, are helping us settle this old argument at more than one level of inquiry.

2 Manuscript Contribution to the FieldThere is an ongoing debate on whether psychiatric disorders are fundamentally different from neurological disorders. We examine this question anew in the context of transcriptome data tracked as human embryonic stem cells differentiate and become neurons. Building upon probabilistic layers of increasing complexity, we describe the dynamics and stochastic trajectories of the full transcriptome and the embedded genes associated with psychiatric and/or neurological disorders. Two fundamentally different types of genes emerge: "lazy genes" with low, odd, and asynchronous variability patterns in expression that would have been, under traditional approaches, considered superfluous gross data, and "active genes" likely included under traditional computational techniques. They give rise to different network architectures and different transitional dynamic states. Active genes have large hubs and a fragile topology, whereas lazy genes show more distributed code during the maturation toward neuronal state. Under these new wholistic approach, the methods reveal that the lazy genes play a fundamental role in differentiating psychiatric from neurological disorders across more than one level of analysis. Including these genes in future interrogation of transcriptome data may open new lines of inquiry across brain genomics in general.

sentences: No influence of antibiotic on locomotion in Drosophila nigrosparsa after recovery, but influence on microbiome, possibly mediating wing-morphology change Antibiotics, such as tetracycline, has been frequently used to cure endosymbiont Wolbachia in arthropods. After the symbionts had been removed, the hosts must be waited for some generations to recover from side effects of the antibiotics. Knowledge of potential long-term effects of the antibiotic is important. Here, we treated Drosophila nigrosparsa with and without antibiotic tetracycline for three generations and two generations recovering time to investigate effects of the tetracycline on the flies concerning locomotion of larvae and adults, wing morphology, and gut microbiome of adults. In addition, gut-microbiome restoration was tested as a solution to reducing potential side effects of tetracycline on the flies microbiome more quickly. We found significant differences in larval and adult locomotion within groups but no significant differences among the control, antibiotic-treated, and gut-restoration groups. We found a slight differentiation of wing morphology into the three groups and significant differences in bacterial abundance among groups. The influence of tetracycline on the gut microbiome may have contributed to wing-morphology differences among groups, which would be an indirect effect of the antibiotic. Together with the absence of an effect on locomotion, this suggests that checking for both direct and indirect effects of tetracycline after a particular recovery time before using tetracycline curing is important. The microbiome of the gut-restoration group was not like that of the control group. Therefore, gut restoration cannot be used to remove effects of tetracycline in D. nigrosparsa, at least in the setup used here.

sentences: Using protein-per-mRNA differences among human tissues in codon optimization Codon usage and nucleotide composition of coding sequences have profound effects on protein expression. However, while it is recognized that different tissues have distinct tRNA profiles and codon usages in their transcriptomes, the effect of tissue-specific codon optimality on protein synthesis remains elusive. Here, we leverage existing state-of-the-art transcriptomics and proteomics datasets from the GTEx project and the Human Protein Atlas to compute the protein-to-mRNA ratios of 36 human tissues. Using this as a proxy of translational efficiency, we build a machine learning model that identifies codons enriched or depleted in specific tissues. In particular, we detect two clusters of tissues with an opposite pattern of codon preferences. We then use the identified patterns for the development of CUSTOM, a codon optimizer algorithm which suggests a synonymous codon design in order to optimize protein production in a tissue-specific manner. In a human cell model, we provide evidence that codon optimization should indeed take into account particularities of the translational machinery of the tissues in which the target proteins are expressed and that our approach can design genes with tissue-optimized expression profiles. Altogether, CUSTOM could benefit biological and biotechnological research, such as the design of tissue-targeted therapies and vaccines.

sentences: Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing The assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

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Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma Immune cell composition within the tumor microenvironment is regulated by tumor-derived factors. Cell Communication Network factor 4 (CCN4/WISP1) is a matricellular protein secreted by cancer cells that promotes metastasis by inducing the epithelial-mesenchymal transition. While metastatic dissemination limits patient survival, the absence of anti-tumor immunity also associates with poor patient out-comes with recent work suggesting these two clinical correlates are linked. Motivated by finding that CCN4 was associated with a dampened anti-tumor immune contexture in patients diagnosed with primary melanoma, we tested for a direct causal link by knocking out CCN4 (CCN4-KO) in the B16F0 and YUMM1.7 mouse models for melanoma. Tumor growth was significantly reduced when CCN4-KO melanoma cells were implanted subcutaneously in immunocompetent C57BL/6 mice but not in immunodeficient NSG mice. Correspondingly, the frequency of total CD45+ tumor-infiltrating leukocytes was significantly increased in CCN4-KO tumors, with increased natural killer (NK) and effector CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC). Additionally, the absence of tumor-derived CCN4 was associated with an impaired splenic generation of suppressive granulocytic MDSC. Among mechanisms linked to local immunosuppression, we found CCN4 directly suppressed antigen-induced IFN{gamma} release by CD8+ T cells, promoted glycolysis and consequent lactate release by melanoma cells, and enhanced tumor secretion of MDSC-attracting chemokines like CCL2 and CXCL1. Finally, CCN4-KO in B16F0 and YUMM1.7 melanoma cells complemented the anti-tumor effect of immune checkpoint blockade (ICB) therapy. Overall, our results suggest that CCN4 promotes tumor-induced immunosuppression and is a potential target for therapeutic combinations with ICB.

Statement of SignificanceGiven emerging interest in understanding the interplay between functional plasticity and anti-tumor immunity, Cell Communication Network factor 4, a secreted matricellular protein linked to promoting metastasis in melanoma, also suppresses anti-tumor immunity.

sentences: Antibiotic-producing Micrococcales govern the microbiome that inhabits the fur of two- and three-toed sloths Sloths have a dense coat on which insects, algae, and fungi coexist in a symbiotic relationship. This complex ecosystem requires different levels of control, however, most of these mechanisms remain unknown. We investigated the bacterial communities inhabiting the hair of two- (Choloepus Hoffmani) and three-toed (Bradypus variegatus) sloths and evaluated their potential for producing antibiotic molecules capable of exerting control over the hair microbiota. The analysis of 16S rRNA amplicon sequence variants (ASVs) revealed that the communities in both host species are dominated by Actinobacteriota and Firmicutes. The most abundant genera were Brevibacterium, Kocuria/Rothia, Staphylococcus, Rubrobacter, Nesterenkonia, and Janibacter. In addition, we isolated nine strains of Brevibacterium and Rothia able to produce substances that inhibited the growth of common mammalian pathogens. The analysis of the biosynthetic gene clusters (BCGs) of these nine isolates suggests that the pathogen-inhibitory activity could be mediated by the presence of siderophores, terpenes, beta-lactones, Type III polyketide synthases (T3PKS), ribosomally synthesized, and post-translationally modified peptides (RiPPs), non-alpha poly-amino acids (NAPAA) like e-Polylysin, ectoine or nonribosomal peptides (NRPs). Our data suggest that Micrococcales inhabiting sloth hair could have a role in controlling microbial populations in that habitat, improving our understanding of this highly complex ecosystem.

sentences: AUTS2 controls neuronal lineage choice through a novel PRC1-independent complex and BMP inhibition Despite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9. Furthermore, using primary cortical neurons, we observed aberrant BMP signaling and dysregulated expression of neuronal genes upon manipulating the AWS complex, indicating that the AWS-CUL4-BMP axis plays a role in regulating neuronal lineage specification in vivo. Thus, our findings uncover a sophisticated cellular signaling network mobilized by a prominent NDD risk factor, presenting multiple potential therapeutic targets for NDD.

sentences: Analysis of Normal Levels of Urine and Plasma Free Glycosaminoglycans in Adults Plasma and urine glycosaminoglycans (GAGs) are long linear sulfated polysaccharides recognized as potential non-invasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition, the so-called GAGome, a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their urine and plasma free GAGomes using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS) method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine, and hemoglobin or erythrocyte counts in plasma. We found higher free GAGome concentration - but not composition - in males. Partitioned by gender, we established reference intervals for all detectable free GAGome features in urine and plasma. We carried out a transference analysis in healthy individuals from two distinct geographical sites, including the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal plasma and urine free GAGomes reference intervals and represents a critical resource for physiology and biomarker research.

sentences: Systematic dissection of a complex gut bacterial community Efforts to model the gut microbiome have yielded important insights into the mechanisms of interspecies interactions, the impact of priority effects on ecosystem dynamics, and the role of diet and nutrient availability in determining community composition. However, the model communities studied to date have been defined or complex but not both, limiting their utility. Here, we construct a defined community of 104 bacterial strains composed of the most common taxa from the human gut microbiota. By propagating this community in growth media missing one amino acid at a time, we show that branched-chain amino acids have an outsize impact on community structure and identify a pathway in Clostridium sporogenes for generating ATP from arginine. We constructed and propagated the complete set of single-strain dropout communities, revealing a sparse network of strain-strain interactions including a novel interaction between C. sporogenes and Lactococcus lactis driven by metabolism. This work forms a foundation for studying strain-strain and strain-nutrient interactions in highly complex defined communities, and it provides a starting point for interrogating the rules of synthetic ecology at the 100+ strain scale.

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Mean-field approximation of network of biophysical neurons driven by conductance-based ion exchange Numerous network and whole brain modeling approaches make use of mean-field models. Their relative simplicity allows studying network dynamics at a large scale. They correspond to lumped descriptions of neuronal assemblies connected via synapses. mean-field models do not consider the ionic composition of the extracellular space, which can change in physiological and pathological conditions, with strong effects on neuron activity. Here we derive a mean-field model of a population of Hodgkin-Huxley type neurons, which links the neuronal intra- and extra-cellular ion concentrations to the mean membrane potential and the mean synaptic input in terms of the synaptic conductance. The model can generate various physiological brain activities including multi-stability at resting states, as well as pathological spiking and bursting behaviors, and depolarization block. The results from the analytical solution of the mean-field model agree with the mean behavior of numerical simulations of large-scale networks of neurons. The mean-field model is analytically exact for non-autonomous ion concentration variables and provides a mean-field approximation in the thermodynamic limit, for locally homogeneous mesoscopic networks of biophysical neurons driven by an ion-exchange mechanism. These results may provide the missing link between high-level neural mass approaches which are used in the brain network modeling and physiological parameters that drive the neuronal dynamics.

Significance StatementIn this study, we applied mathematical formalism to estimate the mean-field behaviors of a large neuronal ensemble taking into account the ion-exchange between the intracellular and extracellular space. The model demonstrates different brain activities including resting state, spiking behavior, and seizure, as a function of the extracellular ion concentration. The relevant parameter regime for different brain activities is extracted mainly in terms of ion concentration and heterogeneity of individual neurons in the network. This neural mass model enables studying the influence of changes in extracellular ionic conditions on whole brain dynamics in health and disease. The effect of external stimulus current and conductance-based coupling of neural masses are also analyzed.

sentences: A retrograde mechanism coordinates memory allocation across brain regions Memories engage ensembles of neurons across different brain regions within a memory system. However, it is unclear whether the allocation of a memory to these ensembles is coordinated across brain regions. To address this question, we used CREB expression to bias memory allocation in one brain region, and rabies retrograde tracing to test memory allocation in connected presynaptic neurons in the other brain regions. We find that biasing allocation of CTA memory in the basolateral amygdala (BLA) also biases memory allocation in presynaptic neurons of the insular cortex (IC). By manipulating the allocation of CTA memory to specific neurons in both BLA and IC, we found that we increased their connectivity and enhanced CTA memory performance. These results - which are corroborated by mathematical simulations, and by studies with auditory fear conditioning - demonstrate that a retrograde mechanism coordinates the allocation of memories across different brain regions.

sentences: No evidence for somatosensory attenuationduring action observation of self-touch The discovery of mirror neurons in the macaque brain in the 1990s triggered investigations on putative human mirror neurons and their potential functionality. The leading proposed function has been action understanding: accordingly, we understand the actions of others by  simulating them in our own motor system through a direct matching of the visual information to our own motor programs. Furthermore, it has been proposed that this simulation involves the prediction of the sensory consequences of the observed action, similar to the prediction of the sensory consequences of our executed actions. Here, we tested this proposal by quantifying somatosensory attenuation behaviorally during action observation. Somatosensory attenuation manifests during voluntary action and refers to the perception of self-generated touches as less intense than identical externally generated touches because the self-generated touches are predicted from the motor command. Therefore, we reasoned that if an observer simulates the observed action and, thus, he/she predicts its somatosensory consequences, then he/she should attenuate tactile stimuli simultaneously delivered to his/her corresponding body part. In three separate experiments, we found a systematic attenuation of touches during executed self-touch actions, but we found no evidence for attenuation when such actions were observed. Failure to observe somatosensory attenuation during observation of self-touch is not compatible with the hypothesis that the putative human mirror neuron system automatically simulates the observed action. In contrast, our findings emphasize a sharp distinction between the motor representations of self and others.

sentences: A Phylogenetic Approach to Inferring the Order in Which Mutations Arise during Cancer Progression SO_SCPLOWUMMARYC_SCPLOWAlthough the role of evolutionary process in cancer progression is widely accepted, increasing attention is being given to the evolutionary mechanisms that can lead to differences in clinical outcome. Recent studies suggest that the temporal order in which somatic mutations accumulate during cancer progression is important. Single-cell sequencing provides a unique opportunity to examine the mutation order during cancer progression. However, the errors associated with single-cell sequencing complicate this task. We propose a new method for inferring the order in which somatic mutations arise within a tumor using noisy single-cell sequencing data that incorporates the errors that arise from the data collection process. Using simulation, we show that our method outperforms existing methods for identifying mutation order in most cases, especially when the number of cells is large. Our method also provides a means to quantify the uncertainty in the inferred mutation order along a fixed phylogeny. We apply our method to empirical data from colorectal and prostate cancer patients.

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Loss of FAS1 function reveals rescue of an aberrant intra-S-phase checkpoint by the G2/M checkpoint regulator SOG1 The WEE1 and ATR kinases represent important regulators of the plant intra-S-phase checkpoint, as evidenced by the hypersensitivity of WEE1KO and ATRKO roots to replication inhibitory drugs. Here, we report on the identification of a defective allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex as a suppressor of WEE1- or ATR-deficient plants. We demonstrate that lack of FAS1 activity results in the activation of an ATM- and SOG1-mediated G2/M-arrest that makes the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for telomere erosion and loss of ribosomal DNA described for the fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 plays a prominent role as secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

One-Sentence SummaryLack of the chromatin assembly factor-1 subunit FAS1 results in a DNA damage response that overrules the need for replication checkpoint activators.

sentences: Tackling the phylogenetic conundrum of Hydroidolina (Cnidaria: Medusozoa: Hydrozoa) by assessing competing tree topologies with targeted high-throughput sequencing. Higher-level relationships of the Hydrozoan subclass Hydroidolina, which encompasses the vast majority of medusozoan cnidarian species diversity, have been elusive to confidently infer. The most widely adopted phylogenetic framework for Hydriodolina based on ribosomal RNA data received low support for several higher level relationships. To address this issue, we developed a set of RNA baits to target more than a hundred loci from the genomes of a broad taxonomic sample of Hydroidolina for high-throughput sequencing. Using these data, we inferred the relationships of Hydroidolina using maximum likelihood and Bayesian approaches. Both inference methods yielded well-supported phylogenetic hypotheses that largely agree with each other. Using maximum likelihood and Baysian hypothesis testing frameworks, we found that several alternate topological hypotheses proposed previously may be rejected in light of the genomic data generated for this study. Both the maximum likelihood and Bayesian topologies inferred herein consistently score well across testing frameworks, suggesting that their consensus represents the most likely phylogenetic hypothesis of Hydroidolina. This phylogenetic framework places Aplanulata as sister lineage to the remainder of Hydroidolina. This is a strong deviation from previous phylogenetic analyses that placed Capitata or Siphonophorae as sister group to the remainder of Hydroidolina. Considering that Aplanulata represents a lineage comprised of species that for the most part possess a life cycle involving a solitary polyp and free-swimming medusa stage, the phylogenetic hypotheses presented herein have potentially large implications for clarifying the evolution of life cycles, coloniality, and the division of labor in Hydrozoa as taxon sampling for phylogenetic analyses becomes more complete.

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Proteomic analysis of mouse kidney tissue associates peroxisomal dysfunction with early diabetic kidney disease BackgroundThe absence of efficient inhibitors for DKD progression reflects the gaps in our understanding of DKD molecular pathogenesis. A comprehensive proteomic analysis was performed on glomeruli and kidney cortex of diabetic mice with subsequent validation of findings in human biopsies and - omics datasets aiming to better understand the underlying molecular biology of early DKD development and progression.

MethodsLC-MS/MS was employed to analyze the kidney proteome of DKD mouse models: Glomeruli of Ins2Akita mice 2 month and 4 month old, and cortex of db/db mice 6 month old. Following label-free quantification, the abundance of detected proteins were correlated with existing kidney datasets and functionally annotated. Tissue sections from 16 DKD patients were analyzed by IHC.

ResultsPathway analysis of differentially expressed proteins in the early and late DKD versus controls predicted dysregulation in DKD hallmarks (such as peroxisomal lipid metabolism, {beta}-oxidation and TCA cycle) supporting the functional relevance of the findings. Comparing the observed protein changes in early and late DKD, consistent upregulation of 21 and downregulation of 18 proteins was detected. Among these were downregulated peroxisomal proteins such as NUDT19, ACOX1, and AMACR and upregulated mitochondrial proteins related to aminoacid metabolism including GLS, GLDC, and GCAT. Several of these changes were also observed in the kidney cortex proteome of db/db mice. IHC of human kidney further confirmed the differential expression of NUDT19, AGPS, AMACR and CAT proteins in DKD.

ConclusionsOur study shows an extensive differential expression of peroxisomal proteins in the early stages of DKD that persists regardless of the disease severity. These proteins therefore represent potential markers of early DKD pathogenesis. Collectively, essential pathways associated with peroxisomes such as lipid {beta}-oxidation, plasmalogen synthesis, aminoacid metabolism and response to oxidative stress are downregulated in early DKD, providing new perspectives and potential markers of diabetic kidney dysfunction.

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cpn60 barcode sequences accurately identify newly defined genera within the Lactobacillaceae The cpn60 barcode sequence is established as an informative target for microbial species identification. Applications of cpn60 barcode sequencing are supported by the availability of "universal" PCR primers for its amplification and a curated reference database of cpn60 sequences, cpnDB. A recent reclassification of lactobacilli involving the definition of 23 new genera provided an opportunity to update cpnDB and to determine if the cpn60 barcode could be used for accurate identification of species consistent with the new framework. Analysis of 275 cpn60 sequences representing 258/269 of the validly named species in Lactobacillus, Paralactobacillus and the 23 newer genera showed that cpn60-based sequence relationships were consistent with the whole-genome-based phylogeny. Aligning or mapping full length barcode sequences or a 150 bp subsequence resulted in accurate and unambiguous species identification in almost all cases. Taken together, our results show that the combination of available reference sequence data, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn60 barcode make it a useful target for the detection and identification of lactobacilli as defined by the latest taxonomic framework.

Significance and Impact of the StudyThe genus Lactobacillus recently underwent a major reorganization resulting in the definition of 23 new genera. Lactobacilli are widespread in environmental and host-associated microbiomes and are exploited in food and biotechnology applications, making methods for their accurate identification desirable. Here we show that the combination of a reference sequence database, "universal" barcode amplification primers, and the inherent sequence diversity within the cpn60 barcode make it a useful target for the detection and identification of lactobacilli as defined by the latest taxonomic framework.

sentences: Early life trauma leads to violent behavior and its inheritance by impairing local thyroid hormone availability in brain Escalated and inappropriate levels of aggressive behavior referred to as pathological in psychiatry can lead to violent outcomes with detrimental impact on health and society. Early life trauma triggers adulthood violence and criminality, though molecular mechanisms remain elusive. Here, we provide prefrontal cortex and hypothalamus specific transcriptome profiles of peripubertal stress (PPS) exposed Balb/c adult male mice exhibiting escalated aggression and adult female mice resilient to such aberrant behavioral responses. We identify transthyretin (TTR) as a key regulator of PPS induced escalated aggression and its intergenerational inheritance. TTR mediated long-term perturbation in hypothalamic thyroid hormone (TH) availability contributed to male aggressive behavior without affecting circulating hormone. Ttr gene ablation in hypothalamus impaired local TH signaling including levels of TH transporters (Mct8, Oatp1c1), deiodinase 2(DIO2) and TH responsive genes (Nrgn, Trh and Hr). Escalated aggressive behavior and impaired TTR-TH signaling was also inherited in F1 male progenies. Further, we deciphered Ttr promoter hyper methylation in hypothalamus of such abnormally aggressive males across generations. Interestingly prefrontal cortex showed opposite pattern of Ttr expression as well as long term epigenetic changes. Also, T4 increase by levothyroxine in PFC did not produce any behavioral changes. Our findings reveal that trauma during puberty trigger lasting escalated aggression by epigenetic programming of TTR and consequent impaired thyroid availability in brain. TTR-TH signaling in brain can serve as potential target in reversal of escalated aggression and related psychopathologies.

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Should deep-sequenced amplicons become the new gold-standard for analysing malaria drug clinical trials? BackgroundRegulatory clinical trials are required to ensure the continued supply and deployment of effective antimalarial drugs. Patient follow-up in such trials typically lasts several weeks as the drugs have long half-lives and new infections often occur during this period. "Molecular correction" is therefore used to distinguish drug failures from new infections. The current WHO-recommend method for molecular correction uses length-polymorphic alleles at highly diverse loci but is inherently poor at detecting low density clones in polyclonal infections. This likely leads to substantial underestimates of failure rates, delaying the replacement of failing drugs with potentially lethal consequences. Deep sequenced amplicons (AmpSeq) substantially increase the detectability of low-density clones and may offer a new "gold standard" for molecular correction.

MethodsPharmacological simulation of clinical trials was used to evaluate the suitability of AmpSeq for molecular correction. We investigated the impact of factors such as the number of amplicon loci analysed, the informatics criteria used to distinguish genotyping  noise from real low density signals, the local epidemiology of malaria transmission, and the potential impact of genetic signals from gametocytes.

ResultsAmpSeq greatly improved molecular correction and provided accurate drug failure rate estimates. The use of 3 to 5 amplicons was sufficient, and simple, non-statistical, criteria could be used to classify recurrent infections as drug failures or new infections.

ConclusionsThese results strongly endorse the deployment of AmpSeq as the standard for molecular correction in regulatory trials, with its potential extension into routine surveillance once the requisite technical support becomes established.

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AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Summary SentenceExtreme ABA resistance conferred by overexpression of AFP2 is not mediated by interactions with E3 ligases, but the dwa background maintains desiccation tolerance despite ABA resistance.

sentences: Chemical inhibition of pathological reactive astrocytes promotes neural protection Disease, injury, and aging induce reactive astrocyte states with pathological functions1-4. In neurodegenerative diseases, inflammatory reactive astrocytes are abundant and contribute to progressive cell loss. Modulating the state or function of these reactive astrocytes thereby represents an attractive therapeutic goal5,6. Leveraging a cellular phenotypic screening platform, we show that chemical inhibitors of HDAC3 effectively block pathological astrocyte reactivity. Inhibition of HDAC3 reduces molecular and functional features of reactive astrocytes in vitro including inflammatory gene expression, cytokine secretion, and antigen presentation. Transcriptional and chromatin mapping studies show that HDAC3 inhibition mediates a switch between pro-inflammatory and anti-inflammatory states, which disarms the pathological functions of reactive astrocytes. Systemic administration of a blood-brain barrier penetrant chemical inhibitor of HDAC3, RGFP966, blocks reactive astrocyte formation and promotes axonal protection in vivo. Collectively, these results establish a platform for discovering chemical modulators of reactive astrocyte states, inform the mechanisms controlling astrocyte reactivity, and demonstrate the therapeutic potential of modulating astrocyte reactivity for neurodegenerative diseases.

sentences: Structure and inhibitor binding characterization of oncogenic MLLT1 mutants Dysfunction of YEATS-domain-containing MLLT1, an acetyl/acyl-lysine dependent epigenetic reader domain, has been implicated in the development of aggressive cancers. Mutations in the YEATS domain have been recently reported as a cause of MLLT1 aberrant reader function. However, structural basis for the reported alterations in affinity for acetyled/acylated histone has remained elusive. Here, we report the crystal structures of both insertion and substitution present in cancer, revealing significant conformational changes of the YEATS-domain loop 8. Structural comparison demonstrates that such alteration not only altered the binding interface for acetylated/acylated histones, but the sequence alterations in the T1 loop may enable dimeric assembly consistent inducing self-association behavior. Nevertheless, we show that also the MLLT1 mutants can be targeted by developed acetyllysine mimetic inhibitors with affinities similarly to wild type. Our report provides a structural basis for the altered behaviors and potential strategy for targeting oncogenic MLLT1 mutants.

sentences: Blocking Gi/o-coupled signaling eradicates cancer stem cells and sensitizes breast tumors to HER2-targeted therapies to inhibit tumor relapse Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that are resistant to anti-tumor therapies, making them a likely origin of tumor relapse after treatment. In many cancers including breast cancer, CSC function is regulated by G protein-coupled receptors (GPCRs), making GPCR signaling an attractive target for new therapies designed to eradicate CSCs. Yet, CSCs overexpress multiple GPCRs that are redundant in maintaining CSC function, so it is unclear how to target all the various GPCRs to prevent relapse. Here, in a model of HER2+ breast cancer (i.e., transgenic MMTV-Neu mice), we were able to block tumorsphere- and tumor-forming capability of CSCs by targeting GPCRs coupled to Gi/o proteins (Gi/o-GPCRs). Similarly, in HER2+ breast cancer cells, blocking signaling downstream of Gi/o-GPCRs in the PI3K/AKT and Src pathways also enhanced HER2-targeted elimination of CSCs. In a proof-of-concept study, when CSCs were selectively ablated (via a suicide gene construct), loss of CSCs from HER2+ breast cancer cell populations mimicked the effect of targeting Gi/o-GPCR signaling, suppressing their capacity for tumor initiation and progression and enhancing HER2-targeted therapy. Thus, targeting Gi/o-GPCR signaling in HER2+ breast cancer is a promising approach for eradicating CSCs, enhancing HER2+ targeted therapy and blocking tumor reemergence.

sentences: Exploring generality of experimental conformational changes with AlphaFold predictions Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the context of available knowledge. LysR-type transcriptional regulators (LTTR) constitute the most common family of bacterial regulators. Intriguingly, their experimental structures are remarkably diverse. The active species, composed of flexible monomers dimerizing through their N- and C-terminal domains in a circular arrangement, differ across LTTR, due to intrinsic sequence differences or because crystals stabilize diverse snapshots of a common dynamic mechanism. We have used AlphaFold2 (AF) to interrogate the experimental AtzR structure in the context of predictions guided towards the different hetero-multimeric conformations known for other LTTR. Our approach drives AF prediction with the structure-based selection of the information input through sequence alignment and template conformation, linked to examination of the energy with PISA and interactions with ALEPH.

sentences: An Approach to Measuring Protein Turnover in Human Induced Pluripotent Stem Cell Organoids by Mass Spectrometry Patient-derived organoids from induced pluripotent stem cells have emerged as a model for studying human diseases beyond conventional two-dimensional (2D) cell culture. Briefly, these three-dimensional organoids are highly complex, capable of self-organizing, recapitulate cellular architecture, and have the potential to model diseases in complex organs, such as the brain. For example, the hallmark of Parkinsons disease - proteostatic dysfunction leading to the selective death of neurons in the substantia nigra - present a subtle distinction in cell type specificity that is simply lost in 2D cell culture models. As such, the development of robust methods to study global proteostasis and protein turnover in organoids will remain a critical need as organoid models evolve. To solve this problem, we have designed a workflow to extract proteins from organoids and measure global protein turnover using mass spectrometry and stable isotope labeling using amino acids in cell culture (SILAC). This allowed us to measure the turnover rates of 844 proteins and compare protein turnover to previously reported data in primary cell cultures and in vivo models. Taken together, this method will facilitate the study of proteostasis in organoid models of human disease and will provide an analytical and statistical framework to measure protein turnover in organoids of all cell types.

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Nonquantal Transmission at the Vestibular Hair Cell-Calyx Synapse: KLV currents Modulate Fast Electrical and Slow K+ potentials in the Synaptic Cleft Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the pre-synaptic hair cell to the post-synaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here we present a computational model that captures salient experimental observations of NQT and identifies overlooked features that corroborate the existence of an electrical potential ({phi}) in the synaptic cleft. We show that changes in cleft{phi} reduce transmission latency and illustrate the relative contributions of both cleft [K+] and{phi} to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.

Significance StatementThe ability of the vestibular system to drive the fastest reflexes in the nervous system depends on rapid transmission of mechanosensory signals at vestibular hair cell synapses. In mammals and other amniotes, afferent neurons form unusually large calyx terminals on certain hair cells, and communication at these synapses includes nonquantal transmission (NQT), which avoids the synaptic delay of quantal transmission. We present a quantitative model that shows how NQT depends on the extent of the calyx covering the hair cell and attributes the short latency of NQT to changes in synaptic cleft electrical potential caused by current flowing through open potassium channels in the hair cell. This previously undescribed mechanism may act at other synapses.

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Attention cueing in rivalry: insights from pupillometry We used pupillometry to evaluate the effects of attention cueing on perceptual bi-stability, as reported by adult human observers. Perceptual alternations and pupil diameter were measured during two forms of rivalry, generated by presenting a white and a black disk to the two eyes (binocular rivalry) or splitting the disks between eyes (interocular grouping rivalry). In line with previous studies, we found that subtle pupil size oscillations (about 0.05 mm) tracked alternations between exclusive dominance phases of the black or white disk. These oscillations were larger for perceptually stronger stimuli: presented to the dominant eye or with physically higher luminance contrast. However, cueing of endogenous attention to one of the rivaling percepts did not affect pupil oscillations during exclusive dominance phases. This was in spite of the reliable effects of endogenous attention on perceptual dominance, which shifted in favor of the cued percept by about 10%. The results were comparable for binocular and interocular grouping rivalry. Cueing only had a marginal modulatory effect on pupil size during mixed percepts in binocular rivalry. This may suggest that, rather than acting by modulating perceptual strength, endogenous attention primarily acts during periods of unresolved competition, which is compatible with attention being automatically directed to the rivaling stimuli during periods of exclusive dominance and thereby sustaining perceptual alternations.

Significance StatementBinocular rivalry depends on attention. When it is diverted away from the stimuli, perceptual alternations slow down; when it is preferentially directed to one stimulus, perception lingers more on it, consistent with attention enhancing the effective strength of the rivaling stimuli. Here we introduce pupillometry as a means to indirectly track changes in effective stimulus strength. We find that pupil size oscillates, accurately tracking perceived luminance during two forms of rivalry: binocular rivalry and interocular grouping rivalry. Both show robust effects of attention cueing on perceptual dominance, but pupil oscillations during exclusive dominance are unaffected by cueing. This suggests that endogenous attention does not affect perceptual strength during exclusive dominance, though it might do so during transition phases.

sentences: Conserved Architecture of Brain Transcriptome Changes between Alzheimer's Disease and Progressive Supranuclear Palsy in Pathologically Affected and Unaffected Regions We identify a striking correlation in the directionality and magnitude of gene expression changes in brain transcriptomes between Alzheimers disease (AD) and Progressive Supranuclear Palsy (PSP). Further, the transcriptome architecture in AD and PSP is highly conserved between the temporal and cerebellar cortices, indicating highly similar transcriptional changes occur in pathologically affected and "unaffected" areas of the brain. These data have broad implications for interpreting transcriptomic data in neurodegenerative disorders.

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Preclinical evaluation of a precision medicine approach to DNA vaccination in Type 1 diabetes Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to be presented with the goal of inducing tolerance. Approaches employing specific epitopes restricted to the subjects MHC haplotypes have multiplied and offer a more focused and tailored way of targeting autoreactive T cells. In addition, the Endotope platform allows endogenously expressed epitopes to be processed and presented on appropriate MHC class I and II molecules. Here, we evaluated the efficacy of a DNA vaccine encoding epitopes selected and tailored for the non-obese diabetic (NOD) mouse compared to the expression of the proinsulin protein, one of the most successful antigens in prevention of NOD disease, and we assessed the influence of several parameters (e.g. route, dosing frequency) on preventing diabetes onset at normoglycemic and dysglycemic stages. First, encoded peptides should be secreted for effective disease prevention. Furthermore, short weekly treatments with Endotope and proinsulin DNA vaccines delay disease onset, but sustained treatments are required for long-term protection, which was more significant with intradermal delivery. Although epitopes can be presented for at least two weeks, reducing the frequency of antigen administration from weekly to every other week reduced efficacy. Finally, both Endotope and proinsulin DNA vaccines were effective in the dysglycemic stage of disease, but proinsulin provided better protection, particularly in subjects with slower progression of disease. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

SIGNIFICANCE STATEMENTAntigen-specific immunotherapy is a targeted approach to treat autoimmune diseases by turning off responses to disease-relevant antigens only, leaving the rest of the immune system unaffected. Protein antigens contain many epitopes, but only a fraction of them can be presented on a specific HLA haplotype and the relative importance of different antigens vary between patients due to disease heterogeneity. Strategies based on specific epitopes do not only consider the HLA haplotype and immune profile of groups of patients but can also include important neoepitopes not present in protein antigens. Here, we provide proof-of-principle that such strategy applied to tolerogenic DNA vaccination is effective in a preclinical model of autoimmune diabetes, paving the way for precision medicine using endogenously encoded epitopes.

sentences: HiIDDD: A high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells DNA damage is a prominent biomarker for numerous diseases, including cancer and aging. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, semiquantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and {gamma}-H2AX. We demonstrate sensitive detection of DNA damage in a wide set of freshly isolated and cryopreserved primary human immune cells, including CD4+ and CD8+ T cells, B cells and monocytes with low inter-assay variability. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

sentences: Recovery from acute SARS-CoV-2 infection and development of anamnestic immune responses in T cell-depleted rhesus macaques Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

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Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae Vibrio coralliilyticus is an extracellular bacterial pathogen and a causative agent of vibriosis in larval oysters. Host mortality rates can quickly reach 100% during vibriosis outbreaks in oyster hatcheries. Type VI Secretion Systems (T6SS) are rapidly polymerizing, contact dependent injection apparatus for prey cell intoxication and play important roles in pathogenesis. DNA sequencing of V. coralliilyticus RE22Sm indicated the likely presence of two functional T6SSs with one on each of two chromosomes. Here, we investigated the antibacterial and anti-eukaryotic roles of the two T6SSs (T6SS1 and T6SS2) against E. coli Sm10 cells and Crassostrea virginica larvae, respectively. Mutations in hcp and vgrG genes were created and characterized for their effects upon bacterial antagonism and eukaryotic host virulence. Mutations in hcp1 and hcp2 resulted in significantly reduced antagonism against E. coli Sm10, with the hcp2 mutation demonstrating the greater impact. In contrast, mutations in vgrG1 or vgrG2 had little effect on E. coli killing. In eastern oyster larval challenge assays, T6SS1 mutations in either hcp1 or vgrG1 dramatically attenuated virulence against C. virginica larvae. Strains with restored wild type hcp or vgrG genes reestablished T6SS-mediated killing to that of wild type V. coralliilyticus RE22Sm. These data suggest that the T6SS1 of V. coralliilyticus RE22Sm principally targets eukaryotes and secondarily bacteria, while the T6SS2 primarily targets bacterial cells and secondarily eukaryotes. Attenuation of pathogenicity was observed in all T6SS mutants, demonstrating the requirement for proper assembly of the T6SS systems to maintain maximal virulence.

ImportanceVibriosis outbreaks lead to large-scale hatchery losses of oyster larvae (product and seed) where Vibrio sp. associated losses of 80 to 100 percent are not uncommon. Practical and proactive biocontrol measures can be taken to help mitigate larval death by Vibrio sp. by better understanding the underlying mechanisms of virulence in V. coralliilyticus. In this study, we demonstrate the presence of two Type VI Secretion Systems (T6SS) in V. coralliilyticus RE22Sm and interrogate the roles of each T6SS in bacterial antagonism and pathogenesis against a eukaryotic host. Specifically, we show that the loss of T6SS1 function results in the loss of virulence against oyster larvae.

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Dissociation of early and late face-related processes in Autism Spectrum Disorder and Williams syndrome Williams syndrome (WS) and Autism Spectrum Disorders (ASD) are psychiatric conditions associated with atypical but opposite face-to-face interactions patterns: WS patients overly stare at others, ASD individuals escape eye contact. Whether these behaviors result from dissociable visual processes within the occipito-temporal pathways is unknown.

Using high-density electroencephalography, multivariate signal processing algorithms and a protocol designed to identify and extract evoked activities sensitive to facial cues, we investigated how WS (N=14), ASD (N=14) and neurotypical subjects (N=14) decode the information content of a face stimulus.

We found two neural components in neurotypical participants, both strongest when the eye region was projected onto the subjects fovea, simulating a direct eye contact situation, and weakest over more distant regions, reaching a minimum when the focused region was outside the stimulus face. The first component peaks at 170ms, an early signal known to be implicated in low-level face features. The second is identified later, 260ms post-stimulus onset and is implicated in decoding salient face social cues.

Remarkably, both components were found distinctly impaired and preserved in WS and ASD. In WS, we could weakly decode the 170ms signal based on our regressor relative to facial features, probably due to their relatively poor ability to process faces morphology, while the late 260ms component was highly significant. The reverse pattern was observed in ASD participants who showed neurotypical like early 170ms evoked activity but impaired late evoked 260ms signal. Our study reveals a dissociation between WS and ASD patients and point at different neural origins for their social impairments.

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Decoupling alignment strategy from feature quantification using a standard alignment incidence data structure SummaryThe abundance of genomic feature such as gene expression is often estimated from observed total number of alignment incidences in the targeted genome regions. We introduce a generic data structure and associated file format for alignment incidence data so that method developers can create novel pipelines comprising models, each optimal for read alignment, post-alignment QC, and quantification across multiple sequencing modalities.

Availability and Implementationalntools software is freely available at https://github.com/churchill-lab/alntools under MIT license.

Contactkb.choi@jax.org or gary.churchill@jax.org

sentences: Dopamine D1-like receptor blockade and stimulation decreases operant responding for nicotine and food in male and female rats Dopamine has been implicated in smoking, but there remains a need for a better understanding of the effects of dopamine D1-like receptor agonists on nicotine intake and the role of sex in the effects of dopaminergic drugs on nicotine intake. This work studied the effects of D1-like receptor stimulation and blockade on operant responding for nicotine and food and locomotor activity in male and female rats. The effects of the D1-like receptor antagonist SCH 23390 (0.003, 0.01, 0.03 mg/kg) and the D1-like receptor agonist A77636 (0.1, 0.3, 1 mg/kg) on responding for nicotine and food, and locomotor activity were investigated. The effects of SCH 23390 were investigated 15 min and 24 h after treatment, and the effects of the long-acting drug A77636 were investigated 15 min, 24 h, and 48 h later. Operant responding for nicotine and food was decreased 15 min, but not 24 h, after treatment with SCH 23390. Operant responding for nicotine was decreased 15 min, 24 h, and 48 h after treatment with A77636, and food responding was decreased 15 min and 24 h later. Locomotor activity was decreased 15 min, but not 24 h, after treatment with SCH 23390. A77636 only decreased locomotor activity 48 h after treatment. There were no sex differences in the effects of SCH 23390 or A77636. In conclusions, the D1-like receptor antagonist SCH 23390 reduces nicotine intake and causes sedation in rats. Stimulation of D1-like receptors with A77636 decreases nicotine intake at time points that the drug is not sedative.

sentences: PLIGHT: A tool to assess privacy risk by inferring identifying characteristics from sparse, noisy genotypes The leakage of identifying information in genetic and omics data has been established in many studies, with single nucleotide polymorphisms (SNPs) shown to carry a strong risk of reidentification for individuals and their genetic relatives. While the ability of thousands or hundreds of thousands of SNPs (especially rare ones) to identify individuals has been demonstrated, here we sought to measure the informativeness of even a sparse set of tens of noisy, common SNPs from an individual, by putting the genotype-based privacy leakage from an individual on quantitative footing. We present a computational tool, PLIGHT ("Privacy Leakage by Inference across Genotypic HMM Trajectories"), that employs a population-genetics-based Hidden Markov Model of recombination and mutation to find piecewise matches of a sparse query set of SNPs to a reference genotype panel. Given the ready availability of auxiliary sources of noisy genotype data - such as acquiring small samples of environmental DNA or learning about someones Mendelian diseases and physical characteristics - inference on sparse data becomes a genuine concern. We explore cases where query individuals are either known to be in databases or not, and consider both simulated "mosaics" of genotypes (i.e. genotypes stitched together from diploid segments sampled from two or more source individuals) and actual genotype data obtained from swabs of coffee cups used by a known individual. Our findings are as follows: (1) Even 10 common SNPs (minor allele frequency > 0.05) often are sufficient to identify individuals in conventional genomic databases. (2) We are able to identify first-order relatives (parents, children and siblings) of query individuals with 20-30 common SNPs. (3) We find some potential for leakage of phenotypic information, based on a simulated attack by combining polygenic risk scores (PRSs) of the piecewise genotypic matches. We also found, for simulated mosaics of two individuals, that 20 common SNPs were often sufficient to find the correct identities of both component individuals. Finally, applying PLIGHT to coffee-cup-derived SNPs, we find that our tool is able identify the individual (when present in the reference database) using as little as 30 SNPs; alternatively, when the individual is not present in the reference database, we reconstruct possible genomes for the individual based on just 30-90 query SNPs by piecewise matching to the reference haplotype database. In this way, we are able to perform a small degree of imputation of unobserved query SNPs. Overall, the tool could be used to determine the value of selectively masking released SNPs, in a way that is agnostic to any explicit assumptions about underlying population membership or allele frequencies.

sentences: Muscle group specific transcriptomic and DNA methylation differences related to developmental patterning in FSHD Muscle groups throughout the body are specialized in function and are specified during development by position specific gene regulatory networks. In developed tissue, myopathies affect muscle groups differently. Facioscapulohumeral muscular dystrophy, FSHD, affects upper body and tibialis anterior (TA) muscles earlier and more severely than others such as quadriceps. To investigate an epigenetic basis for susceptibility of certain muscle groups to disease, we perform DNA methylation and RNA sequencing on primary patient derived myoblasts from TA and quadricep for both control and FSHD2 as well as RNA-seq for myoblasts from FSHD1 deltoid, bicep and TA over a time course of differentiation. We find that TA and quadricep retain methylation and expression differences in transcription factors that are key to muscle group specification during embryogenesis. FSHD2 patients have differences in DNA methylation and expression related to SMCHD1 mutations and FGF signaling. Genes induced specifically in FSHD are more highly expressed in commonly affected muscle groups. We find a set of genes that distinguish more susceptible muscle groups including development-associated TFs and genes involved in WNT signaling. Adult muscle groups therefore retain transcriptional and DNA methylation differences associated with development, which may contribute to susceptibility in FSHD.

sentences: Small Molecule Modulation of the Archetypal UbiB protein COQ8 Small molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize inhibitor and activator molecules for the archetypal UbiB member, COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays, and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Second, using 1H-13C HMQC NMR and hydrogen-deuterium exchange mass spectrometry, we reveal that the CoQ precursor mimetic, 2-propylphenol (2-PP), modulates the quintessential UbiB KxGQ domain to increase COQ8A nucleotide affinity and ATPase activity. Our newfound chemical tools promise to lend new mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.

sentences: Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs Optimizing drug candidates for blood-brain barrier (BBB) penetration in humans remains one of the key challenges and many devastating brain diseases including neurodegenerative diseases still do not have adequate treatments. So far, it has been difficult to establish state-of-the-art human stem cell derived in vitro models that mimic physiological barrier properties including a 3D microvasculature in a format that is scalable enough to screen drugs for BBB penetration in early drug development phases. To address this challenge, we established human induced pluripotent stem cell (iPSC)-derived brain endothelial microvessels in a standardized and scalable multi-well plate format. iPSC-derived brain microvascular endothelial cells (BMECs) were supplemented with primary cell conditioned media and grew to intact microvessels in 10 days of culturing. Produced microvessels show a typical BBB phenotype including endothelial protein expression, tight-junctions and polarized localization of efflux transporter. Microvessels exhibited physiological relevant trans-endothelial electrical resistance (TEER), were leak-tight for 10 kDa dextran-Alexa 647 and strongly limited the permeability of sodium fluorescein (NaF). Permeability tests with reference compounds confirmed the suitability of our model as platform to identify potential BBB penetrating anti-inflammatory drugs. In summary, the here presented brain microvessel platform recapitulates physiological properties and allows rapid screening of BBB permeable anti-inflammatory compounds that has been suggested as promising substances to cure so far untreatable neurodegenerative diseases.

sentences: Age-dependent contribution of domain-general networks to semantic cognition Aging is characterized by a decline of cognitive control. In semantic cognition, this leads to the paradox that older adults usually show poorer task performance than young adults despite their greater semantic knowledge. So far, the underlying neural changes of these behavioral differences are poorly understood. In the current neuroimaging study, we investigated the contribution of domain-general networks to a verbal semantic fluency task in young and older adults. In both age groups, task processing was characterized by a strong positive integration within the multiple-demand as well as between the multiple-demand and the default mode system during semantic fluency. However, the behavioral relevance of strengthened connectivity differed between groups: While within-network functional connectivity in both networks predicted greater efficiency in semantic fluency in young adults, it was associated with a slower performance in older adults. Moreover, only young adults profited from increased connectivity between networks for their semantic memory performance. Our results suggest that the functional coupling of usually anti-correlated networks is critical for successful task processing, independent of age, when access to semantic memory is required. Furthermore, our findings lend novel support to the notion of reduced efficiency in the aging brain, due to neural dedifferentiation in semantic cognition.

sentences: Target Class Profiling of Small Molecule Methyltransferases Target class profiling (TCP) is a chemical biology approach to investigating understudied biological target classes. TCP is achieved by developing a generalizable assay platform and screening curated compound libraries to interrogate the chemical biological space of members of an enzyme family with clinical and biological importance. In this work we took a TCP approach to investigate inhibitory activity across a set of small molecule methyltransferases, a subclass of methyltransferase enzymes, with the goal of creating a launchpad to explore this largely understudied target class. Using the representative enzymes nicotinamide N-methyltransferase (NNMT), phenylethanolamine N-methyltransferase (PNMT), histamine N-methyltransferase (HNMT), glycine N-methyltransferase (GNMT), catechol O-methyltransferase (COMT), and guanidinoacetate N-methyltransferase (GAMT), we optimized high-throughput screening (HTS)-amenable assays to screen 27,574 unique small molecules against all targets. From this dataset we identified a novel inhibitor which selectively inhibits the small molecule methyltransferase HNMT, and demonstrated how this platform approach can be leveraged for a targeted drug discovery campaign using the example of HNMT.

sentences: A Novel in vitro Model for Mature Toxoplasma gondii Bradyzoites Reveals their Metabolome and a Diminished Role of the Mitochondrial Tricarboxylic Acid Cycle The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. However, current in vitro models do not allow long-term culture of these cysts to maturity. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts that are orally infectious to mice and tolerate exposure to a range of antibiotics and temperature stresses. Metabolomic characterization of purified cysts reveals global changes that comprise increased levels of amino acids and decreased abundance of nucleobase- and tricarboxylic acid cycle-associated metabolites. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. Direct access to persistent stages of T. gondii under defined cell culture conditions will be essential for the dissection of functionally important host-parasite interactions and drug evasion mechanisms. It will also facilitate the identification of new strategies for therapeutic intervention.

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Dynamics of global gene expression and chromatin accessibility of the peripheral nervous system in animal models of persistent pain Efforts to understand genetic variability involved in an individuals susceptibility to chronic pain support a role for upstream regulation by epigenetic mechanisms. To examine the transcriptomic and epigenetic basis of chronic pain that resides in the peripheral nervous system, we used RNA-seq and ATAC-seq of the rat dorsal root ganglion (DRG) to identify novel molecular pathways associated with pain hypersensitivity in two well-studied persistent pain models induced by Chronic Constriction Injury (CCI) of the sciatic nerve and intra-plantar injection of Complete Freunds Adjuvant (CFA) in rats. Our RNA-seq studies identify a variety of biological process related to synapse organization, membrane potential, transmembrane transport, and ion binding. Interestingly, genes that encode transcriptional regulators were disproportionately downregulated in both models. Our ATAC-seq data provide a comprehensive map of chromatin accessibility changes in the DRG. A total of 1123 regions showed changes in chromatin accessibility in one or both models when compared to the naive and 31 shared differentially accessible regions (DAR)s. Functional annotation of the DARs identified disparate molecular functions enriched for each pain model which suggests that chromatin structure may be altered differently following sciatic nerve injury and hind paw inflammation. Motif analysis identified 17 DNA sequences known to bind transcription factors in the CCI DARs and 33 in the CFA DARs. Two motifs were significantly enriched in both models. Our improved understanding of the changes in chromatin accessibility that occur in chronic pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG.

SummaryShared transcriptomic and epigenetic changes in two animal models improves our understanding of how chromatin structural changes alter DRG gene expression under persistent pain conditions.

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Relative time constraints improve molecular dating Dating the tree of life is central to understanding the evolution of life on Earth. Molecular clocks calibrated with fossils represent the state of the art for inferring the ages of major groups. Yet, other information on the timing of species diversification can be used to date the tree of life. For example, horizontal gene transfer events and ancient coevolutionary relationships such as (endo)symbioses can imply temporal relationships between two nodes in a phylogeny (Davin et al. 2018). These alternative sources of temporal constraints can be particularly helpful when the geological record is sparse, e.g. for microorganisms, which represent the vast majority of extant and extinct biodiversity.

Here, we present a new method to combine fossil calibrations and relative age constraints to estimate chronograms. We provide an implementation of relative age constraints in RevBayes (Hohna et al. 2016) that can be combined in a modular manner with the wide range of molecular dating methods available in the software.

We use both realistic simulations and empirical datasets of 40 Cyanobacteria and 62 Archaea to evaluate our method. We show that the combination of relative age constraints with fossil calibrations significantly improves the estimation of node ages.

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A novel Rab11-Rab3a cascade required for lysosome exocytosis Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood.

Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis independently of the exocyst complex, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes.

Thus, our studies suggest that Rab11-positive vesicles transport GRAB to activate Rab3a on lysosomes, establishing a Rab11-Rab3 cascade that is essential for lysosome exocytosis.

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Identification of shared bacterial strains in the vaginal microbiota of reproductive-age mothers and daughters using genome-resolved metagenomics It has been suggested that the human microbiome might be vertically transmitted from mother to offspring and that early colonizers may play a critical role in development of the immune system. Studies have shown limited support for the vertical transmission of the intestinal microbiota but the derivation of the vaginal microbiota remains largely unknown. Although the vaginal microbiota of children and reproductive age cis women differ in composition, the vaginal microbiota could be vertically transmitted. To determine whether there was any support for this hypothesis, we examined the vaginal microbiota of daughter-mother pairs from the Baltimore metropolitan area (ages 14-27, 32-51; n=39). We assessed whether the daughters microbiota was similar in composition to their mothers using metataxonomics. Permutation tests revealed that while some pairs did have similar vaginal microbiota, the degree of similarity did not exceed that expected by chance. Genome-resolved metagenomics was used to identify shared bacterial strains in a subset of the families (n=22). We found a small number of bacterial strains that were shared between mother-daughter pairs but identified more shared strains between individuals from different families, indicating that vaginal bacteria may display biogeographic patterns. Earlier-in-life studies are needed to demonstrate vertical transmission of the vaginal microbiota.

ImportanceEarly colonizers of our microbiota are theorized to play an important role in the development of our immune system, yet we know little about how these communities are established. Vertical transmission from mother to offspring at the time of birth is theorized to be a major source of early colonizers but limited evidence supporting this process has only been shown for the intestinal tract microbiota. The provenance of the vaginal microbiota is largely unknown, although some have posited it is similarly vertically transmitted. We examined the vaginal microbiota of mother-daughter pairs and found limited evidence in support of this hypothesis. However, our analysis also revealed putative biogeographic patterns in the distribution of the strains which comprise the vaginal microbiota. Our results give insight into the role of vertical transmission for the vaginal microbiota and motivate future studies on the biogeography of these bacteria.

sentences: Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence Developing complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies--yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.

sentences: Genome size evolution in the beetle genus Diabrotica Diabrocite corn rootworms are one of the most economically significant pests of maize in the United States and Europe and an emerging model for insect-plant interactions. Genome sizes of several species in the genus Diabrotica were estimated using flow cytometry along with that of Acalymma vittatum as an outgroup. Genome sizes ranged between 1.56 - 1.64 gigabase pairs (Gb) and between 2.26-2.59 Gb, respectively, for the Diabrotica subgroups fucata and virgifera; the Acalymma vittatum genome size was around 1.65 Gb. This result indicated that a substantial increase in genome size occurred in the ancestor of the virgifera group. Further analysis of fucata group and virgifera group genome sequencing reads indicated that the genome size difference between the Diabrotica subgroups could be attributed to a higher content of transposable elements, mostly miniature inverted-transposable elements (MITEs) and gypsy-like long terminal repeat (LTR) retroelements.

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

sentences: Antiviral Resistance against Viral Mutation: Praxis and Policy for SARS CoV-2 AO_SCPLOWBSTRACTC_SCPLOWNew tools developed by Moderna, BioNTech/Pfizer, and Oxford/Astrazeneca, among others, provide universal solutions to previously problematic aspects of drug or vaccine delivery, uptake and toxicity, portending new tools across the medical sciences. A novel method is presented based on estimating protein backbone free energy via geometry to predict effective antiviral targets, antigens and vaccine cargos that are resistant to viral mutation. This method, partly described in earlier work of the author, is reviewed and reformulated here in light of the recent proliferation of structural data on the SARS-CoV-2 spike glycoprotein and its latest mutations in the variants of concern and several further variants of interest including all international lineages. Particular attention to structures computed with Cryo Electron Microscopy allows the novel approach of probing the pH dependence of free energy in order to infer function. Key findings include: collections of recurring mutagenic residues occur across strains, presumably through cooperative convergent evolution; the preponderance of mutagenic residues do not participate in backbone hydrogen bonds; metastability of the spike glycoprotein limits the change of free energy from before to after mutation and thereby constrains selective pressure; and there are mRNA or virus-vector cargos which target low free energy peptides proximal to conserved high free energy peptides providing specific recipes for vaccines with greater specificity than the current full-spike approach. These results serve to limit peptides in the spike glycoprotein with high mutagenic potential and thereby provide a priori constraints on viral and attendant vaccine evolution. Scientific and regulatory challenges to nucleic acid therapeutic and vaccine development and deployment are finally discussed.

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Comparative Multiomic Analysis Reveals Low T Cell Infiltration as the Primary Feature of Tobacco Use in HPV(+) Oropharyngeal Cancer PurposeTobacco use is an independent adverse prognostic feature in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). Despite this, the biologic features associated with tobacco use have not been systematically investigated in this population. We sought to characterize the genomic and immunologic features of HPV(+) OPSCC associated with tobacco use and adverse oncologic outcomes.

Experimental DesignWhole exome sequencing of 47 primary HPV(+) OPSCC tumors was performed to investigate mutational differences associated with tobacco exposure. To characterize the tumor immune microenvironment (TIME), targeted mRNA hybridization was performed and immunohistochemical (IHC) staining was used to validate these findings.

ResultsLow expression of transcripts in a T cell-inflamed gene expression profile (TGEP) was associated with tobacco use at the time of diagnosis and lower overall and disease-free survival. Tobacco use was associated with an increased proportion of T>C substitutions and a lower proportion of mutational signatures typically observed in HPV(+) OPSCC tumors, but was not associated with increases in mutational burden or the rate of recurrent oncogenic mutations.

ConclusionsIn HPV(+) OPSCC, low T cell infiltration of primary tumors is associated with current tobacco use and worse oncologic outcomes. Rather than an increased mutational burden, tobaccos primary and clinically relevant association is immunosuppression of the primary TIME. An objective clinical assay like the TGEP, which quantifies immune infiltration of the primary TIME, may have value for HPV(+) OPSCC risk stratification in future clinical trials.

sentences: Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that MetQ is a lipoprotein NmMetQ is a substrate binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To address the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the absence and presence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function/topology, playing a role in NmMetNI-mediated transport at the inner-membrane in addition to moonlighting functions on the bacterial surface.

sentences: Interplay between BALL and CBP maintains H3K27 acetylation on active genes in Drosophila CREB binding protein (CBP) is a multifunctional transcriptional co-activator that interacts with a variety of transcription factors and acts as a histone acetyltransferase. In Drosophila, CBP mediated acetylation of histone H3 lysine 27 (H3K27ac) is a known hallmark of gene activation regulated by trithorax group proteins (trxG). Recently, we have shown that a histone kinase Ballchen (BALL) substantially co-localizes with H3K27ac at trxG target loci and is required to maintain gene activation in Drosophila. Here, we report direct interaction between BALL and CBP, which positively regulates H3K27ac. Analysis of genome-wide binding profile of BALL and CBP reveals major overlap and their co-localization at actively transcribed genes. We show that BALL biochemically interacts with CBP and depletion of BALL results in drastic reduction in H3K27ac. Together, these results demonstrate a previously unknown synergy between BALL and CBP and reveals a potentially new pathway required to maintain gene activation during development.

sentences: Visualizing functional human profilin in cells and in vitro applications Profilin is an essential regulator of actin and microtubule dynamics and therefore a critical control point for the normal division, motility, and morphology of cells. Most studies of profilin have focused on biochemical investigations using purified protein because high cellular concentrations present challenges for conventional imaging modalities. In addition, past studies that employed direct labeling or conventional fusion protein strategies compromised different facets of profilin function. We engineered two versions of tagged profilin that retain native activities with respect to phosphoinositide lipids, actin monomers, formin-mediated actin assembly, and microtubule polymerization. mApple-profilin-1 directly binds to dimers of tubulin (kD = 1.7 {micro}M) and the microtubule lattice (kD = 10 {micro}M) to stimulate microtubule assembly. In cells, Halo-tagged profilin-1 fully rescues profilin-1(-/-) cells from knockout-induced perturbations to cell shape, actin filament architecture, and microtubule arrays. In cells expressing Halo-profilin, we visualized a subset of individual molecules of profilin-1 by titrating fluorescent Halo-ligands. Further, we combined this imaging approach with specific function-disrupting point-mutants in profilin to visualize dynamic profilin associated with microtubules in live-cells. Thus, these tagged profilins are reliable tools for studying the dynamic interactions of profilin with actin or microtubules in live-cell or in vitro applications.

sentences: Combined application of biochar with nitrogen fertilizer  improves soil quality and reduces soil respiration whilst sustaining wheat grain yield in a semiarid environment This study was conducted to investigate the effect of biochar, straw and N fertilizer on soil properties, soil respiration and grain yield of spring wheat (Triticum aestivum L.) in semi-arid Western Loess Plateau of northwestern China. The two carbon sources (straw and biochar) were applied alone or combined with nitrogen fertilizer (urea, 46% nitrogen [N]), whilst the soil without carbon is made up of nitrogen fertilizer applied at 0, 50 and 100 kg N/ha. The experiment was arranged in a randomized complete block design with three replicates and was conducted in 2014, 2015 and 2016 cropping season. Results showed that the greatest grain yields were found with 100 kg N ha-1 fertilization rate under biochar, straw and soils without carbon, but the greatest effect occurred on the biochar amended soils. Biochar amendment produced the greatest grain yield at 1906 kg ha-1, followed by straw treated soils at 1643 kg ha-1, and soils without carbon the lowest at 1553 kg ha-1. This results is supported by the fact that, biochar amended soils (at 0-10 cm) increased soil organic C by 17.14% and 21.65% compared to straw treated soils and soils without carbon respectively. Seasonal soil respirations were between 19.05% and 23.67% lower in BN100 compared with SN50 and CN100. Soil respiration reduced with increasing N fertilization rates under all treatments, but the greatest effect occurred on biochar plots. Biochar amended soils decreased carbon emission by 26.80% and 9.54% compared to straw treated soils and soils without carbon amendment respectively. Increased grain yield and the decreased carbon emission in BN100 translated into greater carbon emission efficiency (2.88 kg kg-1) which was significantly different compared with the other treatments. Combined application of biochar with 100 kg N ha-1 in rainfed spring wheat was a suitable agricultural practice.

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Evolving a mitigation of the stress response pathway to change the basic chemistry of life Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-{beta}-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to {beta}-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to "calm down" its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo.

SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1-3, little is known about the underlying mechanisms.

We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth.

We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution.

Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

sentences: A spatio-spectral approach for movement decoding from cortical and subcortical recordings in patients with Parkinson's disease The application of machine learning to intracranial signal analysis has the potential to revolutionize deep brain stimulation (DBS) by personalizing therapy to dynamic brain states, specific to symptoms and behaviors. Most decoding pipelines for movement decoding in the context of adaptive DBS are based on single channel frequency domain features, neglecting spatial information available in multichannel recordings. Such features are extracted either from DBS lead recordings in the subcortical target and/or from electrocorticography (ECoG). To optimize the simultaneous use of both types of signals, we developed a supervised online-compatible movement decoding pipeline based on multichannel and multiple site recordings. We found that adding spatial information to the model has the potential to improve decoding. In addition, we demonstrate movement decoding from spatio-spectral features derived from cortical and subcortical oscillations. We demonstrate between-patients variability of the spatial neural maps and its relationship to feature decoding performance. This application of spatial filters to decode movement from combined cortical and subcortical recordings is an important step in developing machine learning approaches for intelligent DBS systems.

sentences: A viral toolbox for conditional and transneuronal gene expression in zebrafish The zebrafish is an important model in systems neuroscience but a key limitation is the lack of viral tools to dissect the structure and function of neuronal circuitry. We developed methods for efficient gene transfer and retrograde tracing in adult and larval zebrafish by herpes simplex viruses (HSV1). HSV1 can be combined with the Gal4/UAS system to target cell types with high spatial, temporal and molecular specificity. We also established methods for efficient transneuronal tracing by modified rabies viruses in zebrafish. We demonstrate that HSV1 and rabies viruses can be used to visualize and manipulate genetically or anatomically identified neurons within and across different brain areas of adult and larval zebrafish. An expandable library of viruses is provided to express fluorescent proteins, calcium indicators, optogenetic probes, toxins and other molecular tools. This toolbox creates new opportunities to interrogate neuronal circuits in zebrafish through combinations of genetic and viral approaches.

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Analysis and extension of exact mean-field theory with dynamic synaptic currents Neural mass models such as the Jansen-Rit or Wendling systems provide a practical framework for representing and interpreting electro-physiological activity (1-6) in both local and global brain models (7, 8). However, they are only partly derived from first principles. While the post-synaptic potential dynamics are inferred from data and can be grounded on diffusion physics (9-11), Freemans "wave to pulse" (W2P) sigmoid function (12-14), used to transduce mean population membrane potential into firing rate, rests on a weaker theoretical standing. On the other hand, Montbrio et al (15, 16) derive an exact mean-field theory (MPR) from a quadratic integrate and fire neuron model under some simplifying assumptions, thereby connecting microscale neural mechanisms and meso/macroscopic phenomena. The MPR model can be seen to replace Freemans W2P sigmoid function with a pair of differential equations for the mean membrane potential and firing rate variables--a dynamical relation between firing rate and membrane potential--, providing a more fundamental interpretation of the semi-empirical NMM sigmoid parameters. In doing so, we show it sheds light on the mechanisms behind enhanced network response to weak but uniform perturbations. In the exact mean-field theory, intrinsic population connectivity modulates the steady-state firing rate W2P relation in a monotonic manner, with increasing self-connectivity leading to higher firing rates. This provides a plausible mechanism for the enhanced response of densely connected networks to weak, uniform inputs such as the electric fields produced by non-invasive brain stimulation. This new, dynamic W2P relation also endows the neural mass model with a form of "inertia", an intrinsic delay to external inputs that depends on, e.g., self-coupling strength and state of the system. Next, we complete the MPR model by adding the second-order equations for delayed post-synaptic currents and the coupling term with an external electric field, bringing together the MPR and the usual NMM formalisms into a unified exact mean-field theory (NMM2) displaying rich dynamical features. In the single population model, we show that the resonant sensitivity to weak alternating electric field is enhanced by increased self-connectivity and slow synapses.

O_TEXTBOXSignificance

Several decades of research suggest that weak electric fields influence neural processing. A long-standing question in the field is how networks of neurons process spatially uniform weak inputs that barely affect a single neuron but that produce measurable effects in networks. Answering this can help implement electric field coupling mechanisms in neural mass models of the whole brain, and better represent the impact of electrical stimulation or ephaptic communication. This issue can be studied using local detailed computational models, but the use of statistical mechanics methods can deliver "mean-field models" to simplify the analysis. Following the steps of Montbrio et al (15, 16), we show that the sensitivity to inputs such a weak alternating electric field can be modulated by the intrinsic self-connectivity of a neural population, and produce a more grounded set of equations for neural mass modeling to guide further work.

C_TEXTBOX

sentences: Enabling automated and reproducible spatially resolved transcriptomics at scale Tissue spatial information is an essential component to reach a holistic overview of gene expression mechanisms. The sequencing-based Spatial transcriptomics approach allows to spatially barcode the whole transcriptome of tissue sections using microarray glass slides. However, manual preparation of high-quality tissue sequencing libraries is time-consuming and subjected to technical variability. Here, we present an automated adaptation of the 10x Genomics Visium library construction on the widely used Agilent Bravo Liquid Handling Platform. Compared to the manual Visium library preparation, our automated approach reduces hands-on time by over 80% and provides higher throughput and robustness. Our automated Visium library preparation protocol provides a new strategy to standardize spatially resolved transcriptomics analysis of tissues at scale.

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The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells Epstein-Barr virus (EBV) is a human tumor virus, which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen (EBNA) 2 is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor (EBF) 1, a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. An 1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the 1-helix in EBNA2 can infect and activate B cells efficiently, but the activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F as well as multiple metabolic processes linked to cell cycle progression are impaired in EBV{Delta}1 infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Significance statementEpstein-Barr virus (EBV) infects primary B cells and establishes life-long latent infection in these cells. EBV nuclear antigen (EBNA) 2 drives early processes of B cell activation and cell cycle entry. The surface of the N-terminal dimerization domain of EBNA2 exposes a five amino acid -helix (1) that recruits EBF1 to activate MYC and downstream targets of both MYC and E2F to support critical metabolic processes in infected B cells and to drive them through S phase in the first cell cycle post-infection. Our study demonstrates how EBNA2 exploits EBF1, a key factor of B cell lineage specification to initiate proliferation and high-lights the 1-helix as a potential Achilles heel of the virus at the stage when latent infection is established.

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SARS-CoV-2-Triggered Mast Cell Rapid Degranulation Induces Alveolar Epithelial Inflammation and Lung Injury SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. Here we showed that SARS-CoV-2-triggeed MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation alterred various signaling pathways in alveolar epithelial cells, particularly, led to the production of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/449680v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@899996org.highwire.dtl.DTLVardef@1c26c0eorg.highwire.dtl.DTLVardef@1442cdcorg.highwire.dtl.DTLVardef@dd4204_HPS_FORMAT_FIGEXP  M_FIG C_FIG In BriefSARS-CoV-2 triggers an immediate mast cell (MC) degranulation, which initiates the alveolar epithelial inflammation and disrupts the tight junction. MC stabilizers that block degranulation reduce virus-induced lung inflammation and injury.

HighlightsO_LIThe binding of RBD of Spike protein of SARS-CoV-2-to ACE2 receptor protein triggers an immediate MC degranulation
C_LIO_LIMC degranulation induces transcriptomic changes include an upregulated inflammatory signaling and a downregulated cell-junction signaling
C_LIO_LIMC degranulation leads to alveolar epithelial inflammation and disruption of tight junctions
C_LIO_LIMC stabilizer that inhibits degranulation reduces SARS-CoV-2-induced lung inflammation and injury in vivo
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CZON-cutter: a CRISPR-Cas9 system with multiplexed organelle imaging in a simple unicellular alga The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner.

SummaryAn engineered CRISPR-Cas9 system, named CZON-cutter, for simultaneous genome editing and fluorescent protein labeling of organelles in Cyanidioschyzon merolae can be used to validate intracellular function of a particular gene, even if it is essential.

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Computational model for migration of human osteoblasts in direct current electric field Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Theoretical approaches to study electrotactic cell migration until now mainly used reaction-diffusion models, and did not consider the effect of electric field on single-cell motility, or incorporate spatially dependent cell-to-cell interactions. Here, we present a computational model that takes into account cell interactions and describes cell migration in direct current electric field. We compare this model with in vitro experiments in which human primary osteoblasts are exposed to direct current electric field of varying field strength. Our results show that cell-cell interactions and fluctuations in the migration direction together lead to anode-directed collective migration of osteoblasts.

Author summaryElectrotactic migration of cells involves directed movement of a large number of single cells under the influence of external electric field. Influencing the migration behaviour of osteoblasts by external direct current electric field offers a promising approach towards building highly effective implants for bone regeneration. We present a computational model for electrotactic migration of osteoblasts subject to external direct current electric field. Our model considers individual cells that interact with each other and the external electric field, and, replicates the experimental observations, based on single-cell analysis, of the response of osteoblasts to electrical stimulation of varying strengths for 7 hours. Our results suggest that tracking trajectories of individual cells provide a way of determining the role of various interactions of a cell in collective migration. Our model provides a framework that links single cell response to the large scale collective dynamics.

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

sentences: CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells The CRISPR/Cas9 system is widely used to permanently delete genomic regions by inducing double-strand breaks via dual guide RNAs. However, on-target consequences of Cas9 deletion events have yet to be fully investigated. To characterize Cas9-induced genotypic abnormalities in human cells, we utilized an innovative droplet-based target enrichment approach followed by long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving a genomic duplication and inversion of the target region as well as integrations of exogenous DNA and interchromosomal DNA fragment rearrangements at the double-strand break sites often at the same time. Although these events altered the genomic composition of the on-target region, we found that the aberrant DNA fragments are still functional, marked by active histones and bound by RNA polymerase III. In HAP1 cells, the integration of the target-derived fragments accelerated cell proliferation in deletion clones. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations and rationalize extra caution when interpreting results from a deletion event.

sentences: Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

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Electrocorticography is superior to subthalamic local field potentials for movement decoding in Parkinson's disease Brain signal decoding promises significant advances in the development of clinical brain computer interfaces (BCI). In Parkinsons disease (PD), first bidirectional BCI implants for adaptive deep brain stimulation (DBS) are now available. Brain signal decoding can extend the clinical utility of adaptive DBS but the impact of neural source, computational methods and PD pathophysiology on decoding performance are unknown. This represents an unmet need for the development of future neurotechnology. To address this, we developed an invasive brain-signal decoding approach based on intraoperative sensorimotor electrocorticography (ECoG) and subthalamic LFP to predict grip-force, a representative movement decoding application, in 11 PD patients undergoing DBS. We demonstrate that ECoG is superior to subthalamic LFP for accurate grip-force decoding. Gradient boosted decision trees (XGBOOST) outperformed other model architectures. ECoG based decoding performance negatively correlated with motor impairment, which could be attributed to subthalamic beta bursts in the motor preparation and movement period. This highlights the impact of PD pathophysiology on the neural capacity to encode movement kinematics. Finally, we developed a connectomic analysis that could predict grip-force decoding performance of individual ECoG channels across patients by using their connectomic fingerprints. Our study provides a neurophysiological and computational framework for invasive brain signal decoding to aid the development of an individualized precision-medicine approach to intelligent adaptive DBS.

Significance StatementNeurotechnology will revolutionize the treatment of neurological and psychiatric patients, promising novel treatment avenues for previously intractable brain disorders. However, optimal surgical and computational approaches and their interactions with neurological disorders are unknown. How can recent advances in machine learning and connectomics aid the precision and performance of invasive brain signal decoding strategies? Do the brain disorders treated with such approaches have impact on decoding performance? We propose a real time compatible advanced machine learning pipeline for invasively recorded brain signals in Parkinsons disease (PD) patients. We report optimal movement decoding strategies with respect to signal source, model architecture and connectomic fingerprint and demonstrate that PD pathophysiology significantly and negatively impacts movement decoding. Our study has broad impacts for the development of smart brain implants for the treatment of PD and other brain disorders.

sentences: Immune-responsive biodegradable scaffolds for enhancing neutrophil regeneration Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications. In autologous hematopoietic stem cell transplantation (HSCT), neutropenia is a complication that limits therapeutic efficacy. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior. In mouse models of immune deficiency, we show that the infiltration of functional myeloid-lineage cells, specifically neutrophils, is essential to mediate HA cryogel degradation. Post-HSCT neutropenia in recipient mice delayed degradation of HA cryogels by up to 3 weeks. We harnessed the neutrophil-responsive degradation to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. Sustained release of G-CSF from HA cryogels enhanced post-HSCT neutrophil recovery, comparable to pegylated G-CSF, which, in turn, accelerated cryogel degradation. HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.

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Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their hosts metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the flys resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetses viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetses reproductive fitness. Using a Gff line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Author SummaryEndosymbiotic bacteria regulate numerous aspects of their hosts reproductive physiology. Natural populations of the tsetse fly, Glossina fuscipes fuscipes (Gff), house heterogeneous infections with the bacterium Spiroplasma glossinidia. Infection with the bacterium results in the presentation of several phenotypes in both male and female Gff that would put them at a significant reproductive disadvantage when compared to their counterparts that do not house the bacterium. These Spiroplasma induced phenotypes include changes in sex-biased gene expression in the reproductive organs, a depletion in the availability of metabolically critical lipids in pregnant females that results in delayed larval development, and compromised sperm fitness. These findings indicate that Spiroplasma exerts an overall negative impact on both male and female reproductive fitness and thus likely has a profound effect on fly population structure. This outcome, in conjunction with the fact that Spiroplasma infected tsetse are unusually refractory to infection with pathogenic African trypanosomes, indicates that the bacterium could be experimentally exploited to reduce disease transmission through the fly.

sentences: RNA Helix Thermodynamics: The End Game Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated [Formula] separately from other [Formula] stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37{degrees} C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

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Remorin proteins serve as membrane topology scaffolds in plants Organization of membrane topologies in plants has so far been mainly attributed to the cell wall and the cytoskeleton. Taking rhizobial infections of legume root cells, where plasma membranes undergo dynamic and large-scale topology changes, as an initial model, we challenged this paradigm and tested whether additional scaffolds such as plant-specific remorins that accumulate on highly curved and often wall-less plasma membrane domains, control local membrane dynamics. Indeed, loss-of-function mutants of the remorin protein SYMREM1 failed to develop stabilized membrane tubes as found in colonized cells in wild-type plants, but released empty membrane spheres instead. Expression of this and other remorins in wall-less protoplasts allowed engineering different membrane topologies ranging from membrane blebs to long membrane tubes. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. This function is likely supported by remorin oligomerization into antiparallel dimers and the formation of higher order membrane scaffolding structures. Taken together we describe an evolutionary confined mechanism that allows the stabilization of large-scale membrane conformations and curvatures in plants.

One-sentence summaryThe remorin SYMREM1 evolved as structural membrane scaffold that stabilizes membrane tubulation and curvature during symbiotic intracellular infections.

sentences: The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands DNA can adopt non-canonical structures that have important biological functions while also providing structural diversity for nanotechnology applications. Here, we describe the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed us to characterize and establish structural parameters of d(CGA) triplets in the parallel-stranded duplex form. Our results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C-CH+ base pair.

sentences: Conservation and divergence of poly(A) tail regulation during the mammalian oocyte-to-embryo transition Poly(A) tail length and non-A residues are vital for oocyte-to-embryo transition (OET) in mice and humans1-5. However, the role of poly(A) tail length and non-A residues during OET in other commonly used mammalian animal models for human diseases remains unexplored. In addition, the degree of conservation in maternal mRNA poly(A) tail dynamics during OET across different mammal species is unknown. Here, we conduct a comparative analysis of the poly(A) tails during OET across four species: mice, rats, pigs, and humans. Dynamics during OET found to be conserved across all four species include: maternal mRNA deadenylation during oocyte maturation and re-polyadenylation after fertilization; a fall-rise trend in poly(A) tail length distribution; a rise-fall trend in the ratio of poly(A) tails with non-A residues; higher abundance of non-A residues in poly(A) tails of maternal mRNA than in zygotic genome activation (ZGA) mRNA; maternal mRNA with U residues degrades faster than those without U residues at the stage when ZGA takes place. While in mice and rats maternal mRNA deadenylation is impaired in parthenogenetic embryos and ZGA inhibition leads to blocked maternal mRNA deadenylation in mice and humans. In contrast, the length of consecutive U residues and the duration time of U residues in poly(A) tail diverges across the four species. Together, these findings reveal that the poly(A) tail mediated maternal mRNA post-transcriptional regulation is highly conserved in mammals with unique divergences in the length and life-span of U residues, providing new insights for the further understanding of OET across different mammals.

sentences: Predictions of Skin Permeability Using Molecular Dynamics Simulation from Two-Dimensional Sampling of Spatial and Alchemical Perturbation Reaction Coordinates A molecular level understanding of skin permeation may rationalize and streamline product development, and improve quality and control, of transdermal and topical drug delivery systems. It may also facilitate toxicity and safety assessment of cosmetics, skin care products. Here, we present new molecular dynamics simulation approaches that make it possible to efficiently sample the free energy and local diffusion coefficient across the skins barrier structure and predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates with so-called Accelerated Weight Histograms, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for twenty permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the ob-tained [Formula] mean square difference was 0.8 cm2 h-2.

sentences: Chromatin Bridges, not Micronuclei, Activate cGAS after Drug-induced Mitotic Errors in Human Cells Mitotic errors can activate cGAS and induce type-I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of anti-mitotic drugs to perturb mitosis in fibroblasts and measured abnormal nuclear morphologies, cGAS localization and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2'3-cGAMP to fibroblasts. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and anti-tumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

sentences: Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of -diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere -diversity loss, but can be driven by {beta}-diversity decay across space and time.

sentences: On the distribution of spinal premotor interneurons The activity of flexor and extensor motor neurons is tightly regulated by a network of interneurons in the spinal cord. The introduction of rabies retrograde monosynaptic tracing has provided a powerful method to map interneurons directly connected to motor neurons so as to visualize premotor circuits. Previous strategies have used AAV for complementing rabies glycoprotein expression in motor neurons to obtain selectivity in transsynaptic transfer to identify premotor interneurons innervating specific motor neuron pools These studies revealed differences in the location of flexor and extensor premotor interneurons. Here, we report that by using a genetic approach to complement rabies glycoprotein expression in motor neurons, we did not observe any differences in the distribution of flexor and extensor premotor interneurons. In order to identify possible causes for these paradoxical findings, we discuss advantages and caveats of the experimental designs and suggest ways forward to resolve possible ambiguities. Furthermore, to obtain a complete picture of existing approaches and results we ask for contributions from the scientific community describing the use of additional mouse models, viral constructs, and complementation methods. The aim is to generate an open, comprehensive database to understand the specific organisation of premotor circuits.

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Single nuclei transcriptome of the Lesser Duckweed Lemna minuta reveals cell trajectories for an entire plant The ability to trace every cell in some model organisms has led to the fundamental understanding of development and cellular function. However, in plants the complexity of cell number, organ size and developmental times makes this a challenge even in the diminutive model plant Arabidopsis thaliana. Here we develop the Lesser Duckweed Lemna minuta as a model with a reduced body plan, small genome size and clonal growth pattern that enables simultaneous tracing of cells from the entire plant over the complete developmental cycle. We generated a chromosome-resolved genome for the 360 megabase genome and defined the growth trajectory of the entire plant with single nuclei RNA sequencing. The L. minuta gene complement represents a primarily non-redundant set with only the ancient tau whole genome duplication shared with all monocots, and paralog expansion as a result of tandem duplications related to phytoremediation. Thirteen distinct cell types representing meristem, the leaf-stem fusion called a frond, and root-like tissues were defined using gene orthology with single cell expression from model plants, gene ontology categories, and cell trajectory analysis. Dividing meristem cells give rise to two main branches of root-transition and mesophyll cells, which then give rise to terminally differentiated parenchyma, epidermal and root cells. Mesophyll tissues express high levels of elemental transport genes consistent with this tissue playing a role in L. minuta wastewater detoxification. The L. minuta genome and cell map provide a paradigm to decipher developmental genes and pathways for an entire plant.

Sentence summaryGenome and single nuclei transcriptome of the Lesser Duckweed Lemna minuta enables tracing of all developmental, transitional and terminal cells of an entire plant.

sentences: iDESC: Identifying differential expression in single-cell RNA sequencing data with multiple subjects Single-cell RNA sequencing (scRNA-seq) enables assessment of transcriptome-wide changes at single-cell resolution. However, dominant subject effect in scRNA-seq datasets with multiple subjects severely confounds cell-type-specific differential expression (DE) analysis. We developed iDESC to separate subject effect from disease effect with consideration of dropouts to identify DE genes. iDESC was shown to have well-controlled type I error and high power compared to existing methods and obtained the best consistency between datasets and disease relevance in two scRNA-seq datasets from same disease, suggesting the importance of considering subject effect and dropouts in the DE analysis of scRNA-seq data with multiple subjects.

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Mechanisms that ensure speed and fidelity in eukaryotic translation termination Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

One Sentence SummaryDirect visualization of eukaryotic translation reveals the dynamics of termination at stop codons.

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Threshold Values for the Gini Variable Importance: A Empirical Bayes Approach Random Forests (RF) are a very widely used modelling tool. Lundberg et al. (2019) concludes that no nonlinear model had a more widespread popularity, from health care to academia to industry, than random forests and decision trees. The bounds of the methodology are still being extended. Bayat et al. (2020) give an example with 80 million variables.

It is highly desirable that RF models be made more interpretable and a large part of that is a better understanding of the characteristics of the variable importance measures generated by the RF. Due to its speed and ease of calculation, we consider the mean decrease in node "impurity" (MDI) variable importance (VI) and address the question of setting a significance level.

The report is organized as follows:

O_LIwe first consider the question of multiple testing in the case of multiple measurements made on two groups (the standard microarray set up Efron (2008)). We show that some standard approaches for multiple testing can fail severely due to the correlation structure of the measurement and other modelling failures. We show that deriving the null distribution by permutation does not fix the problem. This point applies to determining the null distribution of variable importances as well as many other statistical tests;
C_LIO_LIwe show that variable correlation can either increase or decrease the (MDI) of variables in different settings. We also show that there is an additional problem with the permutation null due to the functional relationships between the statistics;
C_LIO_LIWe consider the empirical Bayes argument of Efron (2005) and model the VI as a mixture of two distribution, a null and a non-null distribution. We find that unlike the relatively well behaved case considered in Efrons papers, there are a number of issues here:
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- the distribution may be multi-modal, which creates modelling difficulties;
- the null distribution is not of an obvious form, as it is not symmetric.

O_LIwe resolve these issues to derive a fast, plausible, empirical Bayes method for selecting significant variables while controlling the false discovery rate.
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An mRNA SARS-CoV-2 vaccine employing Charge-Altering Releasable Transporters with a TLR-9 agonist induces neutralizing antibodies and T cell memory The SARS-CoV-2 pandemic has necessitated the rapid development of prophylactic vaccines. Two mRNA vaccines have been approved for emergency use by the FDA and have demonstrated extraordinary effectiveness. The success of these mRNA vaccines establishes the speed of development and therapeutic potential of mRNA. These authorized vaccines encode full-length versions of the SARS-CoV-2 spike protein. They are formulated with Lipid Nanoparticle (LNP) delivery vehicles that have inherent immunostimulatory properties. Different vaccination strategies and alternative mRNA delivery vehicles would be desirable to ensure flexibility of future generations of SARS-CoV-2 vaccines and the development of mRNA vaccines in general.

Here, we report on the development of an alternative mRNA vaccine approach using a delivery vehicle called Charge-Altering Releasable Transporters (CARTs). Using these inherently nonimmunogenic vehicles we can tailor the vaccine immunogenicity by inclusion of co-formulated adjuvants such as oligodeoxynucleotides with CpG motifs (CpG-ODN). Mice vaccinated with the mRNA-CART vaccine developed therapeutically relevant levels of RBD-specific neutralizing antibodies in both the circulation and in the lung bronchial fluids. In addition, vaccination elicited strong and long lasting RBD-specific TH1 T cell responses including CD4+ and CD8+ T cell memory.

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Experimental susceptibility of North American raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) to SARS-CoV-2 Skunks and raccoons were intranasally inoculated or indirectly exposed to SARS-CoV-2. Both species are susceptible to infection; however, the lack of, and low quantity of infectious virus shed by raccoons and skunks, respectively, and lack of cage mate transmission in both species, suggest that neither species are competent SARS-CoV-2 reservoirs.

Article Summary LineExperimental SARS-CoV-2 inoculation of North American raccoons and striped skunks showed susceptibility to infection, but transient, low-level shedding suggests that neither species is likely to be a competent natural reservoir.

sentences: Complementary resource preferences spontaneously emerge in diauxic microbial communities Many microbes grow diauxically, utilizing the available resources one at a time rather than simultaneously. The properties of communities of microbes growing diauxically remain poorly understood, largely due to a lack of theory and models of such communities. Here, we develop and study a minimal model of diauxic microbial communities assembling in a serially diluted culture. We find that unlike co-utilizing communities, diauxic community assembly repeatably and spontaneously leads to communities with complementary resource preferences, i.e., communities where species prefer different resources as their top choice. Simulations and theory explain that the emergence of complementarity is driven by the disproportionate contribution of the top choice resource to the growth of a diauxic species. Additionally, we develop a geometric approach for analyzing serially diluted communities, with or without diauxie, which intuitively explains several additional emergent community properties, such as the apparent lack of species which grow fastest on a resource other than their most preferred resource. Overall, our work provides testable predictions for the assembly of natural as well as synthetic communities of diauxically shifting microbes.

sentences: How ATP and dATP act as molecular switches to regulate enzymatic activity in the prototypic bacterial class Ia ribonucleotide reductase Class Ia ribonucleotide reductases (RNRs) are subject to allosteric regulation to maintain the appropriate deoxyribonucleotide levels for accurate DNA biosynthesis and repair. RNR activity requires a precise alignment of its 2 and {beta}2 subunits such that a catalytically-essential radical species is transferred from {beta}2 to 2. In E. coli, when too many deoxyribonucleotides are produced, dATP binding to RNR generates an inactive 4{beta}4 state in which {beta}2 and 2 are separated, preventing radical transfer. ATP binding breaks the -{beta} interface, freeing {beta}2 and restoring activity. Here we investigate the molecular basis for allosteric activity regulation in the prototypic E. coli class Ia RNR. Through the determination of six crystal structures we are able to establish how dATP binding creates a binding pocket for {beta} on  that traps {beta}2 in the inactive 4{beta}4 state. These structural snapshots also reveal the numerous ATP-induced conformational rearrangements that are responsible for freeing {beta}2. We further discover, and validate through binding and mutagenesis studies, a previously unknown nucleotide binding site on the  subunit that is crucial for the ability of ATP to dismantle the inactive 4{beta}4 state. These findings have implications for the design of allosteric inhibitors for bacterial RNRs.

sentences: Antibiotic-induced accumulation of lipid II sensitizes bacteria to antimicrobial fatty acids Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is of major clinical importance for the treatment of Gram-positive bacterial infections. Here we show that unsaturated fatty acids (UFAs) and vancomycin act synergistically to rapidly kill S. aureus, including vancomycin tolerant and resistant populations. Our results suggest that antibiotic-mediated accumulation of lipid II at the septum facilitates membrane invasion by antimicrobial UFAs. UFA-vancomycin dual treatment generates large fluid patches of flexible lipids in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. This mechanism of synergy may be exploited for the development of new antibiotic therapies that target lipid II to combat both antibiotic tolerance and resistance.

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Aberrant Temporal-spatial Patterns to Sad expressions in Major Depressive Disorders via Hidden Markov Model BackgroundThe pathological mechanisms of Major depressive disorders (MDD) is associated with over-expressing of negative emotions, and the overall temporal-spatial patterns underlying over-representation in depression still remained to be revealed to date. We hypothesized that the aberrant spatio-temporal attributes of the process of sad expressions relate to MDD and help to detect depression severity.

MethodsWe enrolled a total of 96 subjects including 47 MDDs and 49 healthy controls (HCs), and recorded their Magnetoencephalography data under a sad expressions recognition task. A hidden Markov model (HMM) was applied to separate the whole neural activity into several brain states, then to characterize the dynamics. To find the disrupted spatial-temporal features, power estimations and fractional occupancy of each state were contrasted between MDDs and HCs.

ResultsThree states were found over the period of emotional stimuli processing procedure. The visual state was mainly distributed in early stage (0 - 270ms) and the limbic state in middle and later stage (270ms - 600ms) of the task, while the fronto-parietal state remained a steady proportion across the whole period. MDDs activated statistically more in limbic system during limbic state (p = 0.0045) and less in frontoparietal control network during fronto-parietal state (p = 5.38*10-5) relative to HCs. Hamilton-Depression-Rating Scale scores was significantly correlated with the predicted severity value using the state descriptors (p = 0.0062, r = 0.3933).

DiscussionAs human brain exhibited varied activation patterns under the negative stimuli, MDDs expressed disrupted temporal-spatial activated patterns across varied stages involving the primary visual perception and emotional contents processing compared to HCs, indicting disordered regulation of brain functions. Furthermore, descriptors built by HMM could be potential biomarkers for identifying the severity of depression disorders.

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Attention Networks and the Intrinsic Network Structure of the Human Brain Attention network theory states that attention is not a unified construct but consists of three independent systems that are supported by separable distributed networks: an alerting network to deploy attentional resources in anticipation of upcoming events, an orienting network to direct attention to a cued location, and a control network to select relevant information at the expense of concurrently available information. Ample behavioral and neuroimaging evidence supports the dissociation of the three attention domains. The strong assumption that each attentional system is realized through a separable network, however, raises the question how these networks relate to the intrinsic network structure of the brain.

Our understanding of brain networks has advanced majorly in the past years due to the increasing focus on brain connectivity. It is well established that the brain is intrinsically organized into several large-scale networks whose modular structure persists across task states. Existing proposals on how the presumed attention networks relate to intrinsic networks rely mostly on anecdotal and partly contradictory arguments. We addressed this issue by mapping different attention networks with highest spatial precision at the level of cifti-grayordinates. Resulting group maps were compared to the group-level topology of 23 intrinsic networks which we reconstructed from the same participants resting state fMRI data. We found that all attention domains recruited multiple and partly overlapping intrinsic networks and converged in the dorsal fronto-parietal and midcingulo-insular network. While we observed a preference of each attentional domain for its own set of intrinsic networks, implicated networks did not match well to those proposed in the literature. Our results indicate a necessary refinement of the attention network theory.

sentences: Aurora B/C-dependent phosphorylation promotesRec8 cleavage in mammalian oocytes To generate haploid gametes, cohesin is removed in a step-wise manner from chromosome arms in meiosis I and the centromere region in meiosis II, to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease Separase[1, 2]. In yeast, Rec8 is kept in a non-phosphorylated state by the action of PP2A-B56, which is localised to the centromere region, thereby preventing cohesin removal from this region in meiosis I[3-5]. However, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and whether centromeric cohesin protection is indeed brought about by dephosphorylation of Rec8 preventing cleavage, in mammalian meiosis. The identity of one or several potential Rec8-specific kinase(s) is also unknown. This is due to technical challenges, as Rec8 is poorly conserved preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals, and additionally, there is no turn-over of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address how Rec8 cleavage is brought about in mammals, we adapted a biosensor for Separase to study Rec8 cleavage in single mouse oocytes by live imaging, and identified phosphorylation sites promoting cleavage. We found that Rec8 cleavage by Separase depends on Aurora B/C kinase activity, and identified a residue promoting cleavage and being phosphorylated in an Aurora B/C kinase-dependent manner. Accordingly, inhibition of Aurora B/C kinase during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation.

sentences: mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

sentences: Internal microbial zonation assists in the massive growth of marimo, a lake ball of Aegagropila linnaei in Lake Akan Marimo (lake ball) is an uncommon ball-like aggregation of the green alga, Aegagropila linnaei. Although A. linnaei is broadly distributed in fresh and brackish waters in the northern hemisphere, marimo colonies are found only in particular habitats. The colonies have been gradually shrinking in recent years. Nevertheless, it is not clear how and why A. linnaei forms such massive spherical aggregations. Here, we report the bacterial microbiomes inside various sizes and aggregating structures of natural marimo collected from Lake Akan, Japan. We observed multi-layers composed of sediment particles only in the sizeable radial-type marimo with a >20 cm diameter, not in the tangled-type marimo. The deeper layers were enriched by Nitrospira, potential novel sulphur-oxidizing bacteria, and sulphate-reducing Desulfobacteraceae bacteria. The sulphur cycle-related bacteria are unique to Lake Akan due to sulphur deposits from the nearby volcanic mountains. Some of them were also recovered from lake sediments. Microorganisms of the multi-layers would form biofilms incorporating nearby sediment, which would function as microbial "seals" within large radial-type marimo. We propose that the layer structure provides habitats for diverse bacterial communities, promotes airtightness of the marimo, and finally contributes to the massive growth of the aggregation. These findings provide a clue to deciphering the massive growth of endangered marimo aggregates.

sentences: Somnotate: An accurate, robust, and flexible sleep stage classifier for the experimentalist Manual sleep stage annotation is a time-consuming but often essential step in the analysis of sleep data. To address this bottleneck we need automated approaches that exhibit high levels of performance, are robust under different experimental conditions, are accessible, and meet the specific needs of sleep scientists. Here we develop an unbiased framework for assessing automated performance against a consensus derived from multiple experienced researchers. We then construct a new sleep stage classifier that combines automated feature extraction using linear discriminant analysis, with inference based on vigilance state-dependent contextual information using a hidden Markov model. This produces annotation accuracies that exceed expert performance on rodent electrophysiological data. We demonstrate that the classifier is robust to errors in the training data, compatible with different recording configurations, and maintains high performance during experimental interventions including sleep deprivation and optogenetic manipulations. Finally, the classifier quantifies and reports its certainty, which can be leveraged to identify ambiguous epochs for further examination. Ambiguous epochs are shown to include unsuccessful transitions between vigilance states, which may offer new insight into the mechanisms underlying sleep-wake dynamics. We call our classifier  Somnotate and make an implementation available to the neuroscience community.

sentences: Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics Interest in CRISPR diagnostics continues to increase. CRISPR-Cas12 and -Cas13 based detection are particularly interesting as they enable highly specific detection of nucleic acids. The fundamental sensitivity limits of Cas12 and Cas13 enzymes are governed by their kinetic rates and are critical to develop amplification-free assays. However, these kinetic rates remain poorly understood and their reporting has been inconsistent. We here measure kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a and LbuCas13a) and evaluate their limits of detection (LoD) for amplification-free target detection. Collectively, we here present quantitation of enzyme kinetics for 14 gRNAs and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. Importantly, we also validate the self-consistency our measurements by comparing trends and limiting behaviors with a Michaelis-Menten, trans-cleavage reaction kinetics model. Our measurements reveal that activated Cas12 and Cas13 enzymes exhibit typical trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. Moreover, for assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, we find most CRISPR enzymes have an amplification-free LoD in the picomolar range. We find also that successful detection of target requires cleavage (by activated CRISPR enzyme) of at least ~0.1% of the fluorescent reporter molecules. This fraction of cleaved reporters is required to differentiate signal from background, and we hypothesize that this fraction is largely independent of the detection method (i.e., endpoint vs reaction velocity). Our results provide a map of the feasible application range and highlight areas of improvement for the emerging field of CRISPR diagnostics.

sentences: Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection Clostridioides difficile is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that C. difficile exhibits heterogeneity in swimming motility and toxin production in vitro through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the flgB operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts C. difficile virulence in vivo remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into C. difficile R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth in vitro, yielding precisely modified mutants suitable for assessing virulence in vivo. In a hamster model of acute C. difficile infection, the phase-locked ON mutant caused greater toxin accumulation than the phase locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of C. difficile to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of C. difficile to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.

sentences: Quercetin prevents insulin dysfunction in hypertensive animals Angiotensin II induced increase in hypertension enhances oxidative stress and compromises insulin action and pancreatic function. Quercetin-rich foods are beneficial for hypertensive and diabetic animals owing to their antioxidant function. The aim of this study was to evaluate the antioxidant effects of quercetin in hypertensive rats on insulin action, signaling, and secretion. Wistar rats were randomly divided into three groups: sham, hypertensive rats (H), and hypertensive rats supplemented with quercetin (HQ). After three months of initial hypertension, quercetin was administered at 50 mg/kg/day for 30 days. Our results indicate that hypertension and serum lipid peroxidation levels were reduced by quercetin supplementation. We observed increased insulin sensitivity in adipose tissue, corroborating the insulin tolerance test, HOMA index, and improvements in lipid profile. Despite normal insulin secretion at 2.8 and 20 mM of glucose, animals treated with quercetin exhibited increased number of islets per section; increased protein expression of muscarinic receptor type 3, VEGF, and catalase in islets; and hepatic mRNA levels of Ide were normalized. In conclusion, supplementation with quercetin improved insulin action and prevented pancreatic and metabolic dysfunction.

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Evidence Graphs: Supporting Transparent and FAIR Computation, with Defeasible Reasoning on Data, Methods and Results IntroductionTransparency of computation is a requirement for assessing the validity of computed results and research claims based upon them; and it is essential for access to, assessment, and reuse of computational components. These components may be subject to methodological or other challenges over time. While reference to archived software and/or data is increasingly common in publications, a single machine-interpretable, integrative representation of how results were derived, that supports defeasible reasoning, has been absent.

MethodsWe developed the Evidence Graph Ontology, EVI, in OWL 2, with a set of inference rules, to provide deep representations of supporting and challenging evidence for computations, services, software, data, and results, across arbitrarily deep networks of computations, in connected or fully distinct processes. EVI integrates FAIR practices on data and software, with important concepts from provenance models, and argumentation theory. It extends PROV for additional expressiveness, with support for defeasible reasoning. EVI treats any computational result or component of evidence as a defeasible assertion, supported by a DAG of the computations, software, data, and agents that produced it.

ResultsWe have successfully deployed EVI for large-scale predictive analytics on clinical time-series data. Every result may reference its evidence graph as metadata, which can be extended when subsequent computations are executed.

DiscussionEvidence graphs support transparency and defeasible reasoning on results. They are first-class computational objects and reference the datasets and software from which they are derived. They support fully transparent computation, with challenge and support propagation. The EVI approach may be extended to include instruments, animal models, and critical experimental reagents.

sentences: Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free, non-invasive way to characterize cell-substrate interactions and heterogeneities in the cell populations. We quantified the critical adhesion strengths of MCF7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower adhesion strengths at {tau}50 than their non-invasive counterparts, HPL1D and MCF7. Detachment dynamics was inversely correlated with cell invasion potentials. A theoretical model, based on {tau}50 values and the distribution of cell areas on substrates, provided good fits to data from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions: non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. These correlations in adhesion strengths with cell invasiveness, demonstrated here, may be useful in cancer diagnostics and other pathologies featuring misregulation in adhesion.

sentences: The challenges of independence: ontogeny of at-sea behaviour in a long-lived seabird The transition to independent foraging represents an important developmental stage in the life cycle of most vertebrate animals. Juveniles differ from adults in various life history traits and tend to survive less well than adults in most long-lived animals. Several hypotheses have been proposed to explain higher mortality including that of inadequate/inferior foraging skills compared to adults, young naive individuals combining lack of experience and physical immaturity. Thus a change in behaviour, resulting in an improvement of skills acquired from growing experience, is expected to occur during a period of learning through the immaturity phase. Very few studies have investigated the ontogeny of foraging behaviour over long periods of time, particularly in long-lived pelagic seabirds, due to the difficulty of obtaining individual tracking data over several years. We investigated the foraging behaviour, through activity patterns, during the successive life stages of the endangered Amsterdam albatross by using miniaturized activity loggers on naive juveniles, immatures and adults. Juvenile naive birds during their first month at sea after leaving their colony exhibited lower foraging effort (greater proportion of time spent sitting on water, higher duration and more numerous bouts on water, and lower duration and less numerous flying bouts). Juveniles reached similar activity values to those of immatures and adults as early as the 2nd-3rd months since independence, suggesting a progressive improvement of foraging performances during the first two months since fledging. We found support for the body-size hypothesis with respect to sex differences in activity parameters according to time elapsed since departure from the colony and month of the year, consistent with the important sexual dimorphism in the Amsterdam albatross. Whatever the life stage considered, activity parameters exhibited temporal variability reflecting the modulation of foraging behaviour possibly linked to both extrinsic (i.e. environmental conditions such as variability in food resources or in wind) and intrinsic (i.e. energetic demands linked to plumage renew during moult) factors.

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The stability landscape of de novo TIM barrels explored by a modular design approach The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. High conformational stability lowers protein sensitivity to mutations and changes in the environment; thus, understanding and manipulating protein stability will expand the applications of de novo proteins. Since the ({beta}/)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

Significance StatementDe novo protein design expands our knowledge about protein structure and stability. The TIM barrel is a highly relevant fold used in nature to host a rich variety of catalytic functions. Here, we follow a modular approach to design and characterize a collection of de novo TIM barrels and subjected them to a thorough folding analysis. Non-additive effects modulate the increase in stability when different regions of the barrel are mutated, showing a wide variety of thermodynamic properties that allow them to navigate an unexplored region of the stability landscape found in natural TIM barrels. The design of stable proteins increases the applicability of de novo proteins and provides crucial information on the molecular determinants that modulate structure and stability.

One Sentence SummaryA family of designed TIM barrels with diverse thermodynamic properties shows epistatic effects on its stability landscape.

sentences: HERMES: a molecular formula-oriented method to target the metabolome Comprehensive metabolome analyses are hampered by low identification rates of metabolites due to suboptimal strategies in MS and MS2 acquisition, and data analysis. Here we present a molecular formula-oriented and peak detection-free method, HERMES, that improves sensitivity and selectivity for metabolite profiling in MS and structural annotation in MS2. An analysis of environmental water, E. coli, and human plasma extracts by HERMES showed increased biological specificity of MS2 scans, leading to improved mass spectral similarity scoring and identification rates when compared to iterative data-dependent acquisition (DDA). HERMES is available as an R package with a user-friendly graphical interface to allow data analysis and interactive tracking of compound annotations.

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Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon ObjectiveTendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency; these tendons are prone to debilitating injuries, the incidence of which increases with ageing. In equine tendons, energy storage is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the positional anterior tibialis and energy storing Achilles tendons, testing the hypothesis that the Achilles IFM has specialised composition and mechanical properties, which are lost with ageing.

MethodsWe used a multidisciplinary combination of mechanical testing, immunolocalisation and proteomics to investigate structure-function specialisations in functionally distinct human tendons and how these are altered with ageing.

ResultsThe IFM in the energy storing Achilles tendon is more elastic and fatigue resistant than the IFM in the positional anterior tibialis tendon, with a trend towards decreased fatigue resistance with age in the Achilles IFM. With ageing, alterations occur predominantly to the proteome of the Achilles IFM.

ConclusionThe Achilles tendon IFM is specialised for energy storage, and changes to its proteome with ageing are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of key energy storing specialisations and their changes with ageing offers insight towards developing effective treatments for tendinopathy.

Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIEnergy storing tendons in animals and humans are particularly prone to tendinopathy and the incidence increases with increasing age.
C_LIO_LIPrevious work in some animal models has shown that the specialisation of tendon properties for energy storage is achieved primarily through adaptation of the interfascicular matrix, with specialisation lost in ageing. However, the structural specialisations that provide the human Achilles tendon with its energy storing ability, and how these are affected by ageing, remain to be established.
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What does this study add?O_LIWe demonstrate that the interfascicular matrix in the human Achilles tendon is specialised for energy storing, with increased elastic recoil and fatigue resistance, and that these specialisations are partially lost with ageing, likely due to alterations to the proteome of the interfascicular matrix.
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How might this impact on clinical practice or future developments?O_LIShort term, the specialist IFM mechanics we have demonstrated can be detected with new developments in ultrasound functional imaging, offering improved opportunities for contextual tendinopathy diagnostics. Personalised rehabilitation programmes can now be explored and designed specifically to target IFM mechanics.
C_LIO_LILonger term, the knowledge of key specialisations in injury prone energy storing tendons and how they are affected by ageing, offers crucial insight towards developing cell or tissue engineering treatments targeted at restoring tendon structure and function post-injury, specifically targeted at the IFM.
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Comprehensive profiling of liver x receptor splicing in triple negative breast cancer reveals existence of novel splice variants that are prognostic for survival The liver x receptors (LXR) alpha and beta are ligand-responsive transcription factors that link homeostatic control of lipid metabolism with cancer pathophysiology and prognosis. LXR activity is elevated in triple negative breast cancer relative to other breast cancer subtypes, driving gene signatures associated with drug resistance and metastasis. The loci encoding LXR and LXR{beta} produce multiple alternatively spliced proteins, but the true range of variants and their relevance to cancer remain poorly defined. Seven splice variants of LXR or LXR{beta} were detected. Three have not been recorded previously and five were prognostic. High expression of full length LXR was associated with shorter disease-free survival but splice variants harbouring truncations of the ligand binding domain were prognostic for improved survival. All LXR{beta} variants were associated with longer disease-free survival. Mechanistically, while full length LXR positively correlated with target gene expression in primary samples, LXR{beta} was inversely correlated. We conclude that canonical LXR function is an oncogenic driver of triple negative tumour pathophysiology that can be countered by high expression of truncated splice variants and/or full length LXR{beta}.

HighlightsO_LIExpression of full length LXR is associated with shorter disease-free survival of triple negative breast cancer patients
C_LIO_LIA systematic evaluation of cell lines and primary tumour samples indicates LXR splicing is extensive in breast cancer
C_LIO_LIConfirmation of three new LXR splice variants at transcript and protein level
C_LIO_LIExpression of full length LXR{beta} or LXR splice variants that harbour truncated ligand binding domains are associated with better prognosis
C_LIO_LIExpression of LXR target genes is positively correlated with LXR in relapsed patients and inversely correlated with LXR{beta} in survivors.
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sentences: Laboratory Yeast Strains Rely On Oxidative Phosphorylation For Efficient ATP Production Even though it is a well-accepted fact that the energy metabolism of yeast is likely to impact all cellular activities, surprising little is known about the ATP homeostasis of particular yeast strains that are commonly used in cell biological studies. Therefore, we determined key parameters such as oxygen consumption and fermentation rates of the lab strain SEY6210. Our data indicated that even at high glucose concentrations, SEY6210 produces 30-50% of cellular ATP from oxidative phosphorylation. Loss of respiration, either by disrupting ATP synthase function or by growth in anaerobic conditions, was not fully compensated by fermentation and as a result affected energy intensive processes such as the maintenance of the plasma membrane proton gradient and the associated import of nutrients.

sentences: Lipid flippase dysfunction as a novel therapeutic target for endosomal anomalies in Alzheimer's disease {beta}-amyloid precursor protein (APP) and their metabolites are deeply involved in the development of Alzheimers disease (AD). Upon the upregulation of {beta}-site APP cleaving enzyme 1 (BACE1), its product, the {beta}-carboxyl-terminal fragment of APP ({beta}CTF), is accumulated in the early stage of sporadic AD brains. {beta}CTF accumulation is currently considered the trigger for endosomal anomalies to form enlarged endosomes, one of the earliest pathologies in AD. However, the details of the underlying mechanism remain largely unclear. In this study, using BACE1 stably-overexpressing cells, we describe that lipid flippase subcomponent TMEM30A interacts with accumulated {beta}CTF. Among the lipid flippases in endosomes, those composed of TMEM30A and active subcomponent ATP8A1 transports phospholipid, phosphatidylserine (PS), to the cytosolic side of the endosomes. The lipid flippase activity and cytosolic PS distribution are critical for membrane fission and vesicle transport. Intriguingly, accumulated {beta}CTF in model cells impaired lipid flippase physiological formation and activity, along with endosome enlargement. Moreover, in the brains of AD model mice before the amyloid-{beta} (A{beta}) deposition, the TMEM30A/{beta}CTF complex formation occurred, followed by lipid flippase dysfunction. Importantly, our novel A{beta}/{beta}CTF interacting TMEM30A-derived peptide "T-RAP" improved endosome enlargement and reduced {beta}CTF levels. These T-RAP effects could result from the recovery of lipid flippase activity. Therefore, we propose lipid flippase dysfunction as a key pathogenic event and a novel therapeutic target for AD.

sentences: Nicotinamide Riboside alleviates exercise intolerance in ANT1-deficient mice. Mitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. We have analyzed the exercise physiology of mice deficient in ANT1, demonstrating a peripheral limitation of skeletal muscle mitochondrial respiration. Upon exercise, lack of Nicotinamide adenine dinucleotide+ (NAD+) results in a substrate limitation and stalling of the TCA cycle and mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD+ levels in skeletal muscle and improved the exercise capacity and mitochondrial respiration. Thus, increasing NAD+ levels with nicotinamide riboside can alleviate the exercise intolerance associated with ANT1-deficiency, indicating the therapeutic potential of NAD+-stimulating compounds in specific mitochondrial myopathies.

sentences: Neural correlates of subsequent memory-related gaze reinstatement Mounting evidence linking gaze reinstatement- the recapitulation of encoding-related gaze patterns during retrieval- to behavioral measures of memory suggests that eye movements play an important role in mnemonic processing. Yet, the nature of the gaze scanpath, including its informational content and neural correlates, has remained in question. In the present study, we examined eye movement and neural data from a recognition memory task to further elucidate the behavioral and neural bases of functional gaze reinstatement. Consistent with previous work, gaze reinstatement during retrieval of freely-viewed scene images was greater than chance and predictive of recognition memory performance. Gaze reinstatement was also associated with viewing of informationally salient image regions at encoding, suggesting that scanpaths may encode and contain high-level scene content. At the brain level, gaze reinstatement was predicted by encoding-related activity in the occipital pole and basal ganglia, neural regions associated with visual processing and oculomotor control. Finally, cross-voxel brain pattern similarity analysis revealed overlapping subsequent memory and subsequent gaze reinstatement modulation effects in the parahippocampal place area and hippocampus, in addition to the occipital pole and basal ganglia. Together, these findings suggest that encoding-related activity in brain regions associated with scene processing, oculomotor control, and memory supports the formation, and subsequent recapitulation, of functional scanpaths. More broadly, these findings lend support to Scanpath Theorys assertion that eye movements both encode, and are themselves embedded in, mnemonic representations.

sentences: Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels Female Aedes aegypti mosquitoes feed on human blood, which they use to develop their eggs. It has been widely noted that some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. Here we tested mosquito attraction to skin odor collected from human subjects and identified people who are exceptionally attractive or unattractive to mosquitoes. Notably, these preferences were stable over several years, indicating consistent longitudinal differences in skin odor between subjects. We carried out gas chromatography/quadrupole time of flight-mass spectrometry to analyze the chemical composition of human skin odor in these subjects and discovered that highly attractive people produce significantly increased levels of carboxylic acids. Mosquitoes could reliably distinguish a highly attractive human from their weakly attractive counterparts unless we substantially diluted the odor of the "mosquito magnet." This is consistent with the hypothesis that odor concentration is a major driver of differential attraction, rather than the less-favored skin odor blend containing repellent odors, although these are not mutually- exclusive. Mosquitoes detect carboxylic acids with a large family of odor-gated ion channels encoded by the Ionotropic Receptor gene superfamily. Mutant mosquitoes lacking any of the Ionotropic Receptor (IR) co-receptors Ir8a, Ir25a, and Ir76b, were severely impaired in attraction to human scent but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in "mosquito-magnet" human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.

sentences: High potency of sequential therapy with only beta-lactam antibiotics Evolutionary adaptation is a major source of antibiotic resistance in bacterial pathogens. Evolution- informed therapy aims to constrain resistance by accounting for bacterial evolvability. Sequential treatments with antibiotics that target different bacterial processes were previously shown to limit adaptation through genetic resistance trade-offs and negative hysteresis. Treatment with homogeneous sets of antibiotics is generally viewed to be disadvantageous, as it should rapidly lead to cross-resistance. We here challenged this assumption by determining the evolutionary response of Pseudomonas aeruginosa to experimental sequential treatments involving both heterogenous and homogeneous antibiotic sets. To our surprise, we found that fast switching between only {beta}-lactam antibiotics resulted in increased extinction of bacterial populations. We demonstrate that extinction is favored by low rates of spontaneous resistance emergence and low levels of spontaneous cross- resistance among the antibiotics in sequence. The uncovered principles may help to guide the optimized use of available antibiotics in highly potent, evolution-informed treatment designs.

sentences: Novel Murine models of Mania and Depression Neuropathological mechanisms triggering manic syndrome or manic episodes in bipolar disorder remain poorly characterised, as the research progress is severely limited by the paucity of appropriate animal models. Here we developed a novel manic mice model by combining a series of chronic unpredictable rhythm disturbances (CURD), which include disruption of circadian rhythm, sleep deprivation, exposure to cone light, with subsequent interference of followed spotlight, stroboscopic illumination, high temperature stress, noise disturbance and foot shock. To validate this novel manic model, we used multiple behavioural and cell biology approaches comparing the CURD-model with healthy controls and depressed mice. The depression model was created by an exposure to an improved chronic unpredictable mild stress, which we defined as chronic unpredictable mild restraint (CUMR). A novel manic mice model induced by environmental stressors and free from genetic or pharmacological interventions will benefit research into pathological mechanisms of mania.

sentences: A horizon for haptic perception The spatial limits of sensory acquisition (its sensory horizon) is a fundamental property of any sensorimotor system. In the present study, we sought to determine the sensory horizon for the human haptic modality. At first blush, it seems obvious that the haptic system is bounded by the space where the body can interact with the environment (e.g., the arm span). However, the human somatosensory system is exquisitely tuned to sensing with tools--blind-cane navigation being a classic example of this. The horizon of haptic perception therefore extends beyond body space, but to what extent is unknown. We first used neuromechanical modelling to determine the theoretical horizon, which we pinpointed as six meters. We then used a psychophysical localization paradigm to behaviorally confirm that humans can haptically localize objects using a six-meter rod. This finding underscores the incredibly flexibility of the brains sensorimotor representations, as they can be adapted to sense with an object many times longer than the users own body.

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Meta-Research: Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts.

sentences: Reversible adhesion by type IV pili leads to formation of irreversible localized clusters Despite the fact a fundamental first step in the physiopathology of many disease-causing bacteria is the formation of long-lived, localized, multicellular clusters, the spatio-temporal dynamics of the cluster formation process, particularly on host tissues, remains poorly understood. Experiments on abiotic surfaces suggest that the colonization of a surface by swimming bacteria requires i) irreversible adhesion to the surface, ii) cell proliferation, and iii) a phenotypic transition from an initial planktonic state. Here, we investigate how Pseudomonas aeruginosa (PA) infects a polarized MDCK epithelium and show that contrary to what has been reported on the colonization of abiotic surfaces, PA forms irreversible bacterial clusters on apoptotic epithelial cell without requiring irreversible adhesion, cell proliferation, or a phenotypic transition. By combining experiments and a mathematical model, we reveal that the cluster formation process is regulated by type IV pili (T4P). Furthermore, we unveil how T4P quantitatively operate during adhesion on the biotic surface, finding that it is a stochastic process that involves an activation time, requires the retraction of pili, and results in reversible adhesion with a characteristic attachment time. Using a simple kinetic model, we explain how such reversible adhesion process leads to the formation of irreversible bacterial clusters and quantify the cluster growth dynamics.

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De-novo chromosome level assembly of plant genomes from long read sequence data Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. The chromosome level assembly of the contigs generated from long read sequencing has involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (PacBio HiFi) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii. a genome that has been used as model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig and the other 6 assembled into 2-4 main contigs. The small number of chromosome breaks appear to be due to highly repetitive regions of ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now seems possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed.

Significance statement (of up to two sentences)De novo assembly of near complete chromosome level plant genomes is now possible using current long read sequencing and assembly tools.

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The gut microbiome is associated with cocaine behavior and predicts addiction vulnerability in adult male rats The gut-brain axis is a bi-directional communication system through which microbial communities in the gut interact with the nervous system, perhaps influencing neuropsychiatric disorders such as drug abuse. This study used behavioral data and biological samples from the Cocaine Biobank to test the hypothesis that the gut microbiota can predict and reflect susceptibility to cocaine reinforcement. Adult male heterogenous rats were catheterized and allowed to self-administer cocaine in short-access sessions (2 hr/day, 10 days, 0.5 mg/kg per intravenous infusion), followed by progressive ratio (PR) tests, long-access sessions (6 hr/day, 14 days), and alternating blocks of PR, long-access, and footshock testing. Fecal samples were collected at three time points and bacterial 16s rRNA genes were sequenced to profile the microbiota. As expected, cocaine-related behavior varied among subjects, such that a quartile split identified low and high responders on each measure, as well as an overall addiction index. Although beta diversity in the microbiota at baseline and after short access did not predict membership in high or low addiction quartiles, linear discriminant analysis (LDA) identified taxa that were more robustly represented in low or high responders. Beta diversity after long access was different among quartiles, as were several specific taxa. Investigation of baseline differences revealed that high relative abundance of Akkermansia muciniphila predicted future low response rates, whereas Ruminococcaceae predicted high response. This study is the first to report that microbiota variability reflects levels of cocaine intake and that microbial profiles might facilitate diagnosis and identify risk factors predictive of drug vulnerability.

Significance StatementMicrobial organisms inhabiting the gut of animals appear to influence organismal function through various signaling pathways, ultimately affecting behavior and disease vulnerability. This experiment investigates links between gut bacteria and vulnerability to addiction-related behaviors in adult male rats. Not only did gut bacterial profiles change as a result of cocaine intake but also gut bacterial profiles before any exposure to cocaine predicted which animals would be high or low addiction-prone individuals. These results suggest that microbial profiles might facilitate diagnosis and identify risk factors predictive of drug addiction.

sentences: Multiple and Dissociable Effects of Sensory History on Working-Memory Performance AO_SCPLOWBSTRACTC_SCPLOWBehavioural reports of sensory information are biased by stimulus history. The nature and direction of such serial-dependence biases can differ between experimental settings - both attractive and repulsive biases towards previous stimuli have been observed. How and when these biases arise in the human brain remains largely unexplored. They could occur either via a change in sensory processing itself, post-perceptual maintenance or decision-making processes, or both. Here, we analysed behavioural and magnetoencephalographic data from a working-memory task in which participants were sequentially presented with two randomly oriented gratings, one of which was cued for recall at the end of the trial. Behavioural responses showed evidence for two distinct biases: 1) a within-trial repulsive bias away from the previously encoded orientation on the same trial, and 2) a between-trial attractive bias towards the task-relevant orientation on the previous trial. Multivariate classification of stimulus orientation revealed that neural representations during stimulus encoding were biased away from the previous grating orientation, regardless of whether we considered the within- or between-trial prior orientation - despite opposite effects on behaviour. These results suggest that repulsive biases occur at the level of sensory processing and can be overturned at post-perceptual stages to result in attractive biases in behaviour.

sentences: 3D reconstruction identifies loci linked to variation in angle of individual sorghum leaves Selection for yield at high planting density has reshaped the leaf canopy of maize, improving photosynthetic productivity in high density settings. Further optimization of canopy architecture may be possible. However, measuring leaf angles, the widely studied component trait of leaf canopy architecture, by hand is a labor and time intensive process. Here, we use multiple, calibrated, 2D images to reconstruct the 3D geometry of individual sorghum plants using a voxel carving based algorithm. Automatic skeletonization and segmentation of these 3D geometries enable quantification of the angle of each leaf for each plant. The resulting measurements are both heritable and correlated with manually collected leaf angles. This automated and scaleable reconstruction approach was employed to measure leaf-by-leaf angles for a population of 366 sorghum plants at multiple time points, resulting in 971 successful reconstructions and 3,376 leaf angle measurements from individual leaves. A genome wide association study conducted using aggregated leaf angle data identified a known large effect leaf angle gene, several previously identified leaf angle QTL from a sorghum NAM population, and novel signals. Genome wide association studies conducted separately for three individual sorghum leaves identified a number of the same signals, a previously unreported signal shared across multiple leaves, and signals near the sorghum orthologs of two maize genes known to influence leaf angle. Automated measurement of individual leaves and mapping variants associated with leaf angle reduce the barriers to engineering ideal canopy architectures in sorghum and other grain crops.

sentences: Analyzing the Perceptual Attributes of Electro-tactile Stimuli as Function of Various Signal Properties This study is designed to determine the proper electrical signals that induce various perceptual qualities. These perceptions are chosen from a wide range of feelings, comfort and even surface qualities such as smoothness or flatness. The goal is to derive the relationship between the electrical signal properties (i.e. voltage, frequency and duty cycle) and the perceived qualities which are understood by human observers and reveal any pattens in their reports. A total of 144 measurements are collected from 8 participants. The results are analyzed using numerous statistical methods. In the end, the governing factors are identified and a signal with specific properties is is determined for each of the perception qualities. A great number of plots are included in this study to facilitate the process of choosing the appropriate signal for each factor.

sentences: Integrin activation is an essential component of SARS-CoV-2 infection Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the I MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally:  inside-out signaling primes the ligand binding function of integrins via a talin dependent mechanism and  outside-in signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by G13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and G13 binding to the cytoplasmic tail of an integrins {beta} subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

sentences: Tuning the Pennes Perfusion Rate to Model Large Vessel Cooling Effects in Hepatic Radiofrequency Ablation Radio-frequency ablation (RFA) has become a popular method for the minimally invasive treatment of liver cancer. However, the success rate of these treatments depends heavily on the amount of experience the clinician possesses. Mathematical modelling can help mitigate this problem by providing an indication of the treatment outcome. Thermal lesions in RFA are affected by the cooling effect of both fine-scale and large-scale blood vessels. The exact model for large-scale blood vessels is advection-diffusion, i.e. a model capable of producing directional effects, which are known to occur in certain cases. In previous research, in situations where directional effects do not occur, the advection term in the blood vessel model has been typically replaced with the Pennes perfusion term, albeit with a higher-than usual perfusion rate. Whether these values of the perfusion rate appearing in literature are optimal for the particular vessel radii in question, has not been investigated so far. The present work aims to address this issue. An attempt has been made to determine, for values of vessel radius between 0.55 mm and 5 mm, best estimates for the perfusion rate which minimize the error in thermal lesion volumes between the perfusion-based model and the advection-based model. The results for the best estimate of the perfusion rate presented may be used in existing methods for fast estimation of RFA outcomes. Furthermore, the possible improvements to the presented methodology have been highlighted.

sentences: Rapid transgenerational adaptation in response to intercropping increases facilitation and reduces competition By capitalising on positive biodiversity-productivity relationships, intercropping provides opportunities to improve agricultural sustainability1. However, intercropping is generally implemented using commercial seeds that were bred for maximal productivity in monocultures, which might limit the benefits of crop diversity on yield2,3. Plants can adapt over generations to the level of surrounding plant diversity, notably through increases in niche differentiation4. However, this adaptation potential and the corresponding yield benefit potential have not been explored in annual crop systems. Here we show that plant-plant interactions among annual crops evolved towards increased facilitation and reduced competition when the plants coexistence history matched their current diversity setting, which led to an increase in overyielding of up to 58%. These higher yield benefits were linked to character convergence between species sharing the same coexistence history for two generations. Notably, the six crop species tested converged towards taller phenotypes with lower leaf dry matter content when grown in mixtures. This study provides the first empirical evidence for the importance of parental diversity affecting plant-plant interactions and ecosystem functioning of the following generations in annual cropping systems. These results have important implications for diversified agriculture as they demonstrate the yield potential of targeted cultivars for intercropping, which can be achieved through specific breeding for mixtures.

sentences: The rescue of epigenomic abnormalities in ICF1 patient iPSCs following DNMT3B correction is incomplete at a residual fraction of H3K4me3-enriched regions Background: Bi-allelic hypomorphic mutations in DNMT3B disrupt DNA methyltransferase activity and lead to Immunodeficiency, Centromeric instability, Facial anomalies syndrome, type 1 (ICF1). While several ICF1 phenotypes have been linked to abnormally hypomethylated repetitive regions, the unique genomic regions responsible for the remaining disease phenotypes remain largely uncharacterized. Here we explored two ICF1 patient-induced pluripotent stem cells (iPSCs) and their CRISPR/Cas9 corrected clones to determine whether gene correction can overcome DNA methylation defects and related/associated changes in the epigenome of non-repetitive regions. Results: Hypomethylated regions throughout the genome are highly comparable between ICF1 iPSCs carrying different DNMT3B variants, and significantly overlap with those in ICF1-peripheral blood and lymphoblastoid cell lines. These regions include large CpG island domains, as well as promoters and enhancers of several lineage-specific genes, in particular immune-related, suggesting that they are pre-marked during early development. The gene corrected ICF1 iPSCs reveal that the majority of phenotype-related hypomethylated regions re-acquire normal DNA methylation levels following editing. However, at the most severely hypomethylated regions in ICF1 iPSCs, which also display the highest increased H3K4me3 levels and enrichment of CTCF-binding motifs, the epigenetic memory persisted, and hypomethylation was uncorrected. Conclusions: Restoring the catalytic activity of DNMT3B rescues the majority of the aberrant ICF1 epigenome. However, a small fraction of the genome is resilient to this reversal, highlighting the challenge of reverting disease states that are due to genome-wide epigenetic perturbations. Uncovering the basis for the persistent epigenetic memory will promote the development of strategies to overcome this obstacle.

sentences: Bidirectional cooperation between Ubtf1 and SL1 determines RNA Polymerase I promoter recognition in cell and is negatively affected in the UBTF-E210K neuroregression syndrome. Transcription of the [~]200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the Taf1b subunit of SL1 causes a striking depletion UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

sentences: Task Demands Change Motion Information Coding in Area MT of the Macaque Neurons in the primate Middle Temporal (MT) area signal information about visual motion and work together with the lateral prefrontal cortex (LPFC) to support memory-guided comparisons of visual motion direction. These areas are reciprocally connected and both contain neurons that signal visual motion direction in the strength of their responses. Previously, LPFC was shown to display marked changes in stimulus coding with altered task demands. Since MT and LPFC work together, we sought to determine if MT neurons display similar changes with heightened task demands. We hypothesized that heightened working-memory task demands would improve the task-relevant information and precipitate memory-related signals in MT. Here we show that engagement in a motion direction comparison task altered non-sensory activity and improved stimulus encoding by MT neurons. We found that this improvement in stimulus information transmission was largely due to preferential reduction in trial-to-trial variability within a sub-population of highly direction-selective neurons. We also found that a divisive normalization mechanism accounted for seemingly contradictory effects of task-demands on a heterogeneous population of neurons.

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Hippocampal Neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism IntroductionThe neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear.

MethodsHere, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions.

ResultsThe acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies.

ConclusionsThese findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

HighlightsO_LIUnder hippocampal Y2R antagonism, place preference memory retrieval is enhanced
C_LIO_LISpatial retrieval enhancement under Y2R blockade is correlated with changes in regional brain energy metabolism
C_LIO_LIEnhanced retrieval associated CCO activity increases in the dorsal DG, while decreasing in the ventral CA1, IL cortex and mammillary bodies
C_LIO_LIY2R exert control over patterns of brain activation that are relevant for spatial memory expression
C_LI

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Human Cytomegalovirus uses a Host Stress Response to Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very Long Chain Fatty Acids Stress and virus infection are known to regulate lipid metabolism in cells. Human cytomegalovirus infection induces fatty acid (FA) elongation and increases the cellular abundance of lipids with very long-chain FA tails (VLCFAs). While reprogramming of metabolism can be stress-related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knockout PKR-like ER kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. We found that in HCMV-infected cells, PERK promotes the increase in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFAs tails. Consistent with the SFA/MUFA lipidome changes, PERK enhances the protein levels of FA elongase 7 (ELOVL7), which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection was independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Consistent with the PUFA lipidome changes, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK--and more broadly, ER stress--may be necessary for membrane biogenesis needed to generate infectious HCMV virions.

IMPORTANCEHCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator, PERK, controls fatty acid (FA) elongation and cellular abundance of several types of lipids following HCMV infection. Specifically, we found that PERK promotes FA elongase 7 synthesis of lipids with saturated/monounsaturated very long-chain FA tails which are important for building the viral membrane of infectious HCMV virions. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.

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Interaction of an α-synuclein epitope with HLA-DRB1*15:01 initiates early enteric features of Parkinson's disease in humanized mice Parkinsons disease (PD) patients possess circulating T cells that recognize specific -synuclein-(-syn)-derived epitopes. One epitope, -syn32-46, interacts strongly with the HLA-DRB1*15:01 allele implicated in multiple autoimmune diseases. Whether this interaction and -syn-specific T cells play roles in PD pathogenesis remains unknown. We report that -syn32-46 immunization of a humanized mouse that expresses HLA-DRB1*15:01 triggers intestinal inflammation. This enteric pathology is characterized by activation of innate and adaptive immune responses and type I interferon signaling as well as loss of enteric dopaminergic neurons in the submucosal plexus, and results in constipation and weight loss. Depletion of CD4+, but not CD8+, T cells partially rescues enteric neurodegeneration. Thus, an interaction between -syn32-46 and HLA-DRB1*15:01 induces gut inflammation and CD4+ T cell-mediated loss of enteric dopaminergic neurons in the humanized mice. These findings suggest a mechanism for the prodromal enteric features of PD and indicate future directions for disease screening and treatment.

HIGHLIGHTS AND eTOC BlurbO_LI-syn32-46 immunization of a HLA-DRB1*15:01 mouse triggers weight loss and constipation.
C_LIO_LI-syn32-46 immunizations induce gut inflammation and loss of enteric dopaminergic neurons.
C_LIO_LIDepletion of CD4+, but not CD8+, T cells partially rescues enteric neurodegeneration.
C_LIO_LIInteraction between -syn32-46 and HLA-DRB1*15:01 are critical for the prodromal phase of PD.
C_LI

Parkinsons disease (PD) patients have circulating T cells that recognize -synuclein-(-syn)-epitopes. One epitope -syn32-46, interacts with the HLA-DRB1*15:01; however, its role in PD pathogenesis remains unknown. Garretti et al. show that -syn32-46 immunization of a humanized mouse expressing HLA-DRB1*15:01 triggers intestinal inflammation, loss of enteric dopaminergic neurons, constipation and weight loss, suggesting a critical role in the prodromal PD.

sentences: In an arms race between host and parasite, a lungworm's ability to infect a toad is determined by host susceptibility, not parasite preference Evolutionary arms races can alter both parasite infectivity and host resistance, and it is difficult to separate the effects of these twin determinants of infection outcomes. Using a co-introduced, invasive host-parasite system (the lungworm Rhabdias pseudosphaerocephala and the cane toad Rhinella marina), we quantified behavioural responses of parasite larvae to skin-chemical cues of toads from different invasive populations, and rates at which hosts became infected following standardised exposure to lungworms. Chemical cues from toad skin altered host-seeking behaviour by parasites, similarly among populations. The number of infection attempts (parasite larvae entering the hosts body) also did not differ between populations, but rates of successful infection (establishment of adult worm in host lungs) was higher for range-edge toads than for range-core conspecifics. Thus, lower resistance to parasite infection in range-edge toads appears to be due to less effective immune defences of the host rather than differential behavioural responses of the parasite. In this ongoing host-parasite arms-race, changing outcomes appear to be driven by shifts in host immunocompetence.

sentences: The impact of mutational processes on structural genomic plasticity in cancer cells Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD through BRCA1 and BRCA2 inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

sentences: Neuronal Dot1l is a broad mitochondrial gene-repressor associated with human brain aging via H3K79 hypermethylation Methylation of histone 3 at lysine 79 (H3K79) and its catalyst, disrupter of telomeric silencing (Dot1l), have been coupled to multiple forms of stress like bioenergetic and ER challenges. However, studies on H3K79 methylation and Dot1l in the aging brain and neurons are very limited. This together with increasing evidence of a dynamic neuroepigenome made us wonder if H3K79 methylation and Dot1l could play unknown roles in brain aging and associated disorders. In aged humans, we found strong and consistent hypermethylation of H3K79 in neurons that accumulate lipofuscine, while neuronal Dot1l transcript abundance reacts to bioenergenic and oxidative challenges. Indeed, in dopaminergic neurons we found rapid global H3K79me turnover (<12h). While shortly after reduction of H3K79 methylation, synaptic transcripts decreased while mitochondrial genes, particularly respiratory chain transcripts increased. Strikingly, 6 months after reduction of Dot1l levels, almost solely a variety of mitochondrial genes linked to aging and Parkinsons disease remained increased. These profiles are in much detail inverse to those described in hallmark PD and aging studies and associate Dot1l and H3K79me with neuronal stress in the aging brain while putting Dot1l forward as dynamic master regulator of mitochondrial transcription in dopamine neurons.

sentences: Identifying synergies and antagonisms between plant defense traits in Asclepias syriaca As a general rule, plants defend against herbivores with multiple traits. The defense synergy hypothesis posits that some traits are more potent when co-expressed with others, compared to their independent potency. However, this hypothesis has rarely been tested outside of synergies within a class of particular phytochemicals, and seldom under field conditions. We tested for synergies between multiple defense traits of common milkweed (Asclepias syriaca) by assaying the performance of two specialist herbivores on plants in natural populations. We employed both standard regression and exploratory analysis using a novel application of Random Forests that allowed us to detect synergies between defense traits. In hypothesis testing, we found the first empirical evidence for a previously hypothesized synergy between one pair of co-expressed defense traits (latex and cardenolides), but not another (latex and trichomes). When exploring all potential interactions between pairs of traits we found eight synergies and five antagonisms in predicting herbivore performance. Half of the identified synergies involved carbon, which is the basis of several defenses including chemical and physical barriers to feeding, and also essential nutrients. Our findings suggest that defense synergies could explain co-expression of latex and cardenolides in milkweeds. This synergy may be common among the diverse plant species that employ latex as a defense. Future studies should test carbon-based synergies, which our work suggests are prevalent, as well as the other synergies identified in our exploratory analysis. Our analytic approach provides a general, flexible framework for more broadly discovering and predicting the coexpression of traits through their synergistic function.

sentences: Probing 3'UTRs as modular regulators of gene expression Genes are commonly abstracted into a coding sequence and cis-regulatory elements (CREs), such as promoters and terminators, and short sequence motifs within these regions. Modern cloning techniques allow easy assembly of synthetic genetic constructs from discrete cis-regulatory modules. However, it is unclear how much the contributions of CREs to gene expression depend on other CREs in the host gene. Using budding yeast, we probe the extent of composability, or independent effects, of distinct CREs. We confirm that the quantitative effect of a terminator on gene expression depends on both promoter and coding sequence. We then explore whether individual cisregulatory motifs within terminator regions display similar context dependence, using transcriptomewide datasets of mRNA decay. To test the extent of composability, we construct reporter genes consisting of combinations of motifs within various terminator contexts, paired with different promoters. Our results show that the effect of a motif on RNA abundance depends both on its host terminator, and also on the associated promoter sequence. This emphasises the need for improved motif inference that includes both local and global context effects, which in turn could aid in the accurate use of CREs for the engineering of synthetic genetic constructs.

sentences: Disease modeling by efficient genome editing using a near PAM-less base editor in vivo. Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing the NRN PAM sequence, we could introduce point mutations for the first time in an animal model and achieved up to 100% efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we applied our method to create a new zebrafish model for melanoma predisposition based on the simultaneous editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

sentences: CAR T cell therapy drives endogenous locoregional T cell dynamics in a responding patient with glioblastoma CAR T cell therapy has transformed clinical care and management of patients with certain hematological cancers. However, it remains unclear whether the success of CAR T cell therapy relies solely on CAR T cell engagement with tumor antigen, or if it also requires the stimulation of an individual patients endogenous T cell response. Here, we performed combined analysis of longitudinal, single cell RNA and T cell receptor sequencing on glioblastoma tumors, peripheral blood (PB), and cerebrospinal fluid (CSF) from a patient with recurrent multifocal glioblastoma that underwent a remarkable response followed by recurrence on IL13RA2-targeted CAR T cell therapy (Brown et al. 2016). Single cell analysis of a tumor resected prior to CAR T cell therapy revealed the existence of an inflamed tumor microenvironment including a CD8+ cytotoxic, clonally expanded and antigen specific T cell population that disappeared in the recurrent setting. Longitudinal tracking of T cell receptors uncovered distinct T cell dynamics classes in the CSF during CAR T cell therapy. These included T cell clones with transient dynamics, representing intraventricular CAR T cell delivery and endogenous T cell recruitment from the PB into the CSF; and a group of T cells in the cerebrospinal fluid, that tracked with clonally expanded tumor resident T cells and whose dynamics contracted concomitantly with tumor volume. Our results suggest the existence of an endogenous T cell population that was invigorated by intraventricular CAR T cell infusions, and combined with the therapy to produce a complete response.

sentences: Systems genetics uncovers microbe-lipid-host connections in the murine gut The molecular bases of how host genetic variation impact gut microbiome remain largely unknown. Here, we used a genetically diverse mouse population and systems genetics strategies to identify interactions between molecular phenotypes, including microbial functions, intestinal transcripts and cecal lipids that influence microbe-host dynamics. Quantitative trait loci (QTL) analysis identified genomic regions associated with variations in bacterial taxa, bacterial functions, including motility, sporulation and lipopolysaccharide production, and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and cecal levels of ornithine lipids (OL). Follow-up studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that OL have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits. Collectively, these results suggest that OL are key players in A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

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Chromatin accessibility of primary human cancers ties regional mutational processes with tissues of origin BackgroundRegional distribution of somatic mutations in cancer genomes associates with DNA replication timing (RT) and chromatin accessibility (CA), however normal tissues and cell lines have contributed these insights while associations with the epigenomes of primary cancers remain uncharacterized.

ResultsHere we model megabase-scale mutation burden in whole cancer genomes using [~]900 CA and RT profiles of primary cancers, normal tissues, and cell lines. CA profiles of primary cancers, rather than normal tissues, predict regional mutagenesis in most cancer types. Regional mutation burden associates with the CA profiles of matching cancer types, indicating tissue-specific determinants of mutagenesis. However, mutagenesis in squamous cell and lymphoid cancers instead associates with RT profiles. Mutational signatures also show tissue-specific associations with cancer epigenomes, especially for carcinogen-induced and unannotated signatures. Lastly, while each cancer type includes certain frequently-mutated genomic regions exceeding epigenome-informed predictions of mutation burden, these regions show a pan-cancer convergence to biological processes involved in development and cancer. Thus, modelling excess mutations using epigenomes highlights known cancer driver genes as well as frequently mutated non-coding regions.

ConclusionsThe dominant association of regional mutation burden with cancer epigenomes suggests that many passenger mutations are determined by the epigenetic landscapes of transformed cells and may occur later in tumor evolution. CA-informed models help find cancer genes and pathways with positive selection and highlight regions where additional mutation burden is contributed by local mutational processes. This study underlines the complex interplay of mutational processes, genome function and evolution in cancer and tissues of origin.

sentences: Versican binds collagen via its G3 domain and regulates the organization and mechanics of collagenous matrices Type I collagen is the most abundant structural protein in the body and, with other fibrillar collagens, forms the fibrous network of the extracellular matrix (ECM). Another group of ECM polymers, the glycosaminoglycans (GAGs) and GAG-modified proteoglycans, play important roles in regulating collagen behaviors and contribute to the compositional, structural and mechanical complexity of the ECM. While the binding between collagen and small leucine-rich proteoglycans (SLRPs) has been studied in detail, the interactions between collagen and the large bottlebrush proteoglycan versican are not well understood. Here, we report that versican binds collagen directly and regulates collagen structure and mechanics. Versican colocalizes with collagen fibers in vivo and binds to collagen via its C-terminal G3 domain (a non-GAG-modified domain present in all known versican isoforms) in vitro; it promotes the deposition of a highly-aligned collagen-rich matrix by fibroblasts. Versican also shows an unexpected effect on the rheology of collagen gels in vitro, causing decreased stiffness and attenuated shear strain stiffening, and the cleavage of versican in liver results in reduced tissue compression stiffening. Thus, versican is an important collagen binding partner, playing a role in modulating collagen organization and mechanics.

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Contribution of innate and adaptive immune cells to the elimination of  Salmonella enterica  serotype Enteritidis infection in young broiler chickens Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans through contaminated poultry products, as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate immune cells including natural killer (NK) cells, macrophages and dendritic cells (DCs) were assessed during 21 days post-infection (dpi). In parallel, numbers and function of {gamma}{delta} T cells, CD8+ and CD4+ T cells as well as antibody titres were determined. SE was observed in the intestine and spleen of SE-infected chickens at 7 dpi. NK and T cells responded first to SE at 1 and 3 dpi as indicated by increased numbers of intestinal IL-2R+ and 20E5+ NK cells, in addition to enhanced activation of intestinal and splenic NK cells. At 7 dpi in the spleen, the presence of macrophages and the expression of activation markers on DCs was increased. At 21 dpi, an increase in intestinal {gamma}{delta} and CD8+ T cell numbers was observed. Furthermore, SE-specific proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in all blood samples of SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells during early stages of infection and it is hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE in infected chickens is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance and prevent SE infection and colonization in young broiler chickens and hence increase food safety for humans.

Author summarySalmonella enterica serotype Enteritidis (SE) causes foodborne zoonotic diseases in humans, as well as a severe disease in young chickens. As a consequence of which health and welfare of humans and chickens are affected, resulting in substantial economic losses. To enable development of immune-mediated prevention strategies in chickens, more insight in the immune responses to SE is needed to understand how the infection is eliminated. For this purpose, we investigated non-specific and specific immune responses upon experimental SE infection in young broiler chickens. In this study, we found SE in the intestine and spleen of SE-infected chickens at 7 days post-infection (dpi). We show that natural killer (NK) cells respond first by enhanced presence and activation, followed by increased presence of macrophages and activation of dendritic cells. These early responses are hypothesized to stimulate the observed subsequent specific T cell and antibody responses. Better understanding of immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance and prevent SE infection and colonization in young chickens and hence reduce SE-related foodborne illness in humans.

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Parallel evolution of amphioxus and vertebrate small-scale gene duplications BackgroundAmphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their "2R" whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genome has duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates.

ResultsWe present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates, and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements.

ConclusionsIn contrast to their slower molecular and morphological evolution, amphioxus small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms.

sentences: Effect of SARS-CoV-2 spike mutations on animal ACE2 usage and in vitro neutralization sensitivity The emergence of SARS-CoV-2 variants poses greater challenges to the control of COVID-19 pandemic. Here, we parallelly investigated three important characteristics of seven SARS-CoV-2 variants, including two mink-associated variants, the B.1.617.1 variant, and the four WHO-designated variants of concerns (B.1.1.7, B.1.351, P.1, and B.1.617.2). We first investigated the ability of these variants to bind and use animal ACE2 orthologs as entry receptor. We found that, in contrast to a prototype variant, the B.1.1.7, B.1.351, and P.1 variants had significantly enhanced affinities to cattle, pig, and mouse ACE2 proteins, suggesting increased susceptibility of these species to these SARS-CoV-2 variants. We then evaluated in vitro neutralization sensitivity of these variants to four monoclonal antibodies in clinical use. We observed that all the variants were partially or completely resistant against at least one of the four tested antibodies, with B.1.351 and P.1 showing significant resistance to three of them. As ACE2-Ig is a broad-spectrum anti-SARS-CoV-2 drug candidate, we then evaluated in vitro neutralization sensitivity of these variants to eight ACE2-Ig constructs previously described in three different studies. All the SARS-CoV-2 variants were efficiently neutralized by these ACE2-Ig constructs. Interestingly, compared to the prototype variant, most tested variants including the variants of concern B.1.1.7, B.1.351, P.1, and B.1.617.2 showed significantly increased (up to [~]15-fold) neutralization sensitivity to ACE2-Ig constructs that are not heavily mutated in the spike-binding interface of the soluble ACE2 domain, suggesting that SARS-CoV-2 evolves toward better utilizing ACE2, and that ACE2-Ig is an attractive drug candidate for coping with SARS-CoV-2 mutations.

sentences: Interactions between genes altered during cardiotoxicity and neurotoxicity in zebrafish revealed using induced network modules analysis Zebrafish has become a prominent model organism to study toxicology due to its genomic similarity to humans, optical clarity, well defined developmental stages, short generation time and cost-effective maintenance. It also provides a shorter time frame for in vivo toxicology evaluation compared to mammalian experimental systems. As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health and the effects of toxicant exposure are emerging as critical public health concerns. Owing to this, in this study, we analyzed the impact of such chemical induced toxicity in zebrafish. We first searched the literature comprehensively for genes that were modulated during neurotoxicity and cardiotoxicity in zebrafish and then studied the interactions between those genes using induced network modules analysis of the database ConsensusPathDB. The induced network modules analysis helps to study gene interactions through various types of interactions like gene regulatory, biochemical, genetic and protein interactions. A constant communication between the heart and the brain is an important physiological phenomenon. Therefore, we also analyzed the interactions among genes that are modulated simultaneously during cardiotoxicity and neurotoxicity. This study has led us to identify some potential predictive biomarkers for neurotoxicity and cardiotoxicity in zebrafish.

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RapidoPGS: A rapid polygenic score calculator for summary GWAS data without a test dataset MotivationPolygenic scores (PGS) aim to genetically predict complex traits at an individual level. PGS are typically trained on genome-wide association summary statistics and require an independent test dataset to tune parameters. More recent methods allow parameters to be tuned on the training data, removing the need for independent test data, but approaches are computationally intensive. Based on fine-mapping principles, we present RapidoPGS, a flexible and fast method to compute PGS requiring summary-level GWAS datasets only, with little computational requirements and no test data required for parameter tuning.

ResultsWe show that RapidoPGS performs slightly less well than two out of three other widely-used PGS methods (LDpred2, PRScs, and SBayesR) for case-control datasets, with median r2 difference: -0.0092, -0.0042, and 0.0064, respectively, but up to 17,000-fold faster with reduced computational requirements. RapidoPGS is implemented in R and can work with user-supplied summary statistics or download them from the GWAS catalog.

Availability and implementationOur method is available with a GPL license as an R package from GitHub.

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Transfer rate of enveloped and non-enveloped viruses between fingerpads and surfaces Fomites can represent a reservoir for pathogens, which may be subsequently transferred from surfaces to skin. In this study we aim to understand how different factors (including virus type, surface type, time since last handwash, and direction of transfer) affect virus transfer rates, defined as the fraction of virus transferred, between fingerpads and fomites. To determine this, 360 transfer events were performed with 20 volunteers using Phi6 (a surrogate for enveloped viruses) and MS2 (a surrogate for non-enveloped viruses), and three clean surfaces (stainless steel, painted wood, and plastic). Considering all transfer events (all surfaces and both transfer directions combined), the mean transfer rates of Phi6 and MS2 were 0.17 and 0.26, respectively. Transfer of MS2 was significantly higher than Phi6 (P<0.05). Surface type was a significant factor that affected the transfer rate of Phi6: Phi6 is more easily transferred to and from stainless steel and plastic than to and from painted wood. Direction of transfer was a significant factor affecting MS2 transfer rates: MS2 is more easily transferred from surfaces to fingerpads than from fingerpads to surfaces. Data from these virus transfer events, and subsequent transfer rate distributions, provide information which can be used to refine quantitative microbial risk assessments. This study is the first to provide a large-scale data set of transfer events with a surrogate for enveloped viruses, which extends the reach of the study to the role of fomites in the transmission of human enveloped viruses like influenza and SARS-CoV-2.

ImportanceThis study created the first large-scale data set for the transfer of enveloped viruses between skin and surfaces. The data set produced by this study provides information on modelling the distribution of enveloped and non-enveloped virus transfer rates, which can aid in the implementation of risk assessment models in the future. Additionally, enveloped and non enveloped viruses were applied to experimental surfaces in an equivalent matrix to avoid matrix effects, so results between different viral species can be directly compared without confounding effects of different matrices. Our results indicating how virus type, surface type, time since last handwash, and direction of transfer affect virus transfer rates can be used in decision-making processes to lower the risk of viral infection from transmission through fomites.

sentences: Frequent paternal mitochondrial inheritance and rapid haplotype frequency shifts in copepod hybrids Mitochondria are assumed to be maternally inherited in most animal species, and this foundational concept has fostered advances in phylogenetics, conservation, and population genetics. Like other animals, mitochondria were thought to be solely maternally inherited in the marine copepod Tigriopus californicus, which has served as a useful model for studying mitonuclear interactions, hybrid breakdown, and environmental tolerance. However, we present PCR, Sanger sequencing, and Illumina Nextera sequencing evidence that extensive paternal mitochondrial DNA (mtDNA) transmission is occurring in inter-population hybrids of T. californicus. PCR on four types of crosses between three populations (total sample size of 376 F1 individuals) with 20% genome-wide mitochondrial divergence showed 2% to 59% of F1 hybrids with both paternal and maternal mtDNA, where low and high paternal leakage values were found in different cross directions of the same population pairs. Sequencing methods further verified nucleotide similarities between F1 mtDNA and paternal mtDNA sequences. Interestingly, the paternal mtDNA in F1s from some crosses inherited haplotypes that were uncommon in the paternal population. Compared to some previous research on paternal leakage, we employed more rigorous methods to rule out contamination and false detection of paternal mtDNA due to non-functional nuclear mitochondrial DNA fragments. Our results raise the potential that other animal systems thought to only inherit maternal mitochondria may also have paternal leakage, which would then affect the interpretation of past and future population genetics or phylogenetic studies that rely on mitochondria as uniparental markers.

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Cell fitness is an omniphenotype Moores law states that computers get faster and less expensive over time. In contrast in biopharma, there is the reverse spelling, Erooms law, which states that drug discovery is getting slower and costing more money every year. At the current pace, we estimate it costing $9.9T and it taking to the year 2574 to find drugs for less than 1% of all potentially important protein-protein interactions. Herein, we propose a solution to this problem. Borrowing from how Bitcoin works, we put forth a consensus algorithm for inexpensively and rapidly prioritizing new factors of interest (e.g., a gene or drug) in human disease research. Specifically, we argue for synthetic interaction testing in mammalian cells using cell fitness - which reflect changes in cell number that could be due many effects - as a readout to judge the potential of the new factor. That is, if we combine perturbing a known factor with perturbing the unknown factor and they produce a synergistic, i.e., multiplicative rather than additive cell fitness phenotype, this justifies proceeding with the unknown gene/drug in more complex models where the known perturbation is already validated. This recommendation is backed by the following evidence we demonstrate herein: 1) human genes currently known to be important to cell fitness involve nearly all classifications of cellular and molecular processes; 2) Nearly all human genes important in cancer - a disease defined by altered cell number - are also important in other common diseases; 3) Many drugs affect a patients condition and the fitness of their cells comparably. Taken together, these findings suggest cell fitness could be a broadly applicable phenotype for understanding gene, disease, and drug function. Measuring cell fitness is robust and requires little time and money. These are features that have long been capitalized on by pioneers using model organisms that we hope more mammalian biologists will recognize.

Short summaryCell fitness is a biological hash function that enables interoperability of biomedical data.

sentences: Double stranded RNA drives innate immune responses, sickness behavior and cognitive impairment dependent on dsRNA length, IFNAR1 expression and age Double stranded RNA is generated during viral replication. The synthetic analogue poly I:C is frequently used to mimic anti-viral innate immune responses in models of psychiatric and neurodegenerative disorders including schizophrenia, autism, Parkinsons disease and Alzheimers disease. Many studies perform limited analysis of innate immunity despite these responses potentially differing as a function of dsRNA molecular weight and age. Therefore fundamental questions relevant to impacts of systemic viral infection on brain function and integrity remain. Here, we studied innate immune-inducing properties of poly I:C preparations of different lengths and responses in adult and aged mice. High molecular weight (HMW) poly I:C (1-6kb, 12 mg/kg) produced more robust sickness behavior and more robust IL-6, IFN-I and TNF responses than poly I:C of <500 bases (low MW) preparations. This was partly overcome with higher doses of LMW (up to 80 mg/kg), but neither circulating IFN{beta} nor brain transcription of Irf7 were significantly induced by LMW poly I:C, despite brain Ifnb transcription, suggesting that brain IFN-dependent gene expression is predominantly triggered by circulating IFN{beta} binding of IFNAR1. In aged animals, poly I:C induced exaggerated IL-6, IL-1{beta} and IFN-I in the plasma and similar exaggerated brain cytokine responses. This was associated with acute working memory deficits selectively in aged mice. Thus, we demonstrate dsRNA length-, IFNAR1- and age-dependent effects on anti-viral inflammation and cognitive function. The data have implications for CNS symptoms of acute systemic viral infection such as those with SARS-CoV-2 and for models of maternal immune activation.

sentences: Functional Determination of Calcium Binding Sites Required for the Activation of Inositol 1,4,5-trisphosphate receptors. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors including IP3, Ca2+ and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring the biphasic regulation by Ca2+ are yet to be ascertained. Using comparative structural analysis of Ca2+ binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single channel open probability (P0) and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues are neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+ binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well conserved activation site is a common underlying mechanism resulted in increased channel activity shared by IP3Rs and RyRs.

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Predicting the presence and abundance of bacterial taxa in environmental communities through flow cytometric fingerprinting Microbiome management research and applications rely on temporally-resolved measurements of community composition. Current technologies to assess community composition either make use of cultivation or sequencing of genomic material, which can become time consuming and/or laborious in case high-throughput measurements are required. Here, using data from a shrimp hatchery as an economically relevant case study, we combined 16S rRNA gene amplicon sequencing and flow cytometry data to develop a computational workflow that allows the prediction of taxon abundances based on flow cytometry measurements. The first stage of our pipeline consists of a classifier to predict the presence or absence of the taxon of interest, with yields an average accuracy of 88.13{+/-}4.78 % across the top 50 OTUs of our dataset. In the second stage, this classifier was combined with a regression model to predict the relative abundances of the taxon of interest, which yields an average R2 of 0.35{+/-}0.24 across the top 50 OTUs of our dataset. Application of the models on flow cytometry time series data showed that the generated models can predict the temporal dynamics of a large fraction of the investigated taxa. Using cell-sorting we validated that the model correctly associates taxa to regions in the cytometric fingerprint where they are detected using 16S rRNA gene amplicon sequencing. Finally, we applied the approach of our pipeline on two other datasets of microbial ecosystems. This pipeline represents an addition to the expanding toolbox for flow cytometry-based monitoring of bacterial communities and complements the current plating- and marker gene-based methods.

ImportanceMonitoring of microbial community composition is crucial for both microbiome management research and applications. Existing technologies, such as plating and amplicon sequencing, can become laborious and expensive when high-throughput measurements are required. Over the recent years, flow cytometry-based measurements of community diversity have been shown to correlate well to those derived from 16S rRNA gene amplicon sequencing in several aquatic ecosystems, suggesting there is a link between the taxonomic community composition and phenotypic properties as derived through flow cytometry. Here, we further integrated 16S rRNA gene amplicon sequencing and flow cytometry survey data in order to construct models that enable the prediction of both the presence and the abundance of individual bacterial taxa in mixed communities using flow cytometric fingerprinting. The developed pipeline holds great potential to be integrated in routine monitoring schemes and early warning systems for biotechnological applications.

sentences: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules towards the nucleus. Capsid uncoating from the viral genome apparently occurs beyond the nuclear pore. Observation of post-entry events via microscopic detection of HIV-1 capsid protein (CA) is challenging, since epitope shielding limits immunodetection, and the genetic fragility of CA hampers other labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes showed that the amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells is consistent with a complete capsid and revealed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.

sentences: Rapid diversification of the Australian Amitermes group during late Cenozoic climate change Late Cenozoic climate change led to the progressive aridification of Australia over the past 15 million years. This gradual biome turnover fundamentally changed Australias ecosystems, opening new niches and prompting diversification of plants and animals. One example is the Australian Amitermes Group (AAG), consisting of the Australian Amitermes and affiliated genera. Although it represents the most speciose and diverse higher termite group in Australia, little is known about its evolutionary history. We used ancestral range reconstruction and diversification analyses to illuminate 1) the origin and phylogenetic relationships of the AAG, 2) biogeographical processes leading to the current continent-wide distribution, and 3) timing and pattern of diversification in the context of late Cenozoic climate change. By estimating the first time-calibrated phylogeny, we show that the AAG is a monophyletic group, whose ancestor arrived ~11-10 million years ago from Southeast Asia. Ancestral range reconstruction indicates that Australias monsoon region was the launching point for a continental radiation that has been shaped by range expansions and within-area speciation rather than vicariance. We found that multiple arid species diversified from mesic and tropical ancestors in the Plio-Pleistocene, but also observed diversification in the opposite direction. Finally, we show that two pulses of rapid diversification coincided with past climate change during the late Miocene and early Pliocene. Consistent with rapid diversification, species accumulation slowed, likely caused by progressive niche saturation. This study provides a stepping stone for predicting the future response of Australias termite fauna in the face of human-mediated climate change.

sentences: Inter-tissue convergence of gene expression during ageing suggests age-related loss of tissue and cellular identity Developmental trajectories of gene expression may reverse in their direction during ageing, a phenomenon previously linked to cellular identity loss. Our analysis of cerebral cortex, lung, liver and muscle transcriptomes of 16 mice, covering development and ageing intervals, revealed widespread but tissue-specific ageing-associated expression reversals. Cumulatively, these reversals create a unique phenomenon: mammalian tissue transcriptomes diverge from each other during postnatal development, but during ageing, they tend to converge towards similar expression levels, a process we term Divergence followed by Convergence, or DiCo. We found that DiCo was most prevalent among tissue-specific genes and associated with loss of tissue identity, which is confirmed using data from independent mouse and human datasets. Further, using publicly available single-cell transcriptome data, we showed that DiCo could be driven both by alterations in tissue cell type composition and also by cell-autonomous expression changes within particular cell types.

sentences: Individualized event structure drives individual differences in whole-brain functional connectivity Resting-state functional connectivity is typically modeled as the correlation structure of whole-brain regional activity. It is studied widely, both to gain insight into the brains intrinsic organization but also to develop markers sensitive to changes in an individuals cognitive, clinical, and developmental state. Despite this, the origins and drivers of functional connectivity, especially at the level of densely sampled individuals, remain elusive. Here, we leverage novel methodology to decompose functional connectivity into its precise framewise contributions. Using two dense sampling datasets, we investigate the origins of individualized functional connectivity, focusing specifically on the role of brain network "events" - short-lived and peaked patterns of high-amplitude cofluctuations. Here, we develop a statistical test to identify events in empirical recordings. We show that the patterns of cofluctuation expressed during events are repeated across multiple scans of the same individual and represent idiosyncratic variants of template patterns that are expressed at the group level. Lastly, we propose a simple model of functional connectivity based on event cofluctuations, demonstrating that group-averaged cofluctuations are suboptimal for explaining participant-specific connectivity. Our work complements recent studies implicating brief instants of high-amplitude cofluctuations as the primary drivers of static, whole-brain functional connectivity. Our work also extends those studies, demonstrating that cofluctuations during events are individualized, positing a dynamic basis for functional connectivity.

sentences: A Human 3D neural assembloid model for SARS-CoV-2 infection Clinical evidence suggests the central nervous system (CNS) is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although mechanisms remain unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points1. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Prior to infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral  replication hubs, with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new  assembloid model2 that supports SARS-CoV-2 entry and replication in neural tissue, and PCCOs serve as an experimental model for neural infection.

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Investigating relative contributions to psychiatric disease architecture fromsequence elements originating across multiple evolutionary time-scales It remains an open question when and why psychiatric and other disease evolved in human evolutionary history. We harness large genome-wide association studies and recent studies that have identified human-gained epigenetic marks, selective sweeps, archaic introgression, and accelerated evolution to help tackle this question by assessing the enrichment of heritability in these sequence elements for 41 complex traits. We find that human-gained epigenetic elements in the fetal brain harbor variants affecting skeletal, dermatological, and respiratory traits early and psychiatric traits later in development, that genomic sequences under selection since our divergence with Neanderthals are enriched in heritability for autism, and that regions depleted in ancestry from other hominids contribute more to heritability for psychiatric than other traits. Psychiatric disease may be a concomitant outcome of the evolutionary processes that made us uniquely human.

One-Sentence SummaryVariants that cause autism and other psychiatric disease today co-locate with sequence elements that have been important during human evolution.

sentences: Endomitosis controls tissue-specific gene expression during development Polyploid cells contain more than two copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either one nucleus (mononucleation) or two or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of polyploidy are largely unknown. Here, we assess the function of multinucleation in C. elegans intestinal cells through specific inhibition of binucleation without altering genome ploidy. Through single worm RNA sequencing, we find that binucleation is important for tissuespecific gene expression, most prominently for genes that show a rapid upregulation at the transition from larval development to adulthood. Regulated genes include vitellogenins, which encode yolk proteins that facilitate nutrient transport to the germline. We find that reduced expression of vitellogenins in mononucleated intestinal cells leads to progeny with developmental delays and reduced fitness. Together, our results show that binucleation facilitates rapid upregulation of intestine-specific gene expression during development, independently of genome ploidy, underscoring the importance of spatial genome organization for polyploid cell function.

sentences: Local rather than global H3K27me3 dynamics associates with differential gene expression in Verticillium dahliae Differential growth conditions typically trigger global transcriptional responses in filamentous fungi. Such fungal responses to environmental cues involve epigenetic regulation, including chemical histone modifications. It has been proposed that conditionally expressed genes, such as those that encode secondary metabolites but also effectors in pathogenic species, are often associated with a specific histone modification, lysine27 methylation of H3 (H3K27me3). However, thus far no analyses on the global H3K27me3 profiles have been reported under differential growth conditions in order to assess if H3K27me3 dynamics governs differential transcriptional. Using ChIP- and RNA-sequencing data from the plant pathogenic fungus Verticillium dahliae grown in three in vitro cultivation media, we now show that a substantial number of the identified H3K27me3 domains globally display stable profiles among these growth conditions. However, we do observe local quantitative differences in H3K27me3 ChIP-seq signal that associate with a subset of differentially transcribed genes between media. Comparing the in vitro results to expression during plant infection suggests that in planta-induced genes may require chromatin remodelling to achieve expression. Overall, our results demonstrate that some loci display H3K27me3 dynamics associated with concomitant transcriptional variation, but many differentially expressed genes are associated with stable H3K27me3 domains. Thus, we conclude that while H3K27me3 is required for transcriptional repression, it does not appear that transcriptional activation requires global erasure of H3K27me3. We propose that the H3K27me3 domains that do not undergo dynamic methylation may contribute to transcription through other mechanisms or may serve additional genomic regulatory functions.

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The effect of substrate stiffness on tensile force transduction in the epithelial monolayers In recent years, the importance of mechanical signaling and the cellular mechanical microenvironment in affecting cellular behavior has been widely accepted. Cells in epithelial monolayers are mechanically connected to each other and the underlying extracellular matrix (ECM), forming a highly connected mechanical system subjected to various mechanical cues from their environment, such as the ECM stiffness. Changes in the ECM stiffness have been linked to many pathologies, including tumor formation. However, our understanding of how ECM stiffness and its heterogeneities affect the transduction of mechanical forces in epithelial monolayers is lacking. To investigate this, we used a combination of experimental and computational methods. The experiments were conducted using epithelial cells cultured on an elastic substrate and applying a mechanical stimulus by moving a single cell by micromanipulation. To replicate our experiments computationally and quantify the forces transduced in the epithelium, we developed a new model that described the mechanics of both the cells and the substrate. Our model further enabled the simulations with local stiffness heterogeneities. We found the substrate stiffness to distinctly affect the force transduction as well as the cellular movement and deformation following an external force. Also, we found that local changes in the stiffness can alter the cells response to external forces over long distances. Our results suggest that this long-range signaling of the substrate stiffness depends on the cells ability to resist deformation. Furthermore, we found that the cells elasticity in the apico-basal direction provides a level of detachment between the apical cell-cell junctions and the basal focal adhesions. Our simulation results show potential for increased ECM stiffness, e.g. due to a tumor, to modulate mechanical signaling between cells also outside the stiff region. Furthermore, the developed model provides a good platform for future studies on the interactions between epithelial monolayers and elastic substrates.

Author summaryCells can communicate using mechanical forces, which is especially important in epithelial tissues where the cells are highly connected. Also, the stiffness of the material under the cells, called the extracellular matrix, is known to affect cell behavior, and an increase in this stiffness is related to many diseases, including cancers. However, it remains unclear how the stiffness affects intercellular mechanical signaling. We studied this effect using epithelial cells cultured on synthetic deformable substrates and developed a computational model to quantify the results better. In our experiments and simulations, we moved one cell to observe how the substrate stiffness impacts the deformation of the neighboring cells and thus the force transduction between the cells. Our model also enabled us to study the effect of local stiffness changes on the force transduction. Our results showed that substrate stiffness has an apparent impact on the force transduction within the epithelial tissues. Furthermore, we found that the cells can communicate information on the local stiffness changes over long distances. Therefore, our results indicate that the cellular mechanical signaling could be affected by changes in the substrate stiffness which may have a role in the progression of diseases such as cancer.

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

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

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Proteomic signature of the Dravet syndrome in the genetic Scn1a-A1783V mouse model BackgroundDravet syndrome is a rare, severe pediatric epileptic encephalopathy associated with intellectual and motor disabilities. Proteomic profiling in a mouse model of Dravet syndrome can provide information about the molecular consequences of the genetic deficiency and about pathophysiological mechanisms developing during the disease course.

MethodsA knock-in mouse model of Dravet syndrome with Scn1a haploinsufficiency was used for whole proteome, seizure and behavioral analysis. Hippocampal tissue was dissected from two-(prior to epilepsy manifestation) and four-(following epilepsy manifestation) week-old male mice and analyzed using LC-MS/MS with label-free quantification. Proteomic data sets were subjected to bioinformatic analysis including pathway enrichment analysis. The differential expression of selected proteins was confirmed by immunohistochemical staining.

ResultsThe findings confirmed an increased susceptibility to hyperthermia-associated seizures, the development of spontaneous seizures, and behavioral alterations in the novel Scn1a-A1873V mouse model of Dravet syndrome. As expected, proteomic analysis demonstrated more pronounced alterations following epilepsy manifestation. In particular, proteins involved in neurotransmitter dynamics, receptor and ion channel function, synaptic plasticity, astrogliosis, neoangiogenesis, and nitric oxide signaling showed a pronounced regulation in Dravet mice. Pathway enrichment analysis identified several significantly regulated pathways at the later time point, with pathways linked to synaptic transmission and glutamatergic signaling dominating the list.

ConclusionIn conclusion, the whole proteome analysis in a mouse model of Dravet syndrome demonstrated complex molecular alterations in the hippocampus. Some of these alterations may have an impact on excitability or may serve a compensatory function, which, however, needs to be further confirmed by future investigations. The proteomic data indicate that, due to the molecular consequences of the genetic deficiency, the pathophysiological mechanisms may become more complex during the course of the disease. Resultantly, the management of Dravet syndrome may need to consider further molecular and cellular alterations. Ensuing functional follow-up studies, this data set may provide valuable guidance for the future development of novel therapeutic approaches.

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The conventional dendritic cell 1 subset primes CD8+ T cells and traffics tumor antigen to drive anti-tumor immunity in the brain The central nervous system (CNS) antigen presenting cell (APC) which primes anti-tumor CD8+ T cell responses remains undefined. Elsewhere, the conventional dendritic cell 1 (cDC1) performs this role. However, steady-state brain cDC1 are rare; cDC localize to choroid plexus and dura. Using preclinical glioblastoma models and cDC1-deficient mice, we explored the role of cDC1 in CNS anti-tumor immunity. We determined that cDC1 mediate checkpoint blockade-induced survival benefit and prime neoantigen-specific CD8+ T cells against brain tumors. We observed that cDC, including cDC1, isolated from the tumor, the dura, and the CNS-draining cervical lymph nodes harbored a traceable fluorescent tumor-antigen. In patient samples, we observed several APC subsets (including the CD141+ cDC1-equivalent) infiltrating glioblastomas, meningiomas, and dura. In these same subsets, we identified a tumor-specific fluorescent metabolite of 5- aminolevulinic acid, which labels tumor cells during fluorescence-guided glioblastoma resection. Together, these data elucidate the specialized behavior of cDC1 and suggest cDC1 play a significant role in CNS anti-tumor immunity.

One Sentence SummaryCNS cDC1 engage in previously undefined behavior to establish immune responses against brain tumors.

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Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loops C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) [~]25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express [~]30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts.

Author SummaryDespite almost 40 years of innovation, an HIV vaccine to induce antibodies that block virus infection remains elusive. Challenges include the unparalleled sequence diversity of HIVs surface spikes and its dense sugar coat that limits antibody access. However, a growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints. To date, these kinds of antibodies have been difficult to induce by vaccination. However, two antibody targets, one at the spike apex and another at the side of the spikes are more forgiving in their  demands for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites for later use as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that the spikes, when expressed on particles, are infectious, allowing us to appraise our vaccine designs in an ideal manner; ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.

sentences: Dynamics of sex-biased gene expression over development in the stick insect Timema californicum AO_SCPLOWBSTRACTC_SCPLOWSexually dimorphic phenotypes are thought to arise primarily from sex-biased gene expression during development. Major changes in developmental strategies, such as the shift from hemimetabolous to holometabolous development, are therefore expected to have profound consequences for the dynamics of sex-biased gene expression. However, no studies have previously examined sex-biased gene expression during development in hemimetabolous insects, precluding comparisons between developmental strategies. Here we characterized sex-biased gene expression at three developmental stages in a hemimetabolous stick insect (Timema californicum): hatchlings, juveniles, and adults. As expected, the proportion of sex-biased genes gradually increased during development, mirroring the gradual increase of phenotypic sexual dimorphism. Sex-biased genes identified at early developmental stages were generally consistently male- or female-biased at later stages, suggesting their importance in sexual differentiation. Additionally, we compared the dynamics of sex-biased gene expression during development in T. californicum to those of the holometabolous fly Drosophila melanogaster by reanalyzing publicly available RNA-seq data from third instar larval, pupal and adult stages. In D. melanogaster, 84% of genes were sex-biased at the adult stage (compared to only 20% in T. californicum), and sex-biased gene expression increased abruptly at the adult stage when morphological sexual dimorphism is manifested. Our findings are consistent with the prediction that the dynamics of sex-biased gene expression during development differ extensively between holometabolous and hemimetabolous insect species.

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Pkd1 mutation has no apparent effects on peroxisome structure or lipid metabolism BackgroundMultiple studies of tissue and cell samples from patients and pre-clinical models of autosomal dominant polycystic kidney disease report abnormal mitochondrial function and morphology and suggest metabolic reprogramming is an intrinsic feature of this disease. Peroxisomes interact with mitochondria physically and functionally, and congenital peroxisome biogenesis disorders can cause various phenotypes, including mitochondrial defects, metabolic abnormalities and renal cysts. We hypothesized that a peroxisomal defect might contribute to the metabolic and mitochondrial impairments observed in autosomal dominant polycystic kidney disease.

MethodsUsing control and Pkd1-/- kidney epithelial cells, we investigated peroxisome abundance, biogenesis and morphology by immunoblotting, immunofluorescent and live cell imaging of peroxisome-related proteins and assayed peroxisomal specific {beta}-oxidation. We further analyzed fatty acid composition by mass spectrometry in kidneys of Pkd1fl/fl; Ksp-Cre mice. We also evaluated peroxisome lipid metabolism in published metabolomics datasets of Pkd1 mutant cells and kidneys. Lastly, we investigated if the C-terminus or full-length polycystin-1 co-localize with peroxisome markers by imaging studies.

ResultsPeroxisome abundance, morphology and peroxisome-related protein expression in Pkd1-/- cells were normal, suggesting preserved peroxisome biogenesis. Peroxisomal {beta}-oxidation was not impaired in Pkd1-/- cells, and there was no obvious accumulation of very long chain fatty acids in kidneys of mutant mice. Re-analysis of published datasets provide little evidence of peroxisomal abnormalities in independent sets of Pkd1 mutant cells and cystic kidneys, while providing further evidence of mitochondrial fatty acid oxidation defects. Imaging studies with either full length polycystin-1 or its C-terminus, a fragment previously shown to go to the mitochondria, showed minimal co-localization with peroxisome markers.

ConclusionsOur studies showed that loss of Pkd1 does not disrupt peroxisome biogenesis nor peroxisomedependent fatty acid metabolism.

Key points- While mitochondrial abnormalities and fatty acid oxidation impairment have been reported in ADPKD, no studies have investigated if peroxisome dysfunction contributes to these defects.
- We investigated peroxisome morphology, biogenesis and function in cell and animal models of ADPKD and investigated whether polycystin-1 co-localized with peroxisome proteins.
- Our studies show that loss of Pkd1 does not disrupt peroxisome biogenesis nor peroxisome-dependent fatty acid metabolism.

sentences: The cellular architecture of microvessels, pericytes and neuronal cell types in organizing regional brain energy homeostasis in mice Cerebrovasculature and its mural cells must meet dynamic energy demands of different neuronal cell types across the brain, but their spatial relationship is largely unknown. Here, we apply brain-wide mapping methods to create a comprehensive cellular-resolution resource comprising the distribution of and quantitative relationship between cerebrovasculature, pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes, as well as simulation-ready vascular tracing data in mice. We discover strikingly high densities of vasculature and pericytes with high blood perfusion in primary motor-sensory cortices compared to association cortices that show significant positive and negative correlation with parvalbumin+ and nNOS+ neurons, respectively. Thalamo-striatal areas linked to primary motor-sensory cortices also contain high densities of vasculature and pericytes compared to association areas. Collectively, our results unveil a finely tuned spatial relationship between cerebrovascular network and neuronal cell composition in meeting regional energy needs of the brain.

sentences: Acoustic detection and identification of individual rooks in field recordings using multi-task neural networks Individual-level monitoring is essential in many behavioural and bioacoustics studies, but collecting these data is costly in human effort. Many studies of bird vocalisations in particular also involve manipulating the animals or human presence during observations, which can bias vocal production. Autonomous recording units can be used to collect large amounts of data without human supervision, largely removing these sources of bias. Moreover, the recent progress of deep learning greatly facilitated analysing these large amounts of data, for instance to extract vocalisations, classify the species, or classify the vocalisation types in recordings. Acoustic individual identification, however, has so far largely remained limited to a single vocalisation type for a given species, preventing the use of these techniques for automated data collection. We developed a deep convolutional neural network to detect vocalisations as well as identify individuals regardless of the vocalisation produced. We tested it in a group of rooks, a Eurasian social corvid with a diverse vocal repertoire, and found the system to work reliably for these tasks. Our system represents a powerful tool to individually monitor groups of animals in both outdoor and indoor settings, even across long periods of time, and regardless of a species vocal complexity.

sentences: Conditional distribution modeling as an alternative method for covariates simulation: comparison with joint multivariate normal and bootstrap techniques Clinical trial simulation (CTS) is a valuable tool in drug development. To obtain realistic scenarios, the subjects included in the CTS must be representative of the target population. Common ways of generating virtual subjects are based upon bootstrap (BS) procedures or multivariate normal distributions (MVND). Here, we investigated the performance of an alternative method based on conditional distributions (CD). Covariates data from a hypertension drug development program were used. The methods were evaluated based on the original dataset (internal evaluation) and on their ability to reproduce an older, unobserved population (extrapolation). Similar results were obtained in the internal evaluation for summary statistics, yet BS was able to preserve the correlation structure of the empirical distribution, which was not adequately reproduced by MVND; CD was in between BS and MVND. BS does not allow to extrapolate to an unobserved population. When the dataset used to inform the extrapolation was well approximated by a MVND, the results from CD and MVND were comparable. However, improved extrapolation performance was observed for CD when deviations from normality assumptions occurred. If CTS is used to simulate within the observed distribution, BS is the preferred method. When extrapolating to new populations, a parametric method like CD/MVND is needed. In case the empirical multivariate distribution is characterized by linearly related covariates and unimodal marginal distributions, MVND can be used because of the simpler statistical framework and well-established use; however, if uncertainty about the MVND assumptions exists, CD will increase the confidence in the simulations compared to MVND.

sentences: c-Rel is required for IL-33-dependent activation of ILC2s Group 2 innate lymphoid cells (ILC2s) are emerging as important cellular regulators of homeostatic and disease-associated immune processes. The cytokine interleukin-33 (IL-33) promotes ILC2-dependent inflammation and immunity, with IL-33 having been shown to activate NF-{kappa}B in a wide variety of cell types. However, it is currently unclear which NF-{kappa}B members play an important role in IL-33-dependent ILC2 biology. Here, we identify the NF-{kappa}B family member c-Rel as a critical component of the IL-33-dependent activation of ILC2s. Although c-Rel is dispensable for ILC2 development, it is critical for ILC2 function in the lung, with c-Rel-deficient (c-Rel-/-) mice resistant to papain- and IL-33-induced lung inflammation. We also show that the absence of c-Rel reduces the IL-33-dependent expansion of ILC2 precursors and lower levels of IL-5 and IL-13 cytokine production by mature ILC2s in the lung. Together, these results identify the IL-33-c-Rel axis as a central control point of ILC2 activation and function.

sentences: Identification of RNA base pairs and complete assignment of nucleobase resonances by proton-detected solid-state NMR spectroscopy at 100 kHz MAS Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid-state NMR (ssNMR) is applicable to high molecular-weight complexes and does not require crystallization; thus, it is well-suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA-protein complex by ssNMR using conventional 13C- and 15N-detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H-detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin-systems together with their site-specific base pair pattern using sub-milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein-RNA complexes of any size.

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

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

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

sentences: Bio-hybrid micro-swimmers propelled by flagella isolated from C. reinhardtii Bio-hybrid micro-swimmers, composed of biological entities integrated with synthetic constructs, actively transport cargo by converting chemical energy into mechanical work in a fluid at low Reynolds number, where viscous drag dominates over inertia. Here, using isolated and demembranated flagella from green algae Chlamydomonas reinhardtii (C. reinhardtii), we build efficient axonemally-driven micro-swimmers that consume ATP to propel micron-sized beads. Depending on the calcium concentration, we observed two main classes of motion: Whereas beads move along curved trajectories at calcium concentrations below 0.03 mM, they are propelled along straight paths when the calcium concentration increases. In this regime, they reached velocities of approximately 20 m/sec, comparable to human sperm velocity in vivo. We relate this transition to the properties of beating axonemes, in particular the reduced static curvature with increasing calcium concentration. To quantify the motion, we used mode decomposition of the flagellar waveform, and we studied both analytically and numerically the propulsion of the bead as a function of the axonemal waveform and bead-axoneme attachment geometry. While our analysis semi-quantitatively describes the experimental results, it also reveals the existence of a counter-intuitive propulsion regime where the speed of the axonemally-driven bead increases with the size of the bead. Moreover, we demonstrated that asymmetric, sideways attachment of the axoneme to the bead can also contribute to the rotational velocity of the micro-swimmer. The uncovered mechanism has potential applications in the fabrication of synthetic micro-swimmers, and in particular, bio-actuated medical micro-robots for targeted drug delivery.

sentences: Probability-based mechanisms in biological networks with parameter uncertainty Mathematical models of biomolecular networks are commonly used to study cellular processes; however, their usefulness to explain and predict dynamic behaviors is often questioned due to parameter uncertainty. Here, we employ a novel dynamic network analysis within a Bayesian statistic context to probabilistically identify dominant reaction paths in signaling networks. We introduce our approach in an extrinsic apoptosis model using fluorescence microscopy data from HeLa cells for parameter inference. While Bayesian optimization yields several parameter vectors with equally good-fit to experimental data, three execution modes emerge upon analysis of the reaction flux throughout the network using a discretization theory approach. Each mode exhibits different behaviors to in silico perturbations through different mechanisms, thus underscoring the need for thorough characterization of model parameters and associated dynamics. We then apply our approach on the JAK2/STAT5 pathway in colony-forming unit-erythroid (CFU-E) cells. Our analysis provides mechanistic explanation for survival responses of CFU-E cell population that was impossible by considering protein concentration dynamics in isolation.

sentences: Sst+ GPi output neurons provide direct feedback to key nodes of the basal ganglia and drive behavioral flexibility. The GPi-LHb pathway is the main output of the basal ganglia suggested to shape motivated behaviors. We show here that Sst+ GPi-LHb neurons send direct feedback to key nodes of the basal ganglia: the GPe, the striatal striosomes, and dopamine neurons in the SNc. Chronic silencing of this pathway did not affect learning or execution of value-guided choices, but severely disrupted the ability to adapt choice-behavior and seek an alternative reward location after task reversal. Calcium imaging revealed that Sst+ GPi neurons did not signal outcome value or value updates during reversal learning. Instead, progressive suppression of the Sst+ GPi activity was linked to increased commitment to one choice, and activity increased during exploration of alternative choices. We propose that GPi Sst+ neurons drive behavioral flexibility through a direct feedback signal to balance the activity of key nodes in the basal ganglia.

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Induced Membrane Curvature Bypasses Clathrin's Essential Endocytic Function During membrane trafficking, flat membrane is rapidly remodeled to produce nanometer-scale vesicles. The mechanisms underlying this shape change are not completely understood, but coat proteins such as clathrin are implicated. Clathrins ability to bind to membranes of many different geometries casts uncertainty on its specific role in curvature generation and stabilization. Here, we used nanopatterning to produce substrates of ideal optical properties for live-cell imaging, with U-shaped features that bend a cells ventral plasma membrane into shapes characteristic of the energetically-unfavorable intermediate of clathrin- mediated endocytosis (CME). This induced plasma membrane curvature recruits the endocytic machinery, promoting productive endocytosis. Upon clathrin or AP2 disruption, CME sites on flat substrates are diminished. However, induced curvature rescues the localization, turnover and transferrin cargo uptake activities of CME sites after clathrin, but not AP2, disruption. These data establish that clathrins essential function during CME is to facilitate the evolution of membrane curvature rather than to scaffold CME protein recruitment.

SummaryCail et al. demonstrate that induced nanoscale membrane curvature recruits endocytic sites and produces vesicles in cells lacking the coat protein clathrin, while the adaptor protein AP2 is still required, showing that clathrins essential function is to facilitate curvature development of the nascent vesicle.

sentences: Small intestine lactobacilli growth promotion and immunomodulation in weaner pigs fed Cyberlindnera jadinii yeast high inclusion diet and exposed to enterotoxigenic Escherichia coli F4+: O149 1Enterotoxigenic Escherichia coli (ETEC) F4+: O149 is a causative agent for the development of post-weaning diarrhoea (PWD) in pigs that contributes to production losses. Yeast cell wall components used as a feed additive can modulate gut immunity and help protect animals from enteric infections. This work investigated how a novel yeast diet with high inclusion of yeast proteins (40% of crude protein) affected the course of ETEC mediated diarrhoea in weaner piglets from a farm with or without a history of post-weaning diarrhoea. We found that immune response to F4ab ETEC infection and appetite of the animals were altered by high inclusion C. jadinii yeast. The results indicate that the novel diet can support the diseased animals either directly through the effect of yeast beta-glucans and mannans or indirectly through the promotion of small intestine lactobacilli or both.

sentences: Glycoproteomics of Haloferax volcanii reveals an extensive glycoproteome and concurrence of different N-glycosylation pathways Glycosylation is one of the most complex post-translational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here we extend the phenotypic characterisation of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project, we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provides new insights into the roles of N-glycosylation in archaeal cell biology.

sentences: Brevican, Neurocan, Tenascin-C and Tenascin-R Act as Important Regulators of the Interplay between Perineuronal Nets, Synaptic Integrity, Inhibitory Interneurons and Otx2 Fast-spiking parvalbumin interneurons are critical for the function of mature cortical inhibitory circuits. Most of these neurons are enwrapped by a specialized extracellular matrix structure (ECM) called perineuronal net (PNN), which can regulate their synaptic input. In this study, we investigated the relationship between PNNs, parvalbumin interneurons and synaptic distribution on these cells in the adult primary visual cortex (V1) of quadruple knockout mice deficient for the ECM molecules brevican, neurocan, tenascin-C and tenascin-R. We used super-resolution structured illumination microscopy (SIM) to analyze PNN structure and associated synapses. Additionally, we examined parvalbumin and calretinin interneuron populations. We observed a reduction in the number of PNN-enwrapped cells and a clear disorganization of the PNN structure in the quadruple knockout V1. This was accompanied by an imbalance of inhibitory and excitatory synapses with a reduction of inhibitory and an increase of excitatory synaptic elements along the PNNs. Also, the number of parvalbumin interneurons was reduced in the quadruple knockout, while calretinin interneurons, which do not wear PNNs did not display differences in number. Interestingly, we found the transcription factor Otx2 homeoprotein positive cell population also reduced. Otx2 is crucial for parvalbumin and PNN maturation and a positive feedback loop between these parameters has been described. Collectively, these data indicate an important role of brevican, neurocan, tenascin-C and tenascin-R in regulating the interplay between PNNs, inhibitory interneurons, synaptic distribution as well as Otx2 in the V1.

sentences: Predicting activatory and inhibitory drug-target interactions based on mol2vec and genetically perturbed transcriptomes A computational approach to identifying drug-target interactions (DTIs) is a credible strategy for accelerating drug development and understanding the mechanisms of action of small molecules. However, current methods are limited to providing simple DTIs without mode of action (MoA), or they show unsatisfactory performance for a limited range of compounds and targets. Here, we propose AI-DTI, a novel method that predicts activatory and inhibitory DTIs by combining the mol2vec and genetically perturbed transcriptomes. We trained the model on large-scale DTIs with MoA and found that our model outperformed a previous model that predicted activatory and inhibitory DTIs. To extend the applicability, we applied the inferential method for the target vector so that our method can learn and predict a wider range of targets. Our method achieved satisfactory performance in an independent dataset where the target was unseen in the training set and a high-throughput screening dataset where positive and negative samples were explicitly defined. Finally, our method successfully rediscovered approximately half of the DTIs for drugs used in the treatment of COVID-19. These results indicate that AI-DTI is a practically useful tool for guiding drug discovery processes and generating plausible hypotheses that can reveal unknown mechanisms of drug action.

sentences: Identification and Targeting Putative G-Quadruplex Sequences in Candida glabrata: A Route to Virulence and Pathogenesis Control? An increase in the number of Candida species that are resistant to antifungal medication and increases worldwide. Even individuals that are never exposed to antibiotics showing resistance to antifungal drugs. The increase in resistant candida species strains requires a search for novel targets for new antifungal agents. Preventing infection caused by candida species is a tremendous challenge in medicine. Although availability and use of antifungal drugs, disseminated candidiasis leads to a high mortality rate of about 40-60%, poor diagnosis, and improper disease management. Interest in G-quadruplexes as a therapeutic target has been increased in recent years, following the implication of this non-canonical G-quadruplex secondary structure in pathological diseases. However, G-quadruplex has been reported in many pathogens contributing to virulence and pathogenesis, including bacterial pathogens such as Staphylcoccus aureus and Enterococcus spp. Etc, viruses such as SARS-CoV2, HIV, HPV etc., and Fungi such as Candida species, Aspergillus fumigatus, etc. The present aim of the study is to identify and targeting G-Quadruplex forming sequences present in the Candida glabrata. PQSFinder (R Package) identified more than 5000 putative G-quadruplex forming sequences. Out of these, we have used PQS present in the SDH1 gene of Candida glabrata. It may be a key target to ameliorate the C.glabrata infection because it encodes a protein that plays a vital role in energy production in C.glabrata cells shown in the figure. The structure was built based on the already current structure that is telomeric G4 AGGG (TTAGGG)3 {PDB ID-4G0F} for identifying its stabilization by Gold carbine derivatives. Molecular docking and ADMET analysis show that Compound A has the highest binding affinity and has the best ADMET properties among the two compounds. The present study represents PQS in the SDH1 gene could be a novel antifungal target.

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The stoichiometric interaction model for mesoscopic molecular dynamics simulations of liquid-liquid phase separation Liquid-liquid phase separation (LLPS) has received considerable attention in recent years for explaining the formation of cellular biomolecular condensates. The fluidity and the complexity of their components make molecular simulation approaches indispensable for gaining structural insights. Domain-resolution mesoscopic model simulations have been explored for case in which condensates are formed by multivalent proteins with tandem domains. One problem with this approach is that interdomain pairwise interactions cannot regulate the valency of the binding domains. To overcome this problem, we propose a new potential, the stoichiometric interaction (SI) potential. First, we verified that the SI potential maintained the valency of the interacting domains for the test systems. We then examined a well-studied LLPS model system containing tandem repeats of SH3 domains and proline-rich motifs. We found that the SI potential alone cannot reproduce the phase diagram of LLPS quantitatively. We had to combine the SI and a pairwise interaction; the former and the latter represent the specific and non-specific interactions, respectively. Biomolecular condensates with the mixed SI and pairwise interaction exhibited fluidity, whereas those with the pairwise interaction alone showed no detectable diffusion. We also compared the phase diagrams of the systems containing different numbers of tandem domains with those obtained from the experiments, and found quantitative agreement in all but one case.

SIGNIFICANCECells organize their interior structures as not only membrane-bound organelles but also as membrane-less organelles. Membrane-less organelles, such as stress granules, Cajal bodies, and postsynaptic density, are biomolecular condensates in which many biomolecules are gathered owing to their interactions. In some cases, biomolecular condensates are formed by tandemly connected multidomain proteins. In such cases, a mesoscopic simulation model representing each domain as a particle is effective; however, the problem with this approach is that a domain-domain pairwise interaction cannot regulate the well-defined valency. To overcome this problem, in this study, we have developed a new potential, viz. the stoichiometric interaction potential, and confirmed that this potential can reproduce the liquid-liquid phase separation of multidomain proteins, a hallmark of the membrane-less organelles.

sentences: Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery. Keratin filaments provide cellular resistance to mechanical stresses, ensure cell and tissue integrity in addition to regulatory functions. Mutations in keratin genes are related to a variety of epithelial tissue diseases that mostly affect skin and hair. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Interestingly, two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Reconstitution of filaments of substantial length from keratin segments uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex architecture of keratin filaments, which demonstrate a remarkable degree of heterogeneity, suggesting that they are highly flexible, dynamic cytoskeletal structures.

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Extent of Molecular Chaperone Association Might Determine Fates of Membraneless Organelles during Aging in C. elegans Proteome imbalance can lead to protein misfolding and aggregation which is associated with pathologies. Protein aggregation can also be an active, organized process and can be exploited by cells as a survival strategy. In adverse conditions, it is beneficial to deposit the proteins in a condensate rather degrading and resynthesizing. Membraneless organelles (MLOs) are biological condensates formed through liquid-liquid phase separation (LLPS), involving cellular components such as nucleic acids and proteins. LLPS is a regulated process, which when perturbed, can undergo a transition from a physiological liquid condensate to pathological solid-like protein aggregates.

To understand how the MLO-associated proteins (MLO-APs) behave during aging, we performed a comparative meta-analysis with age related proteome of C. elegans. We found that the MLO-APs are highly abundant throughout the lifespan. Interestingly, they are aggregating more in long-lived mutant worms compared to the age matched wildtype worms. GO term analysis revealed that the cell cycle and embryonic development are among the top enriched processes in addition to RNP components in insoluble proteome. Considering antagonistic pleotropic nature of these developmental genes and post mitotic status of C. elegans, we assume that these proteins phase transit during post development. As the organism ages, these MLO-APs either mature to become more insoluble or dissolve in uncontrolled manner. However, in the long-lived daf-2 mutant worms, the MLOs may attain protective states due to extended availability and association of molecular chaperones.

sentences: Truly Privacy-Preserving Federated Analytics for Precision Medicine with Multiparty Homomorphic Encryption Using real-world evidence in biomedical research, an indispensable complement to clinical trials, requires access to large quantities of patient data that are typically held separately by multiple healthcare institutions. Centralizing those data for a study is often infeasible due to privacy and security concerns. Federated analytics is rapidly emerging as a solution for enabling joint analyses of distributed medical data across a group of institutions, without sharing patient-level data. However, existing approaches either provide only limited protection of patients privacy by requiring the institutions to share intermediate results, which can in turn leak sensitive patient-level information, or they sacrifice the accuracy of results by adding noise to the data to mitigate potential leakage. We propose FAMHE, a novel federated analytics system that, based on multiparty homomorphic encryption (MHE), enables privacy-preserving analyses of distributed datasets by yielding highly accurate results without revealing any intermediate data. We demonstrate the applicability of FAMHE to essential biomedical analysis tasks, including Kaplan-Meier survival analysis in oncology and genome-wide association studies in medical genetics. Using our system, we accurately and efficiently reproduce two published centralized studies in a federated setting, enabling biomedical insights that are not possible from individual institutions alone. Our work represents a necessary key step towards overcoming the privacy hurdle in enabling multi-centric scientific collaborations.

sentences: Wilson Statistics: Derivation, Generalization, and Applications to Electron Cryomicroscopy The power spectrum of proteins at high frequencies is remarkably well described by the flat Wilson statistics. Wilson statistics therefore plays a significant role in X-ray crystallography and more recently in electron cryomicroscopy (cryo-EM). Specifically, modern computational methods for three-dimensional map sharpening and atomic modelling of macromolecules by single particle cryo-EM are based on Wilson statistics. Here we provide the first rigorous mathematical derivation of Wilson statistics. The derivation pinpoints the regime of validity of Wilson statistics in terms of the size of the macromolecule. Moreover, the analysis naturally leads to generalizations of the statistics to covariance and higher order spectra. These in turn provide theoretical foundation for assumptions underlying the widespread Bayesian inference framework for three-dimensional refinement and for explaining the limitations of autocorrelation based methods in cryo-EM.

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Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome BackgroundBasenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness.

ResultsHere, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection.

ConclusionsThe growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies.

sentences: Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/ bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

sentences: Age-related Cellular and Microstructural Changes in the Rotator Cuff Enthesis Rotator cuff injuries increase with age. The enthesis is the most frequent site of rotator cuff injury and degeneration. Understanding age-related changes of the enthesis are essential to determine the mechanism of rotator cuff injuries, degeneration, and to guide mechanistically driven therapies. In this study, we explored age-related cellular changes of the rotator cuff enthesis in young, mature, and aged rats. Here we found that the aged enthesis is typified by an increased mineralized zone and decreased non-mineralized zone. Proliferation, migration, and colony forming potential of rotator cuff derived cells (RCECs) was attenuated with aging. The tenogenic and chondrogenic potential were significantly reduced, while the osteogenic potential increased in aged RCECs. The adipogenic potential increased in RCECs with age. This study explores the cellular differences found between young, mature, and aged rotator cuff enthesis cells and provides a basis for further delineation of mechanisms and potential therapeutics for rotator cuff injuries.

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The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the {Delta}crhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

HighlightThe cyanobacterial DEAD-box RNA helicase CrhR binds mainly photosynthesis-associated and redox-controlled transcripts connecting its regulation, localization and phenotypes of mutants for the first time with a set of potential RNA targets.

sentences: Mining transcriptomic data to identify  Saccharomyces cerevisiae  signatures related to improved and repressed ethanol production under fermentation Saccharomyces cerevisiae is known for its outstanding ability to produce ethanol in industry. Identifying the dynamic of gene expression in S. cerevisiae in response to fermentation is required for the establishment of any ethanol production improvement program. The goal of this study was to identify the discriminative genes between improved and repressed ethanol production as well as clarifying the molecular responses to this process through mining the transcriptomic data. Through 11 machine learning based algorithms from RapidMiner employed on available microarray datasets related to yeast fermentation performance under Mg2+ and Cu2+ supplementation, 172 probe sets were identified by at least 5 AWAs. Some have been identified as being involved in carbohydrate metabolism, oxidative phosphorylation, and ethanol fermentation. Principal component analysis (PCA) and heatmap clustering were also validated the top-ranked selective probe sets. According to decision tree models, 17 roots with 100% performance were identified. OLI1 and CYC3 were identified as the roots with the best performance, demonstrated by the most weighting algorithms and linked to top two significant enriched pathways including porphyrin biosynthesis and oxidative phosphorylation. ADH5 and PDA1 are also recognized as differential top-ranked genes that contribute to ethanol production. According to the regulatory clustering analysis, Tup1 has a significant effect on the top-ranked target genes CYC3 and ADH5 genes. This study provides a basic understanding of the S. cerevisiae cell molecular mechanism and responses to two different medium conditions (Mg2+ and Cu2+) during the fermentation process.

sentences: Long-range single-molecule mapping of chromatin modification in eukaryotes The epigenetic modifications of histones are essential markers related to the development and pathogenesis of diseases, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches are limited by fragmentation and short-read sequencing represent the average chromatin status in samples and cannot provide information about the long-range chromatin states. We leveraged the advantage of long read sequencing to develop a method "BIND&MODIFY" for profiling the histone modification of individual DNA fibers. Our approach is based on the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrated that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, verify the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in future third-generation sequencing ages.

sentences: Groping in the fog: soaring migrants exhibit wider scatter and do not properly adjust their flight in response to wind under low visibility conditions Atmospheric conditions are known to affect flight propensity, behaviour during flight, and migration route in birds. Yet, the effects of fog have only rarely been studied although they could disrupt orientation and hamper flight. Fog could limit the visibility of migrating birds such that they might not be able to detect landmarks that guide them during their journey. Soaring migrants modulate their flight speed and direction in relation to the wind vector to optimize the cost of transport. Consequently, landmark-based orientation, as well as adjustments of flight speed and direction in relation to wind conditions, could be jeopardized when flying in fog. Using a radar system operated in a migration bottleneck (Strait of Messina, Italy), we studied the behaviour of soaring birds under variable wind and fog conditions over two consecutive springs (2016 and 2017), discovering that migrating birds exhibited a wider scatter of flight directions and responded differently to wind under fog conditions. Birds flying through fog deviated more from the mean migration direction and increased their speed with increasing crosswinds. In addition, airspeed and groundspeed increased in the direction of the crosswind, causing the individuals to drift laterally. Our findings represent the first quantitative empirical evidence of flight behaviour changes when birds migrate through fog and explain why low visibility conditions could risk their migration journey.

sentences: Ozone sensitivity of diverse maize genotypes is associated with differences in gene regulation, not gene content The maize pangenome has demonstrate large amounts of presence/absence variation and it has been hypothesized that presence/absence variation contributes to stress response. To uncover whether the observed genetic variation in physiological response to elevated ozone (a secondary air pollutant that causes significant crop yield losses) concentration is due to variation in genic content, and/or variation in gene expression, we examine the impact of sustained elevated ozone concentration on the leaf tissue from 5 diverse maize inbred genotypes (B73, Mo17, Hp301, C123, NC338). Analysis of long reads from the transcriptomes of the 10 conditions found expressed genes in the leaf are part of the shared genome, with 94.5% of expressed genes from syntenic loci. Quantitative analysis of short reads from 120 plants (twelve from each condition) found limited transcriptional response to sustained ozone stress in the ozone resistant B73 genotype (151 genes), while more than 3,300 genes were significantly differentially expressed in the more sensitive NC338 genotype. The genes underpinning the divergence of B73 from the other 4 genotypes implicates ethylene signaling consistent with some findings in Arabidopsis. For the 82 of the 83 genes differentially expressed among all 5 genotypes and the 788 of 789 genes differentially expressed in 4 genotypes (excluding B73) in sensitivity to ozone is associated with oxidative stress tolerance being associated with a weaker response to a reactive oxygen species (ROS) signal and suggests that genetic variation in downstream processes is key to ozone tolerance.

sentences: The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion is a PS-binding protein responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to bridge interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion secretion and binding to PS. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

sentences: Chitin and cuticle proteins form the cuticular layer in the spinning duct of silk-spinning arthropods Chitin is found in the exoskeleton and peritrophic matrix of arthropods, but recent studies have also identified chitin in the spinning duct of silk-spinning arthropods. Here, we report the presence and function of chitin and cuticle proteins ASSCP1 and ASSCP2 in the spinning duct of silkworms. We show that chitin and these proteins are co-located in the cuticular layer of the spinning duct. Ultrastructural analysis indicates that the cuticular layer has a multilayer structure by layered stacking of the chitin laminae. After knocking down ASSCP1 and ASSCP2, the fine structure of this layer was disrupted, which had negative impacts on the mechanical properties of silk. This work clarifies the function of chitin in the spinning duct of silk-spinning arthropods. Chitin and cuticle proteins are the main components of the hard and rigid cuticular layer, providing the shearing stress during silk fibrillogenesis and regulating the final mechanical properties of silk.

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Plant trait heterosis is quantitatively associated with expression heterosis of plastid ribosomal proteins The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to the known higher photosynthetic capacity of maize hybrids, we analyzed proteomic and transcriptomic data from seedling leaf tissues of the hybrid, B73xMo17, and its inbred parents. Subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above mid-parent and high-parent levels, respectively. Nuclear and plastid-encoded subunits were expressed similarly and in a dominant pattern with B73 as the high-parent for most proteins. The reciprocal hybrid displayed the same pattern with B73 still the dominant parent. Protein expression patterns were mostly the same in the adult leaf blade. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to adult plant heights of 15 hybrids. Expression heterosis (hybrid/mid-parent expression levels) was most positively correlated with plant height heterosis (hybrid/mid-parent plant height) for the plastid-encoded and nuclear-encoded chloroplast ribosomal proteins; the cytosolic ribosomal proteins were slightly less correlated. Ethylene biosynthetic enzymes were expressed below mid-parent levels in the hybrids, and the ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. Levels of jasmonic acid biosynthetic enzymes were reduced in both acs2/acs6 and the hybrid, and expression heterosis levels of these proteins were the most negatively correlated with plant height heterosis.

Significance StatementHeterosis (hybrid vigor) boosts the productivity and resilience of crops and livestock above the levels of both parents, yet its underlying mechanisms remain unknown. We analyzed expression patterns of proteins in maize hybrids and their inbred parents. Differences in several molecular machines and biochemical pathways were found and quantitatively assessed using a panel of 15 hybrids. Seedling leaf chloroplast ribosomal proteins were able to quantitatively infer levels of adult plant heterosis. Expression levels of biosynthetic enzymes for the stress hormone, ethylene, were reduced in hybrids as was previously reported for the dicot Arabidopsis. Mutation of these genes in a maize inbred caused the proteome to resemble a hybrid. Repression of ethylene biosynthesis may be a conserved component of heterosis physiology.

sentences: Interplay between protein stability, binding to ACE2 and escape from neutralizing antibodies determines the natural selection of SARS-CoV-2 receptor binding domain variants Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain on its expression, stability, activity, and antibody escape potential-some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels with the two least stable variants showing lesser expression. Out of the 8 well-expressed mutants, only 2 (N501Y and K417T/E484K/N501Y) showed stronger affinity to ACE2, 4 (Y453F, S477N, T478I and S494P) have similar affinity, whereas the other 2 (K417N and E484K) have weaker affinity when compared to the wild-type. In terms of CC12.1 binding, when compared to the wild-type, 4 variants (K417N, Y453F, N501Y and K417T/E484K/N501Y) have weaker affinity, 2 (S477N and S494P) have similar affinity, and 2 (T478I and E484K) have stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all these factors need be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.

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

sentences: Recruitment of an ancient branching program to suppress carpel development in maize flowers Floral morphology is immensely diverse. One developmental process acting to shape this diversity is growth suppression. For example, grass flowers exhibit extreme diversity in floral sexuality, arising through differential suppression of stamens or carpels. In maize, carpels undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen, and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1; ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ~160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes, and show how an ancient developmental program was redeployed to sculpt floral form.

sentences: RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells. Embryonic stem cell (ESC) fate decisions are regulated by a complex molecular circuitry that requires tight and coordinated gene expression regulations at multiple levels from chromatin organization to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key regulatory pathways that efficiently control stem cell homeostasis. However, the molecular mechanisms underlying the regulation of these pathways remain largely unknown to date. Here, we analyzed the expression, in mouse ESCs, of over 300 genes involved in ribosome biogenesis and we identified RSL24D1 as the most differentially expressed between self-renewing and differentiated ESCs. RSL24D1 is highly expressed in multiple mouse pluripotent stem cell models and its expression profile is conserved in human ESCs. RSL24D1 is associated with nuclear pre-ribosomes and is required for the maturation and the synthesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors, including POU5F1 and NANOG, as well as components of the polycomb repressive complex 2 (PRC2). Consistently, RSL24D1 is required for mouse ESC self-renewal and proliferation. Taken together, we show that RSL24D1-dependant ribosome biogenesis is required to both sustain the expression of pluripotent transcriptional programs and silence developmental programs, which concertedly dictate ESC homeostasis.

sentences: Short-term diet intervention alters the small non-coding RNA (sncRNA) landscape of human sperm Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing the effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects, though less is known about the existence of similar mechanisms in human sperm. Here, we first characterised the baseline sncRNA landscape of human sperm, and then studied the effects of a 6-week diet intervention on their expression profile. Baseline profiling identified 5tRFs, miRNAs and piRNAs to be the most abundant sncRNA subtypes, primarily expressed from regulatory elements like UTRs, CpG-rich regions and promoters. Expression of a subset of these sncRNAs varied with age, BMI and sperm quality of the donor. Diet intervention enriched in vitamin D and omega-3 fatty acids showed a marked increase of these nutrients in circulation and altered the sperm sncRNA expression. These included 3 tRFs, 15 miRNAs and 112 piRNAs, with gene targets involved in fatty acid metabolism, vitamin D response (LXR/RXR activation, TGF-beta and Wnt signaling), and transposable elements. These findings provide evidence that human sperms are sensitive to alterations in exposures such as diet, and sncRNAs capture the epigenetic imprint of this change. Hence changes to paternal nutrition during preconception may improve sperm quality and offspring health outcomes. To benefit future research, we developed iDad_DB, an open access database of baseline and diet-altered sncRNA in human male germline.

sentences: Targeting glucose metabolism sensitizes pancreatic cancer to MEK inhibition Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and identify druggable targets for tumor maintenance. This is especially challenging in the context of PDAC, in which activating mutations of KRAS oncogene (KRAS*) dominate the genetic landscape. By using an inducible KrasG12D-driven p53 deficient PDAC mouse model (iKras model), we demonstrate that RAF-MEK-MAPK signaling is the major effector for oncogenic Kras-mediated tumor maintenance. However, MEK inhibition has minimal therapeutic effect as single agent for PDAC both in vitro and in vivo. Although MEK inhibition partially downregulates the transcription of glycolysis genes, it surprisingly fails to suppress the glycolysis flux in PDAC cell, which is a major metabolism effector of oncogenic KRAS. Accordingly, In vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MAPK inhibition. Furthermore, inhibition of glucose metabolism with low dose 2-deoxyglucose (2DG) in combination with MEK inhibitor dramatically induces apoptosis in KrasG12D-driven PDAC cell in vitro, inhibits xenograft tumor growth and prolongs the overall survival of genetically engineered mouse PDAC model. Molecular and metabolism analyses indicate that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal ER stress. Together, our work suggests that combinatory inhibition of glycolysis and MAPK pathway may serve as an alternative approach to target KRAS-driven PDAC.

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Relationship between systolic blood pressure and decision-making during emotional processing ObjectiveEmotional states are expressed in body and mind through subjective experience of physiological changes. In previous work, subliminal priming of anger prior to lexical decisions increased systolic blood pressure (SBP). This increase predicted the slowing of response times (RT), suggesting that baroreflex-related autonomic changes and their interoceptive (feedback) representations, influence cognition. Alexithymia is a subclinical affective dysfunction characterized by difficulty in identifying emotions. Atypical autonomic and interoceptive profiles are observed in alexithymia. Therefore, we sought to identify mechanisms through which SBP fluctuations during emotional processing might influence decision-making, including whether alexithymia contributes to this relationship.

MethodsThirty-two male participants performed an affect priming paradigm and completed the Toronto Alexithymia Scale. Emotional faces were briefly presented (20ms) prior a short-term memory task. RT, accuracy and SBP were recorded on a trial-by-trial basis. Generalized mixed-effects linear models were used to evaluate the impact of emotion, physiological changes, alexithymia score, and their interactions, on performances.

ResultsA main effect of emotion was observed on accuracy. Participants were more accurate on trials with anger primes, compared to neutral priming. Greater accuracy was related to increased SBP. An interaction between SBP and emotion was observed on RT: Increased SBP was associated with RT prolongation in the anger priming condition, yet this relationship was absent under the sadness priming. Alexithymia did not significantly moderate the above relationships.

ConclusionsOur data suggest that peripheral autonomic responses during affective challenges guide cognitive processes. We discuss our findings in the theoretical framework proposed by Lacey and Lacey (1970).

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The Diameter Factor of Aligned Electrospinning Membranes Facilitates Cutaneous Wound Healing by Promoting Epithelialization Topographical properties (such as pattern and diameter) of biomaterials play important roles in influencing cell activities and manipulating the related immune response during the skin wound healing. We prepared aligned electrospinning membranes with different fiber diameters including 319{+/-}100 nm (A300), 588{+/-}132 nm (A600) and 1048{+/-}130 nm (A1000). The A300 membranes significantly improved cell proliferation and spreading, and facilitated skin wound healing (epithelization and vascularization) with the regeneration of hair follicles. The transcriptome revealed the underlying molecular mechanism that A300 could promote macrophages transformation to reparative phenotypes with highly expressed IL10 and TGF{beta}, subsequently promoting epidermal cell migration via secreting matrix metalloproteinases 12 (MMP12). All the results indicate the A300 to be a potential candidate for guided skin regeneration applications.

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sentences: An ultrasensitive method for analysis of viral spike N-glycoforms Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.

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Leaf shape and size variation in bur oaks: An empirical study and simulation of sampling strategies PREMISELeaf shape and size figure strongly in trees adaptation to their environments. Oaks are notoriously variable in leaf morphology. Our study examines the degree to which within-tree, among-tree, and among-site variation contribute to latitudinal variation in leaf shape and size of bur oak (Quercus macrocarpa: Fagaceae), one of North Americas most geographically widespread oak species.

METHODSSamples were collected from four sites each at northern, central, and southern latitudes of the bur oak range. Ten leaf size traits were measured, and variance in these traits and eight ratios based on these traits was partitioned into tree, population, and latitude components. We then parameterized a series of leaf collection simulations using empirical covariance among leaves on trees and trees at sites. We used the simulations to assess the efficiency of different collecting strategies for estimating among-population differences in leaf shape and size.

KEY RESULTSLeaf size measurements were highly responsive to latitude. Site contributed more than tree to total variation in leaf morphology. Simulations suggest that power to detect among-site variance in leaf shape and size can be estimated most efficiently with increases in either leaves per tree (10-11 leaves from each of 5 trees) or trees per site (5 leaves from each of 10+ trees).

CONCLUSIONSOur study demonstrates the utility of simulating sampling and controlling for variance in sampling for leaf morphology, whether the questions being addressed are ecological, evolutionary, or taxonomic. Simulation code is provided as an R package (traitsPopSim) to help researchers plan morphological sampling strategies.

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Unconventional conservation reveals structure-function relationships in the synaptonemal complex Functional requirements constrain protein evolution, commonly manifesting in conserved primary amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) aligns parental chromosome pairs and regulates exchanges between them. In electron micrographs of meiocytes from all eukaryotic clades, the SC appears as a ~100 nm-wide ladder-like structure with regular striations. Despite the conserved ultrastructure and functions, the proteins that make up the SC are highly divergent in sequence. Here we found that, within the Caenorhabditis genus, SC proteins are significantly more diverged than other proteins. However, SC proteins have highly conserved protein length and coiled-coil domain structure. The same unconventional conservation signature holds true for SC proteins in Drosophila and mammals, suggesting it could be a universal feature of SC proteins. We used this evolutionary signature to identify a novel SC protein in the nematode Pristionchus pacificus, Ppa-SYP-1, which has no significant homology to any protein outside of Pristionchus. Our work suggests that the length and relative arrangement of coiled-coils play a key role in the structure and function of the SC. Furthermore, our analysis implies that expanding sequence analysis beyond measures of per-site identity or similarity can enhance our understanding of protein evolution and function.

Short abstractFunctional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100 nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. We found the same unconventional conservation signature in Drosophila and mammals, and used it to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. Our work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance our understanding of protein evolution and function.

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BIAPSS - BioInformatic Analysis of liquid-liquid Phase-Separating protein Sequences Liquid-liquid phase separation (LLPS) has recently emerged as a cornerstone mechanism underlying the biogenesis of membraneless organelles (MLOs). However, a quantitative molecular grammar of protein sequences that controls the LLPS remains poorly understood. The progress in this field is hampered by the insufficiency of comprehensive databases and associated computational infrastructure for targeting biophysical and statistical analysis of phase separating biopolymers. Therefore, we have created a novel open-source web platform named BIAPSS (BioInformatic Analysis of liquid-liquid Phase-Separating protein Sequences) which contains interactive data analytic tools in combination with a comprehensive repository of bioinformatic data for on-the-fly exploration of sequence-dependent properties of proteins with known LLPS behavior. BIAPSS includes a residue-resolution biophysical analyzer for interrogating individual protein sequences (SingleSEQ tab). The latter allows users to correlate regions prone to phase separation with a large array of physicochemical attributes and various short linear motifs. BIAPSS also includes global statistics derived over the universe of most of the known LLPS-driver protein sequences (MultiSEQ tab) for revealing the regularities and sequence-specific signals driving phase separation. Finally, BIAPSS incorporates an extensive cross-reference section that links all entries to primary LLPS databases and other external resources thereby serving as a central navigation hub for the phase separation community. All of the data used by BIAPSS is freely available for download as well-formatted pre-processed data with detailed descriptions, facilitating rapid implementation in user-defined computational protocols.

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C_FIG Author summaryProteins, especially those with low complexity and intrinsically disordered regions, have recently come into the limelight because of mounting evidence showing that these regions can drive the formation of membraneless organelles (MLOs) in cells. The underlying physical mechanism for forming MLOs is liquid-liquid phase separation (LLPS); a thermodynamically driven process whereby a cellular milieu with a relatively well-mixed distribution of biomolecules gets decomposed into liquid droplets where the concentration of selected biomolecules is higher. Deciphering molecular sequence grammar of phase separation has turned out to be challenging because of the complexity of this process in cells and the vastness of sequence space of LLPS-driver proteins. While the field is still in its infancy the growth of experimental data has already spurred the creation of several major databases which collect and annotate bimolecular systems with confirmed LLPS behavior. What is currently missing is a framework that would leverage the existing databases by integrating them with deep biophysical and bioinformatic analysis for identifying statistically significant features of protein sequences implicated in LLPS. In this work, we have addressed this challenge by creating an open-source web platform named BIAPSS (BioInformatic Analysis of liquid-liquid Phase-Separating protein Sequences) which integrates a comprehensive repository of pre-processed bioinformatic data for LLPS-driver protein sequences with interactive analytic applications for on-the-fly analysis of biophysical features relevant for LLPS behavior. BIAPSS empowers users with novel and effective tools for exploring LLPS-related sequence signals for individual proteins (SingleSEQ tab) and globally by integrating common regularities across subgroups or the entire LLPS sequence superset (MultiSEQ). The long-term plan for BIAPSS is to serve as a unifying hub for the experimental and computational community with a comprehensive set of analytic tools, biophysically featured data, and standardized protocols facilitating the identification of sequence hot spots driving the LLPS, which all can support applications for designing new sequences of biomedical interest.

sentences: Population estimation of a cryptic moss frog using acoustic spatially explicit capture recapture Cryptic amphibians pose a problem for conservation managers as they are difficult to find to assess initial populations, and monitor changes during potentially threatening processes. The rough moss frog, Arthroleptella rugosa, is small and occurs in seepages on a single unprotected mountain in South Africas fire prone, biodiverse fynbos biome. The area is heavily impacted by invasive plants, which dry seepages and increase the frequency and intensity of fires, leading to the assessment of this species as Critically Endangered. We aimed to test the efficacy of acoustic spatially explicit capture recapture (aSCR) to estimate the entire population of calling adult A. rugosa, and assess the impacts by invasive plants. Using aSCR, our estimates suggest that the population of A. rugosa is more than five times that previously estimated using aural calling surveys on the mountain, at [~]2000 individuals. This despite an intense fire over the entire area three years earlier that reduced the calling population to a few tens of individuals. Our vegetation surveys suggest that the ongoing removal of invasive plants from the mountain is successful in areas occupied by A. rugosa, but that adjacent areas invaded by pines and hakea have a negative impact on calling density. The private public conservancy partnership on Klein Swartberg Mountain, is conserving this frog but will require ongoing management and monitoring to ensure conservation in the future.

sentences: Unsupervised clustering of track-weighted dynamic functional connectivity reveals white matter substrates of functional connectivity dynamics The contribution of structural connectivity to functional connectivity dynamics is still far from being fully elucidated. Herein, we applied track-weighted dynamic functional connectivity (tw-dFC), a model integrating structural, functional, and dynamic connectivity, on high quality diffusion weighted imaging and resting-state fMRI data from two independent repositories. The tw-dFC maps were analyzed using independent component analysis, aiming at identifying spatially independent white matter components which support dynamic changes in functional connectivity. Each component consisted of a spatial map of white matter bundles that show consistent fluctuations in functional connectivity at their endpoints, and a time course representative of such functional activity. These components show high intra-subject, inter-subject, and inter-cohort reproducibility. We provided also converging evidence that functional information about white matter activity derived by this method can capture biologically meaningful features of brain connectivity organization, as well as predict higher-order cognitive performance.

sentences: Visuomotor information drives interference between the hands more than dynamic motor information during bimanual reaching During complex bimanual movements, interference can occur in the form of one hand influencing the action of the contralateral hand. Interference likely results from conflicting sensorimotor information shared between brain regions controlling hand movements via neural crosstalk. However, how visual and force-related feedback processes interact with each other during bimanual reaching is not well understood. In this study, four groups experienced either a visuomotor perturbation, dynamic perturbation, combined visuomotor and dynamic perturbation, or no perturbation in their right hand during bimanual reaches, with each hand controlling its own cursor. The left hand was examined for interference as a consequence of the right-hand perturbation. The results indicated that the visuomotor and combined perturbations showed greater interference in the left hand than the dynamic perturbation, but that the combined and visuomotor perturbations were equivalent. This suggests that dynamic sensorimotor and visuomotor processes do not interact between hemisphere-hand systems, and that primarily visuomotor processes lead to interference between the hands.

sentences: Modulation of endometrial E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli The endometrium is a dynamic tissue that undergoes extensive remodelling to attain a receptive state which is further modulated in presence of an embryo for successful initiation of pregnancy. Cadherins are the proteins of junctional complex of which E-cadherin (E-Cad) is crucial for maintaining epithelial cell state and integrity of the epithelial barrier; gain of N-cadherin (N-Cad) in epithelial cells leads to epithelial to mesenchymal transition (EMT). In the present study, we aimed to investigate the expression of E-Cad and N-Cad in the mouse endometrial luminal epithelium and its modulation by estrogen, progesterone and embryonic stimuli. We observed that E-Cad is diffusely expressed in the luminal epithelium of mouse endometrium during the estrus stage and upon estrogen treatment. It is apico-laterally and basolaterally sorted at the diestrus stage and in response to combined treatment of estrogen and progesterone. In 3D spheroids of human endometrial epithelial cells, combined treatment with estrogen and progesterone led to lateral sorting of E-Cad. In the mouse endometrium at the time of embryo implantation, there is loss of E-Cad which was associated with the gain of N-Cad suggestive of EMT in the luminal epithelium. This EMT is possibly driven by embryonic stimuli as treatment with estrogen and progesterone did not lead to gain of N-Cad expression. In conclusion, the present study demonstrates that steroid hormones directly affect E-Cad sorting in the endometrial epithelium.

sentences: The impacts of hydropower on freshwater macroinvertebrate richness: A global meta-analysis Hydroelectric dams and their reservoirs have been suggested to affect freshwater biodiversity. However, studies investigating the consequences of hydroelectric dams and reservoirs on macroinvertebrate richness have reached opposite conclusions. We carried out a meta-analysis devised to elucidate the effects of hydropower dams and their reservoirs on macroinvertebrates richness while accounting for the potential role played by moderators such as biomes, impact types, study designs, sampling seasons and gears. We used a random and mixed effect model, combined with robust variance estimation, to conduct the meta-analysis on 72 pairs of observations (i.e., impacted versus reference) extracted from 17 studies (more than one observation per study). We observed a large range of effect sizes, from very negative to very positive impacts of hydropower. However, according to this meta-analysis, hydropower dams and their reservoirs did not have an overall clear, directional and statistically significant effect on macroinvertebrate richness. We tried to account for the large variability in effect sizes using moderators, but none of the moderators included in the meta-analysis had statistically significant effect. This suggests that some other moderators, which were unavailable for the 17 studies included in this meta-analysis, might be important (e.g., temperature, granulometry, wave disturbance and macrophytes) and that macroinvertebrate richness may be driven by local, smaller scale processes. As new studies become available, it would be interesting to keep enriching this meta-analysis, as well as collecting local habitat variables, to see if we could finally draw statistically significant conclusions about the impacts of hydropower on macroinvertebrate richness.

sentences: mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including ageing, genetics and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), that leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. While the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by Mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

sentences: Age-Related Type I Collagen Modifications Reveal Tissue-Defining Differences Between Ligament and Tendon Tendons and ligaments tend to be pooled into a single category as dense elastic bands of collagenous connective tissue. They do have many similar properties, for example both tissues are flexible cords of fibrous tissue that join bone to either muscle or bone. Tendons and ligaments are both prone to degenerate and rupture with only limited capacity to heal, however; healing outcomes for tendons are often faster than ligament injuries. Type I collagen constitutes about 80% of the dry weight of tendons and ligaments and is principally responsible for the core strength of each tissue. Collagen synthesis is a complex process with multiple steps and numerous post-translational modifications including proline and lysine hydroxylation, hydroxylysine glycosylation and covalent cross-linking. The chemistry, placement and quantity of intramolecular and intermolecular cross-links are believed to be key contributors to the tissue-specific variations in material strength and biological properties of collagens. As tendons and ligaments grow and develop, the collagen cross-links are known to chemically mature, strengthen and change in profile. Accordingly, changes in cross-linking and other post-translational modifications are likely associated with tissue development and degeneration. Using mass spectrometry, we have compared tendon and ligaments from fetal and adult bovine knee joints to investigate changes in collagen post-translational properties. Although hydroxylation levels at the type I collagen helical cross-linking lysine residues were similar in all adult tissues, ligaments had significantly higher levels of glycosylation at these sites compared to tendon. Differences in lysine hydroxylation were also found between the tissues at the telopeptide cross-linking sites. Total collagen cross-linking analysis, including mature trivalent cross-links and immature divalent cross-links, revealed unique cross-linking profiles between tendon and ligament tissues. Tendons were found to have a significantly higher frequency of smaller diameter collagen fibrils compared with ligament, which we propose is a consequence of the unique cross-linking profile of each tissue. Understanding the specific molecular characteristics that define and distinguish these specialized tissues will be important for designing improved orthopedic treatment approaches.

sentences: Profiling of human neural crest chemoattractant activity as replacement of fetal bovine serum for in vitro chemotaxis assays Fetal bovine serum (FBS) is the only known stimulus for migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We confirmed here in an optimized transwell assay that FBS triggers directed migration along a concentration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chemotactic for NCCs in animal models (e.g. VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any factor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepatoma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease- sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.

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Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenic Escherichia coli DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life, extending also to bacterial virulence. Both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. However, the potential regulatory role of DNA methylation mediated by archetypal Type I systems in Escherichia coli has never been studied. We demonstrated that removal of DNA methylated mediated by three different Escherichia coli Type I RMSs in three distinct E. coli strains had no detectable effect on gene expression or growth in a screen of 1190 conditions. Additionally, deletion of the Type I RMS EcoUTI in UTI89, a prototypical cystitis strain of E. coli, which led to loss of methylation at >750 sites across the genome, had no detectable effect on virulence in a murine model of ascending urinary tract infection (UTI). Finally, introduction of two heterologous Type I RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of "regulation avoidance" for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome. Our results provide a clear and (currently) rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli, further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer.

Author summaryDNA methylation is perhaps the most common epigenetic modification, and it is commonly associated with gene regulation (in nearly all organisms) and virulence (particularly well studied in bacteria). Regarding bacterial virulence, the current DNA methylation literature has focused primarily on orphan methyltransferases or phasevariable restriction modification systems (RMSs). Interestingly, no reports have studied the potential regulatory role of the first RMS discovered, the Type I RMS EcoKI. We used transcriptomics, Phenotype Microarrays, and a murine model of urinary tract infection to screen for functional consequences due to Type I methylation in three unrelated strains of E. coli. Remarkably, we found zero evidence for any epigenetic regulation mediated by these Type I RMSs. Thus, these Type I RMSs appear to function exclusively in host defense against incoming DNA (the canonical function of RMSs), while the methylation status of many hundreds of the corresponding recognition sites has no detectable impact on gene expression or any phenotypes. This led us to the concept of "regulation avoidance" by such DNA methyltransferases, which contrasts with the current literature on bacterial epigenetics. Our study hints at the existence of an entire class of regulation avoidant systems, which provides new perspectives on methylation-mediated gene regulation and bacterial genome evolution.

sentences: Persistent acetylation of newly synthesized histones inhibits DNA replication origin activity Histone acetylation is a known regulator of DNA replication dynamics. In the yeast Saccharomyces cerevisiae, newly synthesized histone H3 deposited behind DNA replication forks are acetylated on lysine 56 (H3 K56ac) by the Rtt109 histone acetyltransferase. Two enzymes of the sirtuin family of deacetylases, Hst3 and Hst4, act redundantly to deacetylate this residue throughout chromosomes following S phase. Cells lacking Hst3 and Hst4 present constitutively acetylated H3 K56, which causes sensitivity to replication-blocking genotoxins and slow growth via mechanisms that remain poorly understood. Here, we present the results of a genome-wide screen aimed at identifying haploinsufficient genes that promote cell fitness in response to inhibition of sirtuins by nicotinamide (NAM). We find that heterozygous deletion alleles of genes involved in the regulation of DNA replication origins sensitize cells to NAM. Consistently, haploid cells harboring the hypomorphic temperature sensitive allele cdc7-4 present striking NAM-induced S phase progression defects at their semi-permissive temperature. We further show i) that Rap1-interacting factor 1 (Rif1), an inhibitor of Cdc7-dependent activation of replication origins, causes DNA damage and replication defects in cells exposed to NAM and in hst3{Delta} hst4{Delta} mutants, and ii) that cdc7-4 hst3{Delta} hst4{Delta} triple mutant cells present strong synthetic temperature sensitivity associated with defective activation of replication origin and delayed S phase progression. Our data further demonstrate that such replication defects are not due to intra-S phase checkpoint activation leading to inhibition of origin activity. We finally show that Rtt109-dependent acetylation of histone H3 lysine 56 and its associated Rtt101-Mms1 ubiquitin ligase complex cause the DNA replication defects observed in cdc7-4 hst3{Delta} hst4{Delta} cells. Overall, these results argue that abnormal regulation of nascent chromatin structure negatively influences DNA replication initiation in cells presenting reduced Dbf4-dependent kinase activity including those experiencing replicative stress.

sentences: Inverted genomic regions between reference genome builds in humans impact imputation accuracy and decrease the power of association testing Over the last two decades, the human reference genome has undergone multiple updates as we complete a linear representation of our genome. There are two versions of human references currently used in the biomedical literature, GRCh37/hg19 and GRCh38, and conversions between these versions are critical for quality control, imputation, and association analysis. In the present study we show that genomic coordinates for single nucleotide variants (SNVs) in regions inverted between different builds of the reference genome are erroneously converted by the TOPMed imputation server. Depending on the array type, we estimate the inappropriate conversions of variant coordinates would occur in about 2-5 Mb of the genome. Independent of the imputation server, however, in-house conversions of variant coordinates are routine yet impact of mapped variants to these inverted regions and its downstream analytical consequences are underrecognized. We observed that errors for palindromic variants in these inverted regions cannot be detected by standard quality control procedures and destabilize the local haplotype structure, leading to loss of imputation accuracy and power in association analyses. Though only a small proportion of the genome is affected, we show that these regions include important disease susceptibility variants that would be lost due to poor imputation. For example, we show that a known locus associated with prostate cancer on chr10 would have its association P-value drop from 2.86x10-7 to 0.0011 in a case-control analysis of 20,286 Africans and African Americans (10,643 cases and 9,643 controls). We devise a straight-forward heuristic based on the popular tool liftOver that can easily detect and correct these variants in the inverted regions between genome builds to locally improve imputation accuracy.

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Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions. Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we do not utilize transforming growth factor-beta inhibitors or a passage step for EVT differentiation, or forskolin for STB formation. Strikingly, under these conditions, presence of a single additional extracellular cue - lam-inin-1 - switched the terminal differentiation of hTSCs from STB to the EVT lineage. Activation of the sphingosine-1 receptor 3 receptor (S1PR3) using a chemical agonist could drive EVT differentiation of hTSCs in the absence of exogenous laminin, albeit less efficiently. To illustrate the utility of a chemically defined culture system for mechanistic studies, we examined the role of protein kinase C (PKC) signaling during hTSC differentiation to the EVT lineage. Inhibition of PKC/{beta} signaling significantly reduced HLA-G expression and the formation of HLA-G+ mesen-chymal EVTs during hTSC differentiation mediated by laminin exposure; however, it did not prevent commitment to the EVT lineage or STB differentiation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation, and will enable mechanistic studies in vitro.

SignificanceDespite its importance to a healthy pregnancy, early human placental development remains poorly understood. Mechanistic studies are impeded by restrictions on research with human embryos and fetal tissues, and significant differences in placentation between humans and commonly used animal models. In this context, human trophoblast stem cells (hTSCs) have emerged as attractive in vitro models for the epithelial cytotrophoblast of the early gestation human placenta. Here we describe chemically defined culture conditions for differentiation of hTSCs to the two major differentiated cell types - extravillous trophoblast and syncytiotrophoblast. These culture conditions enable in vitro studies to reveal molecular mechanisms regulating hTSC differentiation.

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Visualizing and quantifying data from timelapse imaging experiments One obvious feature of life is that it is highly dynamic. The dynamics can be captured by movies that are made by acquiring images at regular time intervals, a method that is also known as timelapse imaging. Looking at movies is a great way to learn more about the dynamics in cells, tissue and organisms. However, science is different from Netflix, in that it aims for a quantitative understanding of the dynamics. The quantification is important for the comparison of dynamics and to study effects of perturbations. Here, we provide detailed processing and analysis methods that we commonly use to analyze and visualize our timelapse imaging data. All methods use freely available open-source software and use example data that is available from an online data repository. The step-by-step guides together with example data allow for fully reproducible workflows that can be modified and adjusted to visualize and quantify other data from timelapse imaging experiments.

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Automated speech detection in eco-acoustic data enables privacy protection and human disturbance quantification Eco-acoustic monitoring is increasingly being used to map biodiversity across huge scales, yet little thought is given to the privacy concerns and potential scientific value of inadvertently recorded human speech. Automated speech detection is possible using Voice Activity Detection (VAD) models, but existing approaches have been developed for indoor or urban use cases, rather than diverse natural soundscapes. In this study we used a data augmentation approach to create ecoVAD, a convolutional neural network designed for robust voice detection in eco-acoustic data. We performed playback experiments using speech samples from a woman, man, and child in two ecosystems in Borsa, Norway, and showed that ecoVAD was able to accurately detect voices at distances of up to 20m - at which point the speech was unintelligible. We compared ecoVAD with two existing VAD models and found that ecoVAD consistently outperformed the state-of-the-art (mean F1 scores: ecoVAD, 0.917; pyannote, 0.890; WebRTC VAD, 0.876). Using long-term passive recordings from a popular hiking location in Bymarka, Norway, we found that the frequency of speech detections was linked closely to peak traffic hours (using bus timings) demonstrating how VAD models can be used to quantify human activity with a fine temporal resolution. Anonymising audio data effectively using VAD models will allow eco-acoustic monitoring to continue to deliver invaluable ecological insight at scale, whilst minimising the risk of data misuse. Furthermore, using speech detections as a fine scale measure of human disturbance opens new possibilities for studying subtle human-wildlife interactions on the vast scales made possible by eco-acoustic monitoring technology.

Author SummaryEco-acoustic monitoring (i.e., recording and analysing the sounds of an ecosystem) allows us to monitor biodiversity and ecological health on vast spatial and temporal scales. However, as networks of audio recorders are increasingly being deployed across the world by scientists and land managers, little thought has been given to the implications of inadvertently recorded human speech in these datasets. We developed a neural-network model to detect and remove speech from audio data, and showed it performed more accurately than existing state-of-the-art models when evaluated on eco-acoustic datasets. Furthermore, we showed frequency of voice detections could be used to monitor human pressure on an ecosystem. Our work paves the way for ethical and responsible processing of eco-acoustic data and provides a novel approach to studying human wildlife interactions on a large scale with high temporal resolution.

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Plasticity in astrocyte subpopulations regulates heroin relapse Opioid use disorder (OUD) produces detrimental personal and societal consequences. Astrocytes are a major cell group in the brain that receives little attention in mediating OUD. We determined how astrocytes and the astroglial glutamate transporter, GLT-1, in the nucleus accumbens core adapt and contribute to heroin seeking in rats. Seeking heroin, but not sucrose, produced two transient forms of plasticity in different astroglial subpopulations. Increased morphological proximity to synapses occurred in one subpopulation and increased extrasynaptic GLT-1 expression in another. Augmented synapse proximity by astroglia occurred selectively at D2-dopamine receptor expressing dendrites, while changes in GLT-1 were not neuron-subtype specific. Importantly, mRNA-antisense inhibition of either morphological or GLT-1 plasticity promoted cue-induced heroin seeking. We show that heroin cues induce two distinct forms of transient plasticity in separate astroglial subpopulations that dampen heroin relapse.

TEASERDifferent subpopulations of astrocytes engage with accumbens synapses to dampen heroin relapse.

sentences: Ca2+-inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryo-EM carried out under partially inactivating Ca2+ conditions revealed two conformations of RyR1, an open state and an inactivated state, resolved at 4.0 and 3.3 [A] resolution, respectively. RyR1s were embedded in nanodiscs with two lipids resolved at each inter-subunit crevice. Ca2+ binding to the high affinity site engages the central (CD) and C-terminal domains (CTD) into a quasi-rigid unit, which separates the S6 four-helix bundle and opens the channel. Further out-of-plane rotation of the quasi-rigid unit pushes S6 towards the central axis, closing (inactivating) the channel. The inactivated conformation is characterized by a downward conformation of the cytoplasmic assembly, a tightly-knit subunit interface contributed by a fully occupied and partially remodeled Ca2+ activation site, and two salt bridges between the EF hand domain and the S2-S3 loop of the neighboring subunit validated by naturally-occurring diseasecausing mutations. Ca2+ also bound to ATP, mediating a tighter interaction between S6 and CTD. Our study suggests that the closed-inactivated is a distinctive state of the RyR1 and its transition to the closed-activable state is not a simple reverse of the Ca2+ mediated activation pathway.

sentences: The central role of the interspecific interactions in the evolution of microbial communities The interspecific interactions play an important role in the establishment of a community phenotype. Furthermore, the evolution of a community can not only occur through an evolution of the species composing the community but also of the interactions among them. In this study, we investigated how widespread was the evolution of interspecific interactions in the evolutionary response of eight two-bacterial species communities regarding productivity. We found evidence for an evolution of the interactions in half of the studied communities which gave rise to a mean change of 15% in community productivity as compared to what was expected from the individual responses. Even when the interactions did not evolve themselves, they influenced the evolutionary responses of the bacterial strains within the communities which further affected community response. We found that the evolution within a community often promoted an adaptation of the bacterial strains to the abiotic environment, especially for the dominant strain in a community. Overall, this study suggested that the evolution of the interspecific interactions was frequent and that it could increase community response to evolution. We propose that the existence of an evolution of the interspecific interactions can justify the consideration of the community as a unit of selection.

sentences: IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has nonredundant biological functions, we currently lack the tools to investigate the molecular basis for isoform-specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. We performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed {beta}-actin, we demonstrate that IntAct actins are properly expressed and that their filament incorporation is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin-binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed {gamma}-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for IntAct actins. Together, our data demonstrate that IntAct is a promising tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.

sentences: Unravelling the shared genetic mechanisms underlying 18 autoimmune diseases using a systems approach Autoimmune diseases (AiDs) are complex heterogeneous diseases characterized by hyperactive immune responses against self. Genome-wide association studies have identified thousands of single nucleotide polymorphisms (SNPs) associated with several AiDs. While these studies have identified a handful of pleiotropic loci that confer risk to multiple AiDs, they lack the power to detect shared genetic factors residing outside of these loci. Here, we integrated chromatin contact, expression quantitative trait loci and protein-protein interaction (PPI) data to identify genes that are regulated by both pleiotropic and non-pleiotropic SNPs. The PPI analysis revealed complex interactions between the shared and disease-specific genes. Furthermore, pathway enrichment analysis demonstrated that the shared genes co-occur with disease-specific genes within the same biological pathways. In conclusion, our results are consistent with the hypothesis that genetic risk loci associated with multiple AiDs converge on a core set of biological processes that potentially contribute to the emergence of polyautoimmunity.

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Rules for designing protein fold switches and their implications for the folding code We have engineered switches between the three most common small folds, 3, 4{beta}+, and /{beta}-plait, referred to here as A, B, and S, respectively. Mutations were introduced into the natural S protein until sequences were created that have a stable S-fold in their longer ([~]90 amino acid) form and have an alternative fold (either A or B) in their shorter (56 amino acid) form. Five sequence pairs were designed and key structures were determined using NMR spectroscopy. Each protein pair is 100% identical in the 56 amino acid region of overlap. Several rules for engineering switches emerged. First, designing one sequence with good native state interactions in two folds requires care but is feasible. Once this condition is met, fold populations are determined by the stability of the embedded A- or B-fold relative to the S-fold and the conformational propensities of the ends that are generated in the switch to the embedded fold. If the stabilities of the embedded fold and the longer fold are similar, conformation is highly sensitive to mutation so that even a single amino acid substitution can radically shift the population to the alternative fold. The results provide insight into why dimorphic sequences can be engineered and sometimes exist in nature, while most natural protein sequences populate single folds. Proteins may evolve toward unique folds because dimorphic sequences generate interactions that destabilize and can produce aberrant functions. Thus, two-state behavior may result from natures negative design rather than being an inherent property of the folding code.

Significance StatementWe establish general rules for designing protein fold switches by engineering dimorphic sequences that link the three most common small folds. The fact that switches can be engineered in arbitrary and common protein folds, sheds light on several important questions: 1) What is the generality of fold switching? 2) What types of folds are amenable to switching? 3) What properties are shared by sequences that can fold into two completely different structures? This work has implications for understanding how amino acid sequence encodes structure, how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure.

ClassificationBiological Sciences: Biochemistry; Physical Sciences: Biophysics and Computational Biology

sentences: Four new Scopalina from Southern California: the first Scopalinida (Porifera: Demospongiae) from the temperate Eastern Pacific Sponges (phylum Porifera) are common inhabitants of kelp forest ecosystems in California, but their diversity and ecological importance are poorly characterized in this biome. Here I use freshly collected samples to describe the diversity of the order Scopalinida in California. Though previously unknown in the region, four new species are described here: Scopalina nausicae sp. nov., S. kuyamu sp. nov., S. goletensis sp. nov., and S. jali sp. nov.. These discoveries illustrate the considerable uncharacterized sponge diversity remaining in California kelp forests, and the utility of SCUBA-based collection to improve our understanding of this diversity.

sentences: BrainPalmSeq: A curated RNA-seq database of palmitoylating and de-palmitoylating enzyme expression in the mouse brain Protein S-palmitoylation is a reversible post-translational lipid modification that plays a critical role in neuronal development and plasticity, while dysregulated S-palmitoylation underlies a number of severe neurological disorders. Dynamic S-palmitoylation is regulated by a large family of ZDHHC palmitoylating enzymes, their accessory proteins, and a small number of known de-palmitoylating enzymes. Here, we curated and analyzed expression data for the proteins that mediate S-palmitoylation from publicly available RNAseq datasets, providing a comprehensive overview of their distribution in the mouse nervous system. We developed a web-tool that enables interactive visualization of the expression patterns for these proteins in the nervous system (http://brainpalmseq.med.ubc.ca/), and explored this resource to find region and cell-type specific expression patterns that give insight into the function of palmitoylating and de-palmitoylating enzymes in the brain and neurological disorders. We found coordinated expression of ZDHHC enzymes with their accessory proteins, de-palmitoylating enzymes and other brain-expressed genes that included an enrichment of S-palmitoylation substrates. Finally, we utilized ZDHHC expression patterns to predict and validate palmitoylating enzyme-substrate interactions.

sentences: p53 pro-apoptotic activity is regulated by the G2/M promoting factor Cdk1 in response to DNA damage Exposure to genotoxic stress promotes cell-cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, how p53 activity is precisely regulated is essential to maintain tissue homeostasis and to prevent cancer development. Here we demonstrate that Drosophila p53 pro-apoptotic activity is regulated by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation induced apoptosis. We show that the p53 protein is not able to bind to and to activate the expression of the pro-apoptotic genes in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 pro-apoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 pro-apoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.

sentences: Sexual selection for rare beneficial mutations promotes the evolution of sexual reproduction and adaptation Sexual selection is the process by which traits providing a mating advantage are favoured. Theoretical treatments of the evolution of sex by sexual selection propose that it operates by reducing the load of deleterious mutations. Here, we postulate instead that sexual selection primarily acts through females preferentially mating with males carrying beneficial mutations. We used simulation and analytical modelling to investigate the evolutionary dynamics of beneficial mutations in the presence of sexual selection. We found that female choice for males with beneficial mutations had a much greater impact on genetic quality than choice for males with low mutational load. We also relaxed the typical assumption of a fixed mutation rate. For deleterious mutations, mutation rate should always be minimized, but when rare beneficial mutations can occur, female choice for males with those rare beneficial mutations could overcome a decline in average fitness and allow an increase in mutation rate. We propose that sexual selection for beneficial mutations could overcome the  two-fold cost of sex much more readily than choice for males with low mutational load and may therefore be a more powerful explanation for the prevalence of sexual reproduction than the existing theory. If sexual selection results in higher fitness at higher mutation rates and if the variability produced by mutation itself promotes sexual selection, then a feedback loop between these two factors could have had a decisive role in driving adaptation.

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Function and constraint in enhancers with multiple evolutionary origins MotivationThousands of human gene regulatory enhancers are composed of sequences with multiple evolutionary origins. These evolutionarily "complex" enhancers consist of older "core" sequences and younger "derived" sequences. However, the functional relationship between the sequences of different evolutionary origins within complex enhancers is poorly understood.

ResultsWe evaluated the function, selective pressures, and sequence variation across core and derived components of human complex enhancers. We find that both components are older than expected from the genomic background, and cores are enriched for derived sequences of similar evolutionary ages. Both components show strong evidence of biochemical activity in massively parallel report assays (MPRAs). However, core and derived sequences have distinct transcription factor (TF) binding preferences that are largely stable across evolutionary origins. Given these signatures of function, both core and derived sequences have substantial evidence of purifying selection. Nonetheless, derived sequences exhibit weaker purifying selection than adjacent cores. Derived sequences also tolerate more common genetic variation and are enriched compared to cores for eQTL associated with gene expression variability in human populations.

ConclusionsBoth core and derived sequences have strong evidence of gene regulatory function, but derived sequences have distinct constraint profiles, TF binding preferences, and tolerance to variation compared with cores. We propose that the step-wise integration of younger derived and older core sequences has generated regulatory substrates with robust activity and the potential for functional variation. Our analyses demonstrate that synthesizing study of enhancer evolution and function can aid interpretation of regulatory sequence activity and functional variation across human populations.

Significance StatementThousands of human gene regulatory enhancers are mosaics of sequences from multiple evolutionary origins, yet how these different segments contribute to enhancer function is poorly understood. By dissecting their regulatory functions, transcription factor binding, constraint, and human genetic variation, we show that both older "core" and younger "derived" sequences in complex enhancers have strong evidence of gene regulatory function, but derived sequences are more likely to harbor genetic variants that influence function. Together, our results support a model in which the integration of sequences of different origins generates regulatory substrates with robust activity and the potential for functional variation.

sentences: Getting to know each other: PPIMem, a novel approach for predicting transmembrane protein-protein complexes Because of their considerable number and diversity, membrane proteins and their macromolecular complexes represent the functional units of cells. Their quaternary structure may be stabilized by interactions between the -helices of different proteins in the hydrophobic region of the cell membrane. Membrane proteins also represent potential pharmacological targets par excellence for various diseases. Unfortunately, their experimental 3D structure and that of their complexes with intramembrane interacting partners are scarce due to technical difficulties. To overcome this key problem, we devised PPIMem, a computational approach for the specific prediction of higher-order structures of -helical transmembrane proteins. The novel approach involves identification of the amino acid residues at the interface of complexes with a 3D structure. The identified residues compose then interaction motifs that are conveniently expressed as mathematical regular expressions. These are used for motif search in databases, and for the prediction of intramembrane protein-protein complexes. Our template interface-based approach predicted 21, 544 binary complexes between 1, 504 eukaryotic plasma membrane proteins across 39 species. We compared our predictions to experimental datasets of protein-protein interactions as a first validation method. The PPIMem online database with the annotated predicted interactions is implemented as a web server and can be accessed directly at https://transint.shinyapps.io/transint/.

sentences: Activation of T cell checkpoint pathways during beta-cell antigen presentation by engineered dendritic cells promotes protection of non-obese diabetic mice from type 1 diabetes Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumor therapy. We reported that dendritic cells (DCs) that are engineered to express selective ligands for checkpoint receptors can induce effective T cell tolerance in antigen-immunization models. Here, we show that pancreatic {beta} -cell antigen (BcAg) presentation by engineered tolerogenic-DCs (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared to engineered control-DCs. Non-obese diabetic (NOD) mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycmic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in, and reversal of, hyperglycemia for a significant duration. Immune cells from the tDC treated mice not only produced lower amounts of IFN{gamma} and higher amounts of IL10 and TGF{beta}1 upon BcAg challenge, but also failed to induce hyperglycemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

sentences: PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, andRNA polymerase pause release at select gene targets Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues1. Thus, it is difficult to achieve a mechanistic understanding of transcription factor function using traditional genetic deletion or RNAi methods, because these slow approaches make it challenging to distinguish direct from indirect transcriptional effects. Here, we used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO12-6 to define how the t(2;13)(q35;q14) disrupts normal gene expression programs to trigger cancer. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses, we identified a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Moreover, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. The activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective and often only a single element within a complex super-enhancer was affected. In addition, fusion of the endogenous PAX3-FOXO1 with APEX2 identified proteins in close proximity with PAX3-FOXO1, including ARID1A and MYOD1. We found that continued expression of PAX3-FOXO1 was required to maintain chromatin accessibility and allow neighboring DNA binding proteins and chromatin remodeling complexes to associate with this small number of regulated enhancers. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network.

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Mimicking tumor cell heterogeneity of colorectal cancer in a patient-derived organoid-fibroblast model Patient-derived organoid (PDO) cancer models are generated from epithelial tumor cells. Although they reflect the molecular tumor characteristics, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we present a colorectal cancer (CRC) organoid model that incorporates epithelial cells and stromal fibroblasts from the same patient. Molecular characterization of primary cancer associated fibroblasts (CAFs) and matched normal fibroblasts (NF) revealed proteomic, secretome and gene expression differences in pathways associated with tumor related fibroblast function. Further, CAFs retained higher motility compared to NFs in vitro. Importantly, both CAFs and NFs supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. PDOs grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared to mono-cultures, and closely resembled the in vivo tumor morphology. This was also confirmed by the calculation of cellular proportions of epithelial cell subtypes in organoid mono-versus co-cultures, which were inferred through bioinformatics deconvolution of bulk RNA sequencing data using published single cell RNA sequencing datasets from CRC tissues. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by majorly deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. For the fibroblasts, we observed enhanced expression of tumor induced marker genes and cytokines characteristic for myo- and immunogenic fibroblasts. This model will be vital as a physiological personalized tumor model to study disease mechanisms and therapy response in CRC.

One Sentence SummaryPatient matched fibroblasts support tumor organoid growth in 3D co-culture and maintain intratumoral cellular heterogeneity and histo-morphology.

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Noise correlations for faster and more robust learning. Distributed population codes are ubiquitous in the brain and pose a challenge to downstream neurons that must learn an appropriate readout. Here we explore the possibility that this learning problem is simplified through inductive biases implemented by stimulus-independent noise correlations that constrain learning to task-relevant dimensions. We test this idea in a set of neural networks that learn to perform a perceptual discrimination task. Correlations among similarly tuned units were manipulated independently of overall population signal-to-noise ratio in order to test how the format of stored information affects learning. Higher noise correlations among similarly tuned units led to faster and more robust learning, favoring homogenous weights assigned to neurons within a functionally similar pool, and could emerge through Hebbian learning. When multiple discriminations were learned simultaneously, noise correlations across relevant feature dimensions sped learning whereas those across irrelevant feature dimensions slowed it. Our results complement existing theory on noise correlations by demonstrating that when such correlations are produced without significant degradation of the signal-to-noise ratio, they can improve the speed of readout learning by constraining it to appropriate dimensions.

Significance statementPositive noise correlations between similarly tuned neurons theoretically reduce the representational capacity of the brain, yet they are commonly observed, emerge dynamically in complex tasks, and persist even in well-trained animals. Here we show that such correlations, when embedded in a neural population with a fixed signal to noise ratio, can improve the speed and robustness with which an appropriate readout is learned. In a simple discrimination task such correlations can emerge naturally through Hebbian learning. In more complex tasks that require multiple discriminations, correlations between neurons that similarly encode the task-relevant feature improve learning by constraining it to the appropriate task dimension.

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Automated 3D reconstruction of the fetal thorax in the standard atlas space from motion-corrupted MRI stacks for 21-36 weeks GA range Slice-to-volume registration (SVR) methods allow reconstruction of high-resolution 3D images from multiple motion-corrupted stacks. SVR-based pipelines have been increasingly used for motion correction for T2-weighted fetal MRI since they allow more informed and detailed diagnosis of brain and body anomalies including congenital heart defects (Lloyd et al., 2019). Recently, fully automated rigid SVR reconstruction of the fetal brain in the atlas space was achieved in (Salehi et al., 2019) that used convolutional neural networks (CNNs) for segmentation and pose estimation. However, these CNN-based methods have not yet been applied to the fetal trunk region. Meanwhile, the existing rigid and deformable SVR (DSVR) solutions (Uus et al., 2020) for the fetal trunk region are limited by the requirement of manual input as well the narrow capture range of the classical gradient descent based registration methods that cannot resolve severe fetal motion frequently occurring at the early gestational age (GA). Furthermore, in our experience, the conventional 2D slice-wise CNN-based brain masking solutions are reportedly prone to errors that require manual corrections when applied on a wide range of acquisition protocols or abnormal cases in clinical setting.

In this work, we propose a fully automated pipeline for reconstruction of the fetal thorax region for 21-36 weeks GA range T2-weighted MRI datasets. It includes 3D CNN-based intra-uterine localisation of the fetal trunk and landmark-guided pose estimation steps that allow automated DSVR reconstruction in the standard radiological space irrespective of the fetal trunk position or the regional stack coverage. The additional step for generation of the common template space and rejection of outliers provides the means for automated exclusion of stacks affected by low image quality or extreme motion. The pipeline was evaluated on a series of experiments including fetal MRI datasets and simulated rotation motion. Furthermore, we performed a qualitative assessment of the image reconstruction quality in terms of the definition of vascular structures on 100 early (median 23.14 weeks) and late (median 31.79 weeks) GA group MRI datasets covering 21 to 36 weeks GA range.

sentences: Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

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Still no evidence for disruption of global patterns of nest predation in shorebirds Many shorebird species are rapidly declining (Piersma et al. 2016; Munro 2017; Studds et al. 2017), but it is not always clear why. Deteriorating and disappearing habitat, e.g. due to intensive agriculture (Donal et al. 2001; Kentie et al. 2013; Kentie et al. 2018), river regulation (Nebel et al. 2008) or mudflat reclamation (Ma et al. 2014; Larson 2017), and hunting (Reed et al. 2018; Gallo-Cajiao et al. 2020) are some of the documented causes. A recent study suggests yet another possible cause of shorebird decline: a global increase in nest predation (Kubelka et al. 2018). The authors compiled an impressive dataset on patterns of nest predation in shorebirds and their analyses suggest that global patterns of nest predation have been disrupted by climate change, particularly in the Arctic. They go as far as to conclude that the Arctic might have become an ecological trap (Kubelka et al. 2018). Because these findings might have far-reaching consequences for conservation and related political decisions, we scrutinized the study and concluded that the main conclusions of Kubelka et al. (2018) are invalid (Bulla et al. 2019a). The authors then responded by reaffirming their conclusions (Kubelka et al. 2019b).

Here, we evaluate some of Kubelka et al.s (2019b) responses, including their recent erratum (2020), and show that the main concerns about the original study still hold. Specifically, (1) we reaffirm that Kubelka et al.s (2018) original findings are confounded by study site. Hence, their conclusions are over-confident because of pseudo-replication. (2) We reiterate that there is no statistical support for the assertion that predation rate has changed in a different way in the Arctic compared to other regions. The relevant test is an interaction between a measure of time (year or period) and a measure of geography (e.g., Arctic vs the rest of the world). The effect of such an interaction is weak, uncertain and statistically non-significant, which undermines Kubelka et al.s (2018) key conclusion. (3) We further confirm that the suggested general increase in predation rates over time is at best a weak and uncertain trend. The most parsimonious hypothesis for the described results is that the temporal changes in predation rate are an artefact of temporal changes in methodology and data quality. Using only high-quality data, i.e. directly calculated predation rates, reveals no overall temporal trend in predation rate. Below we elaborate in detail on each of these points.

We conclude that (i) there is no evidence whatsoever that the pattern in the Arctic is different from that in the rest of the world and (ii) there is no solid evidence for an increase in predation rate over time. While we commend Kubelka et al. for compiling and exploring the data, we posit that the data underlying their study, and perhaps all currently available data, are not sufficient (or of sufficient quality) to test their main hypotheses. We call for standardized and consistent data collection protocols and experimental validation of current methods for estimating nesting success.

sentences: How antiporters exchange substrates across the cell membrane? An atomic-level description of the complete exchange cycle in NarK Major facilitator superfamily (MFS) proteins operate via three different mechanisms: uniport, symport, and antiport. Despite extensive investigations, molecular understanding of antiporters is less advanced than other transporters due to the complex coupling between two substrates and the lack of distinct structures. We employ extensive (~300 s) all-atom molecular dynamics simulations to dissect the complete substrate exchange cycle of the bacterial [Formula] antiporter, NarK. We show that paired basic residues in the binding site prevent the closure of unbound protein and ensure the exchange of two substrates. Conformational transition only occurs in the presence of substrate, which weakens the electrostatic repulsion and stabilizes the transporter by ~1.5 {+/-} 0.1 kcal/mol. Furthermore, we propose a state-dependent substrate exchange model, in which the relative spacing between the paired basic residues determines whether [Formula] and [Formula] bind simultaneously or sequentially. Overall, this work presents a general working model for the antiport mechanism within MFS family.

sentences: Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs Human in vitro models of neural tissue with controllable cellular identity, tunable microenvironment, and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing in jammed microgel supports (i.e., granular gels) holds great promise as it allows precise and programmable patterning of extremely soft and compliant tissue constructs. However, in contrast to the vast material landscape available for bulk hydrogels, granular printing support formulations are restricted to a handful of materials without the ability for facile adjustment of biofunctional properties of the cellular microenvironment. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, we present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise freeform patterning of human stem cells and consequent generation and long-term maintenance of mature subtype-specific neurons that extend projections within the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular channels. Due to its modularity, SHAPE biomanufacturing toolbox not only offers a solution for functional modeling of mechanically sensitive neural constructs, but also has potential to be applied to a wide range of biomaterials with different crosslinking mechanisms to model tissues and diseases where recapitulation of complex architectural features and topological cues is essential.

sentences: Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails dramatic epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC.

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Cerebrovascular responses to O2-CO2 exchange ratio under brief breath-hold challenge in patients with chronic mild traumatic brain injury Breath-by-breath O2-CO2 exchange ratio (bER) is a respiratory gas exchange (RGE) metric, which is the ratio of the changes in the partial pressure of O2 ({Delta}PO2) to CO2 ({Delta}PCO2) between endinspiration and end-expiration, has been demonstrated to characterize the cerebrovascular responses to breath-hold challenge in healthy individuals. We aimed to explore if bER could characterize cerebrovascular responses in patients with chronic mild traumatic brain injury (mTBI) under breath-hold challenge. We also investigated the correlation between bER and the severity of post-concussion symptoms.

Blood-oxygenation-level-dependent (BOLD) images were acquired using functional magnetic resonance imaging (fMRI) on eleven patients with chronic mTBI and eleven controls without brain injury history when performing a breath-hold task. Time series of RGE metrics of {Delta}PO2, {Delta}PCO2, and bER were computed, and their cross-correlation with regional change in BOLD ({Delta}BOLD) was calculated. Symptom burden was assessed using the Rivermead Post Concussion Questionnaire (RPQ), and its correlation with RGE changes was also measured.

Compared with controls, a diffuse decrease in the correlation between regional {Delta}BOLD and bER was found in the brain of mTBI patients (pfdr<0.05). No significant difference was found between patients and controls for the correlation of regional {Delta}BOLD with {Delta}PO2 and {Delta}PCO2. The averaged changes in bER ({rho}=0.79, p=0.01) and {Delta}PO2 ({rho}=0.70, p=0.03) in breath-hold epochs decreased with increased symptom severity indicated by RPQ scores.

Our imaging and symptom severity findings suggest that bER can be used to characterize cerebrovascular responses to breath-hold in mTBI patients. RGE may contribute to the post-concussive symptom severity.

sentences: Doppler Slicing for Ultrasound Super-Resolution Without Contrast Agents Much of the information needed for diagnosis and treatment monitoring of diseases like cancer and cardiovascular disease is found at scales below the resolution limit of classic ultrasound imaging. Recently introduced vascular super-localization methods provide more than a ten-fold improvement in spatial resolution by precisely estimating the positions of microbubble contrast agents. However, most vascular ultrasound scans are currently performed without contrast agents due to the associated cost, training, and post-scan monitoring. Here we show that super-resolution ultrasound imaging of dense vascular structures can be achieved using the natural contrast of flowing blood cells. Instead of relying on separable targets, we used Fourier-based decomposition to separate signals arising from the different scales of vascular structures while removing speckle noise using multi-ensemble processing. This approach enabled the use of compressed sensing for super-resolution imaging of the underlying vascular structures, improving resolution by a factor of four. Reconstruction of ultrafast mouse brain scans revealed details that could not be resolved in regular Doppler images, agreeing closely with bubble-based super-localization microscopy of the same fields of view. By combining multi-ensemble Doppler acquisitions with narrowband Fourier decomposition and computational super-resolution imaging, this approach opens new opportunities for affordable and scalable super-resolution ultrasound imaging.

sentences: Understanding double descent through the lens of principal component regression A number of recent papers have studied the double-descent phenomenon: as the number of parameters in a supervised learning model increasingly exceeds that of data points ("second-descent"), the empirical risk curve has been observed to not overfit, instead decreasing monotonically, sometimes to a level even better than the best "first-descent" model (using a subset of features not exceeding the number of data points). Understanding exactly when this happens and why it happens is an important theoretical problem. Focusing on the over-parameterized linear regression setting, a commonly chosen case study in the double-descent literature, we present two theoretical results: 1) final second-descent (regression using all of the predictor variables) and principal component (PC) regression without dimensionality reduction are equivalent; 2) the PCR risk curve can be expected to lower bound not only all linearly transformed first-descent models, but also all linearly transformed second-descent models (including the elimination of features as a special case); 3) if the smallest singular value of the design matrix is "large enough" (we will define mathematically), final second-descent can be expected to outperform any first-descent or second-descent model. These insights have important ramifications for a type of semi-supervised learning problem, a scenario which can explain why a face representation trained on unlabeled faces from one race would be better for later supervised-learning tasks on the same race of faces than for faces from another race - this can both provide a scientific explanation for the other-race effect seen in humans and give hints for how to mitigate similar issues in the domain of ethical AI.

sentences: Identification of potential proteins and microRNAs in Multiple Sclerosis and Huntington's diseases using in silico methods. Neurogenerative diseases like multiple sclerosis, Huntingtons disease are the major roadblocks in the way towards a healthy brain. Neurodegenerative diseases like multiple sclerosis and Huntingtons disease are affected by several factors such as environmental, immunological, genetics, and the worse scenario we can think of is that they are on the rise worldwide. Degenerative diseases specifically target a limited group of neurons at first resulting in the loss of specific functions associated with the specific part of the brain. The early diagnosis of these neurodegenerative diseases is important so that treatments can start from the early stages of these diseases. In this study, we have established a link between Multiple sclerosis and Huntingtons disease, and also we were able to establish the possible microRNAs that were connected to the expression of genes associated with these two diseases. In this present study, we analyzed the microarray datasets obtained from Gene Expression Omnibus and we identified 266 differentially expressed genes tried to identify using in silico methods the Hub genes involved in Multiple sclerosis and Huntingtons disease. After identifying the genes and proteins we tried to identify the microRNAs that are interacting with the Hub genes. In our study, we identified that the protein network has PTPRC, CXCL8, RBM25 proteins that have maximum connectivity. The top Hub genes are then subjected to a database that contains information concerning the microRNAs that are interacting with the Hub proteins as well as with each other. According to our study, the hsa-mir-155-5p has one of the highest degrees in the microRNA network. Our study will be useful in the future for the development of new drug targets for these neurodegenerative diseases.

sentences: Broadening a SARS-CoV-1 neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

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Integrating reward information for prospective behaviour Value-based decision-making is often studied in a static context, where participants decide which option to select from those currently available. However, everyday life often involves an additional dimension: deciding when to select to maximise reward. Recent evidence suggests that agents track the latent reward of an option, updating changes in their latent reward estimate, to achieve appropriate selection timing (latent reward tracking). However, this strategy can be difficult to distinguish from one in which the optimal selection time is estimated in advance, allowing an agent to wait a pre-determined amount of time before selecting, without needing to monitor an options latent reward (distance-to-goal tracking). Here we show that these strategies can in principle be dissociated. Human brain activity was recorded using electroencephalography (EEG) while female and male participants performed a novel decision task. Participants were shown an option and decided when to select it, as its latent reward changed from trial-to-trial. While the latent reward was uncued, it could be estimated using cued information about the options starting value and value growth rate. We then used representational similarity analysis to assess whether EEG signals more closely resembled latent reward tracking or distance-to-goal tracking. This approach successfully dissociated the strategies in this task. Starting value and growth rate were translated into a distance-to-goal signal, far in advance of selecting the option. Latent reward could not be independently decoded. These results demonstrate the feasibility of using high temporal resolution neural recordings to identify internally computed decision variables in the human brain.

Significance StatementReward-seeking behaviour involves acting at the right time. However, the external world does not always tell us when an action is most rewarding, necessitating internal representations that guide action timing. Specifying this internal neural representation is challenging because it might stem from a variety of strategies, many of which make similar predictions about brain activity. This study used a novel approach to test whether alternative strategies could be dissociated in principle. Using representational similarity analysis, we were able to distinguish between candidate internal representations for selection timing. This shows how pattern analysis methods can be used to measure latent decision information in non-invasive neural data.

sentences: Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and aging Astrocytes are resident glia cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question.

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

sentences: Perilipin 5 interacts with Fatp4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (Plin5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and {beta}-oxidation during starvation of myoblasts requires both phosphorylation of Plin5 and an intact Plin5 mitochondrial tethering domain. We further identified the acyl-CoA synthetase, Fatp4 (ACSVL4) as a novel mitochondrial interactor of Plin5. The C-terminal domains of Plin5 and Fatp4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of Plin5, lipolysis, and subsequent channeling of FAs from LDs to Fatp4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

sentences: Analysis of Rod/Cone Gap Junctions from the Reconstruction of Mouse Photoreceptor Terminals Using serial blockface-scanning electron microscopy (SBF-SEM) and focused ion beam-scanning electron microscopy (FIB-SEM), combined with confocal microscopy for the gap junction protein Cx36, we reconstructed mouse photoreceptor terminals and located the gap junctions between them. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules where Cx36 clusters were located, close to the mouth of the synaptic opening. There were approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. Our calculations suggest a mean of 82 Cx36 channels between a rod/cone pair of which 25% are open at rest. Rod/cone gap junctions are modulated by dopamine. Comparing our morphological calculations of maximum coupling to previous physiological results suggests that dopamine antagonists can drive rod/cone gap junctions to a surprisingly high open probability, approaching 100%.

sentences: The F-box protein AFF1 regulates ARF protein accumulation to regulate auxin response Auxin critically regulates nearly every aspect of plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. Although ARF protein stability is regulated via the 26S proteasome, molecular mechanisms underlying ARF stability and turnover are unknown. Here, we report the identification and functional characterization of an F-box E3 ubiquitin ligase, which we have named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). AFF1 directly interacts with ARF19 and regulates its accumulation. Mutants defective in AFF1 display ARF19 protein hyperaccumulation, attenuated auxin responsiveness, and developmental defects. Together, our data suggest a new mechanism, namely control of ARF protein stability, in regulating auxin response.

sentences: AGC: Compact representation of assembled genomes High-quality sequence assembly is the ultimate representation of complete genetic information of an individual. Several ongoing pangenome projects are producing collections of high-quality assemblies of various species. Here, we show how to represent the sequenced genomes in 2-3 orders of magnitude smaller space, allowing easy and fast extraction of any contig or its part.

sentences: Recovery of deleted deep sequencing data sheds more light on the early Wuhan SARS-CoV-2 epidemic The origin and early spread of SARS-CoV-2 remains shrouded in mystery. Here I identify a data set containing SARS-CoV-2 sequences from early in the Wuhan epidemic that has been deleted from the NIHs Sequence Read Archive. I recover the deleted files from the Google Cloud, and reconstruct partial sequences of 13 early epidemic viruses. Phylogenetic analysis of these sequences in the context of carefully annotated existing data further supports the idea that the Huanan Seafood Market sequences are not fully representative of the viruses in Wuhan early in the epidemic. Instead, the progenitor of currently known SARS-CoV-2 sequences likely contained three mutations relative to the market viruses that made it more similar to SARS-CoV-2s bat coronavirus relatives.

sentences: Stable motifs delay species loss in simulated food webs Some three-species motifs (unique patterns of interactions between three species) are both more stable when modeled in isolation and over-represented in empirical food webs. This suggests that these motifs may reduce extinction risk for species participating in them, ultimately stabilizing the food web as a whole. We test whether a species time to extinction following a perturbation is related to its participation in stable and unstable motifs and assess how motif roles co-vary with a species degree or trophic level. We found that species motif roles are related to their times to extinction following a disturbance. Specifically, participating in many omnivory motifs (whether in absolute terms, as a proportion of the species role, or relative to other species in the network) was associated with more rapid extinction, even though omnivory has previously been identified as a stable motif. Participating in the other three stable motifs (three-species chain, apparent competition, and direct competition) was associated with longer times to extinction. While motif roles were associated with extinction risk, they also varied strongly with degree and trophic level. This means that these simpler measures of a species role may be sufficient to roughly predict which species are most vulnerable to disturbance, but the additional information encapsulated in a motif role can further refine predictions of vulnerability. Moreover, where researchers are a priori interested in motif roles, our results confirm that these roles can be interpreted with respect to extinction risk.

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Isolation, screening, degradation characteristics of a quinclorac-degrading bacteria D and its potential of bioremediation for rice field environment polluted by quinclorac Quinclorac (QNC) is a highly selective, hormonal, and low-toxic herbicide with a long duration. And the growth and development of subsequent crops are easily affected by QNC accumulated in the soil. In this paper, a QNC-degrading strain D was isolated and screened from the rice paddy soil. Through morphology, physiological and biochemical tests and 16Sr DNA gene analysis, strain D was identified as Cellulosimicrobium cellulans sp. And the QNC degradation characteristics of strain D were studied. Under the optimal culture conditions, the QNC-degrading rate was 45.9% after culturing for 21 days. The QNC-degrading efficiency of strain D in the field was evaluated by a simulated pot experiment. The results show that strain D can promote the growth of rice and QNC-degrading effectively. This research could provide a new bacterial species for microbial degradation of QNC and lay a theoretical foundation for further research on QNC remediation.

ImportanceAt present, some QNC-degrading bacteria have been isolated from different environments, but there are no reports of Cellulosimicrobium cellulans sp. bacterial that could degrade QNC. In this study, a new QNC-degradation strain was selected from the paddy soil. The degradation characteristics of strain D were studied in detail. The results shown that strain D had a satisfactory quinclorac-degrading efficiency. Two degradation products of QNC by strain D were identified by HPLC-Q-TOF/MS: 3-pyridylacetic acid (138.0548 m/z) and 3-ethylpyridine (108.0805 m/z), which have not been reported before. The strain D had a potential ability of quinclorac-degrading effectively in the quinclorac-polluted paddy field environment.

sentences: Reward learning and working memory: effects of massed versus spaced training and post-learning delay period Neuroscience research has illuminated the mechanisms supporting learning from reward feedback, demonstrating a critical role for the striatum and midbrain dopamine system. However, in humans, short-term working memory that is dependent on frontal and parietal cortices can also play an important role, particularly in commonly-used paradigms in which learning is relatively condensed in time. Given the growing use of reward-based learning tasks in translational studies in computational psychiatry, it is important to understand the degree of working memory contributions and whether gradual learning mechanisms can be better isolated. In our experiments, we manipulated the spacing between repetitions along with a post-learning delay preceding a test phase. We found that learning was slower for stimuli repeated after a long delay (spaced-trained) compared to those repeated immediately (massed-trained), likely reflecting the remaining contribution of feedback learning mechanisms when working memory is not available. Brief interruptions of massed learning led to drops in subsequent performance. Further, individual differences in working memory capacity positively correlated with massed learning performance. Critically, after a delay period but not immediately, relative preferences decayed in the massed condition and increased in the spaced condition. Overall, our results provide additional support for a large role of working memory in reward-based learning in temporally condensed designs. We suggest that spacing training within or between sessions is a promising approach to better isolate and understand mechanisms supporting gradual reward-based learning, with particular importance for understanding potential learning dysfunctions in addiction and psychiatric disorders.

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Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

SummaryKono et al. show the rapid recruitment of nucleoplasmic lamin C to sites of nuclear envelope rupture with Barrier-to-autointegration factor. Lamin A/C is also involved in nuclear DNA sensing with cytoplasmic cGAS at the ruptured sites.

sentences: A structural property for reduction of biochemical networks Large-scale biochemical models are of increasing sizes due to the consideration of interacting organisms and tissues. Model reduction approaches that preserve the flux phenotypes can simplify the analysis and predictions of steady-state metabolic phenotypes. However, existing approaches either restrict functionality of reduced models or do not lead to significant decreases in the number of modelled metabolites. Here, we introduce an approach for model reduction based on the structural property of balancing of complexes that preserves the steady-state fluxes supported by the network and can be efficiently determined at genome scale. Using two large-scale mass-action kinetic models of Escherichia coli, we show that our approach results in a substantial reduction of 99% of metabolites. Applications to genome-scale metabolic models across kingdoms of life result in up to 55% and 85% reduction in the number of metabolites when arbitrary and mass-action kinetics is assumed, respectively. We also show that predictions of the specific growth rate from the reduced models match those based on the original models. Since steady-state flux phenotypes from the original model are preserved in the reduced, the approach paves the way for analysing other metabolic phenotypes in large-scale biochemical networks.

sentences: Bacteriostatic antibiotics promote the evolution of CRISPR-Cas immunity Phage therapy can be used in combination with antibiotics to combat infections with bacterial pathogens1-3. However, bacteria can rapidly evolve phage resistance via receptor mutation, or using their CRISPR-Cas adaptive immune systems4, which insert short phage-derived sequences into CRISPR loci in the bacterial genome5 to guide sequence-specific cleavage of cognate sequences6. Unlike CRISPR-Cas immunity, mutation of the phage receptor leads to attenuated virulence when the opportunistic pathogen Pseudomonas aeruginosa is infected with its phage DMS3vir7, which underscores the need to predict how phage resistance evolves under clinically relevant conditions. Here, using eight antibiotics with various modes of action, we show that bacteriostatic antibiotics (which inhibit cell growth without killing) specifically promote evolution of CRISPR-Cas immunity in P. aeruginosa by slowing down phage development and providing more time for cells to acquire phage-derived sequences and mount an immune response. Our data show that some antimicrobial treatments can contribute to the evolution of phage-resistant pathogens with high virulence.

sentences: Ventral motor thalamic input to prelimbic cortex, but not to striatum, mediates cost-benefit decision-making in rats Both prelimbic corticostriatal neurons and striatal fast-spiking interneurons contribute to decisions that require a trade-off between cost and benefit. We investigated whether ventral motor thalamic input to prelimbic cortex or striatum contributes to cost-benefit decision-making. Optogenetic inhibition of ventral motor thalamic axon terminals in prelimbic cortex biased rats towards a high cost-high benefit option and, in anesthetized rats, decreased neuronal activity in deep layers of prelimbic cortex. Stimulation of ventral motor thalamic nuclei induced a neuronal response in deep layers of prelimbic cortex and simultaneous optogenetic inhibition of layer 1 inhibitory interneurons similarly decreased neuronal activity. Chemogenetic inhibition of striatal-projecting ventral motor thalamic neurons did not affect cost-benefit decision-making. Our results indicate that ventral motor thalamic input to prelimbic cortex, but not striatum, mediates cost-benefit decision-making, probably by regulating prelimbic corticostriatal neuron activity directly as well as indirectly through a network of cortical inhibitory interneurons.

sentences: Distinct mutations and lineages of SARS-CoV-2 virus in the early phase of COVID-19 pandemic and subsequent global expansion A novel coronavirus, SARS-CoV-2, has caused over 190 million cases and over 4 million deaths worldwide since it occurred in December 2019 in Wuhan, China. Here we conceptualized the temporospatial evolutionary and expansion dynamics of SARS-CoV-2 by taking a series of cross-sectional view of viral genomes from early outbreak in January 2020 in Wuhan to early phase of global ignition in early April, and finally to the subsequent global expansion by late December 2020. Based on the phylogenetic analysis of the early patients in Wuhan, Wuhan/WH04/2020 is supposed to be a more appropriate reference genome of SARS-CoV-2, instead of the first sequenced genome Wuhan-Hu-1. By scrutinizing the cases from the very early outbreak, we found a viral genotype from the Seafood Market in Wuhan featured with two concurrent mutations (i.e. M type) had become the overwhelmingly dominant genotype (95.3%) of the pandemic one year later. By analyzing 4,013 SARS-CoV-2 genomes from different continents by early April, we were able to interrogate the viral genomic composition dynamics of initial phase of global ignition over a timespan of 14-week. 11 major viral genotypes with unique geographic distributions were also identified. WE1 type, a descendant of M and predominantly witnessed in western Europe, consisted a half of all the cases (50.2%) at the time. The mutations of major genotypes at the same hierarchical level were mutually exclusive, which implying that various genotypes bearing the specific mutations were propagated during human-to-human transmission, not by accumulating hot-spot mutations during the replication of individual viral genomes. As the pandemic was unfolding, we also used the same approach to analyze 261,323 SARS-CoV-2 genomes from the world since the outbreak in Wuhan (i.e. including all the publicly available viral genomes) in order to recapitulate our findings over one-year timespan. By 25 December 2020, 95.3% of global cases were M type and 93.0% of M-type cases were WE1. In fact, at present all the four variants of concern (VOC) are the descendants of WE1 type. This study demonstrates the viral genotypes can be utilized as molecular barcodes in combination with epidemiologic data to monitor the spreading routes of the pandemic and evaluate the effectiveness of control measures. Moreover, the dynamics of viral mutational spectrum in the study may help the early identification of new strains in patients to reduce further spread of infection, guide the development of molecular diagnosis and vaccines against COVID-19, and help assess their accuracy and efficacy in real world at real time.

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ggtranscript: an R package for the visualization and interpretation of transcript isoforms using ggplot2 MotivationThe advent of long-read sequencing technologies has increased demand for the visualisation and interpretation of transcripts. However, tools that perform such visualizations remain inflexible and lack the ability to easily identify differences between transcript structures. Here, we introduce ggtranscript, an R package that provides a fast and flexible method to visualize and compare transcripts. As a ggplot2 extension, ggtranscript inherits the functionality and familiarity of ggplot2 making it easy to use.

Availability and implementationggtranscript is available at https://github.com/dzhang32/ggtranscript, DOI: https://doi.org/10.5281/zenodo.6374061

sentences: Interacting effects of cold snaps, rain, and agriculture on the fledging success of a declining aerial insectivore Climate change predicts the increased frequency, duration, and intensity of inclement weather periods, such as unseasonably low temperatures (i.e., cold snaps) and prolonged precipitation. Many migratory species have advanced the phenology of important life history stages, and as a result will likely be exposed to these periods of inclement spring weather more often, thus risking reduced fitness and population growth. For declining avian species, including aerial insectivores, anthropogenic landscape changes such as agricultural intensification are another driver of population declines. These landscape changes may affect the foraging ability of food provisioning parents, and reduce the survival of nestlings exposed to inclement weather, through for example pesticide exposure impairing thermoregulation and punctual anorexia. Breeding in agro-intensive landscapes may thus exacerbate the negative effects of inclement weather under climate change. We observed that a significant reduction in the availability of insect prey occurred when daily maximum temperatures fell below 18.3{degrees}C, and thereby defined any day where the maximum temperature fell below this value as a day witnessing a cold snap. We then combined daily information on the occurrence of cold snaps and measures of precipitation to assess their impact on the fledging success of Tree Swallows (Tachycineta bicolor) occupying a nest box system placed across a gradient of agricultural intensification. Estimated fledging success of this declining aerial insectivore was 36.2% lower for broods experiencing four cold snap days during the 12 days post hatching period versus broods experiencing none, and this relationship was worsened when facing more precipitation. We further found that the overall negative effects of a brood experiencing periods of inclement weather was exacerbated in more agro-intensive landscapes. Our results indicate that two of the primary hypothesized drivers of many avian population declines may interact to further increase the rate of declines in certain landscape contexts.

sentences: On-chip light diffraction imaging of nano structures in the guanine platelet Light projection over short distances can minimize the size of photonic devices, e.g., head-mounted displays and lens-free microscopes. Small lenses or light condensers without typical lenses are essential for light control in micron-scale spaces. In this work, micro-platelets floating in water are used for light projection near the image sensor. These platelets, which are made from guanine, have nanohole gratings and demonstrate light diffraction toward specific directions. By setting a thin water layer on the image sensors cover glass, each platelet in water forms column-or bar-code-shaped images on the screen. The projected image shapes and colors are inferred to contain information about nano-structures present in the guanine platelet. The proposed down-sized imaging technique can realize extremely compact and portable imagers for nanoscale object detection.

sentences: Towards a structurally resolved human protein interaction network All cellular functions are governed by complex molecular machines that assemble through protein-protein interactions. Their atomic details are critical to the study of their molecular mechanisms but fewer than 5% of hundreds of thousands of human interactions have been structurally characterized. Here, we test the potential and limitations of recent progress in deep-learning methods using AlphaFold2 to predict structures for 65,484 human interactions. We show that higher confidence models are enriched in interactions supported by affinity or structure based methods and can be orthogonally confirmed by spatial constraints defined by cross-link data. We identify 3,137 high confidence models, of which 1,371 have no homology to a known structure, from which we identify interface residues harbouring disease mutations, suggesting potential mechanisms for pathogenic variants. We find groups of interface phosphorylation sites that show patterns of co-regulation across conditions, suggestive of coordinated tuning of multiple interactions as signalling responses. Finally, we provide examples of how the predicted binary complexes can be used to build larger assemblies. Accurate prediction of protein complexes promises to greatly expand our understanding of the atomic details of human cell biology in health and disease.

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Bioframe: Operations on Genomic Intervals in Pandas Dataframes MotivationGenomic intervals are one of the most prevalent data structures in computational genome biology, and used to represent features ranging from genes, to DNA binding sites, to disease variants. Operations on genomic intervals provide a language for asking questions about relationships between features. While there are excellent interval arithmetic tools for the command line, they are not smoothly integrated into Python, one of the most popular general-purpose computational and visualization environments.

ResultsBioframe is a library to enable flexible and performant operations on genomic interval dataframes in Python. Bioframe extends the Python data science stack to use cases for computational genome biology by building directly on top of two of the most commonly-used Python libraries, numpy and pandas. The bioframe API enables flexible name and column orders, and decouples operations from data formats to avoid unnecessary conversions, a common scourge for bioinformaticians. Bioframe achieves these goals while maintaining high performance and a rich set of features.

Availability and implementationBioframe is open-source under MIT license, cross-platform, and can be installed from the Python package index. The source code is maintained by Open2C on Github at https://github.com/open2c/bioframe.

sentences: RAREsim: A simulation method for very rare genetic variants Identification of rare variant associations is crucial to fully characterize the genetic architecture of complex traits and diseases. Essential in this process is the evaluation of novel methods in simulated data that mirrors the distribution of rare variants and haplotype structure in real data. Additionally, importing real variant annotation enables in silico comparison of methods that focus on putative causal variants, such as rare variant association tests, and polygenic scoring methods. Existing simulation methods are either unable to employ real variant annotation or severely under- or over-estimate the number of singletons and doubletons reducing the ability to generalize simulation results to real studies. We present RAREsim, a flexible and accurate rare variant simulation algorithm. Using parameters and haplotypes derived from real sequencing data, RAREsim efficiently simulates the expected variant distribution and enables real variant annotations. We highlight RAREsims utility across various genetic regions, sample sizes, ancestries, and variant classes.

sentences: Genomic and transcriptomic landscape of advanced renal cell cancer to individualize treatment strategy The genomic landscape of advanced renal cell cancer (RCC) was characterized for 91 patients to identify actionable targets and signatures by combining whole-genome sequencing (WGS) with matched RNA sequencing (RNA-Seq). WGS data were analyzed for somatic small variants, copy-number alterations (CNAs) and structural variants. Somatic aberrations were analyzed for driver genes, CNA drivers, mutational signatures, catastrophic events and fusion genes. For papillary and clear cell RCC, potential actionable drug targets were detected by WGS in 89% and 100% of the patients, respectively. RNA-Seq data of clear cell and papillary RCC were clustered according to a previously developed angio-immunogenic gene signature. WGS and RNA-Seq may improve therapeutic decision making for most patients with advanced RCC, including patients with non-clear cell RCC for whom no standard treatment is available to date. Prospective clinical trials are needed to evaluate the impact of genomic and transcriptomic diagnostics on survival outcome for advanced RCC patients.

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Propranolol reduces sarcoma growth and enhances the response to anti-CTLA4 therapy by modulating the tumor microenvironment The nonselective beta blocker, propranolol, which for decades has been prescribed for treatment of cardiovascular conditions, has recently been used successfully to treat metastatic angiosarcoma. These results have led to an orphan drug designation by the European Medicines Agency for the treatment of soft tissue sarcomas. The anti-tumor effects of propranolol are suggested to involve the reduction of cancer cell proliferation as well as angiogenesis.

Here, we have investigated the anti-angiogenic properties of propranolol in the context of stimulating an anti-tumor immune response. We show that oral administration of propranolol delays tumor progression of MCA205 fibrosarcoma tumors and increases the survival rate of tumor bearing mice. Propranolol works by reducing tumor angiogenesis and facilitating an anti-tumoral microenvironment with increased T cell infiltration and reduced infiltration of myeloid-derived suppressor cells (MDSCs). Using T cell deficient mice, we demonstrate that the full anti-tumor effect of propranolol requires the presence of T cells. Flow cytometry-based analysis and RNA sequencing of FACS-sorted cells show that propranolol-treatment leads to an upregulation of PD-L1 on tumor-associated macrophages (TAMs) and changes in their chemokine expression profile. Lastly, we observe that the co-administration of propranolol significantly enhances the efficacy of anti-CTLA4 therapy.

Our results identify propranolol as an immune modulating agent, which can improve immune checkpoint inhibitor therapies in soft tissue sarcoma patients and potentially in other cancers.

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Virtual deep brain stimulation: Multiscale co-simulation of a spiking basal ganglia model and a whole-brain mean-field model with The Virtual Brain Deep brain stimulation (DBS) has been successfully applied in various neurodegenerative diseases as an effective symptomatic treatment. However, its mechanisms of action within the brain network are still poorly understood. Many virtual DBS models analyze a subnetwork around the basal ganglia and its dynamics as a spiking network with their details validated by experimental data. However, connectomic evidence shows widespread effects of DBS affecting many different cortical and subcortical areas. From a clinical perspective, various effects of DBS besides the motoric impact have been demonstrated. The neuroinformatics platform The Virtual Brain (TVB) offers a modeling framework allowing us to virtually perform stimulation, including DBS, and forecast the outcome from a dynamic systems perspective prior to invasive surgery with DBS lead placement. For an accurate prediction of the effects of DBS, we implement a detailed spiking model of the basal ganglia, which we combine with TVB via our previously developed co-simulation environment. This multiscale co-simulation approach builds on the extensive previous literature of spiking models of the basal ganglia while simultaneously offering a whole-brain perspective on widespread effects of the stimulation going beyond the motor circuit. In the first demonstration of our model, we show that virtual DBS can move the firing rates of a Parkinsons disease patients thalamus - basal ganglia network towards the healthy regime while, at the same time, altering the activity in distributed cortical regions with a pronounced effect in frontal regions. Thus, we provide proof of concept for virtual DBS in a co-simulation environment with TVB. The developed modeling approach has the potential to optimize DBS lead placement and configuration and forecast the success of DBS treatment for individual patients.

Highlights- We implement and validate a co-simulation approach of a spiking network model for subcortical regions in and around the basal ganglia and interface it with mean-field network models for each cortical region.
- Our simulations are based on a normative connectome including detailed tracts between the cortex and the basal ganglia regions combined with subject-specific optimized weights for a healthy control and a patient with Parkinsons disease.
- We provide proof of concept by demonstrating that the implemented model shows biologically plausible dynamics during resting state including decreased thalamic activity in the virtual patient and during virtual deep brain stimulation including normalized thalamic activity and distributed altered cortical activity predominantly in frontal regions.
- The presented co-simulation model can be used to tailor deep brain stimulation for individual patients.

sentences: Interpreting sensory and cognitive signals in the cortical reading network Voxels in the visual cortex contain neurons that are responsive to small regions of the visual field; these regions can be quantified using population receptive field (pRF) analysis. We measured pRFs using several types of patterns: words, visually matched false-fonts, and visually dissimilar checker patterns. In visual field maps pRF estimates using words and false-fonts are similar, but pRF estimates using checkers differ substantially. The pattern of results in the contiguous ventral occipito-temporal reading circuitry is different: word pRF estimates differ from false-fonts and checkers. These findings were replicated at two research sites. We explain these results with a qualitative model, where the response differences in the visual field maps arise from sensory signals, and the differences in the reading circuitry arise from the integration of sensory and cognitive signals.

sentences: GiPS: Genomics-informed parent selection uncovers the breeding value of wheat genetic resources The great efforts spent in the maintenance of past diversity in genebanks are rationalized by the potential role of plant genetic resources in future crop improvement - a concept whose practical implementation has fallen short of expectations. Here, we implement genomics-informed parent selection to expedite pre-breeding without discriminating against non-adapted germplasm. We collect dense genetic profiles for a large winter wheat collection and evaluate grain yield and resistance to yellow rust in representative coresets. Genomic prediction within and across genebanks identified the best parents for PGR x elite derived crosses that outyielded current elite cultivars in multiple field trials.

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Stereo Slant Discrimination of Planar 3D Surfaces: Standard vs. Planar Cross-Correlation Binocular stereo cues are important for discriminating 3D surface orientation, especially at near distances. We devised a single-interval task where observers discriminated the slant of a densely textured planar test surface relative to a textured planar surround reference surface. Although surfaces were rendered with correct perspective, the stimuli were designed so that the binocular cues dominated performance. Slant discrimination performance was measured as a function of the reference slant and the level of uncorrelated white noise added to the test-plane images in the left and right eye. We compared human performance with an approximate ideal observer (planar cross correlation, PCC) and two sub-ideal observers. The PCC observer uses the image in one eye and back projection to predict the test image in the other eye for all possible slants, tilts, and distances. The estimated slant, tilt, and distance are determined by the prediction that most closely matches the measured image in the other eye. The first sub-ideal observer (local PCC, LPCC) applies planar cross correlation over local neighborhoods and then pools estimates across the test plane. The second sub-optimal observer (standard cross correlation, SCC), uses only positional disparity information. We find that the ideal observer (PCC) and the first sub-ideal observer (LPCC) outperform the second sub-ideal observer (SCC), demonstrating the benefits of structural disparities. We also find that all three model observers can account for human performance, if two free parameters are included: a fixed small level of internal estimation noise, and a fixed overall efficiency scalar on slant discriminability.

PrecisWe measured human stereo slant discrimination thresholds for accurately-rendered textured surfaces designed so that performance is dominated by binocular-disparity cues. We compared human performance with an approximate ideal observer and two sub-ideal observers.

sentences: Capturing variation in metagenomic assembly graphs with MetaCortex The assembly of contiguous sequence from metagenomic samples presents a particular challenge, due to the presence of multiple species, often closely related, at varying levels of abundance. Capturing diversity within species, for example viral haplotypes, or bacterial strain-level diversity, is even more challenging. We present MetaCortex, a metagenome assembler based on data structures from the Cortex de novo assembler. MetaCortex captures intra-species diversity by searching for signatures of local variation along assembled sequences in the underlying assembly graph and outputting these sequences in sequence graph format. MetaCortex also implements a novel assembly algorithm for representing intra-species diversity in standard linear format. We show that MetaCortex produces accurate assemblies with higher genome coverage and contiguity than other popular metagenomic assemblers on mock viral communities with high levels of strain level diversity, and on simulated communities containing simulated strains. We also show that accuracy can be increased further by using the sequence graph produced by MetaCortex to create highly accurate single contig sequences.

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Jack-of-all-trades paradigm meets long-term data: generalist herbivores are more widespread and locally less abundant Ecological specialization is one of the most interesting and perplexing attributes of biological systems. While certain macroecological patterns, such as an increase in specialization at lower latitudes, have long been subjects of investigation, there is much yet to be learned about inter-specific variation in specialization within diverse communities. High levels of specialization have been documented for some dominant ecological interactions, such as parasitism and herbivory, but much less is known about the relative abundance of specialists and generalists within those broad functional groups. We examine untested assumptions about the positive association between local abundance and dietary specialization using a 17-year dataset of caterpillar-plant interactions in Ecuador. Our long-term data consist of experimental verification of caterpillar-plant associations and include standardized plot-based samples as well as general, regional collections of caterpillars, allowing for investigations across spatial scales and using different indices of abundance for 1,917 morphospecies of Lepidoptera ("caterpillars") from 33 families. We find that more specialized caterpillars are locally more abundant than generalists, consistent with a key component of the "jack of all trades, master of none" hypothesis, which has otherwise received poor to mixed support from previous studies that have mostly involved fewer species and shorter time series. At larger scales, generalists achieve greater prevalence across the landscape, and we find some evidence for geographic variation in the abundance-diet breadth relationship, in particular among elevational bands. Interspecific variation in abundance had a negative relationship with diet breadth, with specialists having more variable abundances across species. The interesting result that more specialized species can be both rare and common highlights the ecological complexity of specialization.

Statement of authorshipCS wrote the first draft of the manuscript. DMS, MLF and LAD contributed substantially to consequent drafts and revisions. LAD, TRW, SV and DMS collected field data. CS, MLF and LAD generated research questions and designed statistical analyses. CS, DMS, LAD and MLF conducted statistical analyses.

Data statementData supporting the results and conclusions can be found on a website caterpillars.org. Statistical analyses supporting the results are available upon request.

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Targeting mitochondrial double-stranded RNAs ameliorates autoimmune characteristics of Sjogrens syndrome ObjectiveSjogrens syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. This study investigates the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFN response in SS.

MethodsSaliva and tear from SS patients and controls (n=73 for saliva and n=16 for tear), the salivary glands of the SS-prone-non-obese-diabetic mouse, and primary human salivary glandular cells were screened for mt-dsRNAs by RT-qPCR. The human salivary cell line (NS-SV-AC) grown as three-dimensional spheroids were subject to dsRNA stress to measure mt-dsRNA induction and recapitulation of SS glandular inflammatory features. Acetylcholine, SS-IgG, upadacitinib (JAK1 inhibitor), or 2-C'-methyladenosine (mitochondrial transcription inhibitor) were applied to characterize the roles of mt-dsRNAs. To identify endogenous dsRNA-sensor and confirm the mitochondrial origin of cytoplasmic dsRNAs, the immunoprecipitation of dsRNAs was performed.

Resultsmt-dsRNAs were elevated in the SS specimens with salivary ND5 and tear CYTB1 being statistically associated with secretory dysfunction/inflammation and corneal/conjunctival damage, respectively. Stimulation of the spheroids with dsRNA stress of poly I:C induced mt-dsRNAs, p-PKR, and I-IFNS via the JAK1/STAT pathway whereas the inhibition of mt-RNA synthesis or JAK1 attenuated the glandular signature. The inhibitory effect of acetylcholine on mt-dsRNAs and I-IFNS induction was reversed by SS-IgG.

Conclusionmt-dsRNAs amplify the impact of dsRNA stress on SS glandular signatures in vitro, potentially propagating a pseudo-viral signal in the SS target tissue.

SummaryMitochondrial double-stranded RNA levels were elevated in the tear and saliva of SS patients, which was associated with secretory dysfunction and tissue inflammation. These RNAs amplified type I interferon signature as well as glandular phenotypes reported in SS. Inhibitors of mitochondrial RNA transcription or JAK1 in salivary gland acinar cell spheroids attenuated the mitochondrial RNA-mediated changes.

sentences: Characterizing the Behavioral and Neuroendocrine Features of Susceptibility and Resilience to Social Stress Evaluating and coping with stressful social events as they unfold is a critical strategy in overcoming them without long-lasting detrimental effects. Individuals display a wide range of responses to stress, which can manifest in a variety of outcomes for the brain as well as subsequent behavior. Chronic Social Defeat Stress (CSDS) in mice has been widely used to model individual variation following a social stressor. Following a course of repeated intermittent psychological and physical stress, mice diverge into separate populations of social reactivity: resilient (socially interactive) and susceptible (socially avoidant) animals. A rich body of work reveals distinct neurobiological and behavioral consequences of this experience that map onto the resilient and susceptible groups. However, the range of factors that emerge over the course of defeat have not been fully described. Therefore, in the current study, we focused on characterizing behavioral, physiological, and neuroendocrine profiles of mice in three separate phases: before, during, and following CSDS. We found that following CSDS, traditional read-outs of anxiety-like and depression-like behaviors do not map on to the resilient and susceptible groups. By contrast, behavioral coping strategies used during the initial social stress encounter better predict which mice will eventually become resilient or susceptible. In particular, mice that will emerge as susceptible display greater escape behavior on Day 1 of social defeat than those that will emerge as resilient, indicating early differences in coping mechanisms used between the two groups. We further show that the social avoidance phenotype in susceptible mice is specific to the aggressor strain and does not generalize to conspecifics or other strains, indicating that there may be features of threat discrimination that are specific to the susceptible mice. Our findings suggest that there are costs and benefits to both the resilient and susceptible outcomes, reflected in their ability to cope and adapt to the social stressor.

sentences: High throughput genome-wide single cell protein:DNA binding site mapping by targeted insertion of promoters (TIP-seq) Assessing cell to cell, and importantly, chromosome to chromosome, heterogeneity in cellular phenotypes is a central goal of modern cell biology. However, chromatin profiling in single cells has been extremely challenging, and single chromosome profiling has not been achieved. In cases where single cell methods have shown promise, success has been mainly limited to histone proteins and/or require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we have combined the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that does not require specialized equipment and is capable of multiplexing several samples/ target proteins in one experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A (as with CUT&Tag) to insert a T7 RNA polymerase promoter into sites adjacent to an antibody bound to a chromatin protein of interest, followed by linear amplification of flanking DNA with T7 polymerase, cDNA preparation and PCR indexing. Tip-seq provides [~]10-fold higher unique reads and thus higher coverage per single cell compared to state-of-the-art methods. We apply TIP-seq to map histone modifications, RNA PolII and CTCF binding sites in single human and mouse cells. TIP-seq will also be adaptable for other platforms, such as 10X genomics and ICELL8. In summary, TIP-seq provides a high-throughput, low-cost method for single cell protein mapping, that yields substantially higher coverage per cell and signal to noise than existing methods.

sentences: MoTSE: an interpretable task similarity estimator for small molecular property prediction tasks Understanding the molecular properties (e.g., physical, chemical or physiological characteristics and biological activities) of small molecules plays essential roles in biomedical researches. The accumulating amount of datasets has enabled the development of data-driven computational methods, especially the machine learning based methods, to address the molecular property prediction tasks. Due to the high cost of obtaining experimental labels, the datasets of individual tasks generally contain limited amount of data, which inspired the application of transfer learning to boost the performance of the molecular property prediction tasks. Our analyses revealed that simultaneously considering similar tasks, rather than randomly chosen ones, can significantly improve the performance of transfer learning in this field. To provide accurate estimation of task similarity, we proposed an effective and interpretable computational tool, named Molecular Tasks Similarity Estimator (MoTSE). By extracting task-related local and global knowledge from pretrained graph neural networks (GNNs), MoTSE projects individual tasks into a latent space and measures the distance between the embedded vectors to derive the task similarity estimation and thus enhance the molecular prediction results. We have validated that the task similarity estimated by MoTSE can serve as a useful guidance to design a more accurate transfer learning strategy for molecular property prediction. Experimental results showed that such a strategy greatly outperformed baseline methods including training from scratch and multitask learning. Moreover, MoTSE can provide interpretability for the estimated task similarity, through visualizing the important loci in the molecules attributed by the attribution method employed in MoTSE. In summary, MoTSE can provide an accurate method for estimating the molecular property task similarity for effective transfer learning, with good interpretability for the learned chemical or biological insights underlying the intrinsic principles of the task similarity.

sentences: Exosomal miR-769-5p confers cisplatin resistance and tumorigenesis in gastric cancer Cisplatin resistance is the main cause of poor clinical prognosis in patients with gastric cancer (GC). Yet, the exact mechanism of cisplatin resistance remains unclear. Recent studies have suggested that exocrine miRNAs found in the tumor microenvironment participates in tumor metastasis and drug resistance. In this study, we discovered that cisplatin-resistant GC cells communicate with the tumor microenvironment by secreting microvesicles. The biologically active miR-769-5p can be integrated into exosomes and delivered to sensitive cells, thereby spreading cisplatin resistance. Mi769-5p was upregulated in GC tissues and enriched in the serum exosomes of cisplatin-resistant patients. Mechanistically, miR-769-5p promotes cisplatin resistance by targeting CASP9 so as to inhibit the downstream caspase pathway and promote the degradation of the apoptosis-related protein p53 through the ubiquitin-proteasome pathway. Targeting miR-769 with its antagonist to treat cisplatin-resistant GC cells can restore the cisplatin response, confirming that exosomal miR-769-5p can be a key regulator of cisplatin resistance in GC. Therefore, exosomal miR-769-5p derived from drug-resistant cells can be used as a potential therapeutic predictor of anti-tumor chemotherapy to enhance the effect of anti-cancer chemotherapy, which provides a new treatment option for GC.

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The scratch-digging lifestyle of the Permian "microsaur" Batropetes as a model for the exaptative origin of jumping locomotion in frogs Recent studies have shown that the Triassic stem-frog Triadobatrachus lacked the ability to jump off, but nonetheless had the forelimb strength to withstand the impact of landing from a jump. We propose a hypothesis to resolve this pseudoparadox: the strengthened forelimbs are former adaptations to forelimb-based digging that later made jumping possible by exaptation.

Micro-CT data from a skeleton of Batropetes palatinus reveal thin cortical bone, confirming Batropetes as terrestrial. Combining adaptations to walking and digging, confirmed by statistical analyses, Batropetes is thought to have searched for food in leaf litter or topsoil. We interpret Batropetes as having used one forelimb at a time to shove leaf litter aside. Batropetes may thus represent an analog or possibly a homolog of the digging stage that preceded the origin of Salientia.

We discuss the possibility of homology with the digging lifestyles of other "microsaurs" and other amphibians.

sentences: Complex population dynamics in a spatial microbial ecosystem with Physarum polycephalum This research addresses the interactions between the unicellular slime mold Physarum polycephalum and a red yeast in a spatial ecosystem over week-long imaging experiments. An inverse relationship between the growth rates of both species is shown, where P. polycephalum has positive growth when the red yeast has a negative growth rate and vice versa. The data also captures successional/oscillatory dynamics between both species. An advanced image analysis methodology for semantic segmentation is used to quantify population density over time, for all components of the ecosystem. We suggest that P. polycephalum is capable of exhibiting a sustainable feeding strategy by depositing a nutritive slime trail, allowing yeast to serve as a periodic food source. This opens a new direction of P. polycephalum research, where the population dynamics of spatial ecosystems can be readily quantified and complex ecological dynamics can be studied.

sentences: Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, A Potential Oral Drug Candidate for COVID-19 Treatment Preclinical pharmacokinetics (PK) and in vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.2 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

sentences: The growth and expansion of meningeal lymphatic networks are affected in craniosynostosis Congenital skull malformations are associated with vascular anomalies that can impair fluid balance in the central nervous system. We previously reported that humans with craniosynostosis and mutations in TWIST1 have dural venous sinus malformations. It is still unknown whether meningeal lymphatic networks, which are patterned alongside the venous sinuses, are also affected. Using a novel skull flat mounting technique, we show that the growth and expansion of meningeal lymphatics are perturbed in Twist1 craniosynostosis models. Changes to the local meningeal environment, including hypoplastic dura and venous malformations, affect the ability of lymphatic networks to sprout and remodel. Dorsal networks along the transverse sinus are hypoplastic with reduced branching. By contrast, basal networks closer to the skull base are more variably affected, showing exuberant growth in some animals suggesting they are compensating for vessel loss in dorsal networks. Injecting molecular tracers into cerebrospinal fluid reveals significantly less drainage to the deep cervical lymph nodes, indicative of impaired lymphatic function. Collectively, our results show that meningeal lymphatic development is hindered in craniosynostosis, suggesting central nervous system waste clearance may be impeded.

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PhenoComb: A discovery tool to assess complex phenotypes in high-dimension, single-cell datasets MotivationHigh-dimension cytometry assays can simultaneously measure dozens of markers, enabling the investigation of complex phenotypes. However, as manual gating relies on previous biological knowledge, few marker combinations are often assessed. This results in complex phenotypes with potential for biological relevance being overlooked. Here we present PhenoComb, an R package that allows agnostic exploration of phenotypes by assessing all combinations of markers.

DesignPhenoComb uses signal intensity thresholds to assign markers to discrete states (e.g. negative, low, high) and then counts the number of cells per sample from all possible marker combinations in a memory-safe manner. Time and disk space are the only constraints on the number of markers evaluated. PhenoComb also provides several approaches to perform statistical comparisons, evaluate the relevance of phenotypes, and assess the independence of identified phenotypes. PhenoComb allows users to guide analysis by adjusting several function arguments such as identifying parent populations of interest, filtering of low-frequency populations, and defining a maximum complexity of phenotypes to evaluate. We have designed PhenoComb to be compatible with local computer or server-based use.

ResultsIn testing of PhenoCombs performance on synthetic datasets, computation on 16 markers was completed in the scale of minutes and up to 26 markers in hours. We applied PhenoComb to two publicly available datasets: an HIV flow cytometry dataset (12 markers and 421 samples) and the COVIDome CyTOF dataset (40 markers and 99 samples). In the HIV dataset, PhenoComb identified immune phenotypes associated with HIV seroconversion, including those highlighted in the original publication. In the COVID dataset, we identified several immune phenotypes with altered frequencies in infected individuals relative to healthy individuals. Collectively, PhenoComb represents a powerful discovery tool for agnostically assessing high-dimension, single-cell data.

AvailabilityThe PhenoComb R package can be downloaded from https://github.com/SciOmicsLab/PhenoComb

sentences: Structural diversity of the SARS-CoV-2 Omicron spike Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

sentences: Structure of benzothiadiazine at zwitterionic phospholipid cell membranes The use of drugs derived from benzothiadiazine, which is a bicyclic heterocyclic benzene derivative, has become a widespread treatment for diseases such as hypertension (treated with diuretics such as bendroflumethiazide or chlorothiazide), low blood sugar (treated with non-diuretic diazoxide) or the human immunodeficiency virus, among others. In this work we have investigated the interactions of benzothiadiazine with the basic components of cell membranes and solvents such as phospholipids, cholesterol, ions and water. The analysis of the mutual microscopic interactions is of central importance to elucidate the local structure of benzothiadiazine as well as the mechanisms responsible for the access of benzothiadiazine to the interior of the cell. We have performed molecular dynamics simulations of benzothiadiazine embedded in three different model zwitterionic bilayer membranes made by dimyristoilphosphatidylcholine, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoserine and cholesterol inside aqueous sodium-chloride solution in order to systematically examine microscopic interactions of benzothiadiazine with the cell membrane at liquid-crystalline phase conditions. From data obtained through radial distribution functions, hydrogen-bonding lengths and potentials of mean force based on reversible work calculations, we have observed that benzothiadiazine has a strong affinity to stay at the cell membrane interface although it can be fully solvated by water in short periods of time. Furthermore, benzothiadiazine is able to bind lipids and cholesterol chains by means of single and double hydrogen-bonds of different characteristic lengths.

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A phased genome assembly for allele-specific analysis in Trypanosoma brucei Many eukaryotic organisms are diploid or even polyploid, i.e. they harbour two or more independent copies of each chromosome. Yet, to date most reference genome assemblies represent a mosaic consensus sequence in which the homologous chromosomes have been collapsed into one sequence. This procedure generates sequence artefacts and impedes analyses of allele-specific mechanisms. Here, we report the allele-specific genome assembly of the diploid unicellular protozoan parasite Trypanosoma brucei.

As a first step, we called variants on the allele-collapsed assembly of the T. brucei Lister 427 isolate using short-read error-corrected PacBio reads. We identified 96 thousand heterozygote variants across the genome (average of 4.2 variants / kb), and observed that the variant density along the chromosomes was highly uneven. Several long (>100 kb) regions of loss-of-heterozigosity (LOH) were identified, suggesting recent recombination events between the alleles. By analysing available genomic sequencing data of multiple Lister 427 derived clones, we found that most LOH regions were conserved, except for some that were specific to clones adapted to the insect lifecycle stage. Surprisingly, we also found that some Lister 427 clones were aneuploid. We found evidence of trisomy in chromosome five (chr 5), chr 2, chr 6 and chr 7. Moreover, by analysing RNA-seq data, we showed that the transcript level is proportional to the ploidy, evidencing the lack of a general expression control at the transcript level in T. brucei.

As a second step, to generate an allele-specific genome assembly, we used two powerful datatypes for haplotype reconstruction: raw long reads (PacBio) and chromosome conformation (Hi-C) data. With this approach, we were able to assign 99.5% of all heterozygote variants to a specific homologous chromosome, building a 66 Mb long T. brucei Lister 427 allele-specific genome assembly. Hereby, we identified genes with allele-specific premature termination codons and showed that differences in allele-specific expression at the level of transcription and translation can be accurately monitored with the fully phased genome assembly.

The obtained reference-grade allele-specific genome assembly of T. brucei will enable the analysis of allele-specific phenomena, as well as the better understanding of recombination and evolutionary processes. Furthermore, it will serve as a standard to  benchmark much needed automatic genome assembly pipelines for highly heterozygous wild species isolates.

sentences: Translation stalling proline motifs are enriched in slow-growing, thermophilic, and multicellular bacteria Rapid bacterial growth depends on the speed at which ribosomes can translate mRNA into proteins. mRNAs that encode successive stretches of proline can cause ribosomes to stall, substantially reducing translation speed. Such stalling is especially detrimental for species that must grow and divide rapidly. Here we focus on di-prolyl motifs (XXPPX) and ask whether their incidence varies with growth rate. To find out we conducted a broad survey of such motifs in >3000 bacterial genomes across 36 phyla. Indeed, fast-growing species encode fewer motifs than slow-growing species, especially in highly expressed proteins. We also found many di-prolyl motifs within thermophiles, where prolines can help maintain proteome stability. Moreover, bacteria with complex, multicellular lifecycles also encode many di-prolyl motifs. This is especially evident in the slow-growing phylum Myxococcota. Bacteria in this phylum encode many serine-threonine kinases, and many di-prolyl motifs at potential phosphorylation sites within these kinases. Serine-threonine kinases are involved in cell signaling and help regulate developmental processes linked to multicellularity in the Myxococcota. Altogether, our observations suggest that weakened selection on translational rate, whether due to slow or thermophilic growth, may allow di-prolyl motifs to take on new roles in biological processes that are unrelated to translational rate.

sentences: Evolution of innate behavioral strategies through competitive population dynamics Many organism behaviors are innate or instinctual and have been "hard-coded" through evolution. Current approaches to understanding these behaviors model evolution as an optimization problem in which the traits of organisms are assumed to optimize an objective function representing evolutionary fitness. Here, we use a mechanistic birth-death dynamics approach to study the evolution of innate behavioral strategies in a population of organisms in silico. In particular, we performed agent-based stochastic simulations and mean-field analyses of organisms exploring random environments and competing with each other to find locations with plentiful resources. We find that when organism density is low, the mean-field model allows us to derive an effective objective function, predicting how the most competitive phenotypes depend on the exploration-exploitation trade-off between the scarcity of high-resource sites and the increase in birth rate those sites offer organisms. However, increasing organism density alters the most competitive behavioral strategies and precludes the existence of a well-defined objective function. Moreover, there exists a range of densities for which the coexistence of many phenotypes persists for evolutionarily long times.

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Highly-Neutralizing COVID-19-Convalescent-Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters. Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19.

ImportanceConvalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.

sentences: Multivalent binding proteins can drive collapse and reswelling of chromatin in confinement Collapsed conformations of chromatin have been long suspected of being mediated by interactions with multivalent binding proteins, such as CTCF, which can bring together distant sections of the chromatin fiber. In this study, we use Langevin dynamics simulation of coarse grained chromatin polymer to show that the role of binding proteins can be more nuanced than previously suspected. In particular, for chromatin polymer in confinement, entropic forces can drive reswelling of collapsed chromatin with increasing binder concentrations. The reswelling transition happens at physiologically relevant binder concentrations and the extent of reswelling is mediated both by the concentration of binding proteins as well as the strength of confinement. We also study the kinetics of collapse and reswelling and show that both processes occur in similar timescales. We characterise this reswelling of chromatin in biologically relevant regimes and discuss implications for the spatial organisation of the genome.

sentences: Factorbook: an Updated Catalog of Transcription Factor Motifs and Candidate Regulatory Motif Sites The human genome contains roughly 1,600 transcription factors (TFs) (1), DNA-binding proteins recognizing characteristic sequence motifs to exert regulatory effects on gene expression. The binding specificities of these factors have been profiled both in vitro, using techniques such as HT-SELEX (2), and in vivo, using techniques including ChIP-seq (3, 4). We previously developed Factorbook, a TF-centric database of annotations, motifs, and integrative analyses based on ChIP-seq data from Phase II of the ENCODE Project. Here we present an update to Factorbook which significantly expands the breadth of cell type and TF coverage. The update includes an expanded motif catalog derived from thousands of ENCODE Phase II and III ChIP-seq experiments and HT-SELEX experiments; this motif catalog is integrated with the ENCODE registry of candidate cis-regulatory elements to annotate a comprehensive collection of genome-wide candidate TF binding sites. The database also offers novel tools for applying the motif models within machine learning frameworks and using these models for integrative analysis, including annotation of variants and disease and trait heritability. We will continue to expand the resource as ENCODE Phase IV data are released.

sentences: An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

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StrVCTVRE: A supervised learning method to predict the pathogenicity of human genome structural variants BackgroundWhole genome sequencing resolves many clinical cases where standard diagnostic methods have failed. However, at least half of these cases remain unresolved after whole genome sequencing. Structural variants (SVs; genomic variants larger than 50 base pairs) of uncertain significance are the genetic cause of a portion of these unresolved cases. As sequencing methods using long or linked reads become more accessible and structural variant detection algorithms improve, clinicians and researchers are gaining access to thousands of reliable SVs of unknown disease relevance. Methods to predict the pathogenicity of these SVs are required to realize the full diagnostic potential of long-read sequencing.

ResultsTo address this emerging need, we developed StrVCTVRE to distinguish pathogenic SVs from benign SVs that overlap exons. In a random forest classifier, we integrated features that capture gene importance, coding region, conservation, expression, and exon structure. We found that features such as expression and conservation are important but are absent from SV classification guidelines. We leveraged multiple resources to construct a size-matched training set of rare, putatively benign and pathogenic SVs. StrVCTVRE performs accurately across a wide SV size range on independent test sets, which will allow clinicians and researchers to eliminate about half of SVs from consideration while retaining a 90% sensitivity.

ConclusionsWe anticipate clinicians and researchers will use StrVCTVRE to prioritize SVs in patients where no SV is immediately compelling, empowering deeper investigation into novel SVs to resolve cases and understand new mechanisms of disease. StrVCTVRE runs rapidly and is available at https://compbio.berkeley.edu/proj/strvctvre/.

sentences: The landscape of accessible chromatin in quiescent and post-myocardial infarction cardiac fibroblasts After myocardial infarction, quiescent cardiac fibroblasts are activated and undergo multiple proliferation and differentiation events, which contribute to the extracellular matrix remodeling of the infarcted myocardium. We recently found that cardiac fibroblasts of different differentiation states had distinct expression profiles closely related to their functions. Gene expression is directly regulated by chromatin state. However, the role of chromatin reorganization in the drastic gene expression changes during post-MI differentiation of cardiac fibroblast has not been revealed. In this study, the gene expression profiling and genome-wide mapping of accessible chromatin in mouse cardiac fibroblasts isolated from uninjured hearts and the infarcts at different time points were performed by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. ATAC-seq peaks were highly enriched in the promoter area and distal areas where enhancers might be located. A positive correlation was identified between the transcription level and promoter accessibility for many dynamically expressed genes. In addition, it was found that DNA methylation may contribute to the post-MI chromatin remodeling and gene expression in cardiac fibroblasts. Integrated analysis of ATAC-seq and RNA-seq datasets also identified transcription factors that possibly contributed to the differential gene expression between cardiac fibroblasts of different states.

sentences: Distinct Mechanisms of Innate and Adaptive Immune Regulation Underlie Poor Oncologic Outcomes Associated with KRAS-TP53 Co-Alteration in Pancreatic Cancer Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

sentences: High-performance Flexible Microelectrode Array with PEDOT:PSS Coated 3D Micro-cones for Electromyographic Recording High signal-to-noise ratio (SNR) electromyography (EMG) recordings are essential for identifying and analyzing single motor unit activity. While high-density electrodes allow for greater spatial resolution, the smaller electrode area translates to a higher impedance and lower SNR. In this study, we developed an implantable and flexible 3D microelectrode array (MEA) with low impedance that enables high-quality EMG recording. With polyimide micro-cones realized by standard photolithography process and PEDOT:PSS coating, this design can increase effective surface area by up to 250% and significantly improve electrical performance for electrode sites with various geometric surface areas, where the electrode impedance is at most improved by 99.3%. Acute EMG activity from mice was recorded by implanting the electrodes in vivo, and we were able to detect multiple individual motor units simultaneously and with high resolution (SNR >> 100). The charge storage capacity was measured to be 34.2 mC/cm2, indicating suitability of the electrodes for stimulation applications as well.

sentences: Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

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Amino acid transporter SLC38A5 regulates developmental and pathological retinal angiogenesis Amino acid metabolism in vascular endothelium is important for sprouting angiogenesis. SLC38A5 (solute carrier family 38 member 5), an amino acid (AA) transporter, shuttles neutral AAs across cell membrane, including glutamine, which may serve as metabolic fuel for proliferating endothelial cells (ECs) to promote angiogenesis. Here we found that Slc38a5 is highly enriched in normal retinal vascular endothelium, and more specifically in pathological sprouting neovessels. Slc38a5 is suppressed in retinal blood vessels from Lrp5-/- and Ndpy/- mice, both genetic models of defective retinal vascular development with Wnt signaling mutations. Additionally, Slc38a5 transcription is directly regulated by Wnt/{beta}-catenin signaling. Genetic deficiency of Slc38a5 in mice substantially delays retinal vascular development and suppresses pathological neovascularization in oxygen-induced retinopathy modeling ischemic proliferative retinopathies. Inhibition of SLC38A5 in retinal vascular ECs impairs EC proliferation and angiogenic function, suppresses glutamine uptake, and dampens vascular endothelial growth factor receptor 2 (VEGFR2). Together these findings suggest that SLC38A5 is a new metabolic regulator of retinal angiogenesis by controlling AA nutrient uptake and homeostasis in ECs.

Significance StatementAmino acid metabolism in vascular endothelium is important for angiogenesis. SLC38A5 (solute carrier family 38 member 5) is an amino acid (AA) transporter for shuttling neutral AAs such as glutamine across cell membrane. Our work demonstrate that Slc38a5 is highly enriched in retinal vascular endothelium. SLC38A5 regulates endothelial cell glutamine uptake and vascular growth factor receptors to impact blood vessels growth in retinal development and in retinopathies. This work uncovered a novel role of SLC38A5 as a metabolic regulator of retinal angiogenesis by controlling AA nutrient uptake and homeostasis in blood vessel endothelium. Findings from this study also suggest that targeting SLC38A5 or relevant AAs can be a new way to protect against retinopathy.

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Dicer promotes genome stability via the bromodomain transcriptional co-activator Brd4. RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.

SummaryReplication and segregation defects in Dicer1-/- stem cells depend on centromeric transcription by Brd4, and are deeply conserved in fission yeast.

sentences: Altered interaction between RBD and ACE2 receptor contributes towards the increased transmissibility of SARS CoV-2 delta, kappa, beta, and gamma strains with RBD double mutations. The COVID-19 pandemics by SARS-CoV-2 causes catastrophic damage for global human health. The initial step of SARS-CoV-2 infection is the binding of the receptor-binding domain (RBD) in its spike protein to ACE2 receptor in host cell membrane. The evolving of SARS-CoV-2 constantly generates new mutations across its genome including RBD. Besides the well-known single mutation in RBD, the recent new mutation strains with RBD "double mutation" is causing new outbreaks globally, as represented by the delta strain containing RBD L452R/T478K. Although it is considered that the increased transmissibility of the double mutated strains could be attributed to the alteration of mutated RBD to ACE2 receptor, the molecular details remains to be unclear. Using the methods of molecular dynamics simulation, superimposed structural comparison, free binding energy estimation and antibody escaping, we investigated the relationship between ACE2 receptor and the RBD double mutant L452R/T478K (delta), L452R/E484Q (kappa) and E484K/N501Y (beta, gamma). The results demonstrated that each of the three RBD double mutants altered RBD structure, led to enhanced binding affinity of mutated RBD to ACE2 receptor, leading to increased transmissibility of SARS-CoV-2 to the host cells.

sentences: Oscillations support short latency co-firing of neurons during human episodic memory formation. Theta and gamma oscillations in the medial temporal lobe are suggested to play a critical role for human memory formation via establishing synchrony in neural assemblies. Arguably, such synchrony facilitates efficient information transfer between neurons and enhances synaptic plasticity, both of which benefit episodic memory formation. However, to date little evidence exists from humans that would provide direct evidence for such a specific role of theta and gamma oscillations for episodic memory formation. Here we investigate how oscillations shape the temporal structure of neural firing during memory formation in the medial temporal lobe. We measured neural firing and local field potentials in human epilepsy patients via micro-wire electrode recordings to analyze how brain oscillations promote co-incidences of firing between neurons during successful and unsuccessful encoding of episodic memories. The results show that phase-coupling of neurons to faster theta and gamma oscillations correlates with co-firing at short latencies ([~]20-30 ms) and occurs during successful memory formation. Phase-coupling at slower oscillations in these same frequency bands, in contrast, correlates with longer co-firing latencies and occurs during memory failure. Thus, our findings provide evidence which implicate neural oscillations into the synchronization of neural firing in the medial temporal lobe during the encoding of episodic memories.

sentences: Effect of air pockets in drug delivery in jet injections Needle-free jet injections are actuated by a pressure impulse that can be delivered by different mechanisms, and the resultant jets are [Formula](102) m/s. Here, we report on the effect of entrapped air bubbles since filling procedures for pre-filled ampoules can induce bubbles, especially for viscous fluids. We use spring-piston devices as the principal actuation mechanism and vary both the location and size of the initial bubble. We find that the bubble location does have a statistically significant (p < 0.05) effect on the jet exit speed, based upon the volumetric flow rate. However, we reveal subtle features such as intermittent atomization when the gas pockets pass through the orifice and de-pressurize, which leads to spray formation and a temporary increase in jet dispersion, both of which can lead to product loss during an injection. These results have implications for the development of prefilled ampoules for jet injection applications.

sentences: A brain atlas of synapse protein lifetime across the mouse lifespan. Protein turnover is required for synapse maintenance and remodelling and may impact memory duration. We quantified the lifetime of postsynaptic protein PSD95 in individual excitatory synapses across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of protein lifetimes that may extend from a few hours to several months, with distinct spatial distributions in dendrites, neuron types and brain regions. Short protein lifetime (SPL) synapses are enriched in developing animals and in regions controlling innate behaviors, whereas long protein lifetime (LPL) synapses accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. The protein lifetime synaptome architecture is disrupted in an autism model, with synapse protein lifetime increased throughout the brain. These findings add a further layer to synapse diversity in the brain and enrich prevailing concepts in behavior, development, ageing and brain repair.

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Transcriptomic Insights into the Responses in Leaves Storing Lipid Organelles under Different Irradiances To increase the nutritional value of forage, transgenic ryegrass known as High Metabolizable Energy (HME) were previously generated that co-express cysteine-oleosin and diacylglycerol O-acyltransferase. HME not only accumulate lipids in the leaf but also has elevated CO2 assimilation and increased biomass. Shading is one of the most influencing factors for ryegrass growth environments particularly in swards. The aim of this study, therefore, was to determine the influence of irradiance levels on photosynthesis and gene expression in the HME leaves when compared with their corresponding non-transformant (NT). Under low light (150-250 {micro}mol m-2 s-1) and standard light (600-1000 {micro}mol m-2 s-1), the HME accumulated more lipid than NT. The previously reported elevated photosynthesis and increased biomass was observed when the HME were grown under standard light but not under low light. Under both light conditions, compared to NT, the HME had upregulated a number of transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling network while downregulated genes participated in sugar and fructan biosynthesis. We further discuss how the HME differentially manipulated several genes other metabolic pathways including maintenance of redox homeostasis. Combined, the data suggests that the increased photosynthesis capacity in the HME likely corresponds to an increase of micro-lipid sink strength; these are influenced by available light energy and may be related to diffusional and biochemical activities of stomata. Overall, this work provides a clearly understanding of the changes in molecular and biochemical mechanisms underlying the carbon storing as leaf lipid sink of the HME ryegrass.

One sentence summaryShading led to increase leaf lipid accumulation but limit the greater photosynthesis trait of high lipid ryegrass which may be related to limitation of biochemical activities of stomata.

sentences: New Drosophila models to uncover the intrinsic and extrinsic factors mediating the toxicity of the human prion protein Misfolded conformations of the prion protein (PrP) are responsible for devastating neurological disorders in humans and mammals. An unresolved problem is unraveling the mechanisms governing PrP conformational dynamics, misfolding, and the cellular mechanism leading to neurodegeneration. The variable susceptibility of mammals to prion diseases can be exploited to understand the conformational dynamics of PrP. Here we present a new fly model expressing human PrP with robust phenotypes in brain neurons and the eye. Using comparable attP2 insertions, we demonstrate the heightened toxicity of human PrP compared to that of mouse and hamster PrP along with a specific interaction with the amyloid-beta peptide. Using this new highly toxic new model, we started to uncover the intrinsic (sequence / structure) and extrinsic (interactions) factors regulating PrP toxicity. As extrinsic factors, we describe the importance of the PERK - ATF4 branch of the unfolded protein response as a key cellular mechanism mediating the toxicity of human PrP. For intrinsic factors, we introduced point mutations in human PrP (N159D, D167S, N174S) that were partially protective, revealing its high propensity to misfold into toxic conformations.

sentences: Generation and mutational analysis of a transgenic mouse model of human SRY SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRYs three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and the causes of DSD, and present a versatile and powerful system in which to demonstrate causality of SRY variations in DSD, to functionally study the SRY variation database, and to characterize new pathogenic SRY variations found in DSD.

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Mouse mutants in schizophrenia risk genes GRIN2A and AKAP11 show EEG abnormalities in common with schizophrenia patients BACKGROUNDSchizophrenia is a heterogeneous psychiatric disorder with a strong genetic basis, whose etiology and pathophysiology remain poorly understood. Exome sequencing studies have uncovered rare, loss-of-function variants that greatly increase risk of schizophrenia [1], including loss-of-function mutations in GRIN2A (aka GluN2A or NR2A, encoding the NMDA receptor subunit 2A) and AKAP11 (A-Kinase Anchoring Protein 11). AKAP11 and GRIN2A mutations are also associated with bipolar disorder [2], and epilepsy and developmental delay/intellectual disorder [1, 3, 4], respectively. Accessible in both humans and rodents, electroencephalogram (EEG) recordings offer a window into brain activity and display abnormal features in schizophrenia patients. Does loss of Grin2a or Akap11 in mice also result in EEG abnormalities?

METHODSWe monitored EEG in heterozygous and homozygous knockout Grin2a and Akap11 mutant mice compared with their wild-type littermates, at 3- and 6-months of age, across the sleep/wake cycle and during auditory stimulation protocols.

RESULTSGrin2a and Akap11 mutants exhibited increased resting gamma power, attenuated 40-50 Hz auditory steady-state responses (ASSR), and reduced responses to unexpected auditory stimuli during mismatch negativity (MMN) tests. Sleep spindle density was reduced in a gene dose-dependent manner in Akap11 mutants, whereas Grin2a mutants showed increased sleep spindle density.

CONCLUSIONSThe EEG phenotypes of Grin2a and Akap11 mutant mice show a variety of abnormal features that overlap considerably with human schizophrenia patients, reflecting systems-level changes caused by Grin2a and Akap11 deficiency. These neurophysiologic findings further substantiate Grin2a and Akap11 mutants as genetic models of schizophrenia and identify potential biomarkers for stratification of schizophrenia patients.

sentences: Insights into the controls on metabolite distributions along a latitudinal transect of the western Atlantic Ocean Metabolites, or the small organic molecules that are synthesized by cells during metabolism, comprise a complex and dynamic pool of carbon in the ocean. They are an essential form of information, linking genotype to phenotype at the individual, population and community levels of biological organization. Characterizing metabolite distributions inside microbial cells and dissolved in seawater is essential to understanding the controls on their production and fate, as well as their roles in shaping marine microbial food webs. Here, we apply a targeted metabolomics method to quantify particulate and dissolved distributions of a suite of biologically relevant metabolites including vitamins, amino acids, nucleic acids, osmolytes, and intermediates in biosynthetic pathways along a latitudinal transect in the western Atlantic Ocean. We find that, in the euphotic zone, most particulate or intracellular metabolites positively co-vary with the most abundant microbial taxa. In contrast, dissolved metabolites exhibited greater variability with differences in distribution between ocean regions. Although fewer particulate metabolites were detected below the euphotic zone, molecules identified in the deep ocean may be linked to preservation of organic matter or adaptive physiological strategies of deep-sea microbes. Based on the identified metabolite distributions, we propose relationships between certain metabolites and microbial populations, and find that dissolved metabolite distributions are not directly related to their particulate abundances.

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A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients Vibrio cholerae can cause a range of symptoms, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous in V. cholerae DNA repair genes in five out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome, and in one of three patients with isolate genomes sequenced. Second, mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients.

IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

sentences: Dissecting the conformational complexity and flipping mechanism of bacterial heme transport. Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes (1, 2). However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC (3). We provide multi-level evidence that CydDC is a highly specific heme transporter required for assembly and maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and translocation. We found that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degree. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward open states.

sentences: Design of Specific Primer Sets for the Detection of SARS-CoV-2 Variants of Concern B.1.1.7, B.1.351, P.1, B.1.617.2 using Artificial Intelligence As the COVID-19 pandemic continues, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Earlier this year, a variant labelled P.1 with 17 non-synonymous mutations was detected in Brazil. Recently the World Health Organization has raised concern for the variants B.1.617.2 mainly detected in India but now exported worldwide. It is paramount to rapidly develop specific molecular tests to uniquely identify new variants. Using a completely automated pipeline built around deep learning and evolutionary algorithms techniques, we designed primer sets specific to variants B.1.1.7, B.1.351, P.1 and respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for each variant. In-silico tests show that the sequences in the primer sets present high accuracy and are based on 2 mutations or more. In addition, we present an analysis of key mutations for SARS-CoV-2 variants. Finally, we tested the designed primers for B.1.1.7 using RT-PCR. The presented methodology can be exploited to swiftly obtain primer sets for each new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients.

sentences: Raman Signal Denoising using Fully Convolutional Encoder Decoder Network Raman spectroscopy is a vibrational method that gives molecular information rapidly and non-invasively. Despite its advantages, the weak intensity of Raman spectroscopy leads to low-quality signals, particularly with tissue samples. The requirement of high exposure times makes Raman a time-consuming process and diminishes its non-invasive property while studying living tissues. Novel denoising techniques using convolutional neural networks (CNN) have achieved remarkable results in image processing. Here, we propose a similar approach for noise reduction for the Raman spectra acquired with 10x lower exposure times. In this work, we developed fully convolutional encoder-decoder architecture (FCED) and trained them with noisy Raman signals. The results demonstrate that our model is superior (p-value < 0.0001) to the conventional denoising techniques such as the Savitzky-Golay filter and wavelet denoising. Improvement in the signal-to-noise ratio values ranges from 20% to 80%, depending on the initial signal-to-noise ratio. Thus, we proved that tissue analysis could be done in a shorter time without any need for instrumental enhancement.

sentences: Neuron-like function of the nephron central command Interoceptive neurons that sense and regulate our internal milieu have been identified in several organs except in the kidney cortex despite its major importance in maintaining body homeostasis. Here we report that the chief kidney cell type of the macula densa (MD) forms coordinated neural networks in each nephron that resemble peripheral ganglia. A combined in vivo single-cell 4D physiology (sc4DP) and scRNA sequencing approach identified the MD mechanisms of neuronal differentiation, heterogeneity (pacemaker MD cells), sensing of the local and systemic environment via multi-organ crosstalk, and regulation of organ functions by acting as the nephron central command. Consistent with their neuron-like nature, MD cells express the molecular fingerprint of neurodegeneration. Here we put forth the single-cell MD model and concept of local neural networks that control organ and body functions via interoception in normal physiological state and use an integrated mechanism of neurodegeneration in disease.

sentences: Low light intensity delays vegetative phase change in Arabidopsis thaliana Plants that develop under low intensity light (LL) often display a phenotype known as the "shade tolerance syndrome (STS)". This syndrome is similar to the phenotype of plants in the juvenile phase of shoot development, but the basis for this similarity is unknown. We tested the hypothesis that the STS is regulated by the same mechanism that regulates the juvenile vegetative phase by examining the effect of LL on rosette development in Arabidopsis thaliana. We found that LL prolonged the juvenile vegetative phase and that this was associated with an increase the expression of the master regulators of vegetative phase change, miR156 and miR157, and a decrease in the expression of their SPL targets. Exogenous sucrose partially corrected the effect of LL on seedling development and miR156 expression. Our results suggest that the response of Arabidopsis to LL is mediated by an increase in miR156/miR157 expression and by factors that repress SPL gene expression independently of miR156/miR157, and is caused in part by a decrease in carbohydrate production. The effect of LL on vegetative phase change does not require the photoreceptors and transcription factors responsible for the shade avoidance syndrome, implying that light intensity and light quality regulate rosette development by different pathways.

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HIV-linked gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV People living with HIV (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV. To test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines (IL-1{beta}, IL-6, IL-1Ra, IL-10, IL17, IL22, TNF and IFN-{gamma}) in response to different stimuli. We also characterized CD4+ T cell-counts, HIV reservoir and other clinical parameters. Compared to 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels, cytokine production capacity and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-enriched strain was negatively associated with IL-10, IL-6 and TNF production, independent of age, sex and sexual behavior, and positively associated with CD4+ T cell-level, whereas the PLHIV-enriched strain showed no associations. Our findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV.

Novel PointsO_LIWe identified compositional and functional changes in the gut microbiome of PLHIV that were strongly related to sexual behavior.
C_LIO_LIHIV-associated bacterial changes are negatively associated with HIV reservoir. The relative abundance of Firmicutes bacterium CAG 95 and Prevotella sp CAG 5226 both show a negative association with CD4+ T cell-associated HIV-1 DNA.
C_LIO_LIPrevotella copri and Bacteroides vulgatus show association with PBMC production capacity of IL-1{beta} and IL-10 that is independent of age, sex, BMI and sexual behavior.
C_LIO_LIWe observed two genetically different P. copri strains that are enriched in PLHIV and healthy individuals, respectively.
C_LIO_LIThe control-related P. copri strain specifically shows a negative association with IL-10, IL-6 and TNF production and a positive association with CD4+ T cell-level. This suggests it plays a potential protective role in chronic inflammation, which may be related to enrichment of a specific epitope peptide.
C_LI

sentences: MS1Connect: a mass spectrometry run similarity measure Interpretation of newly acquired mass spectrometry data can be improved by identifying, from an online repository, previous mass spectrometry runs that resemble the new data. However, this retrieval task requires computing the similarity between an arbitrary pair of mass spectrometry runs. This is particularly challenging for runs acquired using different experimental protocols. We propose a method, MS1Connect, that calculates the similarity between a pair of runs by examining only the intact peptide (MS1) scans, and we show evidence that the MS1Connect score is accurate. Specifically, we show that MS1Connect outperforms several baseline methods on the task of predicting the species from which a given proteomics sample originated. In addition, we show that MS1Connect scores are highly correlated with similarities computed from fragment (MS2) scans, even though this data is not used by MS1Connect. The MS1Connect software will be made available upon acceptance at https://github.com/bmx8177/MS1Connect.

sentences: Structure and function of diadenylate cyclase DacM from Mycoplasma ovipneumoniae Cyclic diadenosine monophosphate (c-di-AMP) is a second-messenger nucleotide that is produced by many bacteria. C-di-AMP can not only regulate bacterial growth, cell-wall homeostasis, ion transport and gene transcription, but can also be recognized by multiple sensor / receptor proteins in infected host cells to trigger an innate immune response. Mycoplasma ovipneumoniae causes non-progressive pneumonia in both sheep and goats. Here, we analyzed c-di-AMP signaling in M. ovipneumoniae, which is a genome-reduced obligately pathogenic bacterium. Our results demonstrate that these bacteria can produce c-di-AMP, and we could identify the diadenylate cyclase, which was named DacM. The enzyme was found to utilize both ATP and ADP to synthesize c-di-AMP, resembling CdaM from a novel family of diadenylate cyclases first found in Mycoplasma pneumoniae. Furthermore, we present the crystal structures of DacM in the apo state and substrate-bound state at 3 [A] and 1.9 [A] resolution, respectively. Mutation of residues Asp112, Gly113, Tyr128, Phe129, and Arg143 surrounding the active sites to Ala were lethal to DacM enzymatic activity. These structures provide valuable insights into the biochemistry of c-di-AMP, and offer a basis for the structure-based design of new drugs for animal husbandry.

sentences: Specialization and selective social attention establishes the balance between individual and social learning A key question individuals face in any social learning environment is when to innovate alone and when to imitate others. Previous simulation results have found that the best performing groups exhibit an intermediate balance, yet it is still largely unknown how individuals collectively negotiate this balance. We use an immersive collective foraging experiment, implemented in the Minecraft game engine, facilitating unprecedented access to spatial trajectories and visual field data. The virtual environment imposes a limited field of view, creating a natural trade-off between allocating visual attention towards individual search or to look towards peers for social imitation. By analyzing foraging patterns, social interactions (visual and spatial), and social influence, we shine new light on how groups collectively adapt to the fluctuating demands of the environment through specialization and selective imitation, rather than homogeneity and indiscriminate copying of others.

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Genomic and demographic processes differentially influence genetic variation across the X chromosome Mutation, recombination, selection, and demography affect genetic variation across the genome. Increased mutation and recombination both lead to increases in genetic diversity in a region-specific manner, while complex demographic patterns shape patterns of diversity on a more global scale. The X chromosome is particularly interesting because it contains several distinct regions that are subject to different combinations and strengths of these processes, notably the pseudoautosomal regions (PARs) and the X-transposed region (XTR). The X chromosome thus can serve as a unique model for studying how genetic and demographic forces act in different contexts to shape patterns of observed variation. Here we investigate diversity, divergence, and linkage disequilibrium in each region of the X chromosome using genomic data from 26 human populations. We find that both diversity and substitution rate are consistently elevated in PAR1 and the XTR compared to the rest of the X chromosome. In contrast, linkage disequilibrium is lowest in PAR1 and highest on the non-recombining X chromosome, with the XTR falling in between, suggesting that the XTR (usually included in the non-recombining X) may need to be considered separately in future studies. We also observed strong population-specific effects on genetic diversity; not only does genetic variation differ on the X and autosomes among populations, but the effects of linked selection on the X relative to autosomes have been shaped by population-specific history. The substantial variation in patterns of variation across these regions provides insight into the unique evolutionary history contained within the X chromosome.

Significance StatementDemography and selection affect the X chromosome differently from non-sex chromosomes. However, the X chromosome can be subdivided into multiple distinct regions that facilitate even more fine-scaled assessment of these processes. Here we study regions of the human X chromosome in 26 populations to find evidence that recombination may be mutagenic in humans and that the X-transposed region may undergo recombination. Further we observe that the effects of selection and demography act differently on the X chromosome relative to the autosomes across human populations. Together, our results highlight profound regional differences across the X chromosome, simultaneously making it an ideal system for exploring the action of evolutionary forces as well as necessitating its careful consideration and treatment in genomic analyses.

sentences: Gut regulates brain synaptic assembly through neuroendocrine signaling pathway The gut-brain axis plays an essential role in regulating neural development in response to environmental stimuli, such as microbes or nutrients. Defects in gut-brain communication can lead to various neurological disorders. However, it remains unknown whether gut plays any intrinsic role in regulating neuronal development. Through a genetic screen in C. elegans, we uncovered that an intrinsic Wnt-endocrine pathway in gut regulates synaptic development and neuronal activity in brain. Specifically, the Wnt signaling upregulates the expression of the neuropeptide NLP-40 in the gut, which then facilitates presynaptic assembly through the neuronal expressed GPCR AEX-2 receptor during development. The NLP-40 acts most likely through modulating neuronal activity and promoting synaptic protein trafficking. Therefore, this study reveals a novel role of gut in synaptic development in the brain.

sentences: Hyperspectral characterisation of natural illumination in woodland and forest environments Light in nature is complex and dynamic, and varies along spectrum, space, direction, and time. While both spectrally resolved measurements and spatially resolved measurements are widely available, spectrally and spatially resolved measurements are technologically more challenging. Here, we present a portable imaging system using off-the-shelf components to capture the full spherical light environment in a spectrally and spatially resolved fashion. The method relies on imaging the 4{pi}-steradian light field reflected from a mirrored chrome sphere using a commercial hyperspectral camera (400-1000 nm) from multiple directions and an image-processing pipeline for extraction of the mirror sphere, removal of saturated pixels, correction of specular reflectance of the sphere, promotion to a high dynamic range, correction of misalignment of images, correction of intensity compression, erasure of the imaging system, unwrapping of the spherical images, filling-in blank regions, and stitching images collected from different angles. We applied our method to Wytham Woods, an ancient semi-natural woodland near Oxford, UK. We acquired a total of 168 images in two sites with low and high abundance of ash, leading to differences in canopy, leading to a total 14 hyperspectral light probes. Our image-processing pipeline corrected small (<3 {degrees}) field-based misalignment adequately. Our novel hyperspectral imaging method is adapted for field conditions and opens up novel opportunities for capturing the complex and dynamic nature of the light environment.

sentences: Differential dopaminergic modulation of spontaneous cortico-subthalamic activity in Parkinson's disease Pathological oscillations including elevated beta activity in the subthalamic nucleus (STN) and between STN and cortical areas are a hallmark of neural activity in Parkinsons disease (PD). Oscillations also play an important role in normal physiological processes and serve distinct functional roles at different points in time. We characterised the effect of dopaminergic medication on oscillatory whole-brain networks in PD in a time-resolved manner by employing a hidden Markov model on combined STN local field potentials and magnetoencephalography (MEG) recordings from 17 PD patients. Dopaminergic medication led to coherence within the medial and orbitofrontal cortex in the delta/theta frequency range. This is in line with known side effects of dopamine treatment such as deteriorated executive functions in Parkinsons disease. In addition, dopamine caused the beta band activity to switch from an STN-mediated motor network to a frontoparietal-mediated one. In contrast, dopamine did not modify local STN-STN coherence in PD. STN-STN synchrony emerged both on and off medication. By providing electrophysiological evidence for the differential effects of dopaminergic medication on the discovered networks, our findings open further avenues for electrical and pharmacological interventions in PD.

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Processive translocation of cohesive and non-cohesive cohesin in vivo Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. Purified cohesin translocates along DNA in a diffusive fashion that can be propelled by transcribing RNA polymerase. The complex also extrudes DNA loops in a process that consumes ATP. In this study we examine processive genomic translocation of cohesin in vivo. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by transcription in yeast. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore, cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the DNA binding chamber(s) of cohesin. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

Significance StatementCohesin is an architectural protein that brings distant chromosomal DNA sites together. The complex links sister chromatids cohesion but it also binds to single pieces of DNA in ways that do not generate cohesion. One class of non-cohesive complexes organizes chromosomal DNA into loops. All cohesin complexes move on DNA but the constraints on such movement are not fully explored. Here, we use size-calibrated obstacles in yeast to interrogate cohesin and the properties of its movement on DNA. We show that both cohesive and non-cohesive complexes translocate processively on chromosomes. In addition, we show that cohesive and non-cohesive complexes are blocked by obstacles of different size. Lastly, we show that stalled cohesive complexes block passage of non-cohesive complexes.

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Gene selection by incorporating genetic networks into case-control association studies Large-scale genome-wide association studies (GWAS) have been successfully applied to a wide range of genetic variants underlying complex diseases. The network-based penalized regression approach has been developed to overcome the challenges caused by the computational efficiency for analyzing high-dimensional genomic data by incorporating a biological genetic network. In this paper, we propose a gene selection approach by incorporating genetic networks into case-control association studies for DNA sequence data or DNA methylation data. Instead of using traditional dimension reduction techniques such as principal component analyses and supervised principal component analyses, we use a linear combination of genotypes at SNPs or methylation values at CpG sites in each gene to capture gene-level signals. We develop three approaches for the linear combination: optimally weighted sum (OWS), LD-adjusted polygenic risk score (LD-PRS), and beta-based weighted sum (BWS). OWS and LD-PRS are supervised approaches that depend on the effect of each SNP or CpG site on the case-control status, while BWS can be extracted without using the case-control status. After using one of the linear combinations of genotypes or methylation values in each gene to capture gene-level signals, we regularize them to perform gene selection based on the biological network. Simulation studies show that the proposed approaches have higher true positive rates than using traditional dimension reduction techniques. We also apply our approaches to DNA methylation data and UK Biobank DNA sequence data for analyzing rheumatoid arthritis. The results show that the proposed methods can select potentially rheumatoid arthritis related genes that are missed by existing methods.

Author SummaryThere is strong evidence showing that when genes are functionally related to each other in a genetic network, statistical methods utilizing prior biological network knowledge can outperform other methods that ignore genetic network structures. Therefore, statistical methods that can incorporate genetic network information into association analysis in human genetic association studies have been widely used since 2008. Here, we take advantage of recently developed methods to capture the gene-level signals in network-based penalized regression of high-dimensional genetic data. We have shown that the selection performance of our proposed methods can outperform three traditional principal component-based dimension reduction techniques in several simulation scenarios in terms of true positive rates. Meanwhile, by applying our methods in both DNA methylation data and DNA sequence data, the genes identified by our proposed methods can be significantly enriched into the rheumatoid arthritis pathway, such as genes HLA-DMA, HLA-DPB1, and HLA-DQA2 in the HLA region.

sentences: Partial reprogramming restores youthful gene expression through transient suppression of cell identity Transient induction of pluripotent reprogramming factors has been reported to reverse some features of aging in mammalian cells and tissues. However, the impact of transient reprogramming on somatic cell identity programs and the necessity of individual pluripotency factors remain unknown. Here, we mapped trajectories of transient reprogramming in young and aged cells from multiple murine cell types using single cell transcriptomics to address these questions. We found that transient reprogramming restored youthful gene expression in adipocytes and mesenchymal stem cells but also temporarily suppressed somatic cell identity programs. We further screened Yamanaka Factor subsets and found that many combinations had an impact on aging gene expression and suppressed somatic identity, but that these effects were not tightly entangled. We also found that a transient reprogramming approach inspired by amphibian regeneration restored youthful gene expression in aged myogenic cells. Our results suggest that transient pluripotent reprogramming poses a neoplastic risk, but that restoration of youthful gene expression can be achieved with alternative strategies.

sentences: Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

sentences: Quality-controlled R-loop meta-analysis reveals the characteristics of R-Loop consensus regions R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA during transcription. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 693 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate method for R-loop data quality control, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called "R-loop regions" (RL regions). In the process, we revealed the stark divergence between S9.6 and dRNH-based R-loop mapping methods and identified biologically meaningful subtypes of both constitutive and variable R-loops. Taken together, this work provides a much-needed method to assess R-loop data quality and reveals intriguing aspects of R-loop biology.

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Escitalopram Administration, Neuroplastic Effects and Relearning: A Diffusion Tensor Imaging Study in Healthy Individuals BackgroundNeuroplastic processes are influenced by selective serotonergic reuptake inhibitors, while learning in conjunction with the administration of serotonergic agents alters white matter microstructure in humans. The goal of this double-blind, placebo-controlled imaging study was to investigate the influence of escitalopram on white matter plasticity during (re)learning.

MethodsSeventy-one healthy individuals (age = 25.6{+/-}5.0, 43 females) underwent 3 diffusion magnetic resonance imaging sessions: at baseline, after 3-weeks of associative learning (emotional/non-emotional content) and after relearning shuffled associations for an additional 3 weeks. During the relearning phase, subjects received daily escitalopram 10 mg or placebo orally. Statistical analysis was performed with statistical parametric mapping (SPM) and using sandwich estimator.

ResultsA three-way and two-way rmANOVA was conducted to analyze the effects of escitalopram on AD, FA, MD and RD during the learning and relearning period. We found no significant three-way or two-way interactions for each DTI metrics (pFDR > 0.05), thus neither after 3 nor after 6 weeks we found significant changes in white matter microstructure.

ConclusionWe examined neither an effect of escitalopram nor learning (or relearning) interventions on different DTI metrics. The duration and intensity of study interventions (i.e. administration of escitalopram and learning as the relearning task) might have been chosen insufficiently to induce detectable alterations. Previous studies examining the effects of SSRIs on white matter tracts in humans are underrepresented, but do mainly yield towards non-significant findings. The results implicate that escitalopram does not impact white matter microstructures in healthy subjects.

sentences: Gut microbial structural variations as determinants of human bile acid metabolism Bile acids (BAs) facilitate intestinal fat absorption and act as important signaling molecules in host{square}gut microbiota crosstalk. BA-metabolizing pathways in the microbial community have been identified, but how the highly variable genomes of gut bacteria interact with host BA metabolism remains largely unknown. We characterized 8,282 structural variants (SVs) of 55 bacterial species in the gut microbiomes of 1,437 individuals from two Dutch cohorts and performed a systematic association study with 39 plasma BA parameters. Both variations in SV-based continuous genetic makeup and discrete subspecies showed correlations with BA metabolism. Metagenome-wide association analysis identified 797 replicable associations between bacterial SVs and BAs and SV regulators that mediate the effects of lifestyle factors on BA metabolism. This is the first large-scale microbial genetic association analysis to demonstrate the impact of bacterial SVs on human BA composition, and highlights the potential of targeting gut microbiota to regulate BA metabolism through lifestyle intervention.

sentences: Widespread inhibitory projections from the interposed cerebellar nucleus The cerebellum consists of parallel parasagittal modules that contribute to diverse behaviors, spanning motor to cognitive. Recent work employing cell-type specific tracing has identified circumscribed output channels of the cerebellar nuclei that could confer tight functional specificity. These studies have largely focused on excitatory projections of the cerebellar nuclei, however, leaving open the question of whether inhibitory neurons also constitute multiple output modules. We mapped output and input patterns to intersectionally restricted cell types of the interposed and adjacent interstitial nuclei. In contrast to the widespread assumption of primarily excitatory outputs and restricted inferior olive-targeting inhibitory output, we found that inhibitory neurons from this region ramified widely within the brainstem, targeting both motor- and sensory-related nuclei, distinct from excitatory output targets. Despite differences in output targeting, monosynaptic rabies tracing revealed largely shared afferents to both cell classes. We discuss the potential novel functional roles for inhibitory outputs in the context of cerebellar theory.

sentences: Limited reciprocal surrogacy of bird and habitat diversity and inconsistencies in their representation in Romanian protected areas Because it is impossible to comprehensively characterize biodiversity at all levels of organization, conservation prioritization efforts need to rely on surrogates. As species distribution maps of relished groups as well as high-resolution remotely sensed data increasingly become available, both types of surrogates are commonly used. A good surrogate should represent as much of biodiversity as possible, but it often remains unclear to what extent this is the case. Here, we aimed to address this question by assessing how well bird species and habitat diversity represent one another. We conducted our study in Romania, a species-rich country with high landscape heterogeneity where bird species distribution data have only recently started to become available. First, we prioritized areas for conservation based on either 137 breeding bird species or 36 habitat classes, and then evaluated their reciprocal surrogacy performance. Second, we examined how well these features are represented in already existing protected areas. Finally, we identified target regions of high conservation value for the potential expansion of the current network of reserves (as planned under the new EU Biodiversity Strategy for 2030). We found that bird species were a better surrogate for habitat diversity than vice versa. Highly ranked areas based on habitat diversity were represented better than areas based on bird species, which varied considerably between species. Our results highlight that taxonomic and environmental (i.e., habitat types) data may perform rather poorly as reciprocal surrogates, and multiple sources of data are required for a full evaluation of protected areas expansion.

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The effects of physical and temporal certainty on locomotion with discrete underfoot perturbations BackgroundAmbulation over complex terrain requires active control of foot placement to maintain a normal kinematic relationship between the center of mass and base of support. Recent investigations have suggested that foot placement location may be selected to anticipate shifts to the underfoot center of pressure. However, it is unclear whether temporal affordance and physical certainty contribute to the selection of a perturbation-specific anticipatory strategy. This study investigates anticipatory and reactive locomotor strategies for repeated underfoot perturbations with varying levels of temporal certainty, temporal affordance, and physical certainty.

MethodsThirteen healthy adults walked with random underfoot perturbations from a mechanized shoe. Temporal certainty was challenged by presenting the perturbations with or without warning. Temporal affordance was challenged by adjusting the timing of a warning tone before the perturbation. Physical certainty was challenged with conditions that included only eversion perturbations, only inversion perturbations, or both eversion and inversion perturbations. Linear-mixed effects models assessed the effect of each condition on the percent change of margin of stability and step width, respectively.

ResultsFor temporally uncertain perturbations and perturbations with one stride or less of affordance, we observed few changes to step width or margin of stability. As affordance increased to two strides, participants adopted a wider step width in anticipation of the perturbation (p = 0.001). Physical certainty had little effect on gait for the step of the perturbation, but participants recovered normal gait sooner when the physical nature of the perturbation was predictable (p < 0.001).

DiscussionDespite having information about the timing and magnitude of upcoming perturbations, individuals do not develop perturbation specific feedforward strategies but instead rely on feedback control to recover normal gait after a perturbation. However, physical certainty appears to improve the efficiency of the feedback controller and allows individuals to recover normal gait sooner.

sentences: The development of efficient communication in the human connectome. The structure of the human connectome develops from childhood throughout adolescence to middle age, but how this affects the speed of neuronal signaling is not well described. To understand the maturation process of transmission speed across the connectome, we stimulated electrocorticographic (ECoG) electrode pairs in 75 human subjects of 4 to 51 years old. We then measured the latency of cortico-cortical evoked responses in other electrodes. Each year, responses got faster, until transmission speed reached a minimum latency at the age of 29-44 years old. Responses during middle age were about two times faster compared to childhood. These increases in speed were observed in both long- and short-range connections between and within frontal, central, parietal, and temporal areas. These findings indicate that long-range and short-range communication between brain regions increase in efficiency well throughout adolescence.

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Bridging scales from spiking activity to the local field potential through latent dynamics During wakefulness, cortical networks exhibit diverse states of activity arising from fluctuations in modulatory inputs, any of which may shape the recorded neural activity across a range of scales, from local field potential (LFP) to the spiking of individual neurons. Multiple studies have uncovered state-dependent changes in single neuron activity specific to cell type. These changes are heterogeneous across cell types, suggesting that there is a link between changes in states of wakefulness and the dynamic patterns of spiking activity across the population, yet this is currently not well understood. Here, we use an unsupervised statistical modeling framework to link fluctuations in the state of the LFP to the dynamics of spiking activity of populations of neurons in the whisker area of primary somatosensory cortex of awake male mice. By identifying latent states from population spiking activity, we show that these spiking states are more informative of states of the LFP than a direct readout of the spiking activity of single neurons or of the population. Further, we find that the latent spiking states predict trial-by-trial variability in sensory responses as well as one aspect of behavior, whisking activity. Taken together, our approach captures the latent variables that underlie the dynamics of the spiking activity of cortical neuronal populations, providing a framework to relate cortical activity across different scales.

Significance statementDuring wakefulness, variability in cortical activity can arise from shifts in arousal or behavior. We analyze the dynamics of two measures of cortical activity, the spiking activity of local populations of neurons and the local field potential (LFP), typically used to track overall levels of arousal. We show that the latent states that capture variability in population spiking activity are highly predictive of LFP state changes, even more so than spiking activity directly. By linking latent states of spiking activity to changes in the LFP, our novel approach bridges fine-scale microcircuits ensemble activity and the meso-scale LFP. Our latent-state framework opens new avenues for extracting information from the LFP as well as identification of subject-level sources of variability.

sentences: Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex Tuberous Sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in regulation of growth and energy consumption. Biochemical and electron cryomicroscopy (cryoEM) studies of recombinant pTSC have revealed the structure and stoichiometry of the pTSC and have hinted at the possibility that the complex form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab dependent purification of pTSC from membrane solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and up to 1 m in length in linear and web-like organizations prepared from pig brain. These observations suggest polymerization of endogenous pTSC into filamentous super-structures.

sentences: Histone marks retained during epigenetic reprogramming and their roles essential for fish early development Reprograming of epigenetic modifications after fertilization is required for proper embryonic development and cell differentiation. However, histone modifications that escape reprogramming in non-mammalian vertebrates and their potential functional roles are poorly understood. Here, we quantitatively analyzed histone modification dynamics during reprogramming in Japanese Killifish, medaka (Oryzias latipes) embryos, and revealed that H3K27ac, H3K27me3 and H3K9me3 are retained, while H3K4 methylation is completely erased. Furthermore, we experimentally demonstrated the functional roles of such retained modifications at early stages; H3K27ac at promoters is required for proper patterning of H3K4 and H3K27 methylation at zygotic genome activation (ZGA) and specific retention of H3K9me3 at telomeric regions maintains genomic stability during cleavage stage. These results expand the understanding of diversity and conservation of reprogramming in vertebrates and unveil previously uncharacterized functions of histone modifications retained during epigenetic reprogramming.

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Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 m spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization (nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues.

TeaserNano-DESI combined with individual ion mass spectrometry generates images of proteoforms up to 70 kDa.

sentences: Aerobic methoxydotrophy: growth on methoxylated aromatic compounds by Methylobacterium Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C1) compounds. The C1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. In addition, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium. We demonstrated growth on p-hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium, but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to M. nodulans, and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation.

sentences: Genomic Analysis of Human Noroviruses Using Hybrid Illumina-Nanopore Data Whole genome sequence (WGS) analysis of noroviruses is routinely performed by employing a metagenomic approach. While this methodology has several advantages, such as allowing for examination of co-infection, it has some limitations such as the requirement of high viral load to achieve full-length or near full-length genomic sequences. In this study, we used an amplification approach to obtain full-length genomic amplicons from 39 Canadian GII isolates followed by deep sequencing on Illumina and Oxford Nanopore platforms. This approach significantly reduced the required viral titre to obtain full-genome coverage. Herein, we compared the coverage and sequences obtained by both platforms and provided an in-depth genomic analysis of the obtained sequences, including the presence of single nucleotide variants (SNVs) and recombination events.

sentences: The secreted neuronal signal Spock1 regulates the blood-brain barrier The blood-brain barrier (BBB) is comprised of a single layer of endothelial cells with uniquely restrictive properties required for maintaining a tightly controlled homeostatic environment in the brain. Classic quail-chick grafting experiments showed that BBB properties are not intrinsic to brain endothelial cells, but instead are induced by signals from the embryonic brain microenvironment. Here we have identified a neuronally produced signal, Spock1, that specifically regulates BBB functional development in both zebrafish and mice without affecting angiogenesis. Using a combination of mosaic genetic analysis, tracer leakage assays and live imaging we show that Spock1 from neurons can regulate brain vasculature permeability non-cell autonomously. Electron microscopy analyses of spock1 mutants revealed that the leakage arises predominantly through increased endothelial transcytosis of both clathrin-independent small and large vesicles due to altered pericyte-endothelial interactions. Single-cell RNA sequencing analyses revealed a reduction in vascular expression of the cell adhesion molecule mcamb in the spock1 mutants, and this down-regulation of mcamb occurred specifically in regions with increased BBB leakage. These analyses indicate that the neuronal signal Spock1 regulates BBB properties by altering vascular gene expression and cellular interactions.

sentences: Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies.

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Cell patterning by secretion-induced plasma membrane flows Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.

One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

sentences: Exploring of bacterial blight resistance in landraces, mining of resistant gene(s) and genetic divergence of resistant germplasm through molecular markers and quantitative traits. Bacterial blight, one of the oldest and severe diseases of rice poses a major threat towards global rice production and food security. Thereafter, sustainable management of this disease has given paramount importance globally. In the current study, we explored 792 landraces to evaluate their disease reaction status against three highly virulent strains (BXo69, BXo87 and BXo93) of Xanthomonas oryzae pv. oryzae (Xoo). Subsequently, we intended to identify the possible candidate resistant (R) genes responsible for the resistant reaction using six STS markers correspond to Xa4, xa5, Xa7, xa13, Xa21 and Xa23 genes and finally, we evaluated morphological and molecular diversity of the potential bacterial blight resistant germplasm using quantitative traits and ISSR markers. Based on pathogenicity test, a single germplasm was found as highly resistant while, 33 germplasm were resistant and 40 were moderately resistant. Further molecular study on these 74 germplasm divulged that 41 germplasm carried Xa4 gene, 15 carried xa5 gene, 62 carried Xa7 gene, 33 carried xa13 gene, and 19 carried Xa23 gene. Only a single germplasm consisted of Xa21 gene. Interestingly, we found a wide range of gene combinations ranged from 2 to 4 genes among the germplasm resistant to bacterial blight and G3 genotype (Acc. No. 4216; highly resistant) having Xa4, Xa7, xa13, Xa21 and G43 genotype (Acc. No. 1523; resistant) having Xa4, xa5, xa13 and Xa23 gene combination being the most effective against all the Xoo strains. Nonetheless, UPGMA dendrogram and heatmap analysis based on quantitative traits identified two important clusters viz. cluster-III and cluster-VIII with multiple desired traits However, genetic similarity based on ISSR marker data pointed out 3 germplasm, namely, G20 (Acc. No. 4004; contained Xa4, Xa7, xa13 genes), G17 (Acc. No. 3981; contained Xa7, xa13 genes) and G6 (Acc. No. 991; contained Xa4, Xa7, xa13, Xa23 genes) having comparatively lower genetic similarity (0.30, 0.37 and 0.38, respectively) with elite variety BRRI dhan28. Notably, Mantle test of molecular and morphological data indicated that there was a positive correlation (r = 0.113) between them. The outcome of this study would enrich and diversify the rice gene pool and would be useful for the development of durable bacterial blight resistant varieties.

sentences: The rapid, massive growth of COVID-19 authors in the scientific literature We examined the extent to which the scientific workforce in different fields was engaged in publishing COVID-19-related papers. According to Scopus (data cut, August 1, 2021), 210,183 COVID-19-related publications included 720,801 unique authors, of which 360,005 authors had published at least 5 full papers in their career and 23,520 authors were at the top 2% of their scientific subfield based on a career-long composite citation indicator. The growth of COVID-19 authors was far more rapid and massive compared with cohorts of authors historically publishing on H1N1, Zika, Ebola, HIV/AIDS and tuberculosis. All 174 scientific subfields had some specialists who had published on COVID-19. In 109 of the 174 subfields of science, at least one in ten active, influential (top-2% composite citation indicator) authors in the subfield had authored something on COVID-19. 52 hyper-prolific authors had already at least 60 (and up to 227) COVID-19 publications each. Among the 300 authors with the highest composite citation indicator for their COVID-19 publications, most common countries were USA (n=67), China (n=52), UK (n=32), and Italy (n=18). The rapid and massive involvement of the scientific workforce in COVID-19-related work is unprecedented and creates opportunities and challenges. There is evidence for hyper-prolific productivity.

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Field and experimental data together with computational models reveal how cleaner fish adjust decisions in a biological market While it is generally straightforward to quantify individual performance in cognitive experiments, identifying the underlying cognitive processes remains a major challenge. Often, different mechanistic underpinnings yield similar performances, and Lloyd Morgans cannon warrants acceptance of the simpler explanation. Alternatively, when the different mechanisms interact with environmental conditions, variation in performance across environments might allow to statistically infer the mechanism responsible. We illustrate this point by fitting computational models to experimental data on performance by wild-caught cleaner fish Labroides dimidiatus in an ephemeral reward task, as well as cleaner and client fish densities from the locations of capture. Using Bayesian statistics to fit the model parameters to performance data revealed that cleaner fish most likely estimate future consequences of an action, while it appears unlikely that the removal of the ephemeral reward acts as psychological punishment (negative reinforcement). Incorporating future consequences also yields performances that can be considered the result of locally optimal decision-rules, in contrast to the negative reinforcement mechanism. We argue that the combination of computational models with data is a powerful tool to infer the mechanistic underpinnings of cognitive performance.

Author summaryPerformance in behavioural experiments is often used to assess the cognitive abilities of animals. However, animals can get to the same outcome in alternative ways. Thus, the outcome of the experiments does not tell us how animals achieve their performance. In order to overcome this limitation, we used a set of computational models, which provide predictions on how alternative cognitive mechanisms perform in the face of varying environmental conditions, and compare their predictions to performance data from individuals that experienced different conditions throughout their life. Our study system is the cleaner fish Labroides dimidiatus, a coral reef fish that feeds off ectoparasites and dead tissue from the skin of other reef fishes. Cleaner fish often have to choose among alternative clients seeking their cleaning service, and the optimal choice is often hard to achieve. Our performance experiments mimic the natural choices the cleaner fish make. The combination of experiments and computational models points to cleaner fish abilities to account for the future effect of their choices by estimating the long term expected value of those choices. Estimating long term value is a mechanism involved in human foresight.

sentences: Environmental DNA in a Global Biodiversity Hotspot: Lessons from Coral Reef Fish Diversity Across the Indonesian Archipelago Indonesia is the heart of the Coral Triangle, the worlds most diverse marine ecosystem. Preserving the biological and economic value of this marine biodiversity requires efficient and economical ecosystem monitoring, yet our understanding of marine biodiversity in this region remains limited. This study uses environmental DNA (eDNA) to survey fish communities across a pronounced biodiversity gradient in Indonesia. A total of 12,939,690 sequence reads of MiFish 12S rRNA from 39 sites spanning 7 regions of Indonesia revealed 4,146 Amplified Sequence Variants (ASVs). Regional patterns of fish diversity based on eDNA broadly conformed to expectations based on traditional biodiversity survey methods, with the highest fish biodiversity in Raja Ampat and generally lower diversity in Western Indonesia. However, eDNA performed relatively poorly compared to visual survey methods in site-by-site comparisons, both in terms of total number of taxa recovered and ability to assign species names to ASVs. This result stands in a stark contrast to eDNA studies of temperate and tropical ecosystems with lower diversity. Analyses show that while sequencing depth was sufficient to capture all fish diversity within individual seawater samples, variation among samples from individual localities was high, and sampling effort was insufficient to capture all fish diversity at a given sampling site. Interestingly, mean ASVs recovered per one-liter seawater was surprisingly similar across sites, despite substantial differences in total diversity, suggesting a limit to total ASVs (~200) per one-liter eDNA sample. Combined, results highlight two major challenges of eDNA in highly diverse ecosystems such as the Coral Triangle. First, reference databases are incomplete and insufficient for effective ASV taxonomic assignment. Second, eDNA sampling design developed from lower diversity temperate marine ecosystems are inadequate to fully capture diversity of biodiversity hotspots like the Coral Triangle.

sentences: Greater Neural Pattern Dissimilarity and Higher Meaningfulness in the Retrieval Stage are Enhanced by Spaced Learning The question of whether repeated studies bring more variability or less to our brain is a critical problem before scientist continue their further study about memory. In the past ten years, a series of neural pattern representation studies have found that under the condition of spaced learning, the neural pattern similarity(NPS) between two encoding stages increases, and the researchers claimed that these results support the idea that repeated studies bring more similarity along with repetition in our brain, which is conflicted with the encoding variability theory(Feng et al., 2019; Y. Lu et al., 2015; Xue et al., 2010, 2011a). However, we doubt this viewpoint because we think the difference between encoding processing cannot be used to represent the difference between memory states. In current experiments, we used a new experimental paradigm with a longer lag and elaboration learning task to test the encoding variability theory. By comparing the difference between neural pattern dissimilarity(NPDS1)(spaced learning - one-time learning) and NPDS2(massed learning - one-time learning) in the final test (retrieval) stage, we get the result that the NPDS1 was significantly greater than NPDS2 in the parietal lobe of 400ms and the right frontal lobe of 600ms, which is more fitting to the encoding variability mechanism. However, we believe that there is no contradiction between these two experimental evidences. On the contrary, we think they reflect different aspects of the process of spaced learning. We propose that the deficient processing in encoding stage exactly lead to less encoding variability in our memory. More importantly, this result gives us reason to double the paper published ten years ago in Science, which claimed repetition brings greater neural pattern similarity.

sentences: Identification of new leaf intrinsic yield genes using cross-species network analysis in plants Plant leaves differ in their size, form and structure, and the processes of cell division and cell expansion contribute to this diversity. Leaf transcriptional networks covering cell division and cell expansion in Arabidopsis thaliana, maize (Zea mays) and aspen (Populus tremula) were compared to identify candidate genes that are conserved in plant growth and ultimately have the potential to increase biomass (intrinsic yield, IY). Our approach revealed that genes showing strongly conserved co-expression were mainly involved in fundamental leaf developmental processes such as photosynthesis, translation, and cell proliferation. Next, known intrinsic yield genes (IYGs) together with cross-species conserved networks were used to predict novel potential Arabidopsis leaf IYGs. Using an in-depth literature screening, 34 out of 100 top predicted IYGs were confirmed to affect leaf phenotype if mutated or overexpressed and thus represent novel potential IYGs. Globally, these new IYGs were involved in processes mostly covering cell cycle, plant defense responses, gibberellin, auxin and brassinosteroid signaling. Application of loss-of-function lines and phenotypic characterization confirmed two newly predicted IYGs to be involved in leaf growth (NPF6.4 and LATE MERISTEM IDENTITY2). In conclusion, the presented network approach offers an integrative cross-species strategy to identify new yield genes and to accelerate plant breeding.

sentences: Social relationships among captive male pygmy slow lorises (Nycticebus pygmaeus): Is forming male same-sex pairs a feasible management strategy? Little is known about the social behavior of pygmy slow lorises, in particular, the social relationships of same-sex individuals have rarely been investigated. The Slow Loris Conservation Center was built at the Japan Monkey Center to enhance the welfare of confiscated slow lorises, promote their conservation, improve public education and perform scientific research on the species. In the course of improving housing conditions, several same-sex pairs of pygmy slow lorises were formed. We monitored their behaviors and fecal glucocorticoid metabolite (FGM) levels to understand whether male same-sex pairings could be a feasible management strategy. The subjects were 10 male and 6 female lorises for comparison, all of whom were over five years old. We successfully formed five pairs of male lorises after eight formation attempts. Male pairs initially showed some aggressive behaviors; however, the rate decreased approximately 10 days after introduction. All of the male pairs eventually exhibited extensive affiliative social behaviors, including allogrooming and social play, during the dark (active) phase, and sleep site sharing during the light (inactive) phase. The rate of sleep site sharing during the light phase was higher than expected, suggesting that the pairs preferred to stay near each other. There was no evidence of increased stress after a long period of male-male social housing. Female same-sex pairs and male-female pairs demonstrated a high level of affiliative behaviors right after introduction. These results highlight the flexibility and high sociability of this species and indicate that such same-sex pairings are a feasible option for their social management.

sentences: Variable inhibition of unwinding rates of DNA catalyzed by the SARS-Cov-2 (COV19) helicase nsp13 by structurally distinct single DNA lesions. The SARS 2 (Covid 19) helicase nsp13 plays a critically important role in the replication of the Corona virus by unwinding double-stranded RNA (and DNA) with a 5{longrightarrow}3 strand polarity. Here we explored the impact of single, structurally defined covalent DNA lesions on the helicase activity of nsp13 in aqueous solutions, The objectives were to derive mechanistic insights into the relationships between the structures of DNA lesions, the DNA distortions that they engender, and the inhibition of helicase activity. The lesions included two bulky stereoisomeric N2-guanine adducts derived from the reactions of benzo[a]pyrene diol epoxide with DNA. The trans-adduct assumes a minor groove conformation, while the cis-product adopts a base-displaced intercalated conformation. The non-bulky DNA lesions included the intra-strand cross-linked thymine dimers, the cis-syn-cyclobutane pyrimidine dimer, and the pyrimidine (6-4) pyrimidone photoproduct. All four lesions strongly inhibit the helicase activity of nsp13, The UV photolesions feature a 2 - 5-fold smaller inhibition of the nsp13 unwinding activity than the bulky DNA adducts, and the kinetics of these two pairs of DNA lesions are also different. The connections between the structural features of these four DNA lesions and their impact on nsp13 unwinding efficiencies are discussed.

sentences: Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

sentences: Caste, sex, and parasitism influence brain plasticity in a social wasp Phenotypic plasticity is the capacity of a single genotype to exhibit different phenotypes, and can be an adaptive response to specific environmental and social conditions. Social insects are particularly well-suited to study plasticity, because the division of labor amongst females and the different life histories of males and females are associated with specific sensory needs. Here, we take advantage of the social wasp Polistes dominula to explore if brain plasticity is influenced by caste and sex, and the exploitation by the parasite Xenos vesparum. Within sexes, males had proportionally larger optic lobes, while females, regardless of caste, had larger antennal lobes, which is consistent with sensory needs of sex-specific life histories. Within castes, reproductive females had larger calyces, as predicted by their sensory needs for extensive within-colony interactions and forming winter aggregations, than workers who spend more time foraging for nest material and prey. Surprisingly, parasites had different effects on female and male hosts. Female workers were castrated and behaviorally manipulated by female or male parasites, but only showed moderate differences in relative allocation of different brain tissue compared to non-parasitized workers. In contrast, the testes and behavior of parasitized males were essentially unaffected, but they had smaller brains and greater relative volume of most sensory brain regions than non-parasitized males. Our results are consistent with caste and sex mediating brain plasticity in P. dominula and that the parasites manipulation can also drive differential allocation of brain regions depending on host sex.

sentences: Event segmentation reveals working memory forgetting rate We encounter the world as a continuous flow and effortlessly segment sequences of events into episodes. This process of event segmentation engages working memory (WM) for tracking the flow of events and impacts subsequent memory accuracy. WM is limited in how much information is retained (i.e., WM capacity) and for how long the information is retained (i.e., forgetting rate). It is unclear which aspect of WM limitations affects event segmentation. In two separate experiments with multiple tasks, we estimated participants WM capacity and forgetting rate in a dynamic context and evaluated their relationship to event segmentation. The results across tasks show that individuals who reported more movie segments than others (fine-segmenters) have a faster decaying WM. A separate task assessing long-term memory retrieval reveals that the coarse-segmenters have better recognition of temporal order of events in contrast to the fine-segmenters who performed better at free recall. The findings show that event segmentation employs dissociable memory strategies and depends on how long information is retained in WM.

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Cargo surface fluidity reduces inter-motor interference, promotes load-sharing and enhances run-lengths in an ATP dependent manner In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work as teams is unclear. Here we explored the possibility that purely physical mechanisms may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a 3D dynamical simulation of cargo transport along microtubules by teams of canonically non-cooperative kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. We showed explicitly, for the first time, that surface fluidity leads to the reduction of negative mechanical interference between kinesins, enhancing load sharing thereby decreasing single motor off-rates and increasing processivity. Remarkably, we also showed that, independent of fluidity, increasing numbers of bound motors pull the cargo closer to the microtubule, increasing the on-rates of individual unbound motors, resulting in a cooperative increase in bound motor numbers that depends on 3D cargo geometry. At the cargo level, surface fluidity makes more motors available for binding, though this effect is significant only at low ATP or high motor density. Interestingly, we find that the fluidity induced reduction in mechanical interference dominates over the increased availability of motors for typical physiological and in vitro conditions, this allowing us to reconcile different experimental results in different regimes. Finally, we show that these effects can altogether result in enhanced mechanical efficiency and tunable cargo run-lengths for teams of molecular motors over physiological ranges of fluidity with implications for new experimental validation efforts, transport in vivo, artificial cargo design and multi-motor-driven mechanical processes in general.

Author summaryIn cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of protein filaments. How do intrinsically non-cooperative motors function as teams within cells? In this paper, we show, using computer simulations, that the fluid surfaces of cellular cargo reduce the mechanical interference between motors allowing better load sharing and decreasing their detachment rates, thereby increasing the distance over which they can carry cargo. We additionally found that motors pull cargo closer to the filament increasing the attachment rates of other unbound motors. These effects act synergistically with an increased availability of motors due to fluidity to further increase travel distances. Our simulation model accounted for the dynamics of the motors on the cargo surface and their load and ATP dependent kinetics, allowing us to connect single motor properties to overall transport. Our work on understanding how teamwork arises in mechanically coupled motors sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving mechanical efficiency and transport of artificial cargo powered by motor teams.

sentences: Decision formation in parietal cortex transcends a fixed frame of reference Neurons in the lateral intraparietal cortex (area LIP) represent the formation of a decision when it is linked to a specific action, such as an eye movement to a choice target. However, these neurons should be unable to represent a decision that transpires across actions that would disrupt this linkage. We investigated this limitation by recording simultaneously from many neurons. While intervening actions disrupt the representation by single neurons, the ensemble achieves continuity of the decision process by passing information from currently active neurons to neurons that will become active after the action. In this way, the representation of an evolving decision can be generalized across actions and transcends the frame of reference that specifies the neural response fields. The finding extends previous observations of receptive field remapping, thought to support the stability of perception across eye movements, to the continuity of a thought process, such as a decision.

sentences: Microbiome differential abundance methods produce disturbingly different results across 38 datasets Identifying differentially abundant microbes is a common goal of microbiome studies. Multiple methods have been applied for this purpose, which are largely used interchangeably in the literature. Although it has been observed that these tools can produce different results, there have been very few large-scale comparisons to describe the scale and significance of these differences. In addition, it is challenging for microbiome researchers to know which differential abundance tools are appropriate for their study and how these tools compare to one another. Here, we have investigated these questions by analyzing 38 16S rRNA gene datasets with two sample groups for differential abundance testing. We tested for differences in amplicon sequence variants and operational taxonomic units (referred to as ASVs for simplicity) between these groups with 14 commonly used differential abundance tools. Our findings confirmed that these tools identified drastically different numbers and sets of significant ASVs, however, for many tools the number of features identified correlated with aspects of the tested study data, such as sample size, sequencing depth, and effect size of community differences. We also found that the ASVs identified by each method were dependent on whether the abundance tables were prevalence-filtered before testing. ALDEx2 and ANCOM produced the most consistent results across studies and agreed best with the intersect of results from different approaches. In contrast, several methods, such as LEfSe, limma voom, and edgeR, produced inconsistent results and in some cases were unable to control the false discovery rate. In addition to these observations, we were unable to find supporting evidence for a recent recommendation that limma voom, corncob, and DESeq2 are more reliable overall compared with other methods. Although ALDEx2 and ANCOM are two promising conservative methods, we argue that those researchers requiring more sensitive methods should use a consensus approach based on multiple differential abundance methods to help ensure robust biological interpretations.

sentences: Functionalized extracellular matrix scaffolds loaded with endothelial progenitor cells promote neovascularization and diabetic wound healing Diabetic ischemic wound treatment remains a critical clinical challenge. Strategies that enhance angiogenesis and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic condition. We previously identified a cyclic peptide LXW7 that specifically binds to integrin v{beta}3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates VEGF receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a pro-angiogenic molecule LXW7-DS-SILY by conjugating LXW7 to a collagen-binding proteoglycan mimetic DS-SILY and further employed this novel bifunctional ligand to functionalize extracellular matrix (ECM) scaffolds, promote neovascularization and accelerate ischemic wound healing. We established a Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model and found the wounds treated by LXW7-DS-SILY-functionalized ECM scaffolds, with or without EPCs, significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis. These functionalized ECM scaffolds also significantly promoted EPC attachment, growth and survival in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

sentences: Distinct Neural Profiles of Frontoparietal Networks in Boys with ADHD and Boys with Persistent Depressive Disorder Working memory deficits are common in attention-deficit/hyperactivity disorder (ADHD) and depression, two common neurodevelopmental disorders with overlapping cognitive profiles but distinct clinical presentation. Multivariate techniques have previously been utilized to understand working memory processes in functional brain networks in healthy adults, but have not yet been applied to investigate how working memory processes within the same networks differ within typical and atypical developing populations. We used multivariate pattern analysis (MVPA) to identify whether brain networks discriminated between spatial vs. verbal working memory processes in ADHD and Persistent Depressive Disorder (PDD). 36 male clinical participants and 19 typically developing (TD) boys participated in a fMRI scan while completing a verbal and a spatial working memory task. Within a priori functional brain networks (frontoparietal, default mode, salience) the TD group demonstrated differential response patterns to verbal and spatial working memory. The PDD group showed weaker differentiation than TD, with lower classification accuracies observed in primarily the left frontoparietal network. The neural profiles of the ADHD and PDD differed specifically in the SN where the ADHD groups neural profile suggests significantly less specificity in neural representations of spatial and verbal working memory. We highlight within-group classification as an innovative tool for understanding the neural mechanisms of how cognitive processes may deviate in clinical disorders, an important intermediary step towards improving translational psychiatry.

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Utilization of the repeated squat-stand model for studying the directional sensitivity of the cerebral pressure-flow relationship Hysteresis in the cerebral pressure-flow relationship describes the superior ability of the cerebrovasculature to buffer cerebral blood flow changes when mean arterial pressure (MAP) acutely increases compared to when MAP acutely decreases. This phenomenon can be evaluated by comparing the change in middle cerebral artery mean blood velocity (MCAv) per change in MAP during either acute increases or decreases in MAP induced by repeated squat-stands (RSS). However, no real baseline can be employed for this particular protocol as there is no true stable reference point. Herein, we characterized a novel metric using the greatest MAP oscillations induced by RSS without using an independent baseline value and adjusted for time intervals ({Delta}MCAvT/{Delta}MAPT). We also examined whether this metric during each RSS transition were comparable between each other over a 5-min period. {Delta}MCAvT/{Delta}MAPT was calculated using the minimum to maximum MCAv and MAP for each RSS performed at 0.05 Hz and 0.10 Hz. We compared averaged {Delta}MCAvT/{Delta}MAPT during MAP increases and decreases in 74 healthy subjects [9 women; 32 {+/-} 13 years]. {Delta}MCAvT/{Delta}MAPT was lower for MAP increases than MAP decreases at 0.10 Hz RSS only (0.91 {+/-} 0.34 vs. 1.01 {+/-} 0.44 cm{middle dot}s-1/mmHg; p = 0.0013). For both frequency and MAP direction, time during RSS had no effect on {Delta}MCAvT/{Delta}MAPT. This novel analytical method supports the use of the RSS model to evaluate the directional sensitivity of the pressure-flow relationship. These results contribute to the importance of considering the direction of MAP changes when evaluating dynamic cerebral autoregulation.

News & NoteworthyRepeated squat-stand maneuvers are able to examine the directional sensitivity of the cerebral pressure-flow relationship. These maneuvers induce stable physiological cyclic changes where brain blood flow changes with blood pressure increases are buffered more than decreases. These results highlight the importance of considering directional blood pressure changes within cerebral autoregulation.

sentences: Increased intestinal permeability in an orally-reactive peanut allergy model identifies Angiopoietin like-4 as a biomarker Peanut allergy reaction severity correlates with increased intestinal epithelial cell (IEC) barrier permeability. CC027/GeniUnc mice develop peanut allergy by intragastric administration of peanut proteins without adjuvant. We report that peanut-allergic CC027/GeniUnc mice showed increased IEC barrier permeability and systemic peanut allergen Ara h 2 after challenge. Jejunal epithelial cell transcriptomics showed effects of peanut allergy on IEC proliferation, survival, and metabolism, and revealed IEC-predominant angiopoietin like-4 (Angptl4) as a unique feature of CC027/GeniUnc peanut allergy. CC027/GeniUnc mice and peanut-allergic pediatric patients demonstrated significantly higher serum Angptl4 and ANGPTL4 compared to control C3H/HeJ mice and non-peanut-allergic but atopic patients, respectively, highlighting its potential as a biomarker of peanut allergy.

sentences: Mechanism of REST/NRSF Regulation of Clustered Protocadherin Alpha Genes Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5-1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDH genes. Thus, REST/NRSF represses PCDH gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

sentences: MafB, WDR77, and ss-catenin interact with each other and have similar genome association profiles MafB (a bZIP transcription factor), {beta}-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.

sentences: ACCURATE DECODING OF THE SPINAL CORD OUTPUT IN HUMANS WITH IMPLANTED HIGH-DENSITY ELECTRODE ARRAYS Invasive electromyography opened a new window to explore motoneuron behaviour in vivo. However, the technique is limited by the small fraction of active motoneurons that can be concurrently detected, precluding a population analysis in natural tasks. Here, we developed a high-density intramuscular electrode for in vivo human recordings along with a fully automatic methodology that could detect the discharges of action potentials of up to 67 concurrently active motoneurons with 99% accuracy. These data revealed that motoneurons of the same pool receive common synaptic input at frequencies up to 75 Hz and that late recruited motoneurons inhibit the discharges of those recruited earlier. These results constitute an important step in the population coding analysis of the human motor system in vivo.

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OperonSEQer: A set of machine-learning algorithms with threshold voting for detection of operon pairs using short-read RNA-sequencing data Operon prediction in prokaryotes is critical not only for understanding the regulation of endogenous gene expression, but also for exogenous targeting of genes using newly developed tools such as CRISPR-based gene modulation. A number of methods have used transcriptomics data to predict operons, based on the premise that contiguous genes in an operon will be expressed at similar levels. While promising results have been observed using these methods, most of them do not address uncertainty caused by technical variability between experiments, which is especially relevant when the amount of data available is small. In addition, many existing methods do not provide the flexibility to determine whether the stringency with which genes should be evaluated for being in an operon pair. We present OperonSEQer, a set of machine learning algorithms that uses the statistic and p-value from a non-parametric analysis of variance test (Kruskal-Wallis) to determine the likelihood that two adjacent genes are expressed from the same RNA molecule. We implement a voting system to allow users to choose the stringency of operon calls depending on whether your priority is high coverage of operons or high accuracy of the calls. In addition, we provide the code so that users can retrain the algorithm and re-establish hyperparameters based on any data they choose, allowing for this method to be expanded on as additional data is generated and incorporated. We show that our approach detects operon pairs that are missed by current methods by comparing our predictions to publicly available long-read sequencing data. OperonSEQer therefore improves on existing methods in terms of accuracy, flexibility and adaptability.

Author SummaryBacteria and archaea, single-cell organisms collectively known as prokaryotes, live in all imaginable environments and comprise the majority of living organisms on this planet. Prokaryotes play a critical role in the homeostasis of multicellular organisms (such as animals and plants) and ecosystems. In addition, bacteria can be pathogenic, and cause a variety of diseases in these same hosts and ecosystems. In short, understanding the biology and molecular functions of bacteria and archaea and devising mechanisms to engineer and optimize their properties are critical scientific endeavors with significant implications in healthcare, agriculture, manufacturing and climate science among others. One major molecular difference between unicellular and multicellular organisms is the way the express genes - rather than making individual RNA molecules like multicellular organisms, prokaryotes express genes in long contiguous RNA molecules known as operons, which are subsequently processed. Understanding which genes exist within operons is critical for elucidating basic biology and for engineering organisms. In this work, we use a combination of statistical and machine learning-based methods to use next-generation sequencing data to predict operon structure across a range of prokaryotes. Our method provides a easily implemented, robust, accurate and flexible way to determine operon structure in an organism-agnosic manner using readily-available data.

sentences: The crucial role of genome-wide genetic variation in conservation The unprecedented rate of extinction calls for efficient use of genetics to help conserve biodiversity. Several recent genomic and simulation-based studies have argued that the field of conservation biology has placed too much focus on the conservation of genome-wide genetic variation, and that this approach should be replaced with another that focuses instead on managing the subset of functional genetic variation that is thought to affect fitness. Here, we critically evaluate the feasibility and likely benefits of this approach in conservation. We find that population genetics theory and empirical results show that the conserving genome-wide genetic variation is generally the best approach to prevent inbreeding depression and loss of adaptive potential from driving populations towards extinction. Focusing conservation efforts on presumably functional genetic variation will only be feasible occasionally, often misleading, and counterproductive when prioritized over genome-wide genetic variation. Given the increasing rate of habitat loss and other environmental changes, failure to recognize the detrimental effects of lost genome-wide variation on long-term population viability will only worsen the biodiversity crisis.

sentences: Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity Surface ligand activity is a key design parameter for successfully interfacing surfaces with cells - whether in the context of in vitro investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity currently lacks a standardized measurement approach that can be readily paired with live cell investigations. To fill this void, we have developed a concurrent control technique for characterizing in vitro ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for in vitro applications and the other for surface plasmon resonance (SPR) based RGD activity characterization. Recombinant V{beta}3 integrins were injected over the SPR chip surface as mimics of the cellular membrane bound receptors and the resulting binding kinetics parameterized to quantify ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the in vitro chip surfaces on the live cell microscope. We show that the SPR concurrent control approach has multiple advantages based on the facts that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities. Furthermore, by pairing both SPR and in vitro approaches, a comparison of the results can provide biological insights into the nature of cellular adhesion and dynamics.

sentences: Phylogenetic analysis of molecular pathways in Tregs Suppressor Function Treg-mediated suppression of conventional T cells is a fundamental step in regulating the adaptive immune response. It is known that Treg first appeared in vertebrates. However, little is known about the of major suppression pathways mediated by Tregs. We employed artificial intelligence text mining system to highlight the suppression pathways currently known to be utilized by Tregs. Our system identified various pathways mediated by CTLA4, calcium signaling, NfkB and NFAT. We simultaneously employed detailed phylogenetic analysis including multiple sequence alignment, phylogenetic tree building, ancestral sequence reconstruction, neutrality tests and positive selection test to investigate the evolution of Treg mediated pathways. We found that CTLA4 first appeared in vertebrates possibly arising from an IGV containing protein in cartilaginous fish. Conversely, we found that Tregs repurposed ancient pathways such as Calcineurin and CAMP Response Element Modulator. Interestingly, these two pathways were highly conserved between vertebrates and lower invertebrates indicating conservation of function. Taken together, our research indicate that Tregs developed its regulatory systems through evolution in vertebrates as well as reusing conserved ancient regulatory systems that are related to the innate immune system.

sentences: Self-organized stem cell-derived human lung buds with proximo-distal patterning and novel targets of SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic and the lack of therapeutics hinders pandemic control1-2. Although lung disease is the primary clinical outcome in COVID-19 patients1-3, how SARS-CoV-2 induces tissue pathology in the lung remains elusive. Here we describe a high-throughput platform to generate tens of thousands of self-organizing, nearly identical, and genetically matched human lung buds derived from human pluripotent stem cells (hPSCs) cultured on micropatterned substrates. Strikingly, in vitro-derived human lung buds resemble fetal human lung tissue and display in vivo-like proximo-distal coordination of alveolar and airway tissue differentiation whose 3D epithelial self-organization is directed by the levels of KGF. Single-cell transcriptomics unveiled the cellular identities of airway and alveolar tissue and the differentiation of WNThi cycling alveolar stem cells, a human-specific lung cell type4. These synthetic human lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-dependent susceptibilities to infection, intercellular transmission and cytopathology in airway and alveolar tissue in individual lung buds. Interestingly, we detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as targets of SARS-CoV-2. We used this platform to test neutralizing antibodies isolated from convalescent plasma that efficiently blocked SARS-CoV-2 infection and intercellular transmission. Our platform offers unlimited, rapid and scalable access to disease-relevant lung tissue that recapitulate key hallmarks of human lung development and can be used to track SARS-CoV-2 infection and identify candidate therapeutics for COVID-19.

sentences: A new method shows that a minority of liver-localized CD8 T cells display hard-to-detect attraction to Plasmodium-infected hepatocytes Malaria is a disease caused by Plasmodium parasites, resulting in over 200 million infections and 400,000 deaths every year. A critical step of malaria infection is when sporozoites, injected by mosquitoes, travel to the liver and form liver stages. Malaria vaccine candidates which induce large numbers of malaria-specific CD8 T cells in mice are able to eliminate all liver stages, preventing fulminant malaria. However, how CD8 T cells find all parasites in 48 hours of the liver stage lifespan is not well understood. Using intravital microscopy of murine livers, we generated unique data on T cell search for malaria liver stages within a few hours after infection. To detect attraction of T cells to an infection site, we used the von Mises-Fisher distribution in 3D, similar to the 2D von Mises distribution previously used in ecology. Our results suggest that the vast majority (70-95%) of malaria-specific and non-specific liver-localized CD8 T cells did not display attraction towards the infection site, suggesting that the search for malaria liver stages occurs randomly. However, a small fraction (15-20%) displayed weak but detectable attraction towards parasites which already had been surrounded by several T cells. We found that speeds and turning angles correlated with attraction, suggesting that understanding mechanisms that determine the speed of T cell movement in the liver may improve the efficacy of future T cell-based vaccines. Stochastic simulations suggest that a small movement bias towards the parasite dramatically reduces the number of CD8 T cells needed to eliminate all malaria liver stages, but to detect such attraction by individual cells requires data from long imaging experiments which are not currently feasible. Importantly, as far as we know this is the first demonstration of how activated/memory CD8 T cells might search for the pathogen in nonlymphoid tissues a few hours after infection. We have also established a framework for how attraction of individual T cells towards a location in 3D can be rigorously evaluated.

sentences: Detection of specific uncultured bacteriophages by fluorescence in situ hybridisation in pig microbiome Microbial communities have huge impacts on their ecosystems and local environments spanning from marine and soil communities to the mammalian gut. Bacteriophages (phages) are important drivers of population control and diversity in the community, but our understanding of complex microbial communities is halted by biased detection techniques. Metagenomics have provided a method of novel phage discovery independent of in vitro culturing techniques and have revealed a large proportion of understudied phages. Here, five large phage genomes, that were previously assembled in silico from pig faecal metagenomes, are detected and observed directly in their natural environment using a modified phageFISH approach, and combined with methods to decrease bias against large phages. These phages are uncultured with unknown hosts. The specific phages were detected by PCR and fluorescent in situ hybridisation in their original faecal samples as well as across other faecal samples. Co-localisation of bacterial signals and phage signals allowed detection of the different stages of phage life cycle. All phages displayed examples of early infection, advanced infection, burst, and free phages. To our knowledge, this is the first detection of jumbophages in faeces, which were investigated independently of culture, host identification, and size, and based solely on the genome sequence. This approach opens up opportunities for characterisation of novel in silico phages in vivo from a broad range of gut microbiomes.

sentences: A tau class Glutathione-S-Transferase (OsGSTU5) acts as a negative regulator of VirE2 interaction with T-DNA during Agrobacterium infection in rice During Agrobacterium-mediated transformation (AMT), T-DNA along with several virulence proteins like VirD2, VirE2, VirE3, VirD5, and VirF enter into the plant cytoplasm. VirE2 is supposed to serve as single-stranded DNA binding (SSB) protein and assist the cytoplasmic trafficking of T-DNA inside the host cell. In the present study, a rice glutathione-S-transferase (OsGSTU5) that interacts with VirE2 protein in plant cytoplasm has been identified. OsGSTU5 is observed to be involved in post-translational glutathionylation of VirE2 protein (gVirE2). In silico analysis revealed that  gVirE2+ssDNA complex is structurally less stable than  VirE2+ ssDNA complex. The gel shift activity confirms the attenuated SSB property of gVirE2 over VirE2 protein under in vitro condition. Moreover, knock-down and overexpression OsGSTU5 phenotypes of rice showed increased and decreased T-DNA expression, respectively after Agrobacterium infection. The present finding convincingly establishes the role of OsGSTU5 as defense protein in rice that can further serve as an important target for modulation of AMT efficiency in rice.

sentences: THRONCAT: Efficient metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insight into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine-or puromycin analogs allow for the selective visualization and enrichment of the newly synthesized proteins. However, their applications are limited as they require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a novel threonine-derived non-canonical amino acid tagging method based on bioorthogonal threonine analog {beta}-ethynylserine ({beta}ES) that enables efficient and non-toxic labeling of the nascent proteome in complete growth media within minutes. We used THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profiled immediate proteome dynamics of Ramos B-cells in response to receptor activation, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in vivo.

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Facilitation of sensory axon conduction to motoneurons during cortical or sensory evoked primary afferent depolarization (PAD) in humans Sensory and corticospinal (CST) pathways activate spinal GABAergic interneurons with axo-axonic connections onto proprioceptive (Ia) afferents that depolarize these afferents (termed primary afferent depolarization, PAD). In rodents sensory-evoked PAD is produced by GABAA receptors at nodes of Ranvier in Ia-afferents, rather than at presynaptic terminals, and facilitates action potential propagation to motoneurons by preventing branch point failures, rather than causing presynaptic inhibition. Here we examined if PAD likewise facilitates the Ia-afferent mediated H-reflex in humans by evoking PAD with both sensory and CST stimulation. H-reflexes in several lower limb muscles were facilitated by prior conditioning from low-threshold proprioceptive, cutaneous or CST pathways, with a similar time course ([~]200 ms) to the PAD measured in rodent Ia-afferents. Long trains of repeated cutaneous or proprioceptive afferent stimulation produced long-lasting facilitation of the H-reflex for up to 2 minutes, consistent with the tonic depolarization of rodent Ia-afferents mediated by nodal 5-GABA receptors for similar stimulation trains. Facilitation of the conditioned H-reflexes was not mediated by direct facilitation of the motoneurons because isolated stimulation of sensory or CST pathways did not modulate the firing rate of tonically activated motor units in tested muscles. Furthermore, cutaneous conditioning increased the firing probability of a single motor unit during the H-reflex without increasing its firing rate at this time, indicating that the underlying excitatory postsynaptic potential (EPSP) was more probable, but not larger. These results are consistent with sensory and CST pathways activating nodal GABAA receptors that reduce intermittent failure of action potentials propagating into Ia-afferent branches.

Key Points SummaryO_LIThe control of posture and movement requires peripheral sensory feedback, which was previously thought to be inhibited by specialized GABAergic neurons in the spinal cord.
C_LIO_LIBased on new findings in rodents, we provide evidence in humans that sensory and corticospinal pathways that likely activate these GABAergic pathways facilitate, rather than inhibit, the flow of sensory feedback in afferents that carry information about body position, movement and effort.
C_LIO_LIThese new findings of how sensory and descending pathways facilitate this sensory feedback to spinal motor neurons can now be applied to people with injury to the brain or spinal cord where these GABA neurons are affected, allowing us to understand how altered sensory control may affect residual motor function and the production of involuntary muscle spasticity.
C_LI

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Time dependent proinflammatory responses shape virus interference during coinfections of influenza A virus and influenza D virus Both influenza A virus (IAV) and influenza D virus (IDV) are enzootic in pigs. IAV causes approximately 100% morbidity with low mortality, whereas IDV leads to only mild respiratory diseases in pigs. In this study, we performed a series of coinfection experiments in vitro and in vivo to understand how IAV and IDV interact and cause pathogenesis during coinfection. Results showed that IAV inhibited IDV replication when infecting swine tracheal epithelial cells (STEC) with IAV 24- or 48-hours prior to IDV inoculation, and that IDV suppressed IAV replication when IDV preceded IAV inoculation by 48 hours. Virus interference was not identified during simultaneous IAV/IDV infections or with 6 hours between the two viral infections, regardless of their order. The interference pattern at 24- and 48-hours correlated with proinflammatory responses induced by the first infection, which was about 24-hours slower for IDV than IAV. The viruses did not interfere with each other if both infected the cells before proinflammatory responses were induced. Coinfection in pigs further demonstrated that IAV interfered both viral shedding and virus replication of IDV, especially in the upper respiratory tract. Clinically, coinfection of IDV and IAV did not show significant enhancement of disease pathogenesis, compared with the pigs infected with IAV alone. In summary, this study suggests that interference during coinfection of IAV and IDV is primarily due to the proinflammatory response and is therefore dependent on the time between infection, and the order of infection.

ImportanceBoth IAV and IDV are enzootic in pigs, and feral pigs have a higher risk for both IAV and IDV exposures than IDV exposure alone. This study suggests that in coinfection with IAV and IDV either virus can interfere with the replication of the other virus by stimulating proinflammatory responses; however, the proinflammatory response was 24 hours slower for IDV than IAV. In vitro there was no interference during simultaneous coinfection, regardless of infection order. Coinfection of IDV and IAV in pigs did not show enhanced pathogenesis, compared with those infected only with IAV. This study can facilitate our understanding of virus epidemiology and pathogenesis associated with IAV and IDV coinfection.

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Gene family expansion and functional diversification of chitinase and chitin synthase genes in Atlantic salmon (Salmo salar) BackgroundChitin is one of the most abundant polysaccharides in nature, forming important structures in insects, crustaceans, and fungal cell walls. Vertebrates on the other hand are generally considered "non-chitinous" organisms, despite having highly conserved chitin metabolism associated genes. Recent work has revealed that the largest group of vertebrates, the teleosts, have the potential to both synthesize and degrade endogenous chitin. Yet little is still known about the genes and proteins responsible for these dynamic processes. Here we used comparative genomics, transcriptomics, and chromatin accessibility data to characterize the repertoire, evolution, and regulation of genes involved in chitin-metabolism in teleosts, with a particular focus on Atlantic salmon.

ResultsReconstruction of gene family phylogenies provide evidence for an expansion of teleost and salmonid chitinase and chitin synthase genes after multiple whole-genome duplications. Analyses of multi-tissue gene expression data demonstrated a strong bias of gastrointestinal tract expression for chitin metabolism genes, but with different spatial and temporal tissue specificities. Finally, we integrated transcriptomes from a developmental time series of the gastrointestinal tract with chromatin accessibility data to identify putative transcription factors responsible for regulating chitin-metabolism gene expression (CDX1 and CDX2) as well as tissue-specific divergence in the regulation of gene duplicates (FOXJ2). These transcription factors are also potential regulators of multiple glycosyltransferases being co-expressed with the chitin remodeling genes.

ConclusionThe findings presented here add support to the hypothesis that chitin metabolism genes in teleosts play a role in developing and maintaining a chitin-based barrier in the teleost gut and provide a basis for further investigations into the molecular basis of this barrier.

sentences: Release of HIV-1 particles from the viral compartment in macrophages requires an associated cytoskeleton and is driven by mechanical constraints A defining feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named VCC (virus-containing compartments) that is connected to the extracellular medium. The newly formed viral progeny pinches of the membrane and accumulates in the lumen of the VCC. While HIV budding has been extensively studied, very little is known about how viral particles present in the lumen of VCC are released in the extracellular medium. Here we show that the actin dynamics are critical for this process by combining ultrastructural analyses, time-lapse microscopy and perturbations of the actin cytoskeleton. We found that jasplakinolide, which stabilizes actin fibres, inhibited viral release from HIV-1-infected macrophages. Furthermore, in jasplakinolide-treated macrophages, VCC became scattered and no longer co-localized with the integrin CD18, nor the phosphorylated form of the focal adhesion kinase PYK2. Inhibition of PYK2 activity in infected macrophages promoted intracellular retention of viral particles in VCC that were no longer connected to the plasma membrane. Finally, we stimulated the rapid release of viral particles from the VCC by subjecting infected macrophages to frustrated phagocytosis. As macrophages spread on IgG-coated glass surfaces, VCC rapidly migrated to the basal membrane and released their viral content in the extracellular medium, which required their association with CD18 and the actin cytoskeleton. These results highlight that VCC trafficking and virus release are intimately linked to the reorganization of the macrophage actin cytoskeleton in response to external physical cues, suggesting that it might be regulated in tissues by the mechanical stress to which these cells are exposed.

sentences: Multiplexed Cell-Based Diagnostic Devices for Detection of Renal Biomarkers Using Genetic Circuits The number of synthetic biology based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers; urea and uric acid. Using advanced gene expression control strategies we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low cost diagnostic options.

sentences: Convergent evolution of elaborate nests as structural defences in birds The  hanging-basket nests of some weaverbird and icterid species are among the most complex structures built by any animal, but why they have evolved remains to be explained. The precarious attachments and extended entrance tunnels characteristic of these nests are widely speculated to act as structural defences against invasion by nest predators, particularly tree-climbing snakes, but this hypothesis has yet to be systematically tested. We use phylogenetic comparative methods to investigate the role of nest structure in developmental period length, a proxy for offspring mortality, in the weaverbirds (Ploceidae) and icterids (Icteridae), two bird families in which  hanging-basket nests have independently evolved. We find that more elaborate nest designs, particularly those with entrance tunnels, are associated with longer developmental periods in both families. This finding is robust to potentially confounding effects of body mass, phylogenetic relationships and nest location. We provide the first comparative evidence for the role of elaborate nest designs in reducing offspring mortality from nest invasion. More broadly, our findings highlight the importance of offspring mortality risk in shaping diversity in birds nest designs and suggest that complex structures built by animals can buffer against environmental hazards and facilitate the evolution of slower life histories.

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PSK signaling controls ABA homeostasis and signaling genes and maintains shoot growth under osmotic stress Water deficit impairs growth and survival of plants. Many water stress responses are under control of abscisic acid (ABA) but little is known about growth control under osmotic stress. Based on the previously described growth-promoting activity of the peptide hormone phytosulfokine (PSK), we hypothesized that it may contribute to growth regulation under water stress conditions. To test this hypothesis, we analyzed the Arabidopsis thaliana PSK receptor (PSKR) null mutant pskr1-3 pskr2-1 under mannitol and drought stress. In particular under mild water stress, fresht weight and photosynthetic efficiency were more reduced in pskr1-3 pskr2-1 than in wild type. Hydroponic and grafting experiments showed that PSKR signaling was not required for long-distance signaling from mannitol-stressed roots to shoot but rather for cell growth promotion in the shoot. Unlike wild type, pskr1-3 pskr2-1 shoots did not accumulate ABA in response to mannitol, showed misregulation of ABA synthesis genes and elevated expression of ABI1 and ABI2, repressors of ABA signaling whereas application of ABA partially reversed shoot growth inhibition by mannitol in pskr1-3 pskr2-1. In turn, mannitol and ABA induced expression of PSK3 and PSKR1, and ABA promoted expression of PSK2 and PSK4 revealing feedback regulatory loops between PSKR and osmotic stress signaling.

HighlightPhytosulfokine receptor signaling regulates ABA synthesis and signaling genes and promotes ABA accumulation in the shoot of water-stressed plants and maintains leaf growth and photosynthetic efficiency which ensures plant health.

sentences: Effects of blue monochromatic light directed at one eye of pregnant horse mares on gestation, parturition and foal maturity Blue light directed at one eye advances the equine ovulatory season but can also advance foaling. In this study, effects of blue LED light on pregnancy outcome were assessed. Twenty mares with singleton pregnancies were studied over two consecutive years in a cross-over design. In one year, mares received an extended photoperiod using 50 lux of blue LED light (468 nm) directed at a single eye from 08:00 until 23:00 daily via head-worn light masks starting mid-December and in the other year remained untreated as controls. Gestation was shorter in blue LED light-treated (333.2{+/-}1.0 days,{+/-}SEM) than in control pregnancies (337.9{+/-}1.8 days, p<0.05). Colostral IgG content was lower in treated than in control pregnancies (p<0.05) but was adequate to ensure sufficient IgG transfer to newborn foals with a single exception. Foals born to blue LED light-treated mares had lower wither heights (102.5{+/-}1.1 vs. 105.2{+/-}1.0 cm, p<0.01), similar weights (55.0{+/-}1.3 vs. 55.5{+/-}1.1 kg) and took less time to stand after birth than control foals (39{+/-}3 vs. 56{+/-}4 min, p<0.05). The neutrophil/lymphocyte ratio, was higher in foals born to blue LED light-treated mares than controls (3.2{+/-}0.2 vs. 2.7{+/-}0.2; p<0.05). Foals born to blue LED light-treated mares had reduced hair length compared to controls (13.1{+/-}0.8 vs. 20.9{+/-}0.1 mm; p<0.001) and hair regrowth in treated mares was reduced (p<0.05). Maternal plasma prolactin concentration was increased in treated mares (p=0.05) and peaked at foaling (p<0.001). In conclusion, blue LED light directed at one eye advanced foaling and influenced height, hair coat and maturity in foals.

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Bi-directional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current, If, is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. We used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2-5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that {beta}-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously-expressed HCN4 channels and by mathematical models of If. Modelling further suggested that the slow activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Significance StatementCardiac pacemaker cells trigger each heartbeat by virtue of spontaneous oscillations in their membrane voltage. Although the funny current (If) is critical for these oscillations and for setting heart rate, its precise role remains an enigma because it activates mostly outside of the physiological voltage range and quite slowly relative to the pacemaker cycle. Here we show that If is persistently active in pacemaker cells; once opened, the small fraction of ion channels that conduct If do not re-close. Consequently, If flows both inward and outward to help propel the voltage oscillations and it paradoxically conducts a large fraction of the net charge movement. These results establish a new conceptual framework for the role of If in driving cardiac pacemaking.

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Sustained coevolution of phage Lambda and Escherichia coli involves inner as well as outer membrane defenses and counter-defenses Bacteria often evolve resistance to phage through the loss or modification of cell-surface receptors. In Escherichia coli and phage {lambda}, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyze another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair {lambda}s ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that {lambda} evolved an additional counter-defense that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral {lambda}, but some evolved phage grew well on the deletion mutants, indicating they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfills the model of an inverse-gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

IMPACT STATEMENTLaboratory studies of coevolution help us understand how host defenses and pathogen counter-defenses change over time, which is often essential for predicting the future dynamics of host-pathogen interactions. One particular model, termed "inverse-gene-for-gene" coevolution, predicts that coevolution proceeds through alternating steps, whereby hosts lose the features exploited by pathogens, and pathogens evolve to exploit alternative features. Using a classic model system in molecular biology, we describe the nature and timing of a previously overlooked step in the coevolution of E. coli and bacteriophage lambda. Our work demonstrates that this mode of coevolution can profoundly re-shape the interactions between bacteria and phage.

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SARS-CoV-2 harnesses host translational shutoff and autophagy to optimize virus yields: The role of the envelope (E) protein The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to phosphorylation of the translation initiation factor eIF2 and LC3 lipidation that resulted in host translational shutoff. During infection E is highly expressed although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. In support of this, we found that the {gamma}1 34.5 protein of herpes simplex virus 1 (HSV-1) prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S prevented E accumulation. While {gamma}1 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. This negative effect of {gamma}1 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy pathways. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2) and autophagy, to support optimal virus production.

ImportanceIn 2020, a new {beta}-coronavirus, SARS-CoV-2, entered the human population that has caused a pandemic resulting in 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Co-expression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories, and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.

sentences: The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells1. During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood2-4. Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.

sentences: Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor Human mesenchymal stem cells (hMSCs) have great potential in cell-based therapies for tissue engineering and regenerative medicine due to their self-renewal and multipotent properties. Recent studies indicate that Notch1-Dll4 signaling is an important pathway in regulating osteogenic differentiation of hMSCs. However, the fundamental mechanisms that govern osteogenic differentiation are poorly understood due to a lack of effective tools to detect gene expression at single cell level. Here, we established a double-stranded locked nucleic acid (LNA)/DNA (LNA/DNA) nanobiosensor for gene expression analysis in single hMSC in both 2D and 3D microenvironments. We first characterized this LNA/DNA nanobiosensor and demonstrated the Dll4 mRNA expression dynamics in hMSCs during osteogenic differentiation. By incorporating this nanobiosensor with live hMSCs imaging during osteogenic induction, we performed dynamic tracking of hMSCs differentiation and Dll4 mRNA gene expression profiles of individual hMSC during osteogenic induction. Our results showed the dynamic expression profile of Dll4 during osteogenesis, indicating the heterogeneity of hMSCs during this dynamic process. We further investigated the role of Notch1-Dll4 signaling in regulating hMSCs during osteogenic differentiation. Pharmacological perturbation is applied to disrupt Notch1-Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. In addition, the effects of Notch1-Dll4 signaling on hMSCs spheroids differentiation were also investigated. Our results provide convincing evidence supporting that Notch1-Dll4 signaling is involved in regulating hMSCs osteogenic differentiation. Specifically, Notch1-Dll4 signaling is active during osteogenic differentiation. Our results also showed that Dll4 is a molecular signature of differentiated hMSCs during osteogenic induction. Notch inhibition mediated osteogenic differentiation with reduced Alkaline Phosphatase (ALP) activity. Lastly, we elucidated the role of Notch1-Dll4 signaling during osteogenic differentiation in a 3D spheroid model. Our results showed that Notch1-Dll4 signaling is required and activated during osteogenic differentiation in hMSCs spheroids. Inhibition of Notch1-Dll4 signaling mediated osteogenic differentiation and enhanced hMSCs proliferation, with increased spheroid sizes. Taken together, the capability of LNA/DNA nanobiosensor to probe gene expression dynamics during osteogenesis, combined with the engineered 2D/3D microenvironment, enables us to study in detail the role of Notch1-Dll4 signaling in regulating osteogenesis in 2D and 3D microenvironment. These findings will provide new insights to improve cell-based therapies and organ repair techniques.

sentences: HistoClean: Open-source Software for Histological Image Pre-processing and Augmentation to Improve Development of Robust Convolutional Neural Networks The growth of digital pathology over the past decade has opened new research pathways and insights in cancer prediction and prognosis. In particular, there has been a surge in deep learning and computer vision techniques to analyse digital images. Common practice in this area is to use image pre-processing and augmentation to prevent bias and overfitting, creating a more robust deep learning model. Herein we introduce HistoClean; user-friendly, graphical user interface that brings together multiple image processing modules into one easy to use toolkit. In this study, we utilise HistoClean to pre-process images for a simple convolutional neural network used to detect stromal maturity, improving the accuracy of the model at a tile, region of interest, and patient level. HistoClean is free and open-source and can be downloaded from the Github repository here: https://github.com/HistoCleanQUB/HistoClean.

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Direct and indirect cues can enable dual-adaptation, but through different learning processes Switching between motor tasks requires accurate adjustments for changes in dynamics (grasping a cup) or sensorimotor transformations (moving a computer mouse). Dual-adaptation studies have investigated how learning of context-dependent dynamics or transformations is enabled by sensory cues. However, certain cues, such as color, have shown mixed results. We propose that these mixed results may arise from two major classes of cues: "direct" cues, which are part of the dynamic state and "indirect" cues, which are not. We hypothesized that explicit strategies would primarily account for adaptation for an indirect color cue but would be limited to simple tasks while a direct visual separation cue would allow implicit adaptation regardless of task complexity. To test this idea, we investigated the relative contribution of implicit and explicit learning in relation to contextual cue type (colored or visually shifted workspace) and task complexity (one or eight targets) in a dual-adaptation task. We found that the visual workspace location cue enabled adaptation across conditions primarily through implicit adaptation. In contrast, we found that the color cue was largely ineffective for dual-adaptation, except in a small subset of participants who appeared to use explicit strategies. Our study suggests that the previously inconclusive role of color cues in dual-adaptation may be explained by differential contribution of explicit strategies across conditions.

New & NoteworthyWe present evidence that learning of context-dependent dynamics proceeds via different processes depending on the type of sensory cue used to signal the context. Visual workspace location enabled learning different dynamics implicitly, presumably because it directly enters the dynamic state estimate. In contrast, a color cue was only successful where learners were apparently able to leverage explicit strategies to account for changed dynamics. This suggests a unification for the previously inconclusive role of color cues.

sentences: Interplay between external inputs and recurrent dynamics during movement preparation and execution in a network model of motor cortex The primary motor cortex has been shown to coordinate movement preparation and execution through computations in approximately orthogonal subspaces. The underlying network mechanisms, and in particular the roles played by external and recurrent connectivity, are central open questions that need to be answered to understand the neural substrates of motor control. We develop a recurrent neural network model that recapitulates the temporal evolution of single-unit activity recorded from M1 of a macaque monkey during an instructed delayed-reach task. We explore the hypothesis that the observed dynamics of neural covariation with the direction of motion emerges from a synaptic connectivity structure that depends on the preferred directions of neurons in both preparatory and movement-related epochs. We constrain the strength both of synaptic connectivity and of external input parameters by using the data as well as an external input minimization cost. Our analysis suggests that the observed patterns of covariance are shaped by external inputs that are tuned to neurons preferred directions during movement preparation, and they are dominated by strong direction-specific recurrent connectivity during movement execution, in agreement with recent experimental findings on the relationship between motor-cortical and motor-thalamic activity, both before and during movement execution. We also demonstrate that the manner in which single-neuron tuning properties rearrange over time can explain the level of orthogonality of preparatory and movement-related subspaces. We predict that the level of orthogonality is small enough to prevent premature movement initiation during movement preparation; however, it is not zero, which allows the network to encode a stable direction of motion at the population level without direction-specific external inputs during movement execution.

sentences: Modelling altered signalling of G-protein coupled receptors in inflamed environment to advance drug design We previously reported the successful design, synthesis and testing of the prototype opioid painkiller NFEPP that does not elicit adverse side effects. Uniquely, this design was based on mathematical modelling of extracellular interactions between G-protein coupled receptors (GPCRs) and ligands, recognizing that GPCRs function differently under pathological versus healthy conditions. We now present a novel stochastic model of GPCR function that includes intracellular dissociation of G-protein subunits and modulation of plasma membrane calcium channels associated with parameters of inflamed and healthy tissue (pH, radicals). The model is validated against in vitro experimental data for NFEPP and fentanyl ligands at different pH values. We found markedly reduced calcium channel inhibition induced by NFEPP at normal pH compared to lower pH, in contrast to the effect of fentanyl, and enhanced constitutive G-protein activation but lower probability of ligand binding with increasing radical concentrations. By means of molecular dynamics simulations, we also assessed qualitative changes of reaction rates due to additional disulfide bridges inside the GPCR binding pocket. The results suggest that, compared to radicals, low pH is a more important determinant of overall GPCR function in an inflamed environment. Future drug design efforts should take this into account.

sentences: Hidden Transcriptome Signature in Autism Spectrum Disorder (ASD) is Attributed to the Interpatient Heterogeneity Recent studies of ASD have mostly supported the existence of heterogeneity and genomic variation in ASD which have hindered and restrained development of any effective and targetable treatment for a long time. As numerous studies have shown, both genetic and phenotypic heterogeneity is presented in ASD, however, heterogeneity in genetic level is not fully understood which is the key challenges for the further research. Even dozens of ASD susceptibility genes have been discovered which is commonly accounting for 10 to 20 percent of ASD cases, the internal complex combination of mutated genes that determine the epigenetic factors of ASD is still not comprehensively recognized by the recent studies. First by discouraging the traditional method that have been applied in most of the current research of diseases, this research will then focus on dissecting the heterogeneity of polygenic diseases and analyzing with an unconventional approach for acquiring Differently Expressed Genes (DEGs) in Guptas Dataset that provided transcriptome of frontal cortex of ASD patients. Divide categories by using unsupervised learning strategy, the results yielded by analyzing within clusters of ASD have supported the feasibility of the attempts to use heterogeneity to reveal its underlying mechanism. This study puts forward the inference that the heterogeneity of polygenic diseases will obscure the molecular signals related to the disease, and at the same time attempts to use heterogeneity to reveal the underlying mechanism.

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Loss of pod strings in common bean is associated with gene duplication, retrotransposon insertion, and overexpression of PvIND Regulation of fruit development has been central in the evolution and domestication of flowering plants. In common bean (Phaseolus vulgaris L.), a major global staple crop, the two main economic categories are distinguished by differences in fiber deposition in pods: a) dry beans with fibrous and stringy pods; and b) stringless snap/green beans with reduced fiber deposition, but which frequently revert to the ancestral stringy state. To better understand control of this important trait, we first characterized developmental patterns of gene expression in four phenotypically diverse varieties. Then, using isogenic stringless/revertant pairs of six snap bean varieties, we identified strong overexpression of the common bean ortholog of INDEHISCENT (PvIND) in non-stringy types compared to their string-producing counterparts. Microscopy of these pairs indicates that PvIND overexpression is associated with overspecification of weak dehiscence zone cells throughout the entire pod vascular sheath. No differences in PvIND DNA methylation were correlated with pod string phenotype. Sequencing of a 500 kb region surrounding PvIND in the stringless snap bean cultivar Hystyle revealed that PvIND had been duplicated into two tandem repeats, and that a Ty1-copia retrotransposon was inserted between these tandem repeats, possibly driving PvIND overexpression. Further sequencing of stringless/revertant isogenic pairs and diverse materials indicated that these sequence features had been uniformly lost in revertant types and were strongly predictive of pod phenotype, supporting their role in PvIND overexpression and pod string phenotype.

SignificanceFruit dehiscence is a key trait for seed dissemination. In legumes, e.g., common bean, dehiscence relies on the presence of fibers, including pod "strings". Selections during domestication and improvement have decreased (dry beans) or eliminated (snap beans) fibers, but reversion to the fibrous state occurs frequently in snap beans. In this article, we document that fiber loss or gain is controlled by structural changes at the PvIND locus, a homolog of the Arabidopsis INDEHISCENT gene. These changes include a duplication of the locus and insertion/deletion of a retrotransposon, which are associated with significant changes in PvIND expression. Our findings shed light on the molecular basis of unstable mutations and provide potential solutions to an important pod quality issue.

Competing Interest StatementThe authors have no competing interests.

sentences: Loss of TREM2 reduces hyperactivation of progranulin deficient microglia but not lysosomal pathology GRN haploinsufficiency causes frontotemporal lobar degeneration and results in microglial hyperactivation, lysosomal dysfunction and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology we evaluated genetic and pharmacological approaches suppressing TREM2 dependent transition of microglia from a homeostatic to a disease associated state. Trem2 deficiency in Grn KO mice led to a reduction of microglia activation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies allowed a complete rescue of elevated levels of TREM2 together with increased shedding and reduction of TREM2 signaling. Furthermore, antibody-treated PGRN deficient hiMGL showed dampened microglial hyperactivation, reduced TREM2 signaling and phagocytic activity, however, lack of rescue of lysosomal dysfunction. Similarly, lysosomal dysfunction, lipid dysregulation and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Furthermore, NfL, a biomarker for neurodegeneration, was elevated in the Grn/Trem2 KO. These findings suggest that microglia hyperactivation is not necessarily contributing to neurotoxicity, and instead demonstrates that TREM2 exhibits neuroprotective potential in this model.

sentences: Neural representations of stereotype content predict social decisions To guide social interaction, people often rely on expectations about the traits of other people based on markers of social group membership, i.e., stereotypes. Although the influence of stereotypes on social behavior is widespread, key questions remain about how traits inferred from social group membership are instantiated in the brain and incorporated into neural computations that guide social behavior. Here, we show that the human lateral orbitofrontal cortex (OFC) represents the content of stereotypes about members of different social groups in the service of social decision-making. During fMRI scanning, participants decided how to distribute resources across themselves and members of a variety of social groups in a modified Dictator Game. Behaviorally, we replicated our recent finding that perceptions of others traits, captured by a two-dimensional framework of stereotype content (warmth and competence), biased participants monetary allocation choices in a context-dependent manner: recipients warmth increased advantageous inequity aversion and their competence increased disadvantageous inequity aversion. Neurally, representational similarity analysis (RSA) revealed that perceptions of others traits in the two-dimensional space were represented in the temporoparietal junction and superior temporal sulcus, two regions associated with mentalizing, and in the lateral OFC, known to represent latent environmental features during goal-directed outcome inference outside the social domain. Critically, only the latter predicted individual choices, suggesting that the effect of stereotypes on behavior is mediated by inference-based, domain-general decision-making processes in the OFC.

sentences: Subcellular mapping of the protein landscape of SARS-CoV-2 infected cells for target-centric drug repurposing The COVID-19 pandemic has resulted in millions of deaths and affected socioeconomic structure worldwide and the search for new antivirals and treatments are still ongoing. In the search for new drug target and to increase our understanding of the disease, we used large scale immunofluorescence to explore the host cell response to SARS-CoV-2 infection. Among the 602 host proteins studied in this host response screen, changes in abundance and subcellular localization were observed for 97 proteins, with 45 proteins showing increased abundance and 10 reduced abundances. 20 proteins displayed changed localization upon infection and an additional 22 proteins displayed altered abundance and localization, together contributing to diverse reshuffling of the host cell protein landscape. We then selected existing and approved small-molecule drugs (n =123) against our identified host response proteins and identified 3 compounds - elesclomol, crizotinib and rimcazole, that significantly reduced antiviral activity. Our study introduces a novel, targeted and systematic approach based on host protein profiling, to identify new targets for drug repurposing. The dataset of [~]75,000 immunofluorescence images from this study are published as a resource available for further studies.

sentences: Differences in Ion-RNA binding modes due to charge density variations explain the stability of RNA in monovalent salts The stability of RNA increases as the charge density of the alkali metal cations increases. The molecular mechanism for the stability does not exist. To fill this gap, we performed all-atom molecular dynamics (MD) pulling simulations to dissect the microscopic origin of this phenomenon. We first established that the free energy landscape obtained in the simulations is in excellent agreement with the single-molecule optical tweezer experiments. The origin of the stronger stability in Na+ compared to K+ is found to be due to the differences in the charge-density related binding modes. The smaller hydrated Na+ ion preferentially binds to the highly charged phosphates. In contrast, the larger K+ ions interact with the major grooves. As a result, the electrostatic repulsion between the phosphate groups is reduced more effectively by Na+ ions. Because the proposed mechanism is generic, we predict that the same conclusions are valid for divalent alkaline earth metal cations.

sentences: Fluorescence lifetime imaging as an in situ and label-free readout for the chemical composition of lignin Important structures and functions within living organisms rely on naturally fluorescent polymeric molecules such as collagen, keratin, elastin, resilin, or lignin. Theoretical physics predict that fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls and one of the most abundant components of wood. Lignin is composed of different types of phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured wood cell wall composition by classical analytical methods in the wood cell walls of these trees. Using statistical modelling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm plants. Finally, we show how quantitative measurements of lignin chemical composition by FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

sentences: Tracking cell lineages in 3D by incremental deep learning Deep learning is emerging as a powerful approach for bioimage analysis. Its use in cell tracking is limited by the scarcity of annotated data for the training of deep learning models. Moreover, annotation, training, prediction and proofreading currently lack a unified user interface. We present ELEPHANT, an interactive platform for 3D cell tracking that addresses these challenges by taking an incremental approach to deep learning. ELEPHANT provides an interface that seamlessly integrates cell track annotation, deep learning, prediction and proofreading. This enables users to implement cycles of incremental learning starting from a few annotated nuclei. Successive prediction-validation cycles enrich the training data, leading to rapid improvements in tracking performance. We test the softwares performance against state-of-the-art methods and track lineages spanning the entire course of leg regeneration in a crustacean over 1 week (504 timepoints). ELEPHANT yields accurate, fully-validated cell lineages with a modest investment in time and effort.

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

sentences: Characterization of cognitive-motor function in women who have experienced intimate partner violence-related brain injury Intimate partner violence (IPV) affects at least 1 in 3 women worldwide and up to 92% report symptoms consistent with brain injury (BI). Although a handful of studies have examined different aspects of brain structure and function in this population, none has characterized potential deficits in cognitive-motor function. This knowledge gap was addressed in the current study by having participants who had experienced IPV complete the bimanual Object Hit & Avoid (OHA) task on a Kinarm End-Point Lab. BI load, as assessed by the Brain Injury Severity Assessment (BISA) tool as well as measures of comorbidities (PTSD, anxiety, depression, substance use, history of abuse) were also collected. Results demonstrated BI load accounted for a significant amount of variability in the number of targets hit and average hand speed. PTSD, anxiety, and depression also contributed significantly to the variability in these measures as well as to the number and proportion of distractor hits, and the object processing rate. Taken together, these findings suggest IPV-related BI, as well as comorbid PTSD, anxiety, and depression, disrupt the processing required to quickly and accurately hit targets while avoiding distractors. This pattern of results reflects the complex interaction between the physical injuries induced by the episodes of IPV and the resulting impacts these experiences have on mental health.

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Network Propagation-based Prioritization of Long Tail Genes in 17 Cancer Types IntroductionThe diversity of genomic alterations in cancer pose challenges to fully understanding the etiologies of the disease. Recent interest in infrequent mutations, in genes that reside in the "long tail" of the mutational distribution, uncovered new genes with significant implication in cancer development. The study of these genes often requires integrative approaches with multiple types of biological data. Network propagation methods have demonstrated high efficacy in uncovering genomic patterns underlying cancer using biological interaction networks. Yet, the majority of these analyses have focused their assessment on detecting known cancer genes or identifying altered subnetworks. In this paper, we introduce a network propagation approach that entirely focuses on long tail genes with potential functional impact on cancer development.

ResultsWe identify sets of often overlooked, rarely to moderately mutated genes whose biological interactions significantly propel their mutation-frequency-based rank upwards during propagation in 17 cancer types. We call these sets "upward mobility genes" (UMGs, 28-83 genes per cancer type) and hypothesize that their significant rank improvement indicates functional importance. We report new cancer-pathway associations based on UMGs that were not previously identified using driver genes alone, validate UMGs role in cancer cell survival in vitro--alone and compared to other network methods--using extensive genome-wide RNAi and CRISPR data repositories, and further conduct in vitro functional screenings resulting the validation of 8 previously unreported genes.

ConclusionOur analysis extends the spectrum of cancer relevant genes and identifies novel potential therapeutic targets.

sentences: Elucidating the Mechanical Energy for Cyclization of a DNA Origami Tile DNA origami has emerged as a versatile method to synthesize nanostructures with high precision. This bottom-up self-assembly approach can produce not only complex static architectures, but also dynamic reconfigurable structures with tunable properties. While DNA origami has been explored increasingly for diverse applications such as biomedical and biophysical tools, related mechanics are also under active investigation. Here we studied the structural properties of DNA origami and investigated the energy needed to deform the DNA structures. We used a single-layer rectangular DNA origami tile as a model system and studied its cyclization process. This origami tile was designed with an inherent twist by placing crossovers every 16 base-pairs (bp), corresponding to a helical pitch of 10.67 bp/turn which is slightly different from that of native B-form DNA (10.5 bp/turn). We used molecular dynamics (MD) simulations based on a coarse-grained model on an open-source computational platform, oxDNA. We calculated the energies needed to overcome the initial curvature and induce mechanical deformation by applying linear spring forces. We found that the initial curvature may be overcome gradually during cyclization and a total of ~33.1 kcal/mol is required to complete the deformation. These results provide insights into the DNA origami mechanics and should be useful for diverse applications such as adaptive reconfiguration and energy absorption.

sentences: On Riemannian Geometries of Visual Space AO_SCPLOWBSTRACTC_SCPLOWThis is a study of the phenomenal geometries constructed in the Riemannian geometry framework from simulated iso-disparity conics in the horizontal visual plane of the binocular system with the asymmetric eyes (AEs). For the eyes resting vergence posture, which depends on parameters of the AE, the iso-disparity conics are frontal straight lines in physical space. For all other fixations, the simulated iso-disparity conics consist of families of the ellipses or hyperbolas depending on both the AEs parameters and the bifoveal fixation. However, the iso-disparity conics arcs are perceived in the gaze direction as the frontal lines. Thus, geometries of physical and visual spaces are different. An assumption underlying the relevant architecture of the human visual system is combined with results from simulated iso-disparity straight lines, giving the relative depth as a function of the AEs parameters and the distance. This establishes the metric tensor in binocular space of fixations for the eyes resting vergence posture. The resulting geodesics in the gaze direction, give the distance to the horizon and zero curvature. For all other fixations, only the sign of the curvature may be inferred from the global behavior of the simulated iso-disparity conics.

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Tracing cortical circuits in humans and non-human primates from high resolution connectomic, transcriptomic, and temporal dimensions The neural circuits that support human cognition are a topic of enduring interest. Yet, the lack of tools available to map human brain circuits has precluded our ability to trace the human and non-human primate connectome. We harnessed high-resolution connectomic, anatomic, and transcriptomic data to investigate the evolution and development of frontal cortex circuitry. We applied machine learning to RNA sequencing data to find corresponding ages between humans and macaques and to compare the development of circuits across species. We transcriptionally defined neural circuits by testing for associations between gene expression and white matter maturation. We then considered transcriptional and structural growth to test whether frontal cortex circuit maturation is unusually extended in humans relative to other species. We also considered gene expression and high-resolution diffusion MR tractography of adult brains to test for cross-species variation in frontal cortex circuits. We found that frontal cortex circuitry development is extended in primates, and concomitant with an expansion in cortico-cortical pathways compared with mice in adulthood. Importantly, we found that these parameters varied relatively little across humans and studied primates. These data identify a surprising collection of conserved features in frontal cortex circuits across humans and Old World monkeys. Our work demonstrates that integrating transcriptional and connectomic data across temporal dimensions is a robust approach to trace the evolution of brain connectomics in primates.

Significance StatementWe lack appropriate tools to visualize the human brain connectome. We develop new approaches to study connections in the human and non-human primate brains. The integration of transcription with structure offers an unprecedented opportunity to study circuitry evolution. Our integrative approach finds corresponding ages across species and transcriptionally defines neural circuits. We used this information to test for variation in circuit maturation across species and found a surprising constellation of similar features in frontal cortex neural circuits across humans and primates. Integrating across scales of biological organization expands the repertoire of tools available to study connections in primates, which opens new avenues to study connections in health and diseases of the human brain.

sentences: Glycine attenuates impairments of stimulus-evokedgamma oscillation in the ketaminemodel of schizophrenia Although a substantial number of studies suggests some clinical benefit concerning negative symptoms in schizophrenia through the modulation of NMDA-receptor function, none of these approaches achieved clinical approval. Given the large body of evidence concerning glutamatergic dysfunction in a subgroup of patients, biomarkers to identify those with a relevant clinical benefit through glutamatergic modulation are urgently needed. A similar reduction of the early auditory evoked gamma-band response (aeGBR) as found in schizophrenia patients can be observed in healthy subjects in the ketamine-model, which addresses the putative excitation / inhibition (E/I) imbalance of the diseases. Moreover, this change in gamma-band oscillations can be related to the emergence of negative symptoms. Accordingly, this study investigated whether glycine-related increases of the aeGBR accompany an improvement concerning negative symptoms in the ketamine-model. The impact of subanesthetic ketamine doses and the pretreatment with glycine was examined in twenty-four healthy male participants while performing a cognitively demanding aeGBR paradigm with 64-channel electroencephalography. Negative Symptoms were assessed through the Positive and Negative Syndrome Scale (PANSS). Ketamine alone caused a reduction of the aeGBR amplitude associated with more pronounced negative symptoms compared to placebo. Pretreatment with glycine attenuated both, the ketamine-induced alterations of the aeGBR amplitude and the increased PANSS negative scores in glycine-responders, classified based on relative aeGBR increase. Thus, we propose that the aeGBR represents a possible biomarker for negative symptoms in schizophrenia related to insufficient glutamatergic neurotransmission. This would allow to identify patients with negative symptoms, who might benefit from glutamatergic treatment.

sentences: Dehalobium species implicated in 2,3,7,8-tetrachloro-p-dioxin dechlorination in the contaminated sediments of Sydney Harbour Estuary Polychlorinated dibenzo-p-dioxins and furans (PCDD/F) are some of the most environmentally recalcitrant and toxic compounds. They are naturally occurring and by-products of anthropogenic activity. Sydney Harbour Estuary (Sydney, Australia), is heavily contaminated with PCDD/F. Analysis of sediment cores revealed that the contamination source in Homebush Bay continues to have one of the highest levels of PCDD/F contamination in the world (5207 pg WHO-TEQ g-1) with >50% of the toxicity attributed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) the most toxic and concerning of the PCDD/F congeners. Comparison of congener profiles at the contamination source with surrounding bays and historical data provided evidence for the attenuation of 2,3,7,8-TCDD and other congeners at the source. This finding was supported by the detection of di-, mono- and unchlorinated dibenzo-p-dioxin. Microbial community analysis of sediments by 16S amplicon sequencing revealed an abundance of lineages from the class Dehalococcoidia (up to 15% of the community), including the genus Dehalobium (up to 0.5%). Anaerobic seawater enrichment cultures using perchloroethene as a more amenable growth substrate enriched only the Dehalobium population by more than six-fold. The enrichment culture then proved capable of reductively dechlorinating 2,3,7,8-TCDD to 2,3,7-TCDD and octachlorodibenzo-p-dibenzodioxin to hepta and hexa congeners. This work is the first to show microbial reductive dehalogenation of 2,3,7,8-TCDD with a bacterium from outside the Dehalococcoides genus, and one of only a few that demonstrates PCDD/F degradation in a marine environment.

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Multi-layered networks of SalmoNet2 enable strain comparisons of the Salmonella genus on a molecular level Serovars of the genus Salmonella primarily evolved as gastrointestinal pathogens in a wide range of hosts. Some serotypes later evolved further, adopting a more invasive lifestyle in a narrower host range associated with systemic infections. A system-level knowledge of these pathogens has the potential to identify the complex adaptations associated with the evolution of serovars with distinct pathogenicity, host range and risk to human health. This promises to aid the design of interventions and serve as a knowledge base in the Salmonella research community. Here we present SalmoNet2, a major update to SalmoNet, the first multi-layered interaction resource for Salmonella strains, containing protein-protein, transcriptional regulatory and enzyme enzyme interactions. The new version extends the number of Salmonella genomes from 11 to 20, including strains such as S. Typhimurium D23580, an epidemic multidrug-resistant strain leading to invasive non-typhoidal Salmonella Disease (iNTS), and a strain from Salmonella bongori, another species in the Salmonella genus. The database now uses strain specific metabolic models instead of a generalised model to highlight differences between strains. This has increased the coverage of high-quality protein-protein interactions, and enhances interoperability with other computational resources by adopting standardised formats. The resource website has been updated with tutorials to help researchers analyse their Salmonella data using molecular interaction networks from SalmoNet2. SalmoNet2 is accessible at http://salmonet.org/.

ImportanceMulti-layered network databases collate information from multiple sources, and are powerful both as a knowledge base and platform for analysis. Here we present SalmoNet2, an integrated network resource of 20 Salmonella strains, containing protein-protein, transcriptional regulatory, and metabolic interactions. Key improvements to the update include expanding the number of strains, strain-specific metabolic networks, an increase in high quality protein-protein interactions, community standard computational formats to help interoperability, and online tutorials to help users analyse their data using SalmoNet2.

sentences: Single-cell RNA Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

sentences: Imprinting on time-structured acoustic stimuli in ducklings Filial imprinting is a dedicated learning process that lacks explicit reinforcement. The phenomenon itself is narrowly heritably canalized, but its content, the representation of the parental object, reflects the circumstances of the newborn. Imprinting has recently been shown to be even more subtle and complex than previously envisaged, since ducklings and chicks are now known to select and represent for later generalization abstract conceptual properties of the objects they perceive as neonates, including movement pattern, heterogeneity, and inter-component relationships of same or different. Here we investigate whether day-old Mallard ducklings (Anas platyrhynchos) imprint on the temporal separation between duos of brief acoustic stimuli, and whether they generalize such timing information to novel sound types. Subjects did discriminate temporal structure when imprinted and tested on natural duck calls, but not when using white noise for imprinting or testing. Our data confirm that imprinting includes the establishment of neural representations of both primary percepts and abstract properties of candidate objects, meshing together genetically transmitted prior knowledge with selected perceptual input.

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SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential The balance between the mitochondrial respiratory chain activity and the cells needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We verify that molecule bond vibrations assessed by SERS is a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have found that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential and H+ concentration in the intermembrane space to regulate the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cells consumption of ATP and the overall activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens wide perspectives on cell bioenergetics.

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sentences: EgGLUT1 is crucial for the viability of larvae of Echinococcus granulosus sensus lato by involving its glucose uptake Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by infection with the larvae of Echinococcus granulosus sensu lato (s.l.) cluster. It is urgent to identify novel drug targets and develop new drug candidates against CE. Glucose transporter 1 (GLUT1) is mainly responsible for the transmembrane transport of glucose to maintain its constant cellular availability and is a recent research hotspot as a drug target in various diseases. However, presence and role of GLUT1 in E. granulosus s.l. (EgGLTU1) was unknown. In this study, we cloned a conserved GLUT1 homology gene (named EgGLUT1-ss) from E. granulosus sensu stricto (s.s.) and found EgGLUT1-ss was crucial for glucose uptake of the protoscoleces of E. granulosus s.s.. WZB117, a GLUT1 inhibitor, inhibited glucose uptake of E. granulosus s.s. and the viability of the metacestode in vitro. In addition, WZB117 showed potent therapeutic activity in E. granulosus s.s.-infected mice: a 10 mg/kg dose of WZB117 significantly reduced the number and weight of parasite cysts as well as the reference drug, albendazole. Our data have defined EgGLUT1 as a key E. granulosus s.l. vulnerability target, involved in its glucose uptake from the host; this opens a new avenue to identify drugs with an ideal activity profile for the treatment of CE.

sentences: The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism Lipid mobilization through fatty acid {beta}-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where {beta}-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in {beta}-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although {beta}-oxidation occurs mainly in mitochondria in mammals, Arf1s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites.

sentences: Inference and Uncertainty Quantification of Stochastic Gene Expression via Synthetic Models AO_SCPLOWBSTRACTC_SCPLOWEstimating uncertainty in model predictions is a central task in quantitative biology. Biological models at the single-cell level are intrinsically stochastic and nonlinear, creating formidable challenges for their statistical estimation which inevitably has to rely on approximations that trade accuracy for tractability. Despite intensive interest, a sweet spot in this trade off has not been found yet. We propose a flexible procedure for uncertainty quantification in a wide class of reaction networks describing stochastic gene expression including those with feedback. The method is based on creating a tractable coarse-graining of the model that is learned from simulations, a synthetic model, to approximate the likelihood function. We demonstrate that synthetic models can substantially outperform state-of-the-art approaches on a number of nontrivial systems and datasets, yielding an accurate and computationally viable solution to uncertainty quantification in stochastic models of gene expression.

sentences: The PRO model accounts for the anterior cingulate cortex role in risky decision-making and monitoring The anterior cingulate cortex (ACC) has been implicated in a number of functions including performance monitoring and decision making involving effort. The prediction of responses and outcomes (PRO) model has provided a unified account of much human and monkey ACC data involving anatomy, neurophysiology, EEG, fMRI, and behavior. Here we explore the computational nature of ACC with the PRO model, extending it to account specifically for both human and macaque monkey decision-making under risk, including both behavioral and neural data. We show that the PRO model can account for a number of additional effects related to outcome prediction, decision-making under risk, gambling behavior, and we show that the ACC represents the variance of uncertain outcomes, suggesting a link between ACC function and mean-variance theories of decision making. The PRO model provides a unified account of a large set of data regarding the ACC.

sentences: Spatiotemporal organization of movement-invariant and movement-specific signaling in the output layer of motor cortex Motor cortex generates descending output necessary for executing a wide range of limb movements. Although movement-related activity has been described throughout motor cortex, the spatiotemporal organization of movement-specific signaling in deep layers remains largely unknown. Here, we recorded layer 5B population dynamics in the caudal forelimb area of motor cortex while mice performed a forelimb push/pull task and found that most neurons show movement-invariant responses, with a minority displaying movement specificity. Cell-type-specific imaging identified that movement-invariant responses dominated pyramidal tract (PT) neuron activity, with a small subpopulation representing movement type, whereas a larger proportion of intratelencephalic (IT) neurons displayed movement-specific signaling. The proportion of IT neurons decoding movement-type peaked prior to movement initiation, while for PT neurons this occurred during movement execution. Our data suggest that layer 5B population dynamics largely reflect movement-invariant signaling, with information related to movement-type being differentially routed through relatively small, distributed subpopulations of projection neurons.

sentences: CRY2 missense mutations suppress P53 and enhance cell growth Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA repair enzymes. While CRYs lack DNA repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SCFFBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3. Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target gene expression, suggesting that this is the primary mechanism by which they influence cell growth.

sentences: Inhibitory IL-10-producing CD4+ T cells develop in a T-bet-dependent manner and facilitate cytomegalovirus persistence via coexpression of arginase-1 Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity, but the mechanisms that underpin the development of such regulatory networks in vivo have remained obscure. To address this knowledge gap, we performed a comprehensive functional, phenotypic, and transcriptomic analysis of IL-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed at sites of viral persistence in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enabled better immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the coexpression of Arg1.

sentences: Surface interaction patches link non-specific binding and phase separation of antibodies Non-specificity is a key challenge in the successful development of therapeutic antibodies. The tendency for non-specific binding in antibodies is often difficult to reduce via judicious design and, instead, it is necessary to rely on comprehensive screening campaigns. A better understanding of the molecular origins that drive antibody non-specificity is therefore highly desirable in order to prevent non-specific off-target binding. Here, we perform a systematic analysis of the impact of surface patch properties on antibody non-specificity using a designer antibody library as a model system and DNA as a non-specificity ligand. Using an in solution microfluidics approach, we discover patches of surface hydrogen bonding to be causative of the observed non-specificity under physiological salt conditions and suggest them to be a vital addition to the molecular origins of non-specificity. Moreover, we find that a change in formulation conditions leads to DNA-induced antibody liquid-liquid phase separation as a manifestation of antibody non-specificity. We show that this behaviour is driven by a cooperative electrostatic network assembly mechanism enabled by mutations that yield a positively charged surface patch. Together, our study provides a direct link between molecular binding events and macroscopic liquid-liquid phase separation. These findings highlight a delicate balance between surface interaction patches and their crucial role in conferring antibody non-specificity.

sentences: Fungal biodiversity in Arctic paleoecosystems assessed by metabarcoding of lake sedimentary ancient DNA Fungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may therefore help reveal biodiversity changes in past ecosystems. Lake sediments contain DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We developed a novel PCR primer combination for fungal metabarcoding targeting a short amplicon to account for length bias of amplification due to ancient DNA degradation. In-silico PCRs showed higher diversity using this primer combination than using previously established fungal metabarcoding primers. We analyzed existing data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2-22 years and examined degradation effects of ancient DNA and storage time-related bias in fungal communities. Amplicon size differed between fungal divisions, however, we observed no significant effect of sample age on amplicon length and GC content, suggesting robust results. We also found no indication of post-coring fungal growth during storage distorting ancient fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.

sentences: IsoSolve: an integrative framework to improve isotopic coverage and consolidate isotopic measurements by MS and/or NMR Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS)and nuclear magnetic resonance (NMR)spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased, and the coverage of isotopic space improved, by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic dataset by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to i) clarify and improve the coverage of isotopic space, ii) evaluate the complementarity and redundancy of different techniques, iii) consolidate isotopic datasets, iv) design experiments, and v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSysLISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open source software that can be readily integrated into data analysis pipelines.

sentences: The role of oxytocin in modulating self-other distinction brain: a pharmacological fMRI study The self-other distinction is crucial in human social cognition and social interaction. Studies have found that oxytocin (OT) sharpens the self-other perceptual boundary but with mixed results. Further, little is known if the effect of OT on self-resemblance facial perception exists, especially on its neural basis. Moreover, it is unclear if OT would influence the judgment in self-other discrimination when the other is a child or an adult. In the current double-blinded, placebo-controlled study, we investigated the effect of OT on self-face perception at both behavioral and neural levels. We morphed participants faces and strangers faces to create four stimuli conditions. After being treated by either OT or placebo (PL), participants reported whether a morphed face resembles themselves, or was morphed with their own faces, while being scanned with fMRI. Behavioral results showed that people judged adult-morphed faces better than child-morphed faces. fMRI results showed that the OT group exhibited generally increased activities in the visual area and IFG for self-morphed faces. Such difference was more pronounced in the adult face compared to child face conditions. Multivariate fMRI analysis revealed that the OT group showed better classification between self-morphed versus other-morphed faces, indicating that OT increased self-other distinction, especially for adult faces and in the left hemisphere. Our study shows the significant effect of OT on self-referential brain processes, providing evidence for the potential OTs effect on a left hemisphere self network.

sentences: Dimerization mechanism and structural features of human LI-cadherin LI-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, since the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin EC1-4 homodimer. The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between EC2 and EC3. Various biochemical techniques and molecular dynamics (MD) simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed by the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assay.

sentences: Optimizing the live attenuated influenza A vaccine backbone for high-risk patient groups The live attenuated influenza vaccine (LAIV) is approved for intranasal spray application in 2-49 year-old patients with safety concerns limiting its use in younger children and immunocompromised patients, mainly from the higher incidence of adverse events and the possibility of uncontrolled replication and reversion to a pathogenic strain, respectively. Further attenuation of the LAIV could generally improve its safety profile, which might come at the cost of reduced immunogenicity. To solve this dilemma, we took advantage of a recently defined mechanism of ER stress induction by modifying IAV non-structural protein 1 (NS1). The modified LAIV (AAmut/PR8) showed stronger ER stress activation in vitro and replicated to lower titers in vivo compared to its parental strain, without affecting protection against homo-subtypic or hetero-subtypic IAV strains. AAmut/PR8 could pose as a suitable strategy to attend the gap to the current LAIV recommendation guidelines in susceptible target populations.

sentences: Large-scale analysis and computer modeling reveal hidden regularities behind variability of cell division patterns in Arabidopsis thaliana embryogenesis Noise plays a major role in cellular processes and in the development of tissues and organs. Several studies have examined the origin, the integration or the accommodation of noise in gene expression, cell growth and elaboration of organ shape. By contrast, much less is known about variability in cell division plane positioning, its origin and links with cell geometry, and its impact on tissue organization. Taking advantage of the first-stereotyped-then-variable division patterns in the embryo of the model plant Arabidopsis thaliana, we combined 3D imaging and quantitative cell shape and cell lineage analysis together with mathematical and computer modeling to perform a large scale, systematic analysis of variability in division plane orientation. Our results reveal that, paradoxically, variability in cell division patterns of Arabidopsis embryos is accompanied by a progressive reduction of cell shape heterogeneity. The paradox is solved by showing that variability operates within a reduced repertoire of possible division plane orientations that is related to cell geometry. We show that in several domains of the embryo, a recently proposed geometrical division rule recapitulates observed variable patterns, thus suggesting that variable patterns emerge from deterministic principles operating in a variable geometrical context. Our work highlights the importance of emerging patterns in the plant embryo under iterated division principles, but also reveal domains where deviations between rule predictions and experimental observations point to additional regulatory mechanisms.

sentences: Functional peroxisomes are required for heat shock-induced hormesis in Caenorhabditis elegans Exact mechanisms of heat shock induced lifespan extension, while documented across species, are still not well understood. Here we put forth evidence that fully functional peroxisomes are required for the activation of the canonical heat shock response and heat-induced hormesis in C. elegans. While during heat shock the HSP-70 chaperone is strongly upregulated in the wild-type (WT) as well as in the absence of peroxisomal catalase ({Delta}ctl-2 mutant), the small heat shock proteins display modestly increased expression in the mutant. Nuclear localization of HSF-1 is reduced in the{Delta} ctl-2 mutant. In addition, heat-induced lifespan extension, observed in the WT, is absent in the{Delta} ctl-2 mutant. Activation of the antioxidant response, the pentose phosphate pathway and increased triglyceride content are the most prominent changes observed during heat shock in the WT worm, but not in the{Delta} ctl-2 mutant. Involvement of peroxisomes in the cell-wide response to transient heat shock reported here gives new insight into the role of organelle communication in the organisms stress response.

sentences: Temporal filters in response to presynaptic spike trains: Interplay of cellular, synaptic and short-term plasticity time scales Temporal filters, the ability of postsynaptic neurons to preferentially select certain presynaptic input patterns over others, have been shown to be associated with the notion of information filtering and coding of sensory inputs. Short-term plasticity (depression and facilitation; STP) has been proposed to be an important player in the generation of temporal filters. We carry out a systematic modeling, analysis and computational study to understand how characteristic postsynaptic (low-, high- and band-pass) temporal filters are generated in response to periodic presynaptic spike trains in the presence STP. We investi-gate how the dynamic properties of these filters depend on the interplay of a hierarchy of processes, including the arrival of the presynaptic spikes, the activation of STP, its effect on the excitatory synaptic connection efficacy, and the response of the postsynaptic cell. These mechanisms involve the inter-play of a collection of time scales that operate at the single-event level (roughly, during each presynaptic interspike-interval) and control the long-term development of the temporal filters over multiple presynaptic events. These time scales are generated at the levels of the presynaptic cell (captured by the presynaptic interspike-intervals), short-term depression and facilitation, synaptic dynamics and the post-synaptic cellular currents. We develop mathematical tools to link the single-event time scales with the time scales governing the long-term dynamics of the resulting temporal filters for a relatively simple model where depression and facilitation interact at the level of the synaptic efficacy change. We extend our results and tools to account for more complex models. These include multiple STP time scales and non-periodic presynaptic inputs. The results and ideas we develop have implications for the understanding of the generation of temporal filters in complex networks for which the simple feedforward network we investigate here is a building block.

sentences: Identifying Protein Complexes in Protein-protein Interaction Data using Graph Convolution Network Protein complexes are groups of two or more polypeptide chains that join together to build noncovalent networks of protein interactions. A number of means of computing the ways in which protein complexes and their members can be identified from these interaction networks have been created. While most of the existing methods identify protein complexes from the protein-protein interaction networks (PPIs) at a fairly decent level, the applicability of advanced graph network methods has not yet been adequately investigated. In this paper, we proposed various graph convolutional networks (GCNs) methods to improve the detection of the protein functional complexes. We first formulated the protein complex detection problem as a node classification problem. Second, the Neural Overlapping Community Detection (NOCD) model was applied to cluster the nodes (proteins) using a complex affiliation matrix. A representation learning approach, which combines the multi-class GCN feature extractor (to obtain the features of the nodes) and the mean shift clustering algorithm (to perform clustering), is also presented. We have also improved the efficiency of the multi-class GCN network to reduce space and time complexities by converting the dense-dense matrix operations into dense-spares or sparse-sparse matrix operations. This proposed solution significantly improves the scalability of the existing GCN network. Finally, we apply clustering aggregation to find the best protein complexes. A grid search was performed on various detected complexes obtained by applying three well-known protein detection methods namely ClusterONE, CMC, and PEWCC with the help of the Meta-Clustering Algorithm (MCLA) and Hybrid Bipartite Graph Formulation (HBGF) algorithm. The proposed GCN-based methods were tested on various publicly available datasets and provided significantly better performance than the previous state-of-the-art methods. The code and data used in this study are available from https://github.com/Analystharsh/GCN_complex_detection

sentences: Mechanical Forces during Lymph Node Expansion Govern Fibroblastic Reticular Network Remodeling Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes, an immunologically-relevant, adult mammalian tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive tissue expansion upon immune challenge. We find that following immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that CLEC-2/podoplanin signalling regulates the cell surface mechanics of fibroblasts, permitting cell elongation and interdigitation through expedited access to plasma membrane reservoirs. Increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation, restoring homeostatic cellular ratios and tissue structure through expansion.

sentences: Proximity labeling reveals spatial regulation of the anaphase-promoting complex/cyclosome by a microtubule adaptor The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. To assess the spatial importance of this interaction, we developed microtubule-localized proximity biotinylation tools, which revealed that depletion of PLEKHA5 decreased the extent of APC/C association with the microtubule cytoskeleton. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

sentences: Quantitative MRI maps of human neocortex explored using cell type-specific gene expression analysis Quantitative MRI (qMRI) allows extraction of reproducible and robust parameter maps. However, the connection to underlying biological substrates remains murky, especially in the complex, densely packed cortex. We investigated associations in human neocortex between qMRI parameters and neocortical cell types by comparing the spatial distribution of the qMRI parameters longitudinal relaxation rate (R1), effective transverse relaxation rate (R2*), and magnetization transfer saturation (MTsat) to gene expression from the Allen Human Brain Atlas, then combining this with lists of genes enriched in specific cell types found in the human brain. As qMRI parameters are magnetic field strength-dependent, the analysis was performed on MRI data at 3T and 7T. All qMRI parameters significantly covaried with genes enriched in GABA- and glutamatergic neurons, i.e. they were associated with cytoarchitecture. The qMRI parameters also significantly covaried with the distribution of genes enriched in astrocytes (R2* at 3T, R1 at 7T), endothelial cells (R1 and MTsat at 3T), microglia (R1 and MTsat at 3T, R1 at 7T), and oligodendrocytes (R1 at 7T). These results advance the potential use of qMRI parameters as biomarkers for specific cell types.

sentences: Dynamics of Salmonella inoculated during rearing ofblack soldier fly larvae (Hermetia illucens) on chicken feed The black soldier fly is currently the most produced edible insect on industrial scale, with its larval stage being processed into animal feed as the main application. As this insect species enters the feed and food chain, good hygiene and monitoring practices are needed to avoid the entrance of foodborne pathogens via the larvae. However, insufficient data on the risk of such introductions via industrial larvae production are available. To address this gap, a range of rearing trials were conducted in which the substrate, chicken feed, was inoculated with different levels of Salmonella and in which total viable counts and Salmonella counts were determined during the following days. The outgrowth of Salmonella was slower in those experiments with a lower initial contamination level than in experiments with a higher level. No significant reducing effect originating from the larvae on the substrate Salmonella counts was observed, in contrast to previous studies using other substrates. Our study also revealed that airborne transmission of Salmonella is possible under rearing conditions corresponding to those applied at industrial production sites. Based on our results, we recommend insect producers to use substrate ingredients free of Salmonella, and not to count on the antimicrobial activities that BSFL may exert in some situations towards food pathogens. More inoculation studies using other Salmonella serotypes, other zoonotic bacteria, other substrates, larvae of other ages and including variations on rearing protocols are needed in order to obtain a general view on the dynamics of food pathogens in this insect species and to support comprehensive risk assessments.

sentences: LITOS - a versatile LED illumination tool for optogenetic stimulation Optogenetics has become a key tool to manipulate biological processes with high spatio-temporal resolution. Recently, a number of commercial and open-source multi-well illumination devices have been developed to provide throughput in optogenetics experiments. However, available commercial devices remain expensive and lack flexibility, while open-source solutions require programming knowledge and/or include complex assembly processes. We present a LED Illumination Tool for Optogenetic Stimulation (LITOS) based on an assembled printed circuit board controlling a commercially available 32x64 LED matrix as illumination source. LITOS can be quickly assembled without any soldering, and includes an easy-to-use interface, accessible via a website hosted on the device itself. Complex light stimulation patterns can easily be programmed without coding expertise. LITOS can be used with different formats of multi-well plates, petri dishes, and flasks. We validated LITOS by measuring the activity of the MAPK/ERK signaling pathway in response to different dynamic light stimulation regimes using FGFR1 and Raf optogenetic actuators. LITOS can uniformly stimulate all the cells in a well and allows for flexible temporal stimulation schemes. LITOSs ease of use aims at democratizing optogenetics in any laboratory.

sentences: Mitochondrial effects on fertility and longevity in Tigriopus californicus contradict predictions of the mother's curse hypothesis Strict maternal inheritance of mitochondria favors the evolutionary accumulation of sex-biased fitness effects, as mitochondrial evolution occurs exclusively in female lineages. The "mothers curse" hypothesis proposes that male-harming mutations should accumulate in mitochondrial genomes when they have neutral or beneficial effects on female fitness. Rigorous empirical tests have largely focused on Drosophila, where support for the predictions of mothers curse has been mixed. We investigated the impact of mothers curse mutations in Tigriopus californicus, a minute crustacean. Using nonrecombinant backcrosses, we introgressed four divergent mitochondrial haplotypes into two nuclear backgrounds and recorded measures of fertility and longevity. We found that the phenotypic effects of mitochondrial mutations were context-dependent, being influenced by the nuclear-background in which they were expressed, as well as the sex of the individual and rearing temperature. Mitochondrial haplotype effects were greater for fertility than longevity, and temperature effects were greater for longevity. However, in opposition to mothers curse expectations, females had a higher level of mitochondrial genetic variance than males for both fertility and longevity, and there was no evidence for mitochondrial mutations favoring female performance at the cost of male performance. These results indicate that selection has not resulted in the accumulation of male mutation load in Tigriopus californicus.

sentences: Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species Adeno-associated virus (AAV)-induced dorsal root ganglia (DRG) toxicity has been observed in several nonclinical species, where lesions are characterized by neuronal degeneration/necrosis, nerve fiber degeneration, and mononuclear cell infiltration. As AAV vectors become an increasingly common platform for novel therapeutics, non-invasive biomarkers are needed to better characterize and manage the risk of DRG neurotoxicity in both nonclinical and clinical studies. Based on biological relevance, reagent availability, antibody cross-reactivity, DRG protein expression, and assay performance, neurofilament light chain (NF-L) emerged as a promising biomarker candidate. Dose- and time-dependent changes in NF-L were evaluated in male Wistar Han rats and cynomolgus monkeys following intravenous or intrathecal AAV injection, respectively. NF-L profiles were then compared against microscopic DRG lesions on Day 29 post-dosing. In animals exhibiting DRG toxicity, plasma/serum NF-L was strongly associated with the severity of neuronal degeneration/necrosis and nerve fiber degeneration, with elevations beginning as early as Day 8 in rats ([≥]5x1013 vg/kg) and Day 14 in monkeys ([≥]3.3x1013 vg/dose). Consistent with the unique positioning of DRGs outside the blood-brain barrier, NF-L in cerebrospinal fluid was only weakly associated with DRG findings. In summary, circulating NF-L is a promising biomarker of AAV-induced DRG toxicity in nonclinical species.

sentences: Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded by L. monocytogenes in aerobic and anaerobic conditions into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulates anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartment (BMC)-dependent 1,2-propanediol utilization with concomitant production of propionate and propanol. Notably, anaerobic propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigate the impact of B12 on aerobic and anerobic growth of L. monocytogenes on rhamnose, and observed growth stimulation and pdu BMC activation only in anaerobically grown cells with B12 added to the medium. Comparative Caco-2 virulence assays, showed that these pdu BMC induced cells have significantly higher translocation efficiency compared to aerobically grown cells (without and with added B12) and non-induced anaerobically grown cells, while adhesion and invasion capacity is similar for all cells. Comparative proteomics analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL; teichoic acids export ATP-binding protein, TagH; DNA repair and protection proteins RadA and DPS; and glutathione synthase GshAB previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu BMC induced cells. Our results shed new light into B12 impact on L. monocytogenes competitive fitness and virulence.

sentences: Comparative proteome analysis revealed the differences in responses of bovine alveolar macrophages to both M.tb and Mb infection Bovine tuberculosis, caused by both MTB(Mycobacterium tuberculosis) and MB(Mycobacterium bovis), is one of the most serious zoonotic diseases in the world. Both pathogenic MTB and MB interact closely with the host alveolar macrophages. However, the mechanisms of defense response of macrophages to MTB or MB still need further researches. Presently, in-depth comparative proteome analysis was performed on the MB- and MTB- infected macrophage. We identified that macrophages was more sensitive to the MB infection. Both pathogens could induce strong energy metabolism of macrophages. To eliminate MB and protect the host, a bunch of proteins and signaling which involved in autophagy- and inflammatory-related progresses were highly activated in macrophages following MB infection. In contrast, only proteins relevant to energy metabolisms were highly expressed in macrophages following MTB infection. In summary, we proposed that macrophages were more sensitive to MB attacks and could deployed proteins functioned in autophagy- and inflammatory-related progresses to protect the host. Our research provide a novel insight into the different mechanisms of defense responses of macrophages to both MTB and MB infection.

sentences: Periventricular nodular heterotopia is functionally coupled with the neocortex during resting and task states Periventricular nodular heterotopia (PVNH) is a well-defined developmental disorder characterized by failed neuronal migration, which forms ectopic neuronal nodules along the ventricular walls. Previous studies mainly focus on clinical symptoms caused by the PVNH tissue, such as seizures. However, little is known about whether and how neurons in the PVNH tissue functionally communicate with neurons in the neocortex. To probe this, we applied magnetoencephalography (MEG) and stereo-electroencephalography (sEEG) recordings to patients with PVNH during resting and task states. By estimating frequency-resolved phase coupling strength of the source-reconstructed neural activities, we found that the PVNH tissue was spontaneously coupled with the neocortex in the  to {beta} frequency range, which was consistent with the synchronization pattern within the neocortical network. Furthermore, the coupling strength between PVNH and sensory areas effectively modulated the local neural activity in sensory areas. In both MEG and sEEG visual experiments, the PVNH tissue exhibited visual evoked responses, with a similar pattern and latency as the ipsilateral visual cortex. These findings demonstrate that PVNH is functionally integrated into cognition-related cortical circuits, suggesting a co-development perspective of ectopic neurons after their migration failure.

sentences: Mapping the Cell-Membrane Proteome to the Cancer Hallmarks The hallmarks of biological processes that underlie the development of cancer have been long recognized, yet, existing therapeutic treatments cannot prevent cancer from continuing to be one of the leading causes of death worldwide. This work was aimed at exploring the extent to which the cell-membrane proteins are implicated in triggering cancer hallmark processes, and assessing the ability to pinpoint novel therapeutic targets through a combined membrane proteome/cancer hallmark perspective. By using GO annotations, a database of human proteins associated broadly with ten cancer hallmarks was created. Cell-membrane cellular subfractions of SKBR3/HER2+ breast cancer cells used as a model system were analyzed by high resolution mass spectrometry, and high-quality proteins (FDR<3 %) identified by at least two unique peptides were mapped to the cancer hallmark database. Over 1100 experimentally detected cell-membrane or cell-membrane associated proteins, representing ~8 % of the human cell-membrane proteome, were mapped to the hallmark database. Representative membrane constituents such as receptors, CDs, adhesion and transport proteins were distributed over the entire genome and present in every hallmark category. Sustained proliferative signaling/cell cycle, adhesion/tissue invasion, and evasion of immune destruction emerged as prevalent hallmarks represented by the membrane proteins. Construction of protein-protein interaction networks uncovered a high level of connectivity between the hallmark members, with some receptor (EGFR, ERBB2, FGFR, MTOR, CSF1R), antigen (CD44), and adhesion (MUC1) proteins being implicated in most hallmark categories. An illustrative subset of 116 hallmark proteins that included 44 oncogenes, 28 tumor suppressors, and 38 approved drug targets was subjected to a more in-depth analysis. The existing drug targets were implicated mainly in signaling processes. Network centrality analysis revealed that nodes with high degree, rather than betweenness, represent a good resource for informing the selection of putative novel drug targets. Through heavy involvement in supporting cancer hallmark processes, we show that the functionally diverse and networked landscape of cancer cell-membrane proteins fosters unique opportunities for guiding the development of novel therapeutic interventions, including multi-agent, immuno-oncology and precision medicine applications.

sentences: The outer mitochondrial membrane protein TMEM11 is a novel negative regulator of BNIP3/BNIP3L-dependent receptor-mediated mitophagy Mitochondria play critical roles in cellular metabolism and to maintain their integrity, they are regulated by several quality control pathways, including mitophagy. During BNIP3/BNIP3L-dependent receptor-mediated mitophagy, mitochondria are selectively degraded by the direct recruitment of the autophagosome biogenesis protein LC3. BNIP3 and/or BNIP3L are upregulated situationally, for example during hypoxia and developmentally during erythrocyte maturation. However, it is not well understood how they are regulated at steady-state. Here, we find that the poorly characterized mitochondrial cristae morphology regulator TMEM11 unexpectedly localizes to the outer membrane where it forms a complex with BNIP3 and BNIP3L. Loss of TMEM11 causes mitochondrial morphology defects in a BNIP3/BNIP3L-dependent manner and, further, we find that mitophagy is hyper-active in the absence of TMEM11 during both normoxia and hypoxia. Our results reveal a non-canonical role for TMEM11 as a negative regulator of BNIP3/BNIP3L-mediated mitophagy and suggest that the TMEM11/BNIP3/BNIP3L complex coordinately regulates mitochondrial quality control.

sentences: Complementary task representationsin hippocampus and prefrontal cortexfor generalising the structure of problems Few situations in life are completely novel. We effortlessly generalise prior knowledge to solve novel problems, abstracting common structure and mapping it onto new sensorimotor specifics. Here we trained mice on a series of reversal learning problems that shared the same structure but had different physical implementations. Performance improved across problems, demonstrating transfer of knowledge. Neurons in medial prefrontal cortex (mPFC) maintained similar representations across multiple problems, despite their different sensorimotor correlates, whereas hippocampal (dCA1) representations were more strongly influenced by the specifics of each problem. Critically, this was true both for representations of the events that comprised each trial, and those that integrated choices and outcomes over multiple trials to guide subjects decisions. These data suggest that PFC and hippocampus play complementary roles in generalisation of knowledge, with the former abstracting the common structure among related problems, and the latter mapping this structure onto the specifics of the current situation.

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V3 Interneurons Regulate Locomotor Vigor by Recruitment of Spinal Motor Neurons During Fictive Swimming in Larval Zebrafish Survival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude of motor output combine in a multiplicative manner to determine features of locomotion such as distance traveled, speed, force (thrust), and vigor. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is a less thorough mechanistic understanding for how locomotor amplitude is established. Recent evidence indicates that locomotor amplitude is regulated in part by a subset of functionally and morphologically distinct V2a excitatory spinal interneurons (type II, non-bursting) in larval and adult zebrafish. Here we provide direct evidence, for the first time, that most V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. We also show that elimination of the spinal V3-IN population reduces the proportion of active MNs during fictive swimming but does not alter the range of locomotor frequencies produced. These data are consistent with V3-INs providing excitatory drive to spinal motor neurons and contributing to the production of locomotor amplitude, but not frequency, during swimming in larval zebrafish.

SIGNIFICANCE STATEMENTCurrently, there is a limited understanding about the cellular and spinal network properties that produce locomotor amplitude, defined as limb displacement in limbed animals or tail-bend in non-limbed animals during locomotion. Here we show, directly for the first time in a vertebrate, that V3 interneurons (V3-INs) in zebrafish larvae are active during in vivo fictive locomotion, and that targeted ablation of the spinal V3-IN population reduces the proportion of active motoneurons during fictive swimming. Importantly, ablation of V3-INs does not affect locomotor frequency (speed), which clarifies their role in motor control rather than rhythm generation. Thus, we propose that the V3-IN population is a source of excitation in the vertebrate locomotor neural circuitry that regulates locomotor amplitude independently of speed.

sentences: Mapping Interactome Networks of FOSL1 and FOSL2 in Human Th17 Cells Dysregulated function of Th17 cells has implications in immunodeficiencies and autoimmune disorders. Th17 cell-differentiation is orchestrated by a complex network of transcription factors, including several members of the activator protein (AP-1) family. Among these, FOSL1 and FOSL2 influence the effector responses of Th17 cells. However, the molecular mechanisms underlying these functions are unclear, owing to the poorly characterized protein interaction networks of these factors. Here, we establish the first interactomes of FOSL1 and FOSL2 in human Th17 cells, using affinity purification-mass spectrometry analysis. In addition to the known JUN proteins, we identified several novel binding partners of FOSL1 and FOSL2. Gene ontology analysis found a major fraction of these interactors to be associated with RNA binding activity, which suggests new mechanistic links. Intriguingly, 29 proteins were found to share interactions with FOSL1 and FOSL2, and these included key regulators of Th17-fate. We further validated the binding partners identified in this study by using parallel reaction monitoring targeted mass-spectrometry and other methods. Our study provides key insights into the interaction-based signaling mechanisms of FOSL factors that potentially govern Th17 cell-differentiation and associated pathologies.

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N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2 The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in more than 4 million deaths in the 20 months since the virus was discovered. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout and vaccine hesitancy. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of {beta}-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans. We tested GC as a potential countermeasure for COVID-19. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway and lungs. Our findings demonstrate a new application for soluble immunoadjuvants like GC for preventing severe disease associated with SARS-CoV-2.

Single Sentence SummaryThe immunoadjuvant N-dihydrogalactochitosan diminishes SARS-CoV-2 disease in humanized ACE2 mice representing a new countermeasure against COVID-19.

sentences: Dynamic pneumococcal genetic adaptations support bacterial growth and inflammation during coinfection with influenza Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to post-influenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza-infected hosts. The majority of the 32 identified genes are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated single-gene deletion (SGD) mutants of five identified genes: SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effect on in vivo fitness, disease severity, and host immune responses. Growth of SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed that neutrophil phenotype and distribution was altered in the lungs of influenza-SGD coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.

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In silico analyses of penicillin binding proteins in Burkholderia pseudomallei uncovers SNPs with utility for phylogeography, species differentiation, and sequence typing BackgroundBurkholderia pseudomallei causes melioidosis. Sequence typing this pathogen can reveal geographical origin and uncover epidemiological associations. Here, we describe B. pseudomallei genes encoding putative penicillin binding proteins (PBPs) and investigate their utility for determining phylogeography and differentiating closely related species.

Methodology & Principal FindingsWe performed in silico analysis to characterize 10 PBP homologs in B. pseudomallei 1026b. As PBP active site mutations can confer {beta}-lactam resistance in Gram-negative bacteria, PBP sequences in two resistant B. pseudomallei strains were examined for similar alterations. Sequence alignments revealed single amino acid polymorphisms (SAAPs) unique to the multidrug resistant strain Bp1651 in the transpeptidase domains of two PBPs, but not directly within the active sites. Using BLASTn analyses of complete assembled genomes in the NCBI database, we determined genes encoding PBPs were conserved among B. pseudomallei (n=101) and Burkholderia mallei (n=26) strains. Within these genes, single nucleotide polymorphisms (SNPs) useful for predicting geographic origin of B. pseudomallei were uncovered. SNPs unique to B. mallei were also identified. Based on 11 SNPs identified in two genes encoding predicted PBP-3s, a dual-locus sequence typing (DLST) scheme was developed. The robustness of this typing scheme was assessed using 1,523 RefSeq genomes from B. pseudomallei (n=1,442) and B. mallei (n=81) strains, resulting in 32 sequence types (STs). Compared to multi-locus sequence typing (MLST), the DLST scheme demonstrated less resolution to support the continental separation of Australian B. pseudomallei strains. However, several STs were unique to strains originating from a specific country or region. The phylogeography of Western Hemisphere B. pseudomallei strains was more highly resolved by DLST compared to internal transcribed spacer (ITS) typing, and all B. mallei strains formed a single ST.

SignificanceConserved genes encoding PBPs in B. pseudomallei are useful for strain typing, can enhance predictions of geographic origin, and differentiate strains of closely related Burkholderia species.

Author SummaryBurkholderia pseudomallei causes the life-threatening disease melioidosis and is considered a biological threat and select agent by the United States government. This soil-dwelling bacterium is commonly found in regions of southeast Asia and northern Australia, but it is also detected in other tropical and sub-tropical areas around the world. With a predicted global burden of 165,000 annual cases and mortality rate that can exceed 40% without prompt and appropriate antibiotic treatment, understanding the epidemiology of melioidosis and mechanisms of antibiotic resistance in B. pseudomallei can benefit public health and safety. Recently, we identified ten conserved genes encoding putative penicillin binding proteins (PBPs) in B. pseudomallei. Here, we examined B. pseudomallei PBP sequences for amino acid mutations that may contribute to {beta}-lactam resistance. We also uncovered nucleotide mutations with utility to predict the geographical origin of B. pseudomallei strains and to differentiate closely related Burkholderia species. Based on 11 informative single nucleotide polymorphisms in two genes each encoding a PBP-3, we developed a simple, targeted dual-locus typing approach.

sentences: Computation of the mitochondria age distribution along the axon length We describe a compartmental model of mitochondrial transport in axons, which we apply to compute mitochondrial age at different distances from the soma. The model predicts that the average mitochondrial age at the tip of an axon with a length of 1 cm is approximately 22 hours. The mitochondria are youngest closest to the soma and their age scales approximately linearly with distance from the soma. To the best of the authors knowledge, this is the first attempt to predict the spatial distribution of mitochondrial age within an axon. A sensitivity study of the mean age of mitochondria to various model parameters is also presented.

sentences: New fossils of Australopithecus sediba reveal a nearly complete lower back Adaptations of the lower back to bipedalism are frequently discussed but infrequently demonstrated in early fossil hominins. Newly discovered lumbar vertebrae contribute to a near-complete lower back of Malapa Hominin 2 (MH2), offering additional insights into posture and locomotion in Australopithecus sediba. We show that MH2 demonstrates a lower back consistent with human-like lumbar lordosis and other adaptations to bipedalism, including an increase in the width of intervertebral articular facets from the upper to lower lumbar column ("pyramidal configuration"). This contrasts with recent work on lordosis in fossil hominins, where MH2 was argued to demonstrate no appreciable lordosis ("hypolordosis") similar to Neandertals. Our three-dimensional geometric morphometric (3D GM) analyses show that MH2s nearly complete middle lumbar vertebra is human-like in shape but bears large, cranially-directed transverse processes, implying powerful trunk musculature. We interpret this combination of features to indicate that A. sediba used its lower back in both human-like bipedalism and ape-like arboreal positional behaviors, as previously suggested based on multiple lines of evidence from other parts of the skeleton and reconstructed paleobiology of A. sediba.

sentences: The imprinted Zdbf2 gene finely tunes control of feeding and growth in neonates Genomic imprinting refers to the mono-allelic and parent-specific expression of a subset of genes. While long recognized for their role in embryonic development, imprinted genes have recently emerged as important modulators of postnatal physiology, notably through hypothalamus-driven functions. Here, using mouse models of loss, gain and parental inversion of expression, we report that the paternally expressed Zdbf2 gene controls neonatal growth in mice, in a dose-sensitive but parent-of-origin-independent manner. We further found that Zdbf2-KO neonates failed to fully activate hypothalamic circuits that stimulate appetite, and suffered milk deprivation and diminished circulating Insulin Growth Factor 1 (IGF-1). Consequently, only half of Zdbf2-KO pups survived the first days after birth and those surviving were smaller. This study demonstrates that precise imprinted gene dosage is essential for vital physiological functions at the transition from intra- to extra-uterine life, here the adaptation to oral feeding and optimized body weight gain.

sentences: Empirical abundance distributions are more uneven than expected given their statistical baseline Exploring and accounting for the emergent properties of ecosystems as complex systems is a promising horizon in the search for general processes to explain common ecological patterns. For example, the ubiquitous hollow-curve form of the species abundance distribution is frequently assumed to reflect ecological processes structuring communities, but can also emerge as a statistical phenomenon from the mathematical definition of an abundance distribution. Although the hollow curve may be a statistical artefact, ecological processes may induce subtle deviations between empirical species abundance distributions and their statistically most probable forms. These deviations may reflect biological processes operating on top of mathematical constraints and provide new avenues for advancing ecological theory. Examining [~]22,000 communities, we found that empirical SADs are highly uneven and dominated by rare species compared to their statistical baselines. Efforts to detect deviations may be less informative in small communities - those with few species or individuals - because these communities have poorly-resolved statistical baselines. The uneven nature of many empirical SADs demonstrates a path forward for leveraging complexity to understand ecological processes governing the distribution of abundance, while the issues posed by small communities illustrate the limitations of using this approach to study ecological patterns in small samples.

sentences: Interfering with contextual fear memories by post-reactivation administration of propranolol in mice: a series of null findings Post-reactivation amnesia of contextual fear memories by blockade of noradrenergic signaling has been shown to have limited replicability in rodents. This is usually attributed to several boundary conditions that gate the destabilization of memory during its retrieval. How these boundary conditions can be overcome, and what neural mechanisms underlie post-reactivation changes in contextual fear memories remain largely unknown. Here, we report a series of experiments in a contextual fear-conditioning paradigm in mice, that were aimed at solving these issues. We first attempted to obtain a training paradigm that would consistently result in contextual fear memory that could be destabilized upon reactivation, enabling post-retrieval amnesia by administration of propranolol. Unexpectedly, our attempts were unsuccessful to this end. Specifically, over a series of experiments in which we varied different parameters of the fear acquisition procedure, at best small and inconsistent effects were observed. Additionally, we found that propranolol did not alter retrieval-induced neural activity, as measured by the number of c-Fos+ cells in the hippocampal dentate gyrus. To determine whether propranolol was perhaps ineffective in interfering with reactivated contextual fear memories, we also included anisomycin (i.e., a potent and well-known amnesic drug) in several experiments, and measures of synaptic glutamate receptor subunit GluA2 (i.e., a marker of memory destabilization). No post-retrieval amnesia by anisomycin and no altered GluA2 expression by reactivation was observed, suggesting that the memories did not undergo destabilization. The null findings are surprising, given that the training paradigms we implemented were previously shown to result in memories that could be modified upon reactivation. Together, our observations illustrate the elusive nature of reactivation-dependent changes of non-human fear memory.

sentences: Adipocyte Tribbles1 Regulates Plasma Adiponectin and Plasma Lipids in Mice Multiple GWAS have identified SNPs in the 8q24 locus near the TRIB1 gene that significantly associate with plasma lipids and coronary artery disease. While subsequent studies have uncovered roles for hepatic and myeloid Trib1 in contributing to either plasma lipids or atherosclerosis, the causal tissue for these GWAS associations remains unclear. The same 8q24 SNPs significantly associate with plasma adiponectin levels in humans as well, suggesting a role for TRIB1 in adipose tissue. Here, we report that adipocyte-specific Trib1 knockout mice (Trib1_ASKO) have increased plasma adiponectin levels and decreased plasma cholesterol and triglycerides. We demonstrate that loss of Trib1 increases adipocyte production and secretion of adiponectin independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice suggests that alterations in adipocyte function underlie the plasma lipid changes observed in these mice. Secretomics and RNA-seq analysis revealed that Trib1_ASKO mice have increased production of Lpl and decreased production of Angptl4 in adipose tissue, and fluorescent substrate assays confirm an increase in adipose tissue Lpl activity, which likely underlies the observed triglyceride phenotype. In summary, we demonstrate here a novel role for adipocyte Trib1 in regulating plasma adiponectin, total cholesterol, and triglycerides in mice, confirming previous genetic associations observed in humans and providing a novel avenue through which Trib1 regulates plasma lipids and coronary artery disease.

sentences: CRISPR-Cas is associated with fewer antibiotic resistance genes in bacterial pathogens The acquisition of antibiotic resistance genes via horizontal gene transfer is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of antibiotic resistance. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of antibiotic resistance. We analysed ~40,000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens including Enterococcus, Staphylococcus, Acinetobacter and Pseudomonas. We modelled the association between CRISPR-Cas and indicators of horizontal gene transfer, and found that pathogens with a CRISPR-Cas system were less likely to carry antibiotic resistance genes than those lacking this defence system. Analysis of the mobile genetic elements targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of antibiotic resistance. These results suggest a potential "immunocompromised" state for multidrug-resistant strains that may be exploited in tailored interventions that rely on mobile genetic elements, such as phage or phagemids, to treat infections caused by bacterial pathogens.

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Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, a2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to 2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and brimonidine showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Spermidine dampens inflammation by directly inhibiting Th17 cytokine production through a PRDX1 associated antioxidant pathway The activation of IL-17 signaling has been linked to the pathogenesis of many chronic, inflammatory lung diseases including Cystic Fibrosis (CF). Through unbiased single-cell RNAseq screening, we found that IL-17+ T cells highly express Srm and Smox, which encode two key enzymes for spermidine synthesis. Spermidine has been shown to reduce inflammation by regulating macrophage activation and balancing Th17/Treg differentiation, but its direct effects on Th17 cytokine production has not been carefully investigated. Here, using already differentiated Th17 cells from cultured mouse splenocytes, we found that exogenous spermidine directly inhibits IL-1{beta}/IL-23 induced IL-17 production. Blockade of endogenous spermidine synthesis enhanced IL-17 production above native levels, further supporting that spermidine is a direct regulator of cytokine secretion independent of differentiation. In vivo, spermidine alleviates lung inflammation in both PA infection and LPS induced acute lung injury models. Further RNA-seq analysis suggests spermidine suppression of Th17 cytokine production is mediated through its PRDX1 dependent antioxidant activity. Our data establishes that spermidine is a direct regulator of Type-17 T cell cytokine production and has potent anti-inflammatory effects against lung inflammation.

sentences: Serum proteomics identifies immune pathways and candidate biomarkers of coronavirus infection in wild vampire bats The apparent ability of bats to harbor many virulent viruses without showing disease is likely driven by distinct immune responses that coevolved with mammalian flight and the exceptional longevity of this order. Yet our understanding of the immune mechanisms of viral tolerance is restricted to a small number of bat-virus relationships and remains poor for coronaviruses (CoVs), despite their relevance to human health. Proteomics holds particular promise for illuminating the immune factors involved in bat responses to infection, because it can accommodate especially low sample volumes (e.g., sera) and thus can be applied to both large and small bat species as well as in longitudinal studies where lethal sampling is necessarily limited. Further, as the serum proteome includes proteins secreted from not only blood cells but also proximal organs, it provides a more general characterization of immune proteins. Here, we expand our recent work on the serum proteome of wild vampire bats (Desmodus rotundus) to better understand CoV pathogenesis. Across 19 bats sampled in 2019 in northern Belize with available sera, we detected CoVs in oral or rectal swabs from four individuals (21.1% positivity). Phylogenetic analyses identified all vampire bat sequences as novel -CoVs most closely related to known human CoVs. Across 586 identified serum proteins, we found no strong differences in protein composition nor abundance between uninfected and infected bats. However, receiver operating characteristic curve analyses identified seven to 32 candidate biomarkers of CoV infection, including AHSG, C4A, F12, GPI, DSG2, GSTO1, and RNH1. Enrichment analyses using these protein classifiers identified downregulation of complement, regulation of proteolysis, immune effector processes, and humoral immunity in CoV-infected bats alongside upregulation of neutrophil immunity, overall granulocyte activation, myeloid cell responses, and glutathione processes. Such results denote a mostly cellular immune response of vampire bats to CoV infection and identify putative biomarkers that could provide new insights into CoV pathogenesis in wild and experimental populations. More broadly, applying a similar proteomic approach across diverse bat species and to distinct life history stages in target species could improve our understanding of the immune mechanisms by which wild bats tolerate viruses.

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RecPD: A Recombination-Aware Measure of Phylogenetic Diversity A critical step in studying biological features (e.g., genetic variants, gene families, metabolic capabilities, or taxa) underlying traits or outcomes of interest is assessing their diversity and distribution. Accurate assessments of these patterns are essential for linking features to traits or outcomes and understanding their functional impact. Consequently, it is of crucial importance that the metrics employed for quantifying feature diversity can perform robustly under any evolutionary scenario. However, the standard metrics used for quantifying and comparing the distribution of features, such as prevalence, phylogenetic diversity, and related approaches, either do not take into consideration evolutionary history, or assume strictly vertical patterns of inheritance. Consequently, these approaches cannot accurately assess diversity for features that have undergone recombination or horizontal transfer. To address this issue, we have devised RecPD, a novel recombination-aware phylogenetic-diversity metric for measuring the distribution and diversity of features under all evolutionary scenarios. RecPD utilizes ancestral-state reconstruction to map the presence / absence of features onto ancestral nodes in a species tree, and then identifies potential recombination events in the evolutionary history of the feature. We also derive a number of related metrics from RecPD that can be used to assess and quantify evolutionary dynamics and correlation of feature evolutionary histories. We used simulation studies to show that RecPD reliably identifies evolutionary histories under diverse recombination and loss scenarios. We then apply RecPD in a real-world scenario in a preliminary study type III effector protein families secreted by the plant pathogenic bacterium Pseudomonas syringae and demonstrate that prevalence is an inadequate metric that obscures the potential impact of recombination. We believe RecPD will have broad utility for revealing and quantifying complex evolutionary processes for features at any biological level.

AUTHOR SUMMARYPhylogenetic diversity is an important concept utilized in evolutionary ecology which has extensive applications in population genetics to help us understand how evolutionary processes have distributed genetic variation among individuals of a species, and how this impacts phenotypic diversification over time. However, existing approaches for studying phylogenetic diversity largely assume that the genetic features follow vertical inheritance, which is frequently violated in the case of microbial genomes due to horizontal transfer. To address this shortcoming, we present RecPD, a recombination-aware phylogenetic diversity metric, which incorporates ancestral state reconstruction to quantify the phylogenetic diversity of genetic features mapped onto a species phylogeny. Through simulation experiments we show that RecPD robustly reconstructs the evolutionary histories of features evolving under various scenarios of recombination and loss. When applied to a real-world example of type III secreted effector protein families from the plant pathogenic bacterium Pseudomonas syringae, RecPD reveals that horizontal transfer has played an important role in shaping the phylogenetic distributions of aa substantial proportion of families across the P. syringae species complex. Furthermore, we demonstrate that the traditional measures of feature prevalence are unsuitable as a metric for comparing feature diversity.

sentences: Entering into a self-regulated learning mode prevents detrimental effect of feedback removal on memory Recently, we provided causal evidence that self-regulated dopamine signaling enhanced long-term memory formation in the absence of any external feedback or reward (Ripolles et al., 2016, 2018) if a congruent meaning inferred from semantic context (DA-dependent learning), while DA-signals were absent if no congruent meaning could be inferred (DA-independent learning). Here, we investigated the interaction of self-regulated learning with externally-regulated DA-signalling by providing external performance feedback in the first or second half of trials. We found that removing feedback during DA-dependent learning lowered subsequent recognition rates a day later, whereas recognition remained high in the group which received feedback only in the second half. In contrast, feedback modestly enhanced recognition rates for both groups for DA-independent learning. Our findings suggest that external reinforcers can selectively impair DA-dependent memories if internal DA-dependent processes are not already established and highlights the relevance of self-regulated learning in education to support stable memory formation.

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LISA: Learned Indexes for Sequence Analysis BackgroundNext-generation sequencing (NGS) technologies have enabled affordable sequencing of billions of short DNA fragments at high throughput, paving the way for population-scale genomics. Genomics data analytics at this scale requires overcoming performance bottlenecks, such as searching for short DNA sequences over long reference sequences.

ResultsIn this paper, we introduce LISA (Learned Indexes for Sequence Analysis), a novel learning-based approach to DNA sequence search. We focus on accelerating two of the most essential flavors of DNA sequence search--exact search and super-maximal exact match (SMEM) search. LISA builds on and extends FM-index, which is the state-of-the-art technique widely deployed in genomics tools. Experiments with human, animal, and plant genome datasets indicate that LISA achieves up to 2.2 and 10.8 speedups over the state-of-the-art FM-index based implementations for exact search and super-maximal exact match (SMEM) search, respectively.

Code availabilityhttps://github.com/IntelLabs/Trans-Omics-Acceleration-Library/tree/master/LISA.

sentences: Three new species of nematodes from the syconia of Ficus racemosa in southern India SummaryFicus racemosa with an Indo-Australasian distribution, has so far been recorded to harbour in its fruits, nematode species of the aphelenchoid genera Schistonchus, Ficophagus and Martininema, and species of diplogastrid genera Teratodiplogaster and Pristionchus. The Indian species reported so far from Ficus racemosa lack comprehensive details on morphology and molecular characterization. In this paper, we describe three new species of nematodes obtained from syconia (enclosed globular infructescence or fruit) of Ficus racemosa found in southern India. Ficophagus glomerata n. sp. is characterised by small body having b=5.2-9.6, c= 18-23; slightly set-off lip region having well developed cephalic framework; secretory-excretory pore opens near the head; slender stylet with small, rounded/ sloping knobs; ovoid median bulb with relatively posteriorly-placed valve plates; males with sickle-shaped spicules having spatulate or hammer-shaped capitulum, represented by an elongate-ovoid condylus and long digitate rostrum and tail conoid with fine, hair-like terminal spike. Teratodiplogaster glomerata n. sp. is characterised by long tubular and narrow stoma with fractal pieces in prostegostom; long rectangular metacorpus; female reproductive system with conspicuous spermatheca and amoeboid sperms; males having short, arcuate spicules and keeled gubernaculum; genital papillae in the configuration of P1, P2, C, P3, P4, P5d, (P6d, P7), P8d, Ph and tail conoid with a terminal or subterminal mucro. Pristionchus glomerata n. sp. is characterised by four morphotypes mainly with variations in lip region, stoma, spicules, gubernaculum, and the position of genital papillae. Phylogenetic analyses based upon near-full-length small subunit (SSU) and D2-D3 expansion segments of large subunit (LSU) rRNA genes confirmed affinities with sister species of corresponding genera.

sentences: The Tabula Sapiens: a multiple organ single cell transcriptomic atlas of humans Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual.

sentences: Lipid droplets are a metabolic vulnerability in melanoma Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the pigmented cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved in human melanoma specimens. This state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt oxidative metabolism and slow melanoma growth in vivo. Because the pigmented cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.

sentences: PAMP-triggered Genetic Reprogramming Involves Widespread Alternative Transcription Initiation and an Immediate Transcription Factor Wave Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers of the genetic reprogramming required to reach the immune state remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression (CAGE). Our results show that as much as 15% of all PAMP response genes display alternative transcription initiation. In several cases, use of alternative TSSs may be regulatory as it determines inclusion of target peptides or protein domains, or occurrence of upstream open reading frames (uORFs) in mRNA leader sequences. We also find that 60% of PAMP-response genes respond much earlier than previously thought. In particular, a previously unnoticed cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, some examples of known potentiators of PTI, in one case under direct MAP kinase control, support the notion that the rapidly induced transcription factors could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

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Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. In Benin, West Africa, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases.

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe genetic background of individuals influences their susceptibility to infectious diseases. Specific human groups, like the Fulani in Africa, react to malaria parasites (named Plasmodium) in a specific way. Upon infection, Fulani develop a grossly enlarged spleen, and high levels of anti-Plasmodium antibodies in their blood. They also carry smaller numbers of parasites in their blood, and thus are considered partially protected against malaria. The mechanisms underlying this natural protection, different from other natural protective mechanisms such as the sickle cell trait, are not well understood.

Malaria impairs the deformability of red blood cells and the spleen is a key organ to controlling red blood cell quality. We have recently demonstrated that red blood cells containing live malaria parasites accumulate intensely in the spleen of subjects with long term exposure to these parasites. Enhanced retention of infected and uninfected red blood cells in the spleen would explain why the spleen is larger and why lower numbers of parasites are left in circulation. We thus explored whether the retention of infected and uninfected red blood cells could explain why Fulani are partially protected against malaria. Because it is unethical to perform spleen puncture or biopsies for research purposes, our explorations were indirect by carefully analyzing the properties of circulating red blood cells in a large number of subjects and by assessing whether observations could be explained by their genetic make-up.

Added value of this studyIn more than 500 subjects, we confirmed the high frequency of large spleens in Fulani and, through 2 different methods, we demonstrated an enhanced deformability of their circulating red blood cells, that likely stems from the more efficient removal of the less deformable ones. This enhanced deformability was found to be inheritable based on carefully collected family links and refined analysis of genetic markers.

Implications of all the available evidenceOur findings indicate that genes potentially driving the filtration of red blood cells by the spleen likely influence how subjects in specific groups in Africa and elsewhere react to malaria. While most previous hypotheses pointed to conventional immunological mechanisms as the trigger, we propose that a simple physiological mechanism that controls the quality of red blood cells may drive natural protection from malaria even before the intervention of immunological cells. A better understanding of these processes is of great importance in the context of malaria elimination efforts.

These findings may also have an impact on the understanding of other red blood cell-related disorders, such as inherited red cell diseases, in which splenic filtration of abnormal red blood cells may precipitate splenic complications.

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FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma Background and aimsAbout 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration sufficient to ignite oncogenic FF activation. In line, FGFR-specific tyrosine kinase inhibitors (F-TKI) were shown to provide clinical benefit in FF+ iCCA, although responses were partial and/or limited by resistance mechanisms, including the FF V565F gatekeeper mutation. Herein we present an FF-driven murine iCCA model and exploit its potential for pre-clinical studies on FF therapeutic targeting.

MethodsFour iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by intrahepatic/subcutaneous transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies.

ResultsTumors diagnosed as CCA were obtained upon transplantation of FF-expressing liver organoids. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or presence of V565F mutation. Double blockade of FF-Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo.

ConclusionsFF-driven iCCA pathogenesis was successfully modelled in murine Tp53-/- background. This model revealed biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for improving precision-based approaches against human FF+ iCCA.

Abbreviations used in this paper: ANOVA, analysis of variance; Bap1, BRCA1-Associated-Protein 1; Cdkn2a, cyclin-dependent kinase inhibitor 2A; Cftr, cystic fibrosis transmembrane conductance regulator; Ck19, cytokeratin 19; Cyp3A, cytochrome P450, family 3, subfamily A; EGF, Epidermal growth factor; EGFR, Epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; Erk, extracellular signal-regulated kinase; FGFR2, fibroblast growth factor receptor 2; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor-bound 2; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; H&E, hematoxylin and eosin; HepPar1, hepatocyte Paraffin 1; Hnf4-7, hepatocyte nuclear factor 4 alpha; Hprt, hypoxanthine-guanine phosphoribosyl transferase; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NTRK, neurotrophic Tyrosine Kinase; Parp, Poly (ADP-ribose) polymerase; RECIST, response evaluation criteria in solid tumors; SHP2, Src homology phosphatase 2; Ttr, transthyretin.

sentences: Axes of inter-sample variability among transcriptional neighborhoods reveal disease associated cell states in single-cell data As single-cell datasets grow in sample size, there is a critical need to characterize cell states that vary across samples and associate with sample attributes like clinical phenotypes. Current statistical approaches typically map cells to cell-type clusters and examine sample differences through that lens alone. Here we present covarying neighborhood analysis (CNA), an unbiased method to identify cell populations of interest with greater flexibility and granularity. CNA characterizes dominant axes of variation across samples by identifying groups of very small regions in transcriptional space--termed neighborhoods--that covary in abundance across samples, suggesting shared function or regulation. CNA can then rigorously test for associations between any sample-level attribute and the abundances of these covarying neighborhood groups. We show in simulation that CNA enables more powerful and accurate identification of disease-associated cell states than a cluster-based approach. When applied to published datasets, CNA captures a Notch activation signature in rheumatoid arthritis, redefines monocyte populations expanded in sepsis, and identifies a previously undiscovered T-cell population associated with progression to active tuberculosis.

sentences: Optogenetic activation of the neural circuit between dorsal raphe nucleus and the pre-Botzinger complex contributes to inhibition of the seizure-induced respiratory arrest in the DBA/1 mouse SUDEP model Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death among epilepsy patients, occurring even more frequently in cases with anti-epileptic drug resistance. However, the underlying mechanism of SUDEP remains elusive. Our previous study demonstrated that enhancement of serotonin (5-HT) synthesis by intraperitoneal (IP) injection of 5-hydroxytryptophan (5-HTP) significantly reduced the incidence of seizure-induced respiratory arrest (S-IRA) in a DBA/1 mouse SUDEP model. Given that the 5-HT2A receptor (5-HT2AR) plays an important role in mediating the respiration system in the brain, we hypothesized that 5-HT2AR plays a key role in S-IRA and SUDEP. To test this hypothesis, we examined whether the decreased incidence of S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) injection following 5-HTP administration will be blocked by treatment with ketanserin (KET), a selective antagonist of 5HT2AR, in the DBA/1 mouse SUDEP model. We observed that the reduction in S-IRA by 5-HTP was significantly reversed by IP or intracerebroventricular injection of KET. Considering the localization of 5-HT2AR in the pre-Botzinger complex (PBC), which plays a key role in regulating respiratory rhythm, we next examined whether KET acts on 5-HT2AR in the PBC. To test this hypothesis, we activated the neural circuit between the dorsal raphe nucleus (DR) and PBC using optogenetics technology. We observed that stimulation of TPH2-ChETA-expressing neurons in the DR reduced the incidence of S-IRA evoked by PTZ, and this suppressant effect was significantly reversed by administration of KET in the bilateral PBC with no changes in electroencephalogram activity. The neural circuit between the DR and PBC was confirmed by injection of cholera toxin subunit B555 (CTB-555), a nerve tracer, in the DR or PBC separately. Calcium signaling evoked by PTZ within neurons of the PBC during seizures was significantly reduced by photostimulation of the DR. Taken together, our findings suggest that 5-HT2AR plays a critical role in regulating S-IRA and targeting the serotonergic neural circuit between the DR and PBC is a promising approach to preventing SUDEP.

sentences: HNF4A is required to specify glucocorticoid action in the liver The glucocorticoid receptor (GR) is a nuclear hormone receptor critical to the regulation of energy metabolism and the inflammatory response. The actions of GR are highly dependent on cell type and environmental context. Here, we demonstrate the necessity for liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver-specificity of GR action. In normal mouse liver, the HNF4 motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites found within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodelled, with both loss and gain of GR recruitment evident. Lost sites are characterised by HNF4 motifs and weak GRE motifs. Gained sites are characterised by strong GRE motifs, and typically show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is further demonstrated by evidence of an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

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Phase- and State-Dependent Modulation of Breathing Pattern by preBötzinger Complex Somatostatin Expressing Neurons As neuronal subtypes are increasingly categorized, delineating their functional role is paramount. The preBotzinger Complex (preBotC) subpopulation expressing the neuropeptide somatostatin (SST) is classified as mostly excitatory, inspiratory-modulated and not rhythmogenic. We further characterized their phenotypic identity; 87% were glutamatergic and the balance were glycinergic and/or GABAergic. We then used optogenetics to investigate their modulatory role in both anesthetized and freely moving mice. In anesthetized mice, short photostimulation (100 ms) of preBotC SST+ neurons modulated breathing-related variables in a combinatory phase- and state-dependent manner; changes in inspiratory duration, inspiratory peak amplitude (Amp), and phase were different at higher ([≥]2.5 Hz) vs. lower (<2.5 Hz) breathing frequency. Moreover, we observed a biphasic effect of photostimulation during expiration that is probabilistic, i.e., photostimulation given at the same phase in consecutive cycles can evoke opposite responses (lengthening vs. shortening of the phase). This unexpected probabilistic state- and phase-dependent responses to photostimulation exposed properties of the preBotC that were not predicted and cannot be readily accounted for in current models of preBotC pattern generation. In freely moving mice, prolonged photostimulation decreased f in normoxia, hypoxia, or hypercapnia, and increased Amp and produced a phase advance, which was similar to the results in anesthetized mice when f[≥]2.5 Hz. We conclude that preBotC SST+ neurons are a key mediator of the extraordinary and essential lability of breathing pattern.

Key points summaryO_LIWe transfected preBotzinger Complex (preBotC) somatostatin-expressing (SST+) neurons, which modulate respiratory pattern, but are not rhythmogenic, with channelrhodopsin to investigate phase- and state-dependent modulation of breathing pattern in anesthetized and freely behaving mice in normoxia, hypoxia, and hypercapnia.
C_LIO_LIIn anesthetized mice, photostimulation of preBotC SST+ neurons during inspiration increased inspiratory duration and amplitude regardless of baseline breathing frequency, f.
C_LIO_LIIn anesthetized mice with low f (<2.5 Hz), photostimulation of preBotC SST+ neurons during expiration evoked either phase advance or phase delay, whereas in anesthetized mice with high f ([≥]2.5 Hz) and in freely behaving mice in normoxia, hypoxia, or hypercapnia, photostimulation always evoked phase advance.
C_LIO_LIPhase- and state-dependency is a function of overall breathing network excitability.
C_LIO_LIThe f-dependent probabilistic modulation of breathing pattern by preBotC SST+ neurons was unexpected, requiring reconsideration of current models of preBotC function, which neither predict nor can readily account for such responses.
C_LI

sentences: Genome-wide survey of odorant-binding proteins in the dwarf honey bee Apis florea Odorant binding proteins (OBPs) in insects bind to volatile chemical cue and help in their binding to odorant receptors. The odor coding hypothesis states that OBPs may bind with specificity to certain volatiles and aid the insect in various behaviours. Honeybees are eusocial insects with complex behaviour that requires olfactory inputs. Here, we have identified and annotated odorant binding proteins from the genome of the dwarf honey bee, Apis florea using an exhaustive homology-based bioinformatic pipeline and analyzed the evolutionary relationships between the OBP subfamilies. Our study suggests that Minus-C subfamily may have diverged from the Classic subfamily of odorant binding proteins in insects.

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Evolutionary coupling range varies widely among enzymes depending on selection pressure Recent studies proposed that enzyme active sites induce evolutionary constraints at long distances. The physical origin of such long-range evolutionary coupling is unknown. Here, I use a recent biophysical model of evolution to study the relationship between physical and evolutionary couplings on a diverse data set of monomeric enzymes. I show that evolutionary coupling is not universally long-range. Rather, range varies widely among enzymes, from 2[A] to 20[A]. Furthermore, the evolutionary coupling range of an enzyme does not inform on the underlying physical coupling, which is short-range for all enzymes. Rather, evolutionary coupling range is determined by functional selection pressure.

SIGNIFICANCEUntil recently, only residues near enzyme active sites were thought to be evolutionarily constrained. However, recent studies proposed that active sites induce long-range evolutionary constraints. This seems to conflict with the common finding that physical couplings in proteins are short-range. This raises the question of how short-range physical couplings may cause long-range evolutionary couplings. Here, I show that the function that maps physical coupling into evolutionary coupling depends on functional selection pressure. Under weak selection, both couplings are similarly short-range; under strong selection, short-range physical coupling is non-linearly turned into long-range evolutionary coupling. Thus, due to a huge variation of selection pressure, evolutionary coupling range varies widely among enzymes, from very short (2 [A]) to very long (20 [A]).

sentences: Detection of Cell-Cell Interactions via Photocatalytic Cell Tagging Cell-cell interactions drive essential biological processes critical to cell and tissue development, function, pathology, and disease outcome. The growing appreciation of immune cell interactions within disease environments has led to significant efforts to develop protein- and cell-based therapeutic strategies. A better understanding of these cell-cell interactions will enable the development of effective immunotherapies. However, characterizing these complex cellular interactions at molecular resolution in their native biological contexts remains challenging. To address this, we introduce photocatalytic cell tagging (PhoTag), a modality agnostic platform for profiling cell-cell interactions. Using photoactivatable flavin-based cofactors, we generate phenoxy radical tags for targeted labeling at the cell surface. Through various targeting modalities (e.g. MHC-Multimer, antibody, single domain antibody (VHH)) we deliver a flavin photocatalyst for cell tagging within monoculture, co-culture, and peripheral blood mononuclear cells. PhoTag enables highly selective tagging of the immune synapse between an immune cell and an antigen-presenting cell through targeted labeling at the cell-cell junction. This allowed for the ability to profile gene expression-level differences between interacting and bystander cell populations. Given the modality agnostic and spatio-temporal nature of PhoTag, we envision its broad utilization to detect and profile intercellular interactions within an immune synapse and other confined cellular regions for any biological system.

sentences: The Effect of Spatial Frequency on the Visual Category Representation in the Macaque Inferior Temporal Cortex To expand our knowledge about the object recognition, it is critical to understand the role of spatial frequency (SF) in an object representation that occurs in the inferior temporal (IT) cortex at the final stage of processing the visual information across the ventral visual pathway. Object categories are being recognized hierarchically in at least three levels of abstraction: superordinate (e.g., animal), mid-level (e.g., human face), and subordinate (e.g., face identity). Psychophysical studies have shown rapid access to mid-level category information and low SF (LSF) contents. Although the hierarchical representation of categories has been shown to exist inside the IT cortex, the impact of SF on the multi-level category processing is poorly understood. To gain a deeper understanding of the neural basis of the interaction between SF and category representations at multiple levels, we examined the neural responses within the IT cortex of macaque monkeys viewing several SF-filtered objects. Each stimulus could be either intact or bandpass filtered into either the LSF (coarse shape information) or high SF (HSF) (fine shape information) bands. We found that in both High- and Low-SF contents, the advantage of mid-level representation has not been violated. This evidence suggests that mid-level category boundary maps are strongly represented in the IT cortex and remain unaffected with respect to any changes in the frequency content of stimuli. Our observations indicate the necessity of the HSF content for the superordinate category representation inside the IT cortex. In addition, our findings reveal that the representation of global category information is more dependent on the HSF than the LSF content. Furthermore, the lack of subordinate representation in both LSF and HSF filtered stimuli compared to the intact stimuli provide strong evidence that all SF contents are necessary for fine category visual processing.

sentences: The strength of feedback processing is associated with resistance to visual backward masking during illusory contour processing in adult humans. Re-entrant feedback processing is a key mechanism of visual object-recognition, especially under compromised viewing conditions where only sparse information is available and object features must be interpolated. Illusory contour stimuli are commonly used in conjunction with visual evoked potentials (VEP) to study these filling-in processes, with characteristic modulation of the VEP in the ~100-150ms timeframe associated with this re-entrant processing. Substantial inter-individual variability in timing and amplitude of feedback-related VEP modulation is observed, raising the question whether this variability might underlie inter-individual differences in the ability to form strong perceptual gestalts. Backward masking paradigms have been used to study inter-individual variance in the ability to form robust object perceptions before processing of the mask interferes with object-recognition. Some individuals recognize objects when the time between target object and mask is extremely short, whereas others struggle to do so even at longer target-to-mask intervals. We asked whether timing and amplitude of feedback-related VEP modulations were associated with individual differences in resistance to backward masking. Participants (N=40) showed substantial performance variability in detecting illusory contours at intermediate target-to-mask intervals (67ms and 117ms), allowing us to use kmeans clustering to divide the population into four performance groups (poor, low-average, high-average, superior). There was a clear relationship between the amplitude (but not the timing) of feedback-related VEP modulation and illusory contour detection during backward masking. We conclude that individual differences in the strength of feedback processing in neurotypical humans lead to differences in the ability to quickly establish perceptual awareness of incomplete visual objects.

sentences: On the edge of criticality: strength-dependent perturbation unveils delicate balance between fluctuation and oscillation in brain dynamics Despite decades of research, there is still a lack of understanding of the role and generating mechanisms of the ubiquitous fluctuations and oscillations found in recordings of brain dynamics. Here, we used a strength-dependent perturbative framework to provide a causal mechanistic description of how human brain function is perched at the delicate balance between fluctuation and oscillation. Applying local strength-dependent perturbations and subsequently measuring the perturbative complexity index clearly demonstrates that the overall balance of brain dynamics is shifted towards fluctuations for providing much needed flexibility. Importantly, stimulation in the fluctuation regime modulates specific resting state network, thus providing a mechanistic explanation of experimentally reported brain dynamics. Furthermore, this framework generates specific, testable empirical predictions for human stimulation studies using strength-dependent rather than constant perturbation. Overall, the strength-dependent perturbative framework demonstrates how the human brain is poised on the edge of criticality, between fluctuations to oscillations, allowing for maximal flexibility.

sentences: Neuronal variability reflects probabilistic inference tuned to natural image statistics Neuronal activity in sensory cortex fluctuates over time and across repetitions of the same input. This variability is often considered detrimental to neural coding. The theory of neural sampling proposes instead that variability encodes the uncertainty of perceptual inferences. In primary visual cortex (V1), modulation of variability by sensory and non-sensory factors supports this view. However, it is unknown whether V1 variability reflects the statistical structure of visual inputs, as would be required for inferences correctly tuned to the statistics of the natural environment. Here we combine analysis of image statistics and recordings in macaque V1 to show that probabilistic inference tuned to natural image statistics explains the widely observed dependence between spike-count variance and mean, and the modulation of V1 activity and variability by spatial context in images. Our results show that the properties of a basic aspect of cortical responses -- their variability -- can be explained by a probabilistic representation tuned to naturalistic inputs.

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KIMGENS: A novel method to estimate kinship in organisms with mixed haploid diploid genetic systems robust to population structure MotivationKinship estimation is necessary for evaluating violations of assumptions or testing certain hypotheses in many population genomic studies. However, kinship estimators are usually designed for diploid systems and cannot be used in populations with mixed haploid diploid genetic systems. The only estimators for different ploidies require datasets free of population structure, limiting their usage.

ResultsWe present KIMGENS, an estimator for kinship estimation among individuals of various ploidies, that is robust to population structure. This estimator is based on the popular KING-robust estimator but uses diploid relatives of the individuals of interest as references of heterozygosity and extends its use to haploid-diploid and haploid pairs of individuals. We demonstrate that KIMGENS estimates kinship more accurately than previously developed estimators in simulated panmictic, structured and admixed populations, but has lower accuracy when the individual of interest is inbred. KIMGENS also outperforms other estimators in a honeybee dataset. Therefore, KIMGENS is a valuable addition to a population geneticists toolbox.

Availability and ImplementationKIMGENS and its association simulation tool are implemented and available open-source at https://github.com/YenWenWang/HapDipKinship.

ContactYen-Wen Wang Email: ywang883@wisc.edu

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Masked features of task states found in individual brain networks Completing complex tasks requires flexible integration of functions across brain regions. While studies have shown that functional networks are altered across tasks, recent work highlights that brain networks exhibit substantial individual differences. Here we asked whether individual differences are important for predicting brain network interactions across cognitive states. We trained classifiers to decode state using data from single person "precision" fMRI datasets across 5 diverse cognitive states. Classifiers were then tested on either independent sessions from the same person or new individuals. Classifiers were able to decode task states in both the same and new participants above chance. However, classification performance was significantly higher within a person, a pattern consistent across model types, datasets, tasks, and feature subsets. This suggests that individualized approaches can uncover robust features of brain states, including features obscured in cross-subject analyses. Individualized approaches have potential to deepen our understanding of brain interactions during complex cognition.

Citation Diversity StatementRecently, the field of neuroscience has reported a bias in citation practices such that papers from minority groups are more often under-cited relative to the number of papers in the field1. The authors of this paper were proactive in consideration of selecting references that reflect diversity of the field in thought, contribution, and gender. Utilizing previously derived databases 1,2 we obtained the predicted gender of authors referenced in this manuscript. By this measure (and excluding self-citations to the first and last authors of our current paper), our references contain 13.87% woman(first)/woman(last), 23.3% man/woman, 23.3% woman/man, and 39.53% man/man. This method is limited in that a) names, pronouns, and social media profiles used to construct the databases may not, in every case, be indicative of gender identity and b) it cannot account for intersex, non-binary, or transgender people. Second, we obtained the predicted racial/ethnic category of the first and last author of each reference by databases that store the probability of a first and last name being carried by an author of color3. By this measure (and excluding self-citations), our references contain 10.83% author of color (first)/author of color(last), 10.64% white author/author of color, 23.55% author of color/white author, and 54.98% white author/white author. This method is limited in that a) names and Florida Voter Data to make the predictions may not be indicative of racial/ethnic identity, and b) it cannot account for Indigenous and mixed-race authors, or those who may face differential biases due to the ambiguous racialization or ethnicization of their names. We look forward to future work that could help us to better understand how to support equitable practices in science.

sentences: Neural Representations of Task-relevant and Task-irrelevant Features of Attended Objects Visual attention plays an essential role in selecting task-relevant and ignoring task-irrelevant information, for both object features and their locations. In the real world, multiple objects with multiple features are often simultaneously present in a scene. When spatial attention selects an object, how are the task-relevant and task-irrelevant features represented in the brain? Previous literature has shown conflicting results on whether and how irrelevant features are represented in visual cortex. In an fMRI task, we used a modified inverted encoding model (IEM, e.g., Sprague & Serences, 2015) to test whether we can reconstruct the task-relevant and task-irrelevant features of spatially attended objects in a multi-feature (color + orientation), multi-item display. Subjects were briefly shown an array of three colored, oriented gratings. Subjects were instructed as to which feature (color or orientation) was relevant before each block, and on each trial were asked to report the task-relevant feature of the object that appeared at a spatially pre-cued location, using a continuous color or orientation wheel. By applying the IEM, we achieved reliable feature reconstructions for the task-relevant features of the attended object from visual ROIs (V1 and V4v) and Intraparietal sulcus. Preliminary searchlight analyses showed that task-irrelevant features of attended objects could be reconstructed from activity in some intraparietal areas, but the reconstructions were much weaker and less reliable compared with task-relevant features. These results suggest that both relevant and irrelevant features may be represented in visual and parietal cortex but in different forms. Our method provides potential tools to noninvasively measure unattended feature representations and probe the extent to which spatial attention acts as a "glue" to bind task-relevant and task-irrelevant features.

sentences: Protective host-dependent antagonism among 	Pseudomonas in the Arabidopsis phyllosphere The plant microbiome is a rich biotic environment, comprising numerous taxa. The community structure of these colonizers is constrained by multiple factors, including host-microbe and microbe-microbe interactions, as well as the interplay between the two. While much can be learned from pairwise relationships between individual hosts and microbes, or individual microbes with themselves, the ensemble of interrelations between the host and microbial consortia may lead to different outcomes that are not easily predicted from the individual interactions. Their study can thus provide new insights into the complex relationship between plants and microbes. Of particular importance is how strain-specific such plant-microbe-microbe interactions are, and how they eventually affect plant health. Here, we test strain-level interactions in the phyllosphere between groups of co-existing commensal and pathogenic Pseudomonas among each other and with A. thaliana, by employing synthetic communities of genome-barcoded isolates. We found that commensal Pseudomonas prompted a host response leading to a selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of plant protection, however, was dependent on plant genotype, indicating that these effects are host-mediated. There were similar genotype-specific effects on the microbe side, as we could pinpoint an individual Pseudomonas isolate as the predominant cause for this differential interaction. Collectively, our work highlights how within-species genetic differences on both the host and microbe side can have profound effects on host-microbe-microbe dynamics. The paradigm that we have established provides a platform for the study of host-dependent microbe-microbe competition and cooperation in the A. thaliana-Pseudomonas system.

sentences: Population density and plant availability interplay to shape browsing intensity by roe deer in a deciduous forest Browsing damage in forests relies on a complex interaction between herbivore density and both forest understory composition and relative availability. Although variation in the amount of browsed twigs is sometimes used to assess abundance of large herbivores, the potential confounding effect of resource availability on this relationship has not yet been investigated. To fill the gap, we measured how browsing intensity of the woody plants varied in response to changes in both roe deer (Capreolus capreolus) abundance and vegetation availability from an intensive long-term monitoring. We estimated plant availability and consumption by roe deer from a modified Aldous method throughout a 14 yearlong period during which we experimentally manipulated population density. The functional response was strongly non-linear and density-dependent. When plant availability was low (< 12.5%), browsing intensity strongly increased with plant availability with an increasing rate with roe deer density, whereas beyond this threshold, browsing intensity slightly increased with both plant availability and population density in an additive way. Thus, forest susceptibility to browsing increases with increasing competition for food, especially when plant availability is low. Moreover, the interplay between browsing intensity and population density at low plant availability prevents the use of browsing intensity to monitor roe deer abundance when plant availability is low. Our findings provide clear evidence that relying on key ecological concepts such as functional responses improves the accuracy of management tools when monitoring changes of the herbivore-plant system over time.

sentences: A Chromosome-level Assembly of a Wild Castor Genome Provides New Insights into the Adaptive Evolution in a Tropical Desert Wild castor grows in the high-altitude tropical desert of the African Plateau, a region known for high ultraviolet radiations, strong lights and extremely dry conditions. To investigate the potential genetic basis of adaptation to both highland and tropical deserts, we generated a chromosome-level genome sequence of the wild castor accession WT05, with genome size of 316 Mb and scaffold and contig N50 sizes of 31.93 Mb and 8.96 Mb, respectively. Compared with cultivated castor and other Euphorbiaceae species, the wild castor exhibits positive selection and gene family expansion for genes involved in DNA repair, photosynthesis and abiotic stress responses. Genetic variations associated with positive selection were identified in several key genes, such as LIG1, DDB2, and RECG1, involved in nucleotide excision repair. Moreover, a study of genomic diversity among wild and cultivated accessions revealed genomic regions containing selection signatures associated with the adaptation to extreme environments. The identification of the genes and alleles with selection signatures provides insights into the genetic mechanisms underlying the adaptation of wild castor to the high-altitude tropical desert and would facilitate direct improvement of modern castor varieties.

sentences: Cis-regulatory divergence underpins the evolution of C3-C4 intermediate photosynthesis in Moricandia Altered transcript abundances and cell specific gene expression patterns that are caused by regulatory divergence play an important role in the evolution of C4 photosynthesis. How these altered gene expression patterns are achieved and whether they are driven by cis- or trans-regulatory changes is mostly unknown. To address this question, we investigated the regulatory divergence between C3 and C3-C4 intermediates, using allele specific gene expression (ASE) analyses of Moricandia arvensis (C3-C4), M. moricandioides (C3) and their interspecific F1 hybrids. ASE analysis on SNP-level showed similar relative proportions of regulatory effects among hybrids: 36% and 6% of SNPs were controlled by cis-only and trans-only changes, respectively. GO terms associated with metabolic processes and the positioning of chloroplast in cells were abundant in transcripts with cis-SNPs shared by all studied hybrids. Transcripts with cis-specificity expressed bias toward the allele from the C3-C4 intermediate genotype. Additionally, ASE evaluated on transcript-level indicated that [~]27% of transcripts show signals of ASE in Moricandia hybrids. Promoter-GUS assays on selected genes revealed altered spatial gene expression patterns, which likely result from regulatory divergence in their promoter regions. Assessing ASE in Moricandia interspecific hybrids contributes to the understanding of early evolutionary steps towards C4 photosynthesis and highlights the impact and importance of altered transcriptional regulations in this process.

sentences: Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut Host genetic landscapes can shape microbiome assembly in the animal gut by contributing to the establishment of distinct physiological environments. However, the genetic determinants contributing to the stability and variation of these microbiome types remain largely undefined. Here, we use the free-living nematode Caenorhabditis elegans to identify natural genetic variation among wild strains of C. elegans strains that drives assembly of distinct microbiomes. To achieve this, we first established a diverse model microbiome that represents the phylogenetic and functional diversity naturally found in the C. elegans microbiome. Using this community, we show that C. elegans utilizes immune, xenobiotic and metabolic signaling pathways to favor the assembly of different microbiome types. Variations in these pathways were associated with the enrichment for specific commensals, including the Alphaproteobacteria Ochrobactrum. Using RNAi and mutant strains, we showed that host selection for Ochrobactrum is mediated specifically by host insulin signaling pathways. Ochrobactrum recruitment is blunted in the absence of daf-2/IGFR and requires the insulin signaling transcription factors daf-16/FOXO and pqm-1/SALL2. Further, the ability of C. elegans to enrich for Ochrobactrum is correlated positively with host outcomes, as animals that develop faster are larger and have higher gut Ochrobactrum colonization as adults. These results highlight a new role for the highly conserved insulin signaling pathways in the regulation of microbiome composition in C. elegans.

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S. aureus-serine protease-like protein B (SplB) activates PAR2 and induces endothelial barrier dysfunction Staphylococcus aureus (S. aureus) is a major cause of life-threatening systemic infection in humans. To cause blood stream infections such as sepsis and endocarditis, the bacteria must overcome the hosts endothelial barrier. The serine protease-like proteins (Spls) of S. aureus are known to contribute to pneumonia and allergic airway inflammation in animal models, but their role in endothelial damage is unknown. Here we demonstrate that SplB induces proinflammatory cytokine release in primary human vascular endothelial cells (HUVECs) in vitro. Mechanistically, we show that SplB selectively cleaves and activates human proteinase-activated receptor-2 (PAR2), and induces biased signaling via G13, Gi1/oB (NF-k{beta}), and {beta}-arrestin-1/-2. This activation did not trigger Gq/11-mediated calcium release nor ERK phosphorylation. Inhibition of PAR2 in HUVECs reduced the SplB-mediated cytokine release. Intravital microscopy of cremaster muscles in mice demonstrated that administration of SplB causes microvascular leakage. Genetic deletion of PAR2 in mice or neutralization of SplB with a monoclonal antibody preserved the endothelial barrier. This study identifies PAR2 as a receptor and substrate for SplB and highlights its role in mediating endothelial damage.

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Genomic prediction with whole-genome sequence data in intensely selected pig lines BackgroundEarly simulations indicated that whole-genome sequence data (WGS) could improve prediction accuracy and its persistence across generations and breeds. However, results in real datasets have been ambiguous so far. Large data sets that capture most of the genome diversity in a population must be assembled so that allele substitution effects are estimated with higher accuracy. The objectives of this study were to use a large pig dataset to assess the benefits of using WGS for genomic prediction compared to using commercial marker arrays, to identify scenarios in which WGS provides the largest advantage, and to identify potential pitfalls for its effective implementation.

MethodsWe sequenced 6,931 individuals from seven commercial pig lines with different numerical size. Genotypes of 32.8 million variants were imputed for 396,100 individuals (17,224 to 104,661 per line). We used BayesR to perform genomic prediction for 8 real traits and 9 simulated traits with different genetic architectures. Genomic predictions were performed using either data from a marker array or variants preselected from WGS based on linkage disequilibrium, functional annotation, or association tests. Both single and multi-line training sets were explored.

ResultsUsing WGS improved prediction accuracy relative to the marker array, provided that training sets were sufficiently large, especially for traits with high heritability and low number of quantitative trait nucleotides. The performance of each set of predictor variants was not robust across traits and lines. The most robust results were obtained when preselected variants with statistically significant associations were added to the marker array. Under this method, average improvements of prediction accuracy of 2.5 and 4.2 percentage points were observed in within-line and multi-line scenarios, respectively, with training sets of around 80k individuals.

ConclusionsOur results evidenced the potential for WGS to improve genomic prediction accuracy in intensely selected pig lines. Although the prediction accuracy improvements achieved so far were modest at best, we would expect that more robust improvements could be attained with a combination of larger training sets and optimised pipelines.

sentences: Protein-protein interactions on membrane surfaces analysed using pull-downs with supported bilayers on silica beads Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically-tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.

sentences: Small extracellular vesicles from young mice prevent frailty, improve healthspan and decrease epigenetic age in old mice. Aging is associated with an increased risk of frailty, disability, comorbidities, institutionalization, falls, fractures, hospitalization, and mortality. Searching for strategies to delay the degenerative changes associated with aging and frailty is interesting. We treated old animals intravenously with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals, and we found an improvement of several functional parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function. Frailty index analysis showed that 40% of old control mice were frail, whereas none of the old ADSCs-sEVs treated mice were. Molecular and structural benefits in muscle and kidney accompanied this functional improvement. ADSCs-sEVs induced pro-regenerative effects and a decrease in oxidative stress, inflammation, and senescence markers. Moreover, predicted epigenetic age was lower in tissues of old mice treated with ADSCs-sEVs and their metabolome changed to a youth-like pattern. Finally, we gained some insight into the miRNAs contained in sEVs that might be, at least in part, responsible for the effects observed. We propose that young sEVs treatment can be beneficial against frailty and therefore can promote healthy aging.

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LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19.

In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.

HighlightsO_LILY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variants
C_LIO_LINo loss of potency against currently circulating variants
C_LIO_LIBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID database
C_LIO_LIBreadth of neutralizing activity and potency supports clinical development
C_LI

sentences: Modelling the within-host spread of SARS-CoV-2 infection, and the subsequent immune response, using a hybrid, multiscale, individual-based model. Part I: Macrophages. Individual responses to SARS-CoV-2 infection vary significantly, ranging from mild courses of infection that do not require hospitalisation to the development of disease which not only requires hospitalisation but can be fatal. Whilst many immunological studies have revealed fundamental insights into SARS-CoV-2 infection and COVID-19, mathematical and computational modelling can offer an additional perspective and enhance understanding. The majority of mathematical models for the within-host spread of SARS-CoV-2 infection are ordinary differential equations, which neglect spatial variation. In this article, we present a hybrid, multiscale, individual-based model to study the within-host spread of SARS-CoV-2 infection. The model incorporates epithelial cells (each containing a dynamical model for viral entry and replication), macrophages and a subset of cytokines. We investigate the role of increasing initial viral deposition, increasing delay in type I interferon secretion from epithelial cells (as well as the magnitude of secretion), increasing macrophage virus internalisation rate and macrophage activation, on the spread of infection.

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Clustering single-cell RNA-seq data by rank constrained similarity learning MotivationRecent breakthroughs of single-cell RNA sequencing (scRNA-seq) technologies offer an exciting opportunity to identify heterogeneous cell types in complex tissues. However, the unavoidable biological noise and technical artifacts in scRNA-seq data as well as the high dimensionality of expression vectors make the problem highly challenging. Consequently, although numerous tools have been developed, their accuracy remains to be improved.

ResultsHere, we introduce a novel clustering algorithm and tool RCSL (Rank Constrained Similarity Learning) to accurately identify various cell types using scRNA-seq data from a complex tissue. RCSL considers both local similarity and global similarity among the cells to discern the subtle differences among cells of the same type as well as larger differences among cells of different types. RCSL uses Spearmans rank correlations of a cells expression vector with those of other cells to measure its global similarity, and adaptively learns neighbour representation of a cell as its local similarity. The overall similarity of a cell to other cells is a linear combination of its global similarity and local similarity. RCSL automatically estimates the number of cell types defined in the similarity matrix, and identifies them by constructing a block-diagonal matrix, such that its distance to the similarity matrix is minimized. Each block-diagonal submatrix is a cell cluster/type, corresponding to a connected component in the cognate similarity graph. When tested on 16 benchmark scRNA-seq datasets in which the cell types are well-annotated, RCSL substantially outperformed six state-of-the-art methods in accuracy and robustness as measured by three metrics.

AvailabilityThe RCSL algorithm is implemented in R and can be freely downloaded at https://github.com/QinglinMei/RCSL.

Contactguojunsdu@gmail.com, zcsu@uncc.edu

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: An evolutionary divergent thermodynamic brake in ZAP-70 finetunes the kinetic proofreading in T cell T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. The lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation in TCR. Phylogenetic analysis revealed that the evolution of the thermodynamic-brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. Paralogous kinase Syk expressed in B cells, does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.

sentences: Stable m7G Cap-Distal 5'UTR Hairpin Structure Mediates Distinct 40S and 60S Binding Dynamics The 5' untranslated region (UTR) of diverse mRNAs contains secondary structures that can influence protein synthesis by modulating the initiation step of translation. Studies support the ability of these structures to inhibit 40S subunit recruitment and scanning, but the dynamics of ribosomal subunit interactions with mRNA remain poorly understood. Here, we developed a reconstituted Saccharomyces cerevisiae cell-free translation system with fluorescently labeled ribosomal subunits. We applied this extract and single-molecule fluorescence microscopy to monitor, in real time, individual 40S and 60S interactions with mRNAs containing 5 UTR hairpin structures with varying thermostability. In comparison to mRNAs containing no or weak 5'UTR hairpins ({Delta}G >= -5.4 kcal/mol), mRNAs with stable hairpins ({Delta}G <= -16.5 kcal/mol) showed reduced numbers of 60S recruitment to mRNA, consistent with the expectation of reduced translation efficiency for such mRNAs. Interestingly, such mRNAs showed increased numbers of 40S recruitment events to individual mRNAs but with shortened duration on mRNA. Correlation analysis showed that these unstable 40S binding events were nonproductive for 60S recruitment. Furthermore, although the mRNA sequence is long enough to accommodate multiple 40S, individual mRNAs are predominantly observed to engage with a single 40S at a time, indicating the sequestering of mRNA 5 end by initiating 40S. Altogether, these observations suggest that stable cap-distal hairpins in 5 UTR reduce initiation and translation efficiency by destabilizing 40S-mRNA interactions and promoting 40S dissociation from mRNA. The premature 40S dissociation frees mRNA 5'-end accessibility for new initiation events, but the increased rate of 40S recruitment is insufficient to compensate for the reduction of initiation efficiency due to premature 40S dissociation. This study provides the first single-molecule kinetic characterization of 40S/60S interactions with mRNA during cap-dependent initiation and the modulation of such interactions by cap-distal 5 UTR hairpin structures.

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

Graphical abstract

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sentences: Disruption of folate metabolism causes poor alignment and spacing of mouse conceptuses for multiple generations Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with an Mtrrgt hypomorphic mutation. MTRR is necessary for methyl group utilisation from the folate cycle, and the Mtrrgt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrrgt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes (e.g., eccentric chorioallantoic attachment). Notably, we report misalignment of some Mtrrgt conceptuses within their implantation sites from E6.5. The degree of skewed growth occurs across a continuum, with eccentric chorioallantoic attachment now re-characterised as a severe form of conceptus misalignment. Additionally, some Mtrrgt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Embryo development is influenced by skewed growth since developmental delay and heart malformations (but not neural tube defects) associate with severe misalignment of Mtrrgt/gt conceptuses. Patterning of trophoblast lineage markers is largely unaffected in skewed Mtrrgt/gt conceptuses at E8.5 indicating trophoblast differentiation was normal when misaligned. Typically, the uterus guides conceptus orientation. Accordingly, we manipulate the maternal Mtrr genotype and assess conceptus alignment. Mtrr+/gt, and Mtrrgt/gt mothers, plus Mtrr+/+ mothers, exhibit misaligned conceptuses at E6.5. While progesterone and/or BMP2 signalling required for decidualisation might be disrupted, normal gross decidual morphology, patterning, and blood perfusion is evident regardless of conceptus alignment, arguing against a uterine defect. Given the important finding that Mtrr+/+ mothers also display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrrgt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, misalignment is independent of the uterus and instead is attributed to an embryonic mechanism based on blastocyst transfer experiments. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. Our study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

sentences: Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2 Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

sentences: Dynamic decision policy reconfiguration under outcome uncertainty In uncertain or unstable environments, sometimes the best decision is to change your mind. To shed light on this flexibility, we evaluated how the underlying decision policy adapts when the most rewarding action changes. Human participants performed a dynamic two-armed bandit task that manipulated the certainty in relative reward (conflict) and the reliability of action-outcomes (volatility). Continuous estimates of conflict and volatility contributed to shifts in exploratory states by changing both the rate of evidence accumulation (drift rate) and the amount of evidence needed to make a decision (boundary height), respectively. At the trialwise level, following a switch in the optimal choice, the drift rate plummets and the boundary height weakly spikes, leading to a slow exploratory state. We find that the drift rate drives most of this response, with an unreliable contribution of boundary height across experiments. Surprisingly, we find no evidence that pupillary responses associated with decision policy changes. We conclude that humans show a stereotypical shift in their decision policies in response to environmental changes.

sentences: Identical Sequences, Different Behaviors: ProteinDiversity Captured at the Single-Molecule Level The classical "one sequence, one structure, one function" paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements to a few minutes, which prevents to completely characterize a protein individual and, hence, to capture the heterogeneity among molecular populations. Here, we introduce a magnetic tweezers design, which showcases enhanced stability and resolution that allows us to measure the folding dynamics of a single protein during several uninterrupted days with a high temporal and spatial resolution. Thanks to this instrumental development, we do a complete characterization of two proteins with a very different force-response: the talin R3IVVI domain and protein L. Days-long recordings on the same single molecule accumulate several thousands of folding transitions sampled with sub-ms resolution, which allows us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. Our instrumental development offers a new tool for profiling individual molecules, opening the gates to the characterization of biomolecular heterogeneity.

sentences: Doors and corners of variance partitioning in statistical ecology Variance partitioning is a common tool for statistical analysis and interpretation in both observational and experimental studies in ecology. Its popularity has led to a proliferation of methods with sometimes confusing or contradicting interpretations. Here, we present variance partitioning as a general tool in a model based Bayesian framework for summarizing and interpreting regression-like models. To demonstrate our approach we present a case study comprising of a simple occupancy model for a metapopulation of the Glanville fritillary butterfly. We pay special attention to the thorny issue of correlated covariates and random effects, and highlight uncertainty in variance partitioning. We recommend several alternative measures of variance, which jointly can be used to better interpret variance partitions. Additionally, we extend the general approach to encompass partitioning of variance within and between groups of observations, an approach very similar to analysis of variance. While noting that many troublesome issues relating to variance partitioning, such as uncertainty quantification, have been neglected in the literature, we likewise feel that the rather general applicability of the methods as an extension of statistical model-based analyses has not been fully utilized by the ecological research community either.

sentences: Morphogenesis of a complex glial niche requires an interplay between cellular growth and fusion Neural stem cells (NSCs) are embedded in a multi-layered, intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of the glial network of a NSC niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, eventually spanning the entire tissue while partitioning the NSC population. Elaborate proliferative mechanisms convert these cells into syncytia rich in cytoplasmic bridges. Unexpectedly, CG syncytia further undergo homotypic cell-cell fusion, relying on defined molecular players of cell fusion such as cell surface receptors and actin regulators. Exchange of cellular components is however dynamic in space and time, a previously unreported unique mechanism. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, the coordination of growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide novel insights into how a niche forms and organises while developing intimate contacts with a stem cell population.

sentences: CIP2A is a prime synthetic-lethal target for BRCA-mutated cancers BRCA1/2-mutated cancer cells must adapt to the genome instability caused by their deficiency in homologous recombination. Identifying and targeting these adaptive mechanisms may provide new therapeutic strategies. Here we present the results of genome-scale CRISPR/Cas9-based synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells that identified the gene encoding CIP2A as essential in a wide range of BRCA1- and BRCA2-mutated cells. Unlike PARP inhibition, CIP2A-deficiency does not cause accumulation of replication-associated DNA lesions that require homologous recombination for their repair. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient cells and tumors, indicating that targeting this mitotic chromosome stability process represents an attractive synthetic-lethal therapeutic strategy for BRCA1- and BRCA2-mutated cancers.

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Feed efficiency of lactating Holstein cows was not as repeatable across diets as within diet over subsequent lactation stages BackgroundImproving feed efficiency has become a common target for dairy farmers to meet the requirement of producing more milk with fewer resources. To improve feed efficiency, a prerequisite is to ensure that the cows identified as most or least efficient will remain as such, independently of diet composition. Therefore, the current research analysed the ability of lactating dairy cows to maintain their feed efficiency while changing the energy density of the diet by changing its concentration in starch and fibre. A total of 60 lactating Holstein cows, including 33 primiparous cows, were first fed a high starch diet-low fibre (diet S+F-), then switched over to a low starch diet-high fibre (diet S-F+). To know if diet affect feed efficiency, we compared the ability of feed efficiency to be maintained within a diet over subsequent lactation stages, known as repeatability of feed efficiency, with its ability to be maintained across diets, known as reproducibility of feed efficiency. To do so we used two indicators: the estimation of the error of repeatability/reproducibility, which is commonly used in metrology, and the coefficient of correlation of concordance (CCC), which is used in biology. The effect of diet change could also lead to a change in cows sorting behaviour which could potentially affect feed efficiency if for example the most efficient cows select more concentrate than the least efficient. We therefore analysed the relationship between the differences in individual feed refusals composition and the differences in feed efficiency. To do so, the composition of each feed refusal was described with its near infra-red (NIR) spectroscopy and was performed on each individual feed ingredient, diet and refusals and used as composition variable. The variability of the NIR spectra of the refusals was described with its principal components thanks to a principal component analysis (PCA). The Pearson correlation was estimated to check the relationship between feed efficiency and refusals composition, i.e. sorting behaviour.

ResultsThe error of reproducibility of feed efficiency across diets was 2.95 MJ/d. This error was significantly larger than the errors of repeatability estimated within diet, which were 2.01 MJ/d within diet S-F+ and 2.40 MJ/d within diet S+F-. The CCC was 0.64 between feed efficiency estimated within diet S+F- and feed efficiency estimated within diet S-F+. This CCC was smaller than the one observed for feed efficiency estimated within diet between two subsequent lactation stages (CCC = 0.72 within diet S+F- and 0.85 within diet S-F+). Feed efficiency was poorly correlated to the first two principal components, which explained 90% of the total variability of the NIR spectra of the individual refusals. This suggests that feed sorting behaviour did not explain differences in feed efficiency.

ConclusionsFeed efficiency was significantly less reproducible across diets than repeatable within the same diet over subsequent lactation stages, but cows ranking for feed efficiency was not significantly affected by diet change. This loss in repeatability across diets could be due to a more pronounced feed sorting subsequent to the change in diet composition. However, the differences in sorting behaviour between cows were not associated to feed efficiency differences in this trial neither with the S+F- diet nor with the S-F+ diet. Those results have to be confirmed on diets having different forage to concentrate ratios to ensure that the least and most efficient cows will not change.

sentences: Present and Future Ecological Niche Modeling of Rift Valley fever in East Africa in Response to Climate Change Rift Valley fever (RVF) has been linked with recurrent outbreaks among humans and livestock in several parts of the globe. Predicting RVFs habitat suitability under different climate scenarios offers vital information for developing informed management schemes. The present study evaluated the probable impacts of climate change on the distribution of RVF disease in East Africa (E. A.), using the maximum entropy (MaxEnt) model and the disease outbreak cases. Considering the potential of the spread of the disease in the East Africa region, we utilized two representative concentration pathways (RCP 4.5 and RCP 8.5) climate scenarios in the 2050s and 2070s (average for 2041-2060, and 2061-2080), respectively. All models had satisfactory AUC values of more than 0.809, which are considered excellent. Jackknife tests revealed that Bio4 (temperature seasonality), land use, and population density were the main factors influencing RVF distribution in the region. From the risk maps generated, we infer that, without regulations, this disease might establish itself across more extensive areas in the region, including most of Rwanda and Burundi. The ongoing trade between East African countries and changing climates could intensify RVF spread into new geographic extents with suitable habitats for the important zoonosis. The predicted suitable areas for RVF in eastern Kenya, southern Tanzania, and Somalia overlaps to a large extent where cattle keeping and pastoralism are highly practiced, thereby signifying the urgency to manage and control the disease. This work validates RVF outbreak cases effectiveness to map the diseases distribution, thus contributing to enhanced ecological modeling and improved disease tracking and control efforts in East Africa.

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The Ecology of Palm Genomes: Repeat-associated genome size expansion is constrained by aridity O_LIGenome size varies 2,400-fold across plants, influencing their evolution through changes in cell size and cell division rates which impact plants environmental stress tolerance. Repetitive element expansion explains much genome size diversity, and the processes structuring repeat  communities are analogous to those structuring ecological communities. However, which environmental stressors influence repeat community dynamics has not yet been examined from an ecological perspective.
C_LIO_LIWe measured genome size and leveraged climatic data for 91% of genera within the ecologically diverse palm family (Arecaceae). We then generated genomic repeat profiles for 141 palm species, and analysed repeats using phylogenetically-informed linear models to explore relationships between repeat dynamics and environmental factors.
C_LIO_LIWe show that palm genome size and repeat  community composition are best explained by aridity. Specifically, EnSpm CACTA repeats were more abundant in palm species from wetter environments, which generally had larger genomes (>2.15Gbp/1C), suggesting amplification. In contrast, Ty1-copia Angela elements were more abundant in drier environments.
C_LIO_LIOur results suggest water stress inhibits the expansion of repeats through selection on upper genome size limits. However, Ty1-copia Angela elements, which may associate with stress-response genes, have amplified in arid-adapted palm species. Overall, we provide novel evidence of climate influencing the assembly of repeat  communities.
C_LI

sentences: Spinal cord injury reprograms muscle fibro-adipogenic progenitors to formheterotopic bones within muscles The cells-of-origin of neurogenic heterotopic ossifications (NHO), which develop frequently in the periarticular muscles following spinal cord injuries (SCI) and traumatic brain injuries, remain unclear because the skeletal muscle harbors two progenitor cell populations: satellite cells (SCs) which are myogenic, and fibro-adipogenic progenitors (FAPs) which are mesenchymal. Lineage-tracing experiments using the Cre recombinase /LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in the skeletal muscles under the control of the Pax7 or Prrx1 gene promotors respectively. These experiments demonstrate that following a muscle injury, SCI causes the upregulation of PDGFR on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with instead reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHO. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury suggesting that the cells-of-origin of NHO are locally derived from the injured muscle. We validated these findings in the human pathology using human NHO biopsies. PDGFR+ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice whereas CD56+ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to uncontrolled proliferation and differentiation into osteoblasts.

sentences: Cystic fibrosis patient-derived bronchial organoids unveil druggable pathways against Mycobacterium abscessus infection. Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. Both our knowledge on and models to investigate the host and bacterial determinants that drive Mabs pathology in CF patients remain rudimentary. Here, we evaluated whether the lung organoid technology from CF patients is appropriate for modelling Mabs infection and whether antioxidant treatment is a candidate therapeutic approach in the context of CF disease. We derived airway organoids (AOs) from lung biopsy of a CF patient and characterized these AO by assessing CF transmembrane conductance regulator (CFTR) function, mucus and reactive oxygen species (ROS) production, lipid peroxidation, and cell death. We microinjected smooth (S-) or rough (R-)Mabs in the lumen of AOs to evaluate its fitness, responses of AOs to infection, and treatment efficacy by colony forming unit assay, qPCR and microscopy. We show that CF patient-derived AOs exhibited low residual CFTR function, enhanced mucus accumulation, and increased oxidative stress, lipid peroxidation, and cell death at basal state. While in AOs, S Mabs formed biofilm, R Mabs formed cord serpentines and displayed a higher virulence. S and R Mabs replicated more efficiently in CF AOs than in AOs derived from healthy lung. Pharmacological activation of antioxidant pathways resulted in better control of Mabs growth. In conclusion, we have established CF patient-derived AOs as a suitable human system to decipher mechanisms of CF-enhanced respiratory infection by Mabs and confirmed antioxidant approaches as a potential host-directed strategy to improve Mabs infection control.

sentences: PLEKHA5 regulates mitotic progression by promoting APC/C localization to microtubules The anaphase-promoting complex/cyclosome (APC/C) coordinates advancement through mitosis via temporally controlled polyubiquitination of effector proteins. Despite the long-appreciated spatial organization of key events in mitosis mediated largely by cytoskeletal networks, the spatial regulation of APC/C, the major mitotic E3 ligase, is poorly understood. Here, we describe a microtubule-resident protein, PLEKHA5, as an interactor of APC/C and spatial regulator of its activity in mitosis. PLEKHA5 knockdown delayed mitotic progression, causing accumulation of APC/C substrates dependent upon the PLEKHA5-APC/C interaction. To assess the spatial importance of this interaction, we developed microtubule-localized proximity biotinylation tools, which revealed that depletion of PLEKHA5 decreased the extent of APC/C association with the microtubule cytoskeleton. This decreased APC/C microtubule-localization in turn prevented efficient loading of APC/C with its co-activator CDC20, leading to defects in E3 ligase catalytic activity. We propose that PLEKHA5 functions as an adaptor of APC/C that promotes its subcellular localization to microtubules and facilitates its activation by CDC20, thus ensuring the timely turnover of key mitotic APC/C substrates and proper progression through mitosis.

sentences: Chromosome-level de novo genome assembly of Telopea speciosissima (New South Wales waratah) using long-reads, linked-reads and Hi-C Telopea speciosissima, the New South Wales waratah, is an Australian endemic woody shrub in the family Proteaceae. Waratahs have great potential as a model clade to better understand processes of speciation, introgression and adaptation, and are significant from a horticultural perspective. Here, we report the first chromosome-level genome for T. speciosissima. Combining Oxford Nanopore long-reads, 10x Genomics Chromium linked-reads and Hi-C data, the assembly spans 823 Mb (scaffold N50 of 69.0 Mb) with 97.8 % of Embryophyta BUSCOs complete. We present a new method in Diploidocus (https://github.com/slimsuite/diploidocus) for classifying, curating and QC-filtering scaffolds, which combines read depths, k-mer frequencies and BUSCO predictions. We also present a new tool, DepthSizer (https://github.com/slimsuite/depthsizer), for genome size estimation from the read depth of single copy orthologues and estimate the genome size to be approximately 900 Mb. The largest 11 scaffolds contained 94.1 % of the assembly, conforming to the expected number of chromosomes (2n = 22). Genome annotation predicted 40,158 protein-coding genes, 351 rRNAs and 728 tRNAs. We investigated CYCLOIDEA (CYC) genes, which have a role in determination of floral symmetry, and confirm the presence of two copies in the genome. Read depth analysis of 180  Duplicated BUSCO genes suggest almost all are real duplications, increasing confidence in protein family analysis using annotated protein-coding genes, and highlighting a possible need to revise the BUSCO set for this lineage. The chromosome-level T. speciosissima reference genome (Tspe_v1) provides an important new genomic resource of Proteaceae to support the conservation of flora in Australia and further afield.

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QT-AMP: Quanti-Tray-based amplicon sequencing for simultaneous quantification and identification of enterococci for microbial source tracking Enterococcus is ubiquitous in human feces and has been adopted as a useful indicator of human fecal pollution in water. Although regular enterococci monitoring only examines their numbers, identification of human-specific Enterococcus species or genotypes could help in the discrimination of human fecal contamination from other environmental sources. We documented a new approach to characterize enterococci using an amplicon sequencing platform from Quanti Trays after following the counting of most probable numbers (MPN) of enterococci. We named this method as QT-AMP (Quanti-Tray-based amplicon sequencing). We tested surface water samples collected from three rivers in southwest Florida. We detected 11 Enterococcus species from 45 samples in 1.1 million sequence reads. The method detected three rare species and eight cosmopolitan species (Enterococcus faecalis, E.faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. avium, and E. durans) which have been commonly documented in various enterococci isolation studies. It is likely that the approximate detection level of QT-AMP is four orders of magnitude higher than regular 16S rRNA gene amplicon sequencing. QT-AMP revealed that a majority of Enterolert positive signals are actually the mixture of both enterococci and other facultative aerobes and anaerobes. QT-AMP may have the potential to monitor not only enterococci but also other pathogenic bacteria commonly found in natural environments. This QT-AMP could be a powerful tool to streamline the quantification and identification of enterococci and allows us to do more accurate and efficient microbial source tracking in various water management projects and human health risk assessment.

HighlightsO_LIA selected primer set (27f-519r) can differentiate over 50 Enterococcus species and is suitable for Illumina amplicon sequencing.
C_LIO_LIThe median of the relative contribution of enterococci reads among total sequencing reads was 82.7% and ranged between 0% and 100%.
C_LIO_LIEnterolert signals are most likely the mixture of enterococci and other facultative aerobes and anaerobes.
C_LIO_LIWe identified eight cosmopolitan enterococci species and three rare species.
C_LIO_LIThe median of the relative contribution of non-enterococci reads among total sequencing reads was 17.3%, respectively, and ranged between 0% and 100%.
C_LI

sentences: Single-cell genome-wide association reveals a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus Diversity in the human genome is one factor that confers resistance and susceptibility to infectious diseases. This is observed most dramatically during pandemics, where individuals exhibit large differences in risk and clinical outcomes against a pathogen infecting large portions of the worlds populations. Here, we developed scHi-HOST (single cell High-throughput Human in vitrO Susceptibility Testing), a method for rapidly identifying genetic variants that confer resistance and susceptibility to pathogens. scHi-HOST leverages scRNA-seq (single-cell RNA-sequencing) to simultaneously assign genetic identity to individual cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral entry and replication, and identify expression quantitative trait loci (eQTLs). Applying scHi-HOST to influenza A virus (IAV), we identified eQTLs at baseline and in genes that are induced by IAV infection. Integration of scHi-HOST with a human IAV challenge study (Prometheus) revealed that a missense variant in ERAP1 (Endoplasmic reticulum aminopeptidase 1; rs27895) was associated with IAV burden in cells and human volunteers. Functional studies using RNA interference, ERAP1 inhibitor, and overexpression of alternative alleles demonstrated that ERAP1 is exploited by IAV to promote infection. Specifically, the nonsynonymous substitution, which results in a glycine to aspartate substitution at ERAP1 residue 348, would disrupt the substrate binding pocket of ERAP1, likely resulting in a significantly altered preference for substrates, poorer catalytic efficiency, or both. Finally, rs27895 exhibits substantial population differentiation, with the higher frequency of the minor T allele in two African populations likely contributing to the greater permissivity of cells from these populations to IAV infection. scHi-HOST is an important resource for understanding susceptibility to influenza and is a broadly applicable method for decoding human genetics of infectious disease.

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Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic Pseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus (P) limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglycosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of a cystic fibrosis patient. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.

ClassificationIntegrated genomics and post-genomics approaches in microbial ecology

sentences: CCR2-dependent monocyte-derived cells restrict SARS-CoV-2 infection SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19) and current evidence suggests severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts viral burden in the lung. We find that a recently developed mouse-adapted MA-SARS-CoV-2 strain, as well as the emerging B. 1.351 variant, trigger an inflammatory response in the lung characterized by expression of pro-inflammatory cytokines and interferon-stimulated genes. scRNA-seq analysis of lung homogenates identified a hyper-inflammatory monocyte profile. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes infiltration of classical monocytes into the lung and expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.

sentences: From motif to function:Inferring the functions of long zinc finger proteins through combinatorial selection C2H2 zinc finger proteins (ZFPs) comprise of the largest group of DNA-binding proteins in human genome, and many of them contain long, tandem array of fingers, making the motif discovery, prediction of in vivo cis-regulatory elements (CREs), and understanding their functions particularly challenging. Previous work established that due to the dependent recognition between sub-motifs, the simple, additive recognition model impedes motif discovery and compromises our understanding about how ZFPs work. This work uses ZFP3, a 13-finger long ZFP with no known function, as case example to address the reverse question---given the full-length motif learned through in vitro experiments, like Spec-seq and HT-SELEX, how to reliably identify its in vivo cis-regulatory elements (CREs) and further predict this genes functions. Through sorting of all possible sites within the ChIP-seq peaks with similar predicted binding energy into groups and comparing the aggregate ChIP-seq signals between groups, it is evident that either its full-length or individual sub-motif alone fails to correctly identify all high-affinity specific sites without false-positives, thus it is necessary to revise current algorithm, and use both the core and upstream motifs as separate components to improve the prediction accuracy. Furthermore, significant number of regulatory elements of ZFP3 are found to be proximal to genes associated with microtubules organization and ciliogenesis, which coincides with the fact that ZFP3 is specifically upregulated in multiple ciliated cells. At last, local chromatin accessibility and active chromatin marks like H3K27ac are found to positively associate with the differential binding of ZFP3 between tested cell lines. Overall, this work establishes a novel "From motif to function" strategy for long ZFPs, and the data analysis workflows are implemented through R package TFCookbook for reuse onto other ZFPs.

sentences: Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling Accurate chromosome segregation during cell division requires amphitelic chromosome attachment to the spindle apparatus. It is ensured by the combined activity of the Spindle Assembly Checkpoint1 (SAC), a signaling mechanism that delays anaphase onset in response to unattached chromosomes, and an error correction mechanism that eliminates syntelic attachments2. The SAC becomes active when Mps1 kinase sequentially phosphorylates the kinetochore protein Spc105/KNL1 and the signaling proteins that Spc105/KNL1 recruits to facilitate the production of the Mitotic Checkpoint Complex3-8 (MCC). The error correction mechanism is regulated by the Aurora B kinase, but Aurora B also promotes SAC signaling via indirect mechanisms9-12. Here we present evidence that Aurora B kinase activity directly promotes MCC production by working downstream of Mps1 in budding yeast and human cells. Using the ectopic SAC activation (eSAC) system, we find that the conditional dimerization of Aurora B in budding yeast, and an Aurora B recruitment domain in HeLa cells, with either Bub1 or Mad1, but not the phosphodomain of Spc105/KNL1, leads to ectopic MCC production and mitotic arrest13-16. Importantly, Bub1 must recruit both Mad1 and Cdc20 for this ectopic signaling activity. These and other data show that Aurora B cooperates with Bub1 to promote MCC production, but only after Mps1 licenses Bub1 recruitment to the kinetochore. This direct involvement of Aurora B in SAC signaling may maintain SAC signaling even after Mps1 activity in the kinetochore is lowered.

sentences: Reagent- and actuator-free analysis of individual erythrocytes using three-dimensional quantitative phase imaging and capillary microfluidics Histopathological examination of blood cells plays a crucial role in the diagnosis of various diseases. However, it involves time-consuming and laborious staining procedures required for microscopic review by medical experts and is not directly applicable for point-of-care diagnosis in resource-limited locations. This study reports a dilution-, actuation- and label-free method for the analysis of individual red blood cells (RBCs) using a capillary microfluidic device and quantitative phase imaging. Blood, without any sample treatment, is directly loaded into a micrometer-thick channel such that it forms a quasi-monolayer inside the channel. The morphological and biochemical properties of RBCs, including hemoglobin concentration, hemoglobin content, and corpuscular volume, were retrieved using the refractive index tomograms of individual RBCs measured using 3D quantitative phase imaging. The deformability of individual RBCs was also obtained by measuring the dynamic membrane fluctuations. The proposed framework applies to other imaging modalities and biomedical applications, facilitating rapid and cost-effective diagnosis and prognosis of diseases.

sentences: VCP protects neurons from proteopathic seeding Uptake and spread of proteopathic seeds, such as S, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of S seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase S seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of S seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to S since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

sentences: Surviving hypothermia by ferritin-mediated iron detoxification How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian Fth1/ferritin. Moreover, we show that inducing expression of Fth1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/Fth1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/Fth1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia.

sentences: Spatial proteomics reveals profound subcellular reorganization in human keratinocytes exposed to UVA light The effects of UV light on the skin have been extensively investigated. However, systematic information about how exposure to UVA light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

sentences: UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS Amyotrophic Lateral Sclerosis (ALS) is a fatal, neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS is caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate a function of UBQLN2 in regulating activity of the domesticated gag-pol retrotransposon  paternally expressed gene 10 (PEG10) in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated  nucleocapsid fragment, which uniquely localizes to the nucleus and changes expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

sentences: QTL mapping in an interspecific sorghum population uncovers candidate regulators of salinity tolerance Salt stress impedes plant growth and disrupts normal metabolic processes, resulting in decreased biomass and increased leaf senescence. Therefore, the ability of a plant to maintain biomass when exposed to salinity stress is critical for the production of salt tolerant crops. To identify the genetic basis of salt tolerance in an agronomically important grain crop, we used a recombinant inbred line (RIL) population derived from an interspecific cross between domesticated Sorghum bicolor (inbred Tx7000) and a wild relative, Sorghum propinquum. A high-density genetic map was generated from 177 F3:5 RILs and covered the 10 Sorghum chromosomes with 1991 markers. The genetic map was used to identify 19 total QTL related to plant growth and overall health in optimal and saline conditions. Of these 19 QTL detected, 10 were specific to the salt stress response. The salt-responsive QTL contained numerous genes that have been previously shown to play a role in ionic tolerance, tissue tolerance, and osmotic tolerance, including many aquaporins.

sentences: A heuristic underlies the search for relief in fruit flies Humans rely on multiple systems of sensory information to make decisions. However, strategies that shorten decision-making time by taking into account fewer but more essential elements of information are preferred to strategies involving complex analyses. These "shortcuts to decision" are also termed "heuristics". The identification of heuristic principles in species phylogenetically distant to humans would shed light on the evolutionary origin of speed-accuracy trade-offs and offer the possibility to investigate the brain representations of such trade-offs, urgency, and uncertainty. During experiments on spatial learning, we acknowledged that the search strategies of the invertebrate Drosophila melanogaster, the common fruit fly, resembled a spatial heuristic. Here we show that the fruit fly applies a heuristic termed the "Nearest Neighbour Rule" to avoid bitter taste (a negative stimulation). That is, the fly visits the salient location closest to its current position to hopefully stop the negative stimulation. Only if this strategy proves unsuccessful, the animal uses other learned associations to avoid bitter taste. The acknowledgement of a heuristic in D. melanogaster supports the view that invertebrates can leverage on  economic principles when making choices and that the existence of heuristics in evolution dates to at least 600 million years ago.

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A Compressive Sensing Approach to Inferring Cognitive Representations with Reverse Correlation Uncovering high-level cognitive representations of categories such as faces is an elusive goal that has been frequently sought using reverse correlation, a technique employed in fields ranging from neurophysiology to cognitive psychology. In reverse correlation, subjects are asked to make perceptual judgments (e.g., "do you see a face?") about richly varying stimuli (e.g., white noise), and observed responses are then regressed against stimuli to yield reconstructions of the underlying cognitive representation. However, many thousands of stimulus-response pairs are frequently required, which severely limits the breadth of studies that are feasible using this powerful method. Techniques that are currently employed to improve efficiency, such as filtering the reconstruction, nevertheless bias the outcome. Here, we show that an advanced signal processing technique for improving sampling efficiency - compressive sensing - is directly compatible with reverse correlation. A trio of simulations are performed to demonstrate that compressive sensing can reduce the required stimulus-response pairs by up to 90% without biasing the reconstruction or can retrospectively improve the accuracy of the reconstructions on existing data. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

Significance StatementUncovering cognitive representations is an elusive goal that is increasingly pursued using the reverse correlation method, wherein human subjects make judgments about vague stimuli. Employing reverse correlation often entails collecting thousands of stimulus-response pairs, which severely limits the breadth of studies that are feasible using the method. Here we show that this methodological barrier can be overcome using compressive sensing, an advanced signal processing technique designed to improve sampling efficiency. Three sets of simulations are performed to demonstrate that compressive sensing can improve the accuracy of reconstructed cognitive representations and dramatically reduce the required number of stimulus-response pairs. This work concludes by outlining the potential of compressive sensing to improve representation reconstruction throughout the field of neuroscience and beyond.

sentences: Spontaneous parthenogenesis in the parasitoid wasp Cotesia typhae: low frequency anomaly or evolving process? Hymenopterans are haplodiploids and unlike most other Arthropods they do not possess sexual chromosomes. Sex determination typically happens via the ploidy of individuals: haploids become males and diploids become females. Arrhenotoky is believed to be the ancestral reproduction mode in Hymenopterans, with haploid males produced parthenogenetically, and diploid females produced sexually. However, a number of transitions towards thelytoky (diploid females produced parthenogenetically) have appeared in Hymenopterans, and in most cases populations or species are either totally arrhenotokous or totally thelytokous. Here we present the case of Cotesia typhae (Fernandez-Triana), a Braconidae that produces parthenogenetic females at a low frequency. The phenotyping of two laboratory strains and one natural population showed that this frequency is variable, and that this rare thelytokous phenomenon also happens in the wild. Moreover, mated females from one of the laboratory strains produce a few parthenogenetic daughters among a majority of sexual daughters. The analysis of daughters of heterozygous virgin females allowed us to show that a mechanism similar to automixis with central fusion is very likely at play in C. typhae. This mechanism allows some parts of the genome to remain heterozygous, especially at the chromosomes centromeres, which can be advantageous depending on the sex determination system involved. Lastly, in most species, the origin of thelytoky is either bacterial or genetic, and an antibiotic treatment as well as PCR experiments did not demonstrate a bacterial cause in C. typhae. The unusual case of low parthenogenetic frequency described in this species constitutes another example of the fascinating diversity of sex determination systems in Arthropods.

sentences: Transcriptomic similarities and differences between mouse models and human Alzheimer's Disease Alzheimers disease (AD) is a multifactorial pathology with most cases having a sporadic presentation. Yet, preclinical research studies relied for decades on transgenic models overexpressing human genes found in AD autosomal dominant patients. Recently, knock-in (KI) models, such as the novel hA{beta}-KI mouse, were introduced; however, its translational potential is still being addressed. To unveil core molecular programs in AD, we compared hippocampal transcriptomic profiles of transgenic (5xFAD and APP/PS1) and KI (hA{beta}-KI) mouse models with early- (EOAD) and late- (LOAD) onset AD patients. Comparison of differential expression analyses showed seven genes (S100A6, C1QB, CD33, CD14, SLC11A1, KCNK1 and SST) consistently already among models and human AD. Experimental validation of these genes revealed four altered in mice (SLC11A1, S100A6, CD14, CD33, C1QB) and three in humans (S100A6, SLC11A1, KCNK). Our analysis revealed that all three mouse models presented more Gene Ontology biological processes (GOBP) terms and enriched signaling pathways in common with LOAD than with EOAD subjects. Furthermore, semantic similarity of enriched GOBP terms highlighted model-specific biological alterations that might be further explored. Finally, we identified 17 transcription factors potentially acting as master regulators of AD in all models. Our cross-species exploratory analyses evidenced that the three mouse models presented a remarkable specificity of GOBP to LOAD, but the representation of processes in each model may differ. Thus, the molecular and cellular mechanisms under study should be a factor during mouse model selection in AD.

sentences: Locational memory of macrovessel vascular cells is transcriptionally imprinted The locational predisposition of vascular pathologies illustrates the need for a better insight into vascular heterogeneity. To investigate the transcriptomic basis of angiodiversity, we isolated and analyzed transcriptomes from endothelial cells and vascular smooth muscle cells from nine different adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We identified both reported and novel expression patterns defining specialized adult blood vessels. Our findings also show that adult vascular cells in culture express a remarkably high number of transcription factors crucial to organ development in the embryo. The persistent expression of these genes in culture indicates that these genes are not regulated by the flow or surrounding cell types but are rather fixed in the molecular memory. Therefore, our findings prompt the re-thinking of the extrapolation of results from single-origin endothelial cell systems.

sentences: Controlling homology-directed repair outcomes in human stem cells with dCas9 Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive  dCas9was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

sentences: Prevalence of mutualism in a simple model of microbial co-evolution Evolutionary transitions among ecological interactions are widely known, although their detailed dynamics remain absent for most population models. Adaptive dynamics has been used to illustrate how the parameters of population models might shift through evolution, but within an ecological regime. Here we use adaptive dynamics combined with a generalised logistic model of population dynamics to show that transitions of ecological interactions might appear as a consequence of evolution. To this purpose we introduce a two-microbial toy model in which population parameters are determined by a bookkeeping of resources taken from (and excreted to) the environment, as well as from the byproducts of the other species. Despite its simplicity, this model exhibits all sorts of ecological transitions, some of which resemble those found in nature. Overall, the model shows a clear trend toward the emergence of mutualism.

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A 1-bp deletion in bovine QRICH2 causes low sperm count and immotile sperm with multiple morphological abnormalities BackgroundSemen quality and male fertility are monitored in artificial insemination bulls to ensure high insemination success rates. Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced twelve ejaculates with an aberrantly low number of sperm (0.2{+/-}0.2 x 109 sperm per ml) which were mostly immotile due to multiple morphological abnormalities.

ResultsThe genome of the bull was sequenced at 12-fold coverage to investigate a suspected genetic cause. Comparing the sequence variant genotypes of the bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence of QRICH2 encoding glutamine rich 2 as a compelling candidate causal variant. The 1-bp deletion causes a frameshift in translation and induces a premature termination codon (ENSBTAP00000018337.1:p.Cys1644AlafsTer52). The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subjected to nonsense-mediated mRNA decay. The 1-bp deletion resides on a 675 kb haplotype spanning 181 SNPs from the Illumina BovineHD Bead chip. The haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. This analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities primarily affecting the sperm flagellum and, to a lesser extent, the sperm head.

ConclusionsA recessive loss-of-function allele of bovine QRICH2 likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.

sentences: Transcriptomes of Electrophysiologically Recorded Dbx1-derived Inspiratory Neurons of the preBötzinger Complex in Neonatal Mice Breathing depends on interneurons in the preBotzinger complex (preBotC) derived from Dbx1-expressing precursors. Here we investigate whether rhythm- and pattern-generating functions reside in discrete classes of Dbx1 preBotC neurons. In a slice model of breathing with [~]5 s cycle period, putatively rhythmogenic Type-1 Dbx1 preBotC neurons activate 100-300 ms prior to Type-2 neurons, putatively specialized for output pattern, and 300-500 ms prior to the inspiratory motor output. We sequenced Type-1 and Type-2 transcriptomes and identified differential expression of 123 genes including ionotropic receptors (Gria3 and Gabra1) that may explain their preinspiratory activation profiles and Ca2+ signaling (Cracr2a, Sgk1) involved in inspiratory and sigh bursts. Surprisingly, neuropeptide receptors that influence breathing (e.g., {micro}-opioid and bombesin-like peptide receptors) were only sparsely expressed, which suggests that cognate peptides and opioid drugs exert their profound effects on a small fraction of the preBotC core. These data in the public domain help explain breathings neural origins.

sentences: Reclassification of Shigella species as later heterotypic synonyms of Escherichia coli in the Genome Taxonomy Database Members of the genus Shigella have high genomic similarity to Escherichia coli and are often considered to be atypical members of this species. In an attempt to retain Shigella species as recognizable entities, they were reclassified as Escherichia species in the Genome Taxonomy Database (GTDB) using an operational average nucleotide identity (ANI)-based approach nucleated around type strains. This resulted in nearly 80% of E. coli genomes being reclassified to new species including the common laboratory strain E. coli K-12 (to  E. flexneri) because it is more closely related to the type strain of Shigella flexneri than it is to the type strain of E. coli. Here we resolve this conundrum by treating Shigella species as later heterotypic synonyms of E. coli, present evidence supporting this reclassification, and show that assigning E. coli/Shigella strains to a single species is congruent with the GTDB-adopted genomic species definition.

sentences: RECIPROCALLY INHIBITORY CIRCUITS OPERATING WITH DISTINCT MECHANISMS ARE DIFFERENTLY ROBUST TO PERTURBATION AND MODULATION What features are important for circuit robustness? Reciprocal inhibition is a building block in many circuits. We used dynamic clamp to create reciprocally inhibitory circuits from pharmacologically isolated neurons of the crab stomatogastric ganglion by injecting artificial synaptic (ISyn) and hyperpolarization-activated inward (IH) currents. There are two mechanisms of antiphase oscillations in these circuits: "escape" and "release". In release, the active neuron primarily controls the off/on transitions. In escape, the inhibited neuron controls the transitions. We characterized the robustness of escape and release circuits to alterations in circuit parameters, temperature, and neuromodulation. We found that escape circuits rely on tight correlations between synaptic and H conductances to generate bursting but are resilient to temperature increase. Release circuits are robust to variations in synaptic and H conductances but fragile to temperature increase. The modulatory current (IMI) restores oscillations in release circuits but has little effect in escape circuits. Thus, the same perturbation can have dramatically different effects depending on the circuits mechanism of operation that may not be observable from basal circuit activity.

sentences: Identifying Differential Methylation in Cancer Epigenetics via a Bayesian Functional Regression Model DNA methylation plays an essential role in regulating gene activity, modulating disease risk, and determining treatment response. Researchers can obtain insight into methylation patterns at a single nucleotide level utilizing next-generation sequencing technologies. However, complex features inherent in the data obtained via these technologies pose challenges beyond the typical big data problems. Identifying differentially methylated cytosines (dmc) or regions is one of such challenges. Current methodologies for identifying dmcs fall short in handling low read-depth data and missing values, capturing functional data patterns, granting multiple covariates (categorical, continuous, or combination), and multiple group comparisons. We have developed an efficient method to identify dmcs based on a Bayesian functional regression approach, termed DMCFB, that tackles these shortcomings. Through simulation studies, we establish that DMCFB outperforms current methods and results in better smoothing, and efficient imputation. We apply the proposed method to analyze a dataset containing patients with acute promyelocytic leukemia and control samples. With DMCFB, we discovered many new dmcs, and more importantly, exhibited enhanced consistency of differential methylation within islands and at their adjacent shores. Furthermore, we detected differential methylation at more of the binding sites of the fused gene involved in this cancer.

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Loci for prediction of penicillin and tetracycline susceptibility in Neisseria gonorrhoeae: a genome wide association study BackgroundWhile Neisseria gonorrhoeae poses an urgent public health threat because of increasing antimicrobial resistance, much of the circulating population remains susceptible to historical treatment regimens. Point-of-care diagnostics that report susceptibility could allow for reintroduction of these regimens, but development of such diagnostics has been limited to ciprofloxacin, for which susceptibility can be predicted from a single locus.

MethodsWe assembled a dataset of 12,045 N. gonorrhoeae genomes with phenotypic resistance data for tetracycline (n = 3,611) and penicillin (n = 6,935). Using conditional genome wide association studies (GWAS), we sought to define genetic variants associated with susceptibility to penicillin and tetracycline. We evaluated the sensitivity and specificity of these variants for predicting susceptibility and non-resistance in our collection of gonococcal genomes.

FindingsIn our conditional penicillin GWAS, the presence of a genetic variant defined by a non-mosaic penA allele without an insertion at codon 345 was significantly associated with penicillin susceptibility and had the highest negative effect size of significant variants (p = 5.0 x 10-14, {beta} = -2.5). In combination with the absence of blaTEM, this variant predicted penicillin susceptibility with high specificity (99.8%) and modest sensitivity (36.7%). For tetracycline, the wild type allele at rpsJ codon 57, encoding valine, was significantly associated with tetracycline susceptibility (p = 5.6 x 10-16, {beta} = -1.6) after conditioning on the presence of tetM. The combination of rpsJ codon 57 allele and tetM absence predicted tetracycline susceptibility with high specificity (97.2%) and sensitivity (88.7%).

InterpretationAs few as two genetic loci can predict susceptibility to penicillin and tetracycline in N. gonorrhoeae with high specificity. Molecular point-of-care diagnostics targeting these loci have the potential to increase available treatments for gonorrhea.

FundingNational Institute of Allergy and Infectious Diseases, the National Science Foundation, and the Smith Family Foundation

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Neisseria gonorrhoeae" and "diagnostic" or "assay" plus "penicillin" or "tetracycline" for reports in any language published up to July 1, 2021. We additionally searched for "Neisseria gonorrhoeae" and "genome wide association study". We found that previously proposed molecular diagnostics for penicillin and tetracycline susceptibility either exclusively focused on plasmid-mediated resistance (i.e., targeting blaTEM or tetM) or did not include variants in genes encoding antibiotic targets (e.g., did not include penA or rpsJ). Targets for molecular surveillance have focused on resistance-associated alleles rather than susceptibility-associated alleles. We did not find any previous penicillin or tetracycline conditional genome wide association studies (GWAS) in N. gonorrhoeae.

Added value of this studyTo identify targets for molecular diagnostics that predict penicillin and tetracycline susceptibility, we conducted GWAS conditioning on the presence of plasmid-mediated resistance determinants to detect chromosomal loci with the highest association with susceptibility. We discovered a sequence (penA_01) that differentiates susceptible isolates from those with a resistance-associated insertion at codon 345 and from those with mosaic penA alleles, which is associated with penicillin susceptibility. We also found that rpsJ codon 57 was the chromosomal locus contributing the most to tetracycline susceptibility. The combination of these chromosomal loci and the absence of plasmid encoded determinants predicts penicillin and tetracycline susceptibility with high specificity in both a large global collection of N. gonorrhoeae and a validation dataset consisting of recently published genomes from CDCs Gonococcal Isolate Surveillance Program (GISP) surveillance collected in 2018.

Implications of all the available evidenceThe chromosomal loci penA_01 and rpsJ codon 57 in combination with plasmid loci blaTEM and tetM are candidates for the development of point-of-care molecular diagnostics for penicillin and tetracycline susceptibility. The loci may be combined with the currently available ciprofloxacin susceptibility diagnostics to predict susceptibility to multiple antibiotics. Additionally, our study suggests that conditional GWAS focused on variants associated with susceptibility may be a promising approach to identify minimal sets of loci for molecular diagnostics and surveillance.

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Gestational insulin resistance is mediated by the gut microbiome-indoleamine 2,3-dioxygenase axis Background and aimsNormal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM - maternal metabolism interaction.

MethodsThe GM and plasma metabolome of CD1, NIH-Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT).

ResultsSignificant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post- pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy.

ConclusionsGM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

sentences: Host Induced Gene Silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

sentences: A structured observation distribution for generative biological sequence prediction and forecasting Generative probabilistic modeling of biological sequences has widespread existing and potential application across biology and biomedicine, from evolutionary biology to epidemiology to protein design. Many standard sequence analysis methods preprocess data using a multiple sequence alignment (MSA) algorithm, one of the most widely used computational methods in all of science. However, as we show in this article, training generative probabilistic models with MSA preprocessing leads to statistical pathologies in the context of sequence prediction and forecasting. To address these problems, we propose a principled drop-in alternative to MSA preprocessing in the form of a structured observation distribution (the ``MuE" distribution). The MuE is a latent alignment model in which not only the alignment variable but also the regressor sequence can be latent. We prove theoretically that the MuE distribution comprehensively generalizes popular methods for inferring biological sequence alignments, and provide a precise characterization of how such biological models have differed from natural language latent alignment models. We show empirically that models that use the MuE as an observation distribution outperform comparable methods across a variety of datasets, and apply MuE models to a novel problem for generative probabilistic sequence models: forecasting pathogen evolution.

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Urbanization, Bird Species Richness And Abundance Within Ibadan Metropolis, Nigeria Urbanisation is considered as one of the most profound threat to wildlife, with habitat loss and fragmentation being predominant. This study assessed the impacts of urbanisation on richness, abundance of bird species within Ibadan metropolis, Nigeria. A uniform grid of 500 square meters was installed on the map of Ibadan Metropolis using QGIS to produce 499 grid points distributed across the five urban local governments. 100 grids were selected randomly, identified with mapinR software application and surveyed with 5 point counts within each gird, established at 200m interval to avoid double counting. Each point count was observed for 5 minutes using a pair of 8x42mm binoculars within a 50m radius. Habitat variables like number of buildings, trees, paved roads, communication masts were also recorded.

A total number of 56 species of birds were observed at the end of the assessment, classified into 30 families. The test of statistics showed that there was no statistically significant difference in bird species richness between the Local Governments. The test of statistics showed that there was no statistically significant difference in bird species abundance between the local governments. The test of between-subjects effects revealed that there were no statistical significant effects when all the habitat variables were computed in the model on species richness. The number of paved roads and number of vehicles showed a significant effect on bird species abundance while others variables in the model did not exact statistically significant effects on bird species abundance.

The study therefore concluded that habitat actions due to urbanisation have not affected the richness and abundance of birds species found in all the local governments. The only habitat variables that have currently exacted significant effect on species abundance within the metropolis are number of vehicles and paved roads.

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Using hypertemporal Sentinel-1 data to predict forest growing stock volume In this study, we assess the potential of long time series of Sentinel-1 SAR data to predict forest growing stock volume and evaluate the temporal dynamics of the predictions. The boreal coniferous forests study site is located near the Hyytiala forest station in central Finland and covers an area of 2,500 km2 with nearly 17,000 stands. We considered several prediction approaches (linear, support vector and random forests regression) and fine-tuned them to predict growing stock volume in several evaluation scenarios. The analyses used 96 Sentinel-1 images acquired over three years. Different approaches for aggregating SAR images and choosing feature (predictor) variables were evaluated. Our results demonstrate considerable decrease in RMSEs of growing stock volume as the number of images increases. While prediction accuracy using individual Sentinel-1 images varied from 85 to 91 m3/ha RMSE (relative RMSE 50-53%), RMSE with combined images decreased to 75.6 m3/ha (relative RMSE 44%). Feature extraction and dimension reduction techniques facilitated achieving the near-optimal prediction accuracy using only 8-10 images. When using assemblages of eight consecutive images, the GSV was predicted with the greatest accuracy when initial acquisitions started between September and January.

HighlightsO_LITime series of 96 Sentinel-1 images is analysed over study area with 17,762 forest stands.
C_LIO_LIRigorous evaluation of tools for SAR feature selection and GSV prediction.
C_LIO_LIImproved periodic seasonality using assemblages of consecutive Sentinel-1 images.
C_LIO_LIAnalysis of combining images acquired in "frozen" and "dry summer" conditions.
C_LIO_LICompetitive estimates using calculation of prediction errors with stand-area weighting.
C_LI

sentences: DrosoPhyla: genomic resources for drosophilid phylogeny and systematics The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4000 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the groups and genera in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred well-supported group and species trees for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

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

sentences: Cohesin-mediated loop anchors confine the location of human replication origins DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability across dividing cells in metazoans. It is currently unknown how the location of replication origins and the timing of their activation is determined in the human genome. Here, we dissect the role for G1 phase topologically associating domains (TADs), subTADs, and loops in the activation of replication initiation zones (IZs). We identify twelve subtypes of self-interacting chromatin domains distinguished by their degree of nesting, the presence of corner dot structures indicative of loops, and their co-localization with A/B compartments. Early replicating IZs localize to boundaries of nested corner-dot TAD/subTADs anchored by high density arrays of co-occupied CTCF+cohesin binding sites with divergently oriented motifs. By contrast, late replicating IZs localize to weak TADs/subTAD boundaries devoid of corner dots and most often anchored by singlet CTCF+cohesin sites. Upon global knock-down of cohesin-mediated loops in G1, early wave focal IZs replicate later in S phase and convert to diffuse placement along the genome. Moreover, IZs in mid-late S phase are delayed to the final minutes before entry into G2 when cohesin-mediated dot-less boundaries are ablated. We also delete a specific loop anchor and observe a sharp local delay of an early wave IZ to replication in late S phase. Our data demonstrate that cohesin-mediated loops at genetically-encoded TAD/subTAD boundaries in G1 phase are an essential determinant of the precise genomic placement of human replication origins in S phase.

sentences: GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo, and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro compared to unsorted and GP2- fractions, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminated the formation of teratoma in vivo. This study establishes that the GP2-enriched PPs represent a safe option for T1D treatment.

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Mutation in Abl kinase with altered drug binding kinetics indicates a novel mechanism of imatinib resistance Protein kinase inhibitors are potent anti-cancer therapeutics (1). For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for Chronic Myeloid Leukemia (CML) by 80% (2, 3), but 22-41% of patients acquire resistance to imatinib (4). About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain (5), in which more than a hundred different mutations have been identified (6-8). Some mutations are located near the imatinib binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug binding site (9) and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies (10). Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease-relevant or resistance-causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than two-fold compared to Abl wild type. Surprisingly, one-third of mutations in Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified a clinical Abl mutation that binds imatinib with wild type-like affinity but dissociates from imatinib three times faster. Given the relevance of residence time for drug efficacy (11-14), mutations that alter binding kinetics could cause resistance in the non-equilibrium environment of the body where drug export and clearance play critical roles.

SignificanceWe performed the first in cell screen of imatinib binding against a library of Abl kinase mutants derived from patients with imatinib-resistant CML. The majority of mutations readily bind imatinib, posing the question of how these mutations cause resistance in patients. We identified a kinetic mutant that binds imatinib with wild type affinity but dissociates considerably faster from the mutant kinase. Using NMR and molecular dynamics, we found that this mutation increases the conformational dynamics of the mutant protein, linking conformational dynamics of the protein to drug dissociation. The results underline the importance of drug dissociation kinetics for drug efficacy and propose a novel kinetic resistance mechanism that may be targetable by altering drug treatment schedules.

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Gammadelta T Cells Are Required for CD8+ T Cell Response to Vaccinia Viral Infection Vaccinia virus (VV) is the most studied member of the poxvirus family, is responsible for the successful elimination of smallpox worldwide, and has been developed as a vaccine vehicle for infectious diseases and cancer immunotherapy. We have previously shown that the unique potency of VV in the activation of CD8+ T cell response is dependent on efficient activation of the innate immune system through Toll-like receptor (TLR)-dependent and -independent pathways. However, it remains incompletely defined what regulate CD8+ T cell response to VV infection. In this study, we showed that {gamma}{delta}T cells play an important role in promoting CD8+ T cell response to VV infection. We found that {gamma}{delta}T cells can directly present viral antigens in the context MHC-I for CD8+ T cell activation to VV in vivo, and we further demonstrated that cell-intrinsic MyD88 signaling in {gamma}{delta}T cells is required for activation of {gamma}{delta}T cells and CD8+ T cells. These results illustrate a critical role for {gamma}{delta}T cells in the regulation of adaptive T cell response to viral infection and may shed light on the design of more effective vaccine strategies based on manipulation of {gamma}{delta}T cells.

ImportanceTargeting the immune systems has powerful potentials to treat many disorders, such as some cancers and viral infections. By understanding how the immune system responds to model infections, we can better determine strategies to manipulate our immune systems. Vaccinia virus is responsible for the worldwide elimination of smallpox and produces one of the longest immune responses known in humans. We know from previous findings that NK cells are required for initial immune response and CD8+ T cells are required for the elimination of the virus. How CD8+ T cells are activated in response to Vaccinia virus is not fully understood. This manuscript found that {gamma}{delta}T cells activate CD8+ T cells in response to Vaccinia virus infection through MyD88 pathway

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Evolution of tunnels in alpha/beta-hydrolase fold proteins - what can we learn from studying epoxide hydrolases? The evolutionary variability of a proteins residues is highly dependent on protein region and function. Solvent-exposed residues, excluding those at interaction interfaces, are more variable than buried residues whereas active site residues are considered to be conserved. The abovementioned rules apply also to /{beta}-hydrolase fold proteins - one of the oldest and the biggest superfamily of enzymes with buried active sites equipped with tunnels linking the reaction site with the exterior. We selected soluble epoxide hydrolases as representative of this family to conduct the first systematic study on the evolution of tunnels. We hypothesised that tunnels are lined by mostly conserved residues, and are equipped with a number of specific variable residues that are able to respond to evolutionary pressure. The hypothesis was confirmed, and we suggested a general and detailed way of the tunnels evolution analysis based on entropy values calculated for tunnels residues. We also found three different cases of entropy distribution among tunnel-lining residues. These observations can be applied for protein reengineering mimicking the natural evolution process. We propose a  perforation mechanism for new tunnels design via the merging of internal cavities or protein surface perforation. Based on the literature data, such a strategy of new tunnel design could significantly improve the enzymes performance and can be applied widely for enzymes with buried active sites.

Author SummarySo far very little is known about proteins tunnels evolution. The goal of this study is to evaluate the evolution of tunnels in the family of soluble epoxide hydrolases - representatives of numerous /{beta}-hydrolase fold enzymes. As a result two types of tunnels evolution analysis were proposed (a general and a detailed approach), as well as a  perforation mechanism which can mimic native evolution in proteins and can be used as an additional strategy for enzymes redesign.

sentences: No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

sentences: Innate heuristics and fast learning support escape route selection in mice When faced with imminent danger, animals must rapidly take defensive actions to reach safety. Mice can react to innately threatening stimuli in less than 250 milliseconds [1] and, in simple environments, use spatial memory to quickly escape to shelter [2,3]. Natural habitats, however, often offer multiple routes to safety which animals must rapidly identify and choose from to maximize the chances of survival [4]. This is challenging because while rodents can learn to navigate complex mazes to obtain rewards [5,6], learning the value of different routes through trial-and-error during escape from threat would likely be deadly. Here we have investigated how mice learn to choose between different escape routes to shelter. By using environments with paths to shelter of varying length and geometry we find that mice prefer options that minimize both path distance and path angle relative to the shelter. This choice strategy is already present during the first threat encounter and after only ~10 minutes of exploration in a novel environment, indicating that route selection does not require experience of escaping. Instead, an innate heuristic is used to assign threat survival value to alternative paths after rapidly learning the spatial environment. This route selection process is flexible and allows quick adaptation to arenas with dynamic geometries. Computational modelling of different classes of reinforcement learning agents shows that the observed behavior can be replicated by model-based agents acting in an environment where the shelter location is rewarding during exploration. These results show that mice combine fast spatial learning with innate heuristics to choose escape routes with the highest survival value. They further suggest that integrating priors acquired through evolution with knowledge learned from experience supports adaptation to changing environments while minimizing the need for trial-and-error when the errors are very costly.

sentences: Olfactory sensory preconditioning in Drosophila: role of memory forgetting in gating S1/S2 associations Learning and memory storage is a complex process that has proven challenging to tackle. It is likely that, in real nature, the instructive value of reinforcing experiences is acquired rather than innate. The association between seemingly neutral stimuli increases the gamut of possibilities to create meaningful associations and increases the predictive power of moment-by-moment experiences. Here we report physiological and behavioral evidence of olfactory unimodal sensory preconditioning in fruit flies. We show that the presentation of a pair of odors (S1 and S2) before one of them (S1) is associated with electric shocks elicits a conditional response not only to the trained odor (S1) but to the odor previously paired with it (S2). This occurs even if the S2 odor was never presented in contiguity with the aversive stimulus. In addition, we show that inhibition of the small G protein and known forgetting regulator Rac1 facilitates the association between S1/S2 odors. These results indicate that flies can infer value to non-paired odor based on the previous associative structure between odors, and inhibition of Rac1 lengthens the time of olfactory "sensory buffer," allowing linking of neutral odors presented in sequence.

sentences: Demographic Inferences and Loci Under Selection in a Recently Expanded Coral Population Coral poleward range expansions in response to warming oceans have been historically observed, however contemporary expansion rates of some coral species have become more rapid as global temperatures rise at unprecedented rates. Range expansion can lead to reduced genetic diversity and surfing of deleterious mutations in expanding populations, potentially limiting the ability for adaption and persistence in novel environments. Successful expansions that overcome these founder effects and colonize new habitat have been attributed to multiple introductions from different sources, hybridization with native populations, or rapid adaptive evolution. Here, we investigate population genomic patterns of the reef-building coral Acropora hyacinthus along a latitudinal cline that includes a well-established range expansion front in Japan using 2b-RAD sequencing. A total of 184 coral samples were collected across seven sites spanning from [~]24{degrees}N to near its northern range front at [~]33{degrees}N. We uncover the presence of three cryptic lineages of A. hyacinthus, which occupy discrete areas within this region. Only one lineage is present at the expansion front and we find evidence of its historical occupation of marginal habitats. Within this lineage we also find evidence of bottleneck pressures associated with expansion events including higher clonality, increased linkage disequilibrium, and lower genetic diversity in range edge populations compared to core populations. Asymmetric migration between populations was also detected with lower migration from edge sites. Lastly, we describe genomic signatures of local adaptation potentially attributed to lower winter temperatures experienced at the more recently expanded northern populations. Together these data illuminate the genomic consequences of range expansion in a coral and highlight how adaptation to colder temperatures along the expansion front may facilitate further range expansion in this coral lineage.

sentences: Diverse DNA modification in marine prokaryotic and viral communities Chemical modifications of DNA, including methylation, play an important role in prokaryotes and viruses. However, our knowledge of the modification systems in environmental microbial communities, typically dominated by members not yet cultured, is limited. Here, we conducted  metaepigenomic analyses by single-molecule real-time sequencing of marine microbial communities. In total, 233 and 163 metagenomic assembly genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTases were not associated with the defense mechanism. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative motifs in the methylation system that are highly conserved in Alphaproteobacteria, illuminating the co-evolutionary history of the methylation system and host genome. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses.

sentences: The predictive potential of key adaptation parameters and proxy fitness traits between benign and stressful thermal environments Understanding the adaptive potential of a species is key when predicting whether a species can contend with climate change. Adaptive capacity depends on the amount of genetic variation within a population for relevant traits. However, genetic variation changes in different environments, making it difficult to predict whether a trait will respond to selection when not measured directly in that environment. Here, we investigated how genetic variances, and phenotypic and genetic covariances, between a fitness trait and morphological traits changed between thermal environments in two closely-related Drosophila. If morphological traits strongly correlate with fitness, they may provide an easy-to-measure proxy of fitness to aid in understanding adaptation potential. We used a parent-offspring quantitative genetic design to test the effect of a benign (23{degrees}C) and stressful (28{degrees}C) thermal environment on genetic variances of fecundity and wing size and shape, and their phenotypic and genetic covariances. We found genetic variances were higher within the stressful environment for fecundity but lower within the stressful environment for wing size. We did not find evidence for significant phenotypic correlations. Phenotypic and genetic correlations did not reveal a consistent pattern between thermal environments or within or between species. This corroborates previous research and reiterates that conclusions drawn in one environment about the adaptive potential of a trait, and the relationship of that trait with fitness, cannot be extrapolated to other environments or within or between closely-related species. This confirms that researchers should use caution when generalising findings across environments in terms of genetic variation and adaptive potential.

sentences: Temporally robust occupancy frequency distributions in riverine ecosystems explained by local biodiversity regulation The distribution of species site occupancy--the occupancy frequency distribution (OFD)--is a fundamental pattern in spatial ecology. Despite decades of research, the mechanisms responsible for the shape of the distribution remain incompletely understood. Here, we simultaneously examine both spatial and temporal patterns of site occupancy in communities of macroinverterbrates, macrophytes and diatoms. We find that the shape of the OFD at the catchment scale is typically time invariant despite substantial turnover in composition and species proportional occupancy over the 30 year time series. This can been seen as evidence in favour of an ecological mechanism for the shape of the OFD driven by local regulation of species richness and other biodiversity properties. We show that a simple, locally saturated patch occupancy model explains both the shape of the OFD and the temporal turnover rate in English rivers. This model is mathematically analogous to Hubbells neutral theory however concerns patches occupied by a given species rather its population size. Our analysis supports the view that metacommunities exist in a dynamic steady state arising from local ecological constraints and that this intrinsic regulation can drives the emergence of a wide variety of macroecological patterns. We also show that interspecific differences are unlikely to play a dominant role in driving large scale ecosystem structure.

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Arabidopsis Mediator subunit 17 connects transcription with DNA repair after UV-B exposure Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the Nucleotide Excision DNA Repair system. We here analyzed the role of MED17 in Arabidopsis plants exposed to UV-B radiation, which role has not been previously described. Comparison of med17 mutant transcriptome to that of WT plants showed that almost one third of transcripts with altered expression in med17 plants are also changed by UV-B exposure in WT plants. To validate the role of MED17 in UV-B irradiated plants, plant responses to UV-B were analyzed, including flowering time, DNA damage accumulation and programmed cell death in the meristematic cells of the root tips. Our results show that med17 and OE MED17 plants have altered responses to UV-B; and that MED17 participates in various aspects of the DNA damage response (DDR). Increased sensitivity to DDR after UV-B in med17 plants can be due to altered regulation of UV-B responsive transcripts; but additionally MED17 physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we here also show that MED17 is necessary to regulate the DDR activated by ATR, and that PDCD5 overexpression reverts the deficiencies in DDR shown in med17 mutants. Together, the data presented demonstrates that MED17 is an important regulator of the DDR after UV-B radiation in Arabidopsis plants.

One sentence summaryIn Arabidopsis, MED17 regulates the DNA damage response after UV-B exposure transcriptionally modulating the expression of genes and possibly also physically interacting with DNA repair proteins.

sentences: Defining dual-axis landscape gradients of human influence for studying ecological processes Ecological processes are strongly shaped by human landscape modification, and understanding the reciprocal relationship between ecosystems and modified landscapes is critical for informed conservation. Single axis measures of spatial heterogeneity proliferate in the contemporary gradient ecology literature, though they are unlikely to capture the complexity of ecological responses. Here, we develop a standardized approach for defining multi-dimensional gradients of human influence in heterogeneous landscapes and demonstrate this approach to analyze landscape characteristics of ten ecologically distinct US cities. Using occupancy data of a common human-adaptive songbird collected in each of the cities, we then use our dual-axis gradients to evaluate the utility of our approach. Spatial analysis of landscapes surrounding ten US cities revealed two important axes of variation that are intuitively consistent with the characteristics of multi-use landscapes, but are often confounded in single axis gradients. These were, a hard-to-soft gradient, representing transition from developed areas to non-structural soft areas; and brown-to-green, differentiating between two dominant types of soft landscapes: agriculture (brown) and natural areas (green). Analysis of songbird data demonstrated that occupancy responds to both hard-to-soft (decreasing with development intensity) and brown-to-green gradient (increasing with more natural area). Overall, our results reveal striking consistency in the dominant sources of variation across ten geographically distinct cities and suggests that our approach advances how we relate variation in ecological responses to human influence. Our case study demonstrates this: robins show a remarkably consistent response to a gradient differentiating agricultural and natural areas, but city-specific responses to the more traditional gradient of development intensity, which would be overlooked with a single gradient approach. Managing ecological communities in human dominated landscapes is extremely challenging due to a lack of standardized approaches and a general understanding of how socio-ecological systems function, and our approach offers promising solutions.

sentences: A CNN model for predicting binding affinity changes between SARS-CoV-2 spike RBD variants and ACE2 homologues The cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the association of its receptor binding domain (RBD) with human angiotensin converting enzyme 2 (hACE2) as the first crucial step. Efficient and reliable prediction of RBD-hACE2 binding affinity changes upon amino acid substitutions can be valuable for public health surveillance and monitoring potential spillover and adaptation into non-human species. Here, we introduce a convolutional neural network (CNN) model trained on protein sequence and structural features to predict experimental RBD-hACE2 binding affinities of 8,440 variants upon single and multiple amino acid substitutions in the RBD or ACE2. The model achieves a classification accuracy of 83.28% and a Pearson correlation coefficient of 0.85 between predicted and experimentally calculated binding affinities in five-fold cross-validation tests and predicts improved binding affinity for most circulating variants. We pro-actively used the CNN model to exhaustively screen for novel RBD variants with combinations of up to four single amino acid substitutions and suggested candidates with the highest improvements in RBD-ACE2 binding affinity for human and animal ACE2 receptors. We found that the binding affinity of RBD variants against animal ACE2s follows similar trends as those against human ACE2. White-tailed deer ACE2 binds to RBD almost as tightly as human ACE2 while cattle, pig, and chicken ACE2s bind weakly. The model allows testing whether adaptation of the virus for increased binding with other animals would cause concomitant increases in binding with hACE2 or decreased fitness due to adaptation to other hosts.

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Functional networks of the human bromodomain-containing proteins BackgroundBromodomains are a structurally conserved epigenetic reader domain that bind to acetylated lysine residues in both histone and non-histone proteins. Bromodomain-containing proteins (BRD proteins) function are established scaffolds in the assembly of multi-protein complexes to regulate diverse biological processes. BRD proteins have been classified based on biological and functional similarity, however the functions of many BRD proteins remains unknown. PPI network analysis is useful for revealing organizational roles, identifying functional clusters, and predicting function for BRD proteins.

ResultsWe used available data to construct protein-protein interaction networks (PPINs) to study the properties of the human bromodomain protein family. The network properties of the BRD PPIN establishes that the BRD proteins serve as hub proteins that are enriched near the global center to form an inter-connected PPIN. We identified dense subgraphs formed by BRD proteins and find that different BRD proteins share topological similarity and functional associations. We explored the functional relationships through clustering and Hallmark pathway gene set enrichment analysis and identify potential biological roles for different BRD proteins.

ConclusionsIn our network analysis we confirmed that BRD proteins are conserved central nodes in the human PPI network and function as scaffolds to form distinctive functional clusters. Overall, this study provides detailed insight into the predictive functions of BRD proteins in the context of functional complexes and biological pathways.

sentences: Toll-like receptor 5 as a novel receptor for fungal zymosan Microbial pathogens carry specific structural patterns which were termed pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) as key elements for the recognition of microbial pathogens are necessary for the activation of innate immune pathways. TLRs are activated by binding PAMPs of bacteria, viruses and fungi and initiate a signaling pathway resulting in the activation of transcription factors which modulate the production of various proinflammatory cytokines. It is not fully clear in detail which microbial pattern is recognized by which TLR. Here we show for the first time that TLR5 is a strong receptor for the yeast particle zymosan. We have generated stable human cell lines with combinations of TLR2 and TLR5 knock in/knock out together with stable nuclear factor kappaB (NF-{kappa}B) luciferase reporters. We found that both receptors TLR5 and TLR2 lead to an independent activation of the NF-{kappa}B pathway when simulated with zymosan. Our results demonstrate that TLR5 is a receptor for the fungal particle zymosan in addition to bacterial fragments like flagellin. Distinct cytokine patterns might suggest that TLR5 is potentially important for the differentiation in the recognition of the specific type of the foreign microorganisms and in the specific host defense response.

sentences: Detailed phylogeographic analysis of sticklebacks exposes challenges in genetic studies of admixed populations Introgressive hybridization is an important process contributing to adaptation but challenging to identify, undermining the efforts to understand its role in evolution. On the other hand, many analytical methods assume direct descent from a single common ancestor, and multiple waves of colonisation and admixture can violate their assumptions and lead to seriously biased results. A detailed analysis of 888 whole-genome-sequenced nine-spined sticklebacks revealed a complex pattern of population ancestry involving multiple waves of colonization and introgression across northern Europe. The secondary contact zone between the two recognized lineages was found to be wider than anticipated, displaying a smooth gradient of foreign ancestry with some curious exceptions attributable to freshwater isolates preserving footprints of prehistoric colonisation events. The results provide a back-drop against which introgression and its role in local adaptation can be studied and highlight methodological challenges associated with hidden admixture events.

sentences: A draft genome of Alliaria petiolata (garlic mustard) as a model system for invasion genetics The emerging field of invasion genetics examines the genetic causes and consequences of biological invasions, but few study systems are available that integrate deep ecological knowledge with genomic tools. Here we report on the de novo assembly and annotation of a genome for the biennial herb Alliaria petiolata (M. Bieb.) Cavara & Grande (Brassicaceae), which is widespread in Eurasia and invasive across much of temperate North America. Our goal was to sequence and annotate a genome to complement resources available from hundreds of published ecological studies, a global field survey, and hundreds of genetic lines maintained in Germany and Canada. We sequenced a genotype (EFCC-3-20) collected from the native range near Venice, Italy and sequenced paired-end and mate pair libraries at ~70 x coverage. A de novo assembly resulted in a highly continuous draft genome (N50 = 121 Mb; L50 = 2) with 99.7 % of the 1.1 Gb genome mapping to scaffolds of at least 50 Kb in length. A total of 64,770 predicted genes in the annotated genome include 99 % of plant BUSCO genes and 98 % of transcriptome reads. Consistent with previous reports of (auto)hexaploidy in western Europe, we found that almost one third of BUSCO genes (390/1440) mapped to two or more scaffolds despite < 2 % genome-wide average heterozygosity. The continuity and gene space quality of our draft assembly will enable molecular and functional genomic studies of A. petiolata to address questions relevant to invasion genetics and conservation strategies.

sentences: Natural variation further increases resilience of sorghum bred for chronically drought-prone environments Climate-change-associated shifts in rainfall distribution together with a looming worldwide water crisis make drought resilience of central importance to food security. Even for relatively drought resilient crops such as sorghum, moisture stress is nonetheless one of the major constraints for production. Here, we explore the potential to use natural genetic variation to build on the inherent drought tolerance of an elite cultivar (Teshale) bred for Ethiopian conditions including chronic drought, evaluating a backcross nested-association mapping (BC-NAM) population using 12 diverse founder lines crossed with Teshale under three drought-prone environments in Ethiopia. All twelve populations averaged higher head exsertion and lower leaf senescence than the recurrent parent in the two highest-stress environments, reflecting new drought resilience mechanisms from the donors. A total of 154 QTLs were detected for eight drought responsive traits - the validity of these were supported in that 100 (64.9%) overlapped with QTLs previously detected for the same traits, concentrated in regions previously associated with  stay-green traits as well as the flowering regulator Ma6 and drought resistant gene P5CS2. Allele effects show that some favorable alleles are already present in the Ethiopian cultivar, however the exotic donors offer rich scope for increasing drought resilience. Using model-selected SNPs associated with eight traits in this study and three in a companion study, phenotypic prediction accuracies for grain yield were equivalent to genome-wide SNPs and were significantly better than random SNPs, indicating that these studied traits are predictive of sorghum grain yield. Rich scope for improving drought resilience even in cultivars bred for drought-prone regions, together with phenotypic prediction accuracy for grain yield, provides a foundation to enhance food security in drought-prone areas like the African Sahel.

sentences: Single-cell transcriptome analysis of the zebrafish embryonic trunk During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.

sentences: Decision making in auditory externalization perception:model predictions for static conditions Under natural conditions, listeners perceptually attribute sounds to external objects in their environment. This core function of perceptual inference is often distorted when sounds are produced via hearing devices such as headphones or hearing aids, resulting in sources being perceived unrealistically close or even inside the head. Psychoacoustic studies suggest a mixed role of various monaural and interaural cues contributing to the externalization process. We developed a model framework for perceptual externalization able to probe the contribution of cue-specific expectation errors and to contrast dynamic versus static strategies for combining those errors within static listening environments. Effects of reverberation and visual information were not considered. The model was applied to various acoustic distortions as tested under various spatially static conditions in five previous experiments. Most accurate predictions were obtained for the combination of monaural and interaural spectral cues with a fixed relative weighting (approximately 60% of monaural and 40% of interaural). That model version was able to reproduce the externalization rating of the five experiments with an average error of 12% (relative to the full rating scale). Further, our results suggest that auditory externalization in spatially static listening situations underlie a fixed weighting of monaural and interaural spectral cues, rather than a dynamic selection of those auditory cues.

sentences: C. elegans enteric motor neurons fire synchronized action potentials underlying the defecation motor program The C. elegans nervous system was thought to be strictly analog, constituted solely by graded neurons. We recently discovered neuronal action potentials in the sensory neuron AWA; however, the extent to which the C. elegans nervous system relies on analog or digital neural signaling and coding is unclear. Here we report that the enteric motor neurons AVL and DVB fire all-or-none calcium-mediated action potentials that play essential roles in the rhythmic defecation behavior in C. elegans. Both AVL and DVB synchronously fire giant action potentials to faithfully execute all-or-none expulsion following the intestinal pacemaker. AVL fires unusual compound action potentials with each positive calcium-mediated spike followed by a potassium-mediated negative spike. The depolarizing calcium spikes in AVL are mediated by a CaV2 calcium channel UNC-2, while the negative potassium spikes are mediated by a repolarization-activated potassium channel EXP-2. Whole-body behavior tracking and simultaneous neural imaging in free-moving animals suggest that action potentials initiated in AVL in the head propagate along its axon to the tail and activate DVB through the INX-1 gap junction. Synchronized action potential spikes between AVL and DVB, as well as the negative spike and long-lasting afterhyperpolarization in AVL, play an important function in executing expulsion behavior. This work provides the first evidence that in addition to sensory coding, C. elegans motor neurons also use digital coding scheme to perform specific functions including long-distance communication and temporal synchronization, suggesting further, unforeseen electrophysiological diversity remains to be discovered in the C. elegans nervous system.

sentences: TrpA1 is a shear stress mechanosensing channel regulating intestinal homeostasis in Drosophila Adult stem cells are essential for maintaining normal tissue homeostasis and supporting tissue repair. Although genetic and biochemical programs controlling adult stem cell behavior have been extensively investigated, how mechanosensing regulates stem cells and tissue homeostasis is not well understood. Here, we show that shear stress can activate enteroendocrine cells, but not other gut epithelial cell types, to regulate intestine stem cell-mediated gut homeostasis. This shear stress sensing is mediated by transient receptor potential A1 (TrpA1), a Ca2+-permeable ion channel that expressed only in enteroendocrine cells among all gut epithelial cells. Genetic depletion of TrpA1 or modification of its shear stress sensing function causes reduced intestine stem cell proliferation and intestine growth. We further show that among the TrpA1 splice variants, only select isoforms are activated by shear stress. Altogether, our results suggest the naturally occurring mechanical force such as fluid passing generated shear stress regulates intestinal stem cell-mediated tissue growth by activating enteroendocrine cells, and Drosophila TrpA1 as a new shear stress sensor.

sentences: A Wake-Up Call: Determination of Antibiotics Residue Level in Raw Meat in Abattoir and Selected Slaughterhouses in Five Local Government in Kano State, Nigeria. The frequent use of antibiotics may result in drug residues that can be discovered at varying quantities in animal products such as milk or meat. The presence of pharmaceutical residues in food above the MRLs has been perceived globally by various persons. Antibiotics residues are present in food, which can endanger human health by causing antibiotic sensitivity, allergic reactions, microflora imbalance, bacterial resistance to antibiotics in microorganisms, and financial loss to the food industry. Farmers around the world utilize them on a sporadic basis for both preventative and curative purposes. This study assessed the antibiotics residues in raw meat sold in 6 slaughter houses in Kano States. The study is a descriptive cross-sectional study involving six (6) major slaughter house in Kano state. Muscle, Kidney and liver samples were collected from each slaughterhouse. The antibiotic residues in the meat samples were analysed using high performance liquid chromatography (HPLC) for tetracycline, ciprofloxacin and oxytetracycline residue results were presented in charts and tables. Out of a total of 18 beef samples analyzed during this study, 15 (83%) of the total samples had detectable levels of tetracycline residues from which 6(33.3%) had tetracycline residues at violative levels above the WHO/FAO maximum residue limits (MRLs), out of those 18 beef samples analyzed during this study, 6(33%) of the total samples had detectable levels of oxytetracycline residues from which 3(17%) had oxytetracycline residues at violative levels above the WHO/FAO maximum residue limits (MRLs) and out of those 18 beef samples analyzed during this study, 12(67%) of the total samples had detectable levels of ciprofloxacin, all levels are below the WHO/FAO maximum residue limits (MRLs). This high level of tetracycline and oxytetracycline residues in greater proportion of meat destined for human consumption at violative levels could be as a result of the indiscriminate use and misuse of veterinary drugs as commonly practiced among livestock producers and marketers without observing withdrawal period prior to slaughter. These results indicate that consumers may be predisposed to health hazards and hinder international meat trade from Nigeria. Regulatory authorities should therefore ensure compliance with good agricultural practices including withdrawal period of drugs used for treatment of food animals.

sentences: GNNQQNY: Methodology for biophysical and structural understanding of aggregation GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

sentences: Joint analysis of microsatellites and flanking sequences enlightens complex demographic history of interspecific gene flow and vicariance in rear-edge oak populations. Inference of recent population divergence requires fast evolving markers and necessitates to differentiate shared genetic variation caused by ancestral polymorphism and gene flow. Theoretical research shows that the use of compound marker systems integrating linked polymorphisms with different mutational dynamics, such as a microsatellite and its flanking sequences, can improve estimation of population structure and inference of demographic history, especially in the case of complex population dynamics. However, empirical application in natural populations has so far been limited by lack of suitable methods for data collection. A solution comes from the development of sequence-based microsatellite genotyping which we used to study molecular variation at 36 sequenced nuclear microsatellites in seven Quecus canariensis and four Quercus faginea rear-edge populations across Algeria, to decipher their taxonomic relationship, past evolutionary history and recent demographic trajectory. First, we compare the estimation of population genetics parameters and simulation-based inference of demographic history from microsatellite sequence alone, flanking sequence alone or the combination of linked microsatellite and flanking sequence variation. Second, we measure variable importance from random forest approximate Bayesian computation to identify which of these sequence types is most informative. Whereas the analysis of microsatellite variation alone indicates recent interspecific gene flow, additional information gained from combining with nucleotide variation in flanking sequences, by reducing homoplasy, suggests ancient interspecific gene flow followed by drift in isolation instead. The weight of each polymorphism in the inference also demonstrates the value of linked variations with contrasted mutation dynamic to improve estimation of both demographic and mutational parameters.

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Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and Synthetic Biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed "Super-Resolution Imaging" techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have however not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform Single-Molecule Localization Microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photo-convertible fluorescent protein mEos3.2, enabling Photo-Activated Localization Microscopy (PALM) in most Mycoplasma species. We also describe the application of direct STochastic Optical Reconstruction Microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development SMLM strategies to study these organisms in the future.

ImportanceMycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small size of these bacteria, well below the micron, limits the usefulness of traditional fluorescence imaging methods as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art "Super-Resolution Microscopy" techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photo-convertible fluorescent protein; and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nanometers. Our work paves the way toward a better understanding of mycoplasmas biology through imaging of subcellular structures at the nanometer scale.

sentences: Frog nest foam as a drug delivery system Foams have frequently been used as systems for the delivery of cosmetic and therapeutic molecules; however, there is high variability in the foamability and long-term stability of synthetic foams. The development of pharmaceutical foams that exhibit desirable foaming properties, delivering appropriate amounts of the active pharmaceutical ingredient (API) and that have excellent biocompatibility is of great interest. The production of stable foams is rare in the natural world; however, certain species of frogs have adopted foam production as a means of providing a protective environment for their eggs and larvae from a predators and parasites, to prevent desiccation, to control gaseous exchange, temperature extremes, and to reduce UV damage. These foams show great stability (up to 10 days in tropical environments) and are highly biocompatible due to the sensitive nature of amphibian skin. This work demonstrates for the first time, that nests of the Tungara frog (Engystomops pustulosus) is stable ex situ with useful physiochemical and biocompatible properties and is capable of encapsulating a range of compounds, including antibiotics. These protein foam mixtures share some properties with pharmaceutical foams and may find utility in a range of pharmaceutical applications such as a topical drug delivery systems (DDS).

sentences: The Backbone Network of Dynamic FunctionalConnectivity 1.Temporal networks have become increasingly pervasive in many real-world applications, including the functional connectivity analysis of spatially separated regions of the brain. A major challenge in analysis of such networks is the identification of noise confounds, which introduce temporal ties that are non-essential, or links that are formed by chance due to local properties of the nodes. Several approaches have been suggested in the past for static networks or temporal networks with binary weights for extracting significant ties whose likelihood cannot be reduced to the local properties of the nodes. In this work, we propose a data-driven procedure to reveal the irreducible ties in dynamic functional connectivity of resting state fRMI data with continuous weights. This framework includes a null model that estimates the latent characteristics of the distributions of temporal links through optimization, followed by a statistical test to filter the links whose formation can be reduced to the activities and local properties of their interacting nodes. We demonstrate the benefits of this approach by applying it to a resting state fMRI dataset, and provide further discussion on various aspects and advantages of it.

sentences: Forebrain Shh overexpression improves cognitive function in a Down syndrome mouse model and euploid littermates People with Down syndrome (DS) have intellectual disability, early-onset dementia, and cerebellar hypoplasia. Trisomic cerebellar granule cell precursors from Ts65Dn, a mouse model of DS, had a deficit in mitogenic response to Sonic hedgehog (Shh) in vitro, and newborn Ts65Dn mice received a single subcutaneous injection of the Shh signaling agonist SAG had normalized cerebellar morphology and improved spatial learning and hippocampal synaptic plasticity at adult. However, cognitive effects of Shh overexpression in vivo and where SAG acts to improve cognitive outcomes of trisomy are unknown. Here, we created an inducible human Shh (hShh) knock-in mouse, TRE-bi-hShh-Zsgreen1 (TRE-hShh), expressing dually-lipidated Shh-Np in the presence of transactivator (tTA). Double transgenic mice, Camk2a-tTA;TRE-hShh (Camk2a-hShh) and Pcp2-tTA;TRE-hShh (Pcp2-hShh), increased Shh signaling in forebrain and cerebellum, respectively. Forebrain Shh overexpression normalized hyperactivity, and spatial learning and memory deficits in 3-month-old Ts65Dn, while Shh overexpression in cerebellum had no effect. Further, Camk2a-hShh delayed early-onset severe cognitive impairment in 7-month-old Ts65Dn and enhanced spatial cognition in euploid (Eu) and showed no effect on the longevity of Eu or Ts65Dn, and MRI demonstrated that Pcp2-hShh mitigated disproportionately small cerebellum in Ts65Dn. Finally, Ts65Dn at postnatal day 6 had reduced Gli1 levels in hippocampus and cerebellum, which could be at least partially rescued by Camk2a-hShh and Pcp2-hShh, respectively. Our findings suggest restoration of impaired Shh signaling in forebrain from the perinatal and early postnatal period improves cognitive function.

sentences: Autophagosomes fuse to phagosomes and facilitate the degradation of apoptotic cells in Caenorhabditis elegans Autophagosomes are double-membrane intracellular vesicles that degrade protein aggregates, intracellular organelles, and other cellular components. During the development of the nematode Caenorhabditis elegans, many somatic and germ cells undergo apoptosis. These cells are engulfed and degraded by their neighboring cells. We discovered a novel role of autophagosomes in facilitating the degradation of apoptotic cells using a real-time imaging technique. Specifically, the double-membrane autophagosomes in engulfing cells are recruited to the surfaces of phagosomes containing apoptotic cells and subsequently fuse to phagosomes, allowing the inner vesicle to enter the phagosomal lumen. Mutants defective in the production of autophagosomes display significant defects in the degradation of apoptotic cells, demonstrating the importance of autophagosomes to this process. The signaling pathway led by the phagocytic receptor CED-1, the adaptor protein CED-6, and the large GTPase dynamin (DYN-1) promotes the recruitment of autophagosomes to phagosomes. Moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex components. Further observations suggest that autophagosomes provide apoptotic cell-degradation activities in addition to and in parallel of lysosomes. Our findings reveal that, unlike the single-membrane, LC3-associated phagocytosis (LAP) vesicles reported for mammalian phagocytes, the canonical double-membrane autophagosomes facilitate the clearance of C. elegans apoptotic cells. These findings add autophagosomes to the collection of intracellular organelles that contribute to phagosome maturation, identify novel crosstalk between the autophagy and phagosome maturation pathways, and discover the upstream signaling molecules that initiate this crosstalk.

sentences: Global Epigenetic Analysis Reveals H3K27 Methylation as a Mediator of Double Strand Break Repair The majority of cancer patients is treated with ionizing radiation (IR), a relatively safe and effective treatment considered to target tumors by inducing DNA double strand breaks (DSBs). Despite clinical interest in increasing the efficacy of IR by preventing successful DSB repair, few effective radio-adjuvant therapies exist. Extensive literature suggests that chromatin modifiers play a role in the DSB repair and thus may represent a novel class of radiosensitizers. Indeed, chromatin has both local and global impacts on DSB formation, recognition of breaks, checkpoint signaling, recruitment of repair factors, and timely DSB resolution, suggesting that epigenetic deregulation in cancer may impact the efficacy of radiotherapy. Here, using tandem mass spectrometry proteomics to analyze global patterns of histone modification in MCF7 breast cancer cells following IR exposure, we find significant and long-lasting changes to the epigenome. Our results confirm that H3K27 trimethylation (H3K27me3), best known for mediating gene repression and regulating cell fate, increases after IR. H3K27me3 changes rapidly, accumulating at sites of DNA damage. Inhibitors of the Polycomb related complex subunit and H3K27 methyltransferase EZH2 confirm that H3K27me3 is necessary for DNA damage recognition and cell survival after IR. These studies provide an argument for evaluating EZH2 as a radiosensitization target and H3K27me3 as a marker for radiation response in cancer. Proteomic data are available via ProteomeXchange with identifier PXD019388.

sentences: Developmental neuroanatomy of the Rosy Bitterling Rhodeus ocellatus (Teleostei: Cypriniformes)--A microCT study Bitterlings are a group of teleost fish (Cyprinifromes: Acheilanathidae) notable for their brood parasitic lifestyle. Bitterling embryos develop as parasites inside the gill chamber of their freshwater mussel hosts. However, little is known about brain development in this species. Here, we have imaged the development of the brain of the Rosy Bitterling (Rhodeus ocellatus) at four embryonic stages (165, 185, 210, 235 hours post-fertilization) using micro-computed tomography (microCT) with special emphasis on developmental regionalization and brain ventricular organization. We provide a detailed neuroanatomical account of the development of the brain divisions with reference to The Atlas of Early zebrafish Brain Development and the updated prosomeric model. Segmentation and three-dimensional visualization of the ventricular system were performed in order to clarify changes in the longitudinal brain axis as a result of cephalic flexure during development. During early embryonic and larval development, we find that histological differentiation, tissue boundaries, periventricular proliferation zones, and ventricular spaces are all recognizable using microCT. Importantly, our approach is validated by the fact that the profile of CT values displayed here in the bitterling brain are consistent with genoarchitecture identified in previous studies. We also find developmental heterochrony of the inferior lobe in the Rosy Bitterling compared to the zebrafish. Our study provides a foundation for future studies of the brain development in the Rosy Bitterling, a valuable model species for studying the evolutionary adaptations associated with brood parasitism.

sentences: SpotClean adjusts for spot swapping in spatial transcriptomics data Spatial transcriptomics (ST) is a powerful and widely-used approach for profiling genome-wide gene expression across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind mRNA. Ideally, unique molecular identifiers at a spot measure spot-specific expression, but this is often not the case owing to bleed from nearby spots, an artifact we refer to as spot swapping. We propose SpotClean to adjust for spot swapping and, in doing so, to increase the sensitivity and precision with which downstream analyses are conducted.

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Multi-channel 4D parametrized Atlas of Macro- and Microstructural Neonatal Brain Development Structural and diffusion MRI provide complimentary anatomical and microstructural characterization of early brain maturation. The existing models of the developing brain in time include only either structural or diffusion channels. Furthermore, there is a lack of tools for combined analysis of structural and diffusion MRI in the same reference space.

In this work we propose methodology to generate multi-channel (MC) continuous spatio-temporal parametrized atlas of brain development based on MC registration driven by both T2-weighted and orientation distribution functions (ODF) channels along with the Gompertz model (GM) fitting of the signals and spatial transformations in time. We construct a 4D MC atlas of neonatal brain development during 38 to 44 week PMA range from 170 normal term subjects from developing Human Connectomme Project. The resulting atlas consists of fourteen spatio-temporal microstructural indices and two parcellation maps delineating white matter tracts and neonatal transient structures. We demonstrate applicability of the atlas for quantitative region-specific comparison of 140 term and 40 preterm subjects scanned at the term-equivalent age. We show multi-parametric microstructural differences in multiple white matter regions, including the transient compartments. The atlas and software will be available after publication of the article.

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An evolutionarily conserved odontode gene regulatory network underlies head armor formation in suckermouth armored catfish Odontodes, i.e., teeth and tooth-like structures, consist of a pulp cavity and dentine covered by a mineralized cap. These structures first appeared on the outer surface of vertebrate ancestors and were repeatedly lost and gained across vertebrate clades; yet, the underlying genetic mechanisms and trajectories of this recurrent evolution remain long-standing mysteries. Here, we established suckermouth armored catfish (Ancistrus sp.; Loricariidae), which have uniquely evolved dermal odontodes (dermal denticles) all over most of their body surface, as an experimental model animal amenable to genetic manipulation for studying odontode development. Our histological analysis showed that suckermouth armored catfish develop dermal denticles through the previously defined odontode developmental stages. De novo transcriptomic profiling identified the conserved odontode genetic regulatory network (oGRN) as well as unique expression of paired like homeodomain 2 (pitx2), previously characterized as an early regulator of oGRN in teeth, in developing dermal denticles. Knockdown of pitx2 perturbed formation of the epithelial placode of dermal denticles and altered expression oGRN genes. By comprehensively identifying the genetic program for dermal odontode development in suckermouth armored catfish, this work illuminates how dermal odontodes independently evolved and diverged in distinct teleost lineages.

Summary statementCranial dermal denticles in suckermouth armored catfish develop via an evolutionarily conserved and unique odontode genetic regulatory network.

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Shaping information processing: the role of oscillatory dynamics in a working-memory task Neural oscillations are thought to reflect low-level operations that can be employed for higher-level cognitive functions. Here, we investigated the role of brain rhythms in the 1-30 Hz range by recording MEG in human participants performing a visual delayed match-to-sample paradigm in which orientation or spatial frequency of sample and probe gratings had to be matched. A cue occurring before or after sample presentation indicated the to-be-matched feature. We demonstrate that alpha/beta power decrease tracks the presentation of the informative cue and indexes faster responses. Moreover, these faster responses coincided with an augmented phase alignment of slow oscillations, as well as phase-amplitude coupling between slow and fast oscillations. Importantly, stimulus decodability was boosted by both low alpha power and high beta power. In summary, we provide support for a comprehensive framework in which different rhythms play specific roles: slow rhythms control input sampling, while alpha (and beta) gates the information flow, beta recruits task-relevant circuits, and the timing of faster oscillations is controlled by slower ones.

Significance statementBrain oscillations reflect low-level operations, building blocks, that control the flow of information through the brain. We propose and test a novel comprehensive framework in which slow oscillations control input sampling, alpha gates information flow, beta recruits task-relevant circuits, and the timing of faster oscillations is controlled by slower ones. We collected MEG data while participants performed a visual delayed match-to-sample task with pre- & retro-cues. Phase alignment of slow oscillations, governing input sampling, indexed faster responses. Alpha/beta power, gating information flow, boosted behavior & tracked informative cues. Low alpha (gating) & high beta (circuit-setup) power boosted signal information content. This is an essential step towards a more unified framework regarding the role of oscillatory dynamics in shaping information processing.

sentences: Reduced neutralization of SARS-CoV-2 variants by convalescent plasma and hyperimmune intravenous immunoglobulins for treatment of COVID-19 Hyperimmune immunoglobulin (hCoV-2IG) preparations generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in several clinical trials of hospitalized COVID-19 patients. Here we explored the antibody epitope repertoire, antibody binding and virus neutralizing capacity of six hCoV-2IG batches as well as nine convalescent plasma (CP) lots against SARS-CoV-2 and emerging variants of concern (VOC). The Gene-Fragment Phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike including NTD, RBD, S1/S2 cleavage site, S2-fusion peptide and S2-heptad repeat regions. Antibody binding to the immunodominant epitopes was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay (PsVNA) and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of SARS-CoV-2 VOCs from around the globe were reduced to different levels by hCoV-2IG lots. The most significant loss of neutralizing activity was seen against the B.1.351 (9-fold) followed by P.1 (3.5-fold), with minimal loss of activity against the B.1.17 and B.1.429 ([≤]2-fold). Again, the CP showed more pronounced loss of cross-neutralization against the VOCs compared with hCoV-2IG. Significant reduction of hCoV-2IG binding was observed to the RBD-E484K followed by RBD-N501Y and minimal loss of binding to RBD-K417N compared with unmutated RBD. This study suggests that post-exposure treatment with hCoV-2IG is preferable to CP. In countries with co-circulating SARS-CoV-2 variants, identifying the infecting virus strain could inform optimal treatments, but would likely require administration of higher volumes or repeated infusions of hCOV-2IG or CP, in patients infected with the emerging SARS-CoV-2 variants.

sentences: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling Mini-G proteins are engineered thermostable variants of G subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in {beta}-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of G subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

sentences: The cytoplasmic incompatibility Cif proteins from prophage WO modify sperm genome integrity Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally-transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers, however Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host Modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.

sentences: Functional Architecture of Executive Control and Associated Event-Related Potentials Medial frontal cortex enables executive control by monitoring relevant information and using it to adapt behavior. In macaques performing a saccade countermanding (stop-signal) task, we recorded EEG over and neural spiking across all layers of the supplementary eye field (SEF). We report the laminar organization of concurrently activated neurons monitoring the conflict between incompatible responses and the timing of events serving goal maintenance and executive control. We also show their relation to coincident event-related potentials (ERP). Neurons signaling response conflict were largely broad-spiking found across all layers. Neurons signaling the interval until specific task events were largely broad-spiking neurons concentrated in L3 and L5. Neurons predicting the duration of control and sustaining the task goal until the release of operant control were a mix of narrow- and broad-spiking neurons confined to L2/3. We complement these results with the first report of a monkey homologue of the N2/P3 ERP complex associated with response inhibition. N2 polarization varied with error likelihood and P3 polarization varied with the duration of expected control. The amplitude of the N2 and P3 were predicted by the spike rate of different classes of neurons located in L2/3 but not L5/6. These findings reveal important, new features of the cortical microcircuitry supporting executive control and producing associated ERP.

sentences: Social Housing Status Impacts Rhesus Monkeys' Affective Responding in Classic Threat Processing Tasks The established literature clearly demonstrates that whether or not monkeys are socially reared has long term consequences for their affective behavior. Yet, in the context of behavioral neuroscience and pharmacological studies, social context of adult animals is often ignored. When social context has been studied in adult monkeys, such studies have typically focused on welfare-related issues, as social isolation often leads to the development of abnormal behavior, rather than the impact on outcomes in behavioral neuroscience studies. Variation in social housing conditions for adult animals could have an impact on affective responding and may have significant implications for the interpretation of data from biopsychiatry and behavioral neuroscience studies. We evaluated the affective reactivity of rhesus monkeys (Macaca mulatta) maintained in one of four housing conditions (individually-housed, grate-paired, intermittently-paired, and continuously-paired) using two classic threat processing tasks--a test of responsivity to objects and the Human Intruder Test. Individually-housed monkeys exhibited consistently blunted sensitivity to ostensibly threatening stimuli as compared to socially-housed monkeys. Within the three socially-housed conditions, intermittently- and continuously-paired monkeys behaved similarly to each other and grate-paired monkeys exhibited relatively enhanced sensitivity to threatening stimuli. These findings suggest that the adult housing conditions of monkeys can robustly modulate affective responding in a way that may be consistent with behavioral phenotypes observed in human psychiatric conditions. Results are considered in the context of the broad behavioral and psychiatric neuroscience literatures, which have historically used individually-housed animals, pointing to the potential need to reconsider inferences drawn from those studies.

sentences: In silico Computed Clusters of Prostate Luminal Progenitors Match FACS-Enriched LSCmed cells Several groups recently published single-cell (sc) expression atlases of the adult mouse prostate cells based on RNA sequencing (scRNA-seq) data. All studies identified one computerized cluster of non-secretory luminal progenitor cells enriched in luminal and stemness-related gene transcripts. The actual correspondence between these luminal progenitor cell clusters has not been investigated. In addition, the presence of Krt4 (encoding cytokeratin 4) in these in silico-identified luminal progenitors suggested the overlap with FACS-enriched LSCmed luminal progenitor cells earlier identified as a stem-like, castration-tolerant and tumor-initiating cell population. Here, we used a unified bioinformatics pipeline to re-analyze published prostate scRNA-seq datasets and perform various pan-transcriptomic comparisons including the LSCmed cell signature. Our study demonstrates that i) the mouse prostate luminal progenitor cell clusters identified in the different scRNA-seq studies largely overlap and can be defined by a common 15-gene signature including Krt4, ii) mouse LSCmed cells match both mouse and human luminal progenitors identified by scRNA-seq analysis. Bridging these in silico-identified and ex vivo-characterized prostate luminal progenitor subsets should benefit our understanding of their actual involvement in prostate diseases.

sentences: Remote immune processes revealed by immune-derived circulating cell-free DNA Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

sentences: Cytoskeletal vimentin regulates cell size and autophagy through mTORC1 signaling The nutrient-activated mTORC1 (mechanistic target of rapamycin kinase complex 1) signaling pathway determines cell size by controlling mRNA translation, ribosome biogenesis, protein synthesis, and autophagy. Here we show that vimentin, a cytoskeletal intermediate filament protein that we know to be important for wound healing and cancer progression, determines cell size through mTORC1 signaling, an effect that is also manifested at the organism level in mice. We found that vimentin maintains normal cell size by supporting mTORC1 activation and through inhibition of autophagic flux. This regulation is manifested at all levels of downstream target activation and regulation of protein synthesis. We show that vimentin controls mTORC1 mobility by allowing access to lysosomes. Vimentin inhibits the autophagic flux in normal fibroblasts even under starved conditions, indicating a growth factor-independent inhibition of autophagy at the level of mTORC1. Our findings demonstrate that vimentin couples cell size signaling and autophagy with the biomechanic, sensing, and kinetic functions of the cytoskeleton.

sentences: Ten-eleven translocation 1 Mediated-DNA Hydroxymethylation is Required for Myelination and Remyelination in the Mouse Brain Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and DNA hydroxymethylation profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2 impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.

sentences: piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells There is a need of introducing large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop an approach to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to produce dsDNA ligation products containing variant library sequence flanked by piggyBac inverted terminal repeat sequences (ITRs) at both ends. The ligation products were then cotransfected with a piggyBac transposase expression vector into mammalian cells for library integration. The clone formation assay results showed that about 29,000 resistance clones were generated from a single well of 6-well-plate transfected HEK293T cells. As a proof of principle of using the strategy for DMS, a GFP chromophore random variant library was integrated into the HEK293T cell genome. By FACS selection, we enriched GFP-positive cells. The next-generation sequencing (NGS) results showed that the GFP-chromophore amino acid sequences were restored after selection.

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3D atlas of the pituitary gland of the model fish medaka In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their role during the animal life. Using medaka as the model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba- and pomca-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its developmental plasticity.

Highlights- We offer the first 3D atlas of a teleost pituitary, which presents a valuable resource to the endocrinology and model fish community.
- The atlas reveals the 3D spatial distribution of the seven endocrine cell types and blood vessels in the juvenile/adult male and female pituitary.
- Gene expression for tshba and pomca, as well as the population size of cells expressing these genes, displays obvious sexual dimorphism in the adult medaka pituitary.
- Multi-color in situ hybridization and single cell RNA-seq reveal the existence of bi-hormonal cells, co-expressing lhb-fshb, fshb-tshba, lhb-sl, and a few multi-hormonal cells.
- An online version of the atlas is available at https://www.nmbu.no/go/mpg-atlas.

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Real-time environmental monitoring of contaminants using living electronic sensors Real-time chemical sensing is needed to counter the global threats posed by pollution. We combine synthetic biology and materials engineering to develop a living bioelectronic sensor platform with minute detection times. Escherichia coli was programmed to reduce an electrode in a chemical-dependent manner using a modular, eight-component, synthetic electron transport chain. This strain produced significantly more current upon exposure to thiosulfate, an anion that causes microbial blooms. Incorporating a protein switch into the synthetic pathway and encapsulation of microbes with electrodes and conductive nanomaterials yielded a living bioelectronic sensor that could detect an endocrine disruptor within two minutes in riverine water, implicating the signal as mass transfer limited. These findings provide a new platform for miniature, low-power sensors that safeguard ecological and human health.

One Sentence SummaryChemicals are detected electrically using an allosterically-regulated electron transfer pathway in designer microbes.

sentences: Biased orientation representations can be explained by experience with non-uniform training set statistics Visual acuity is better for vertical and horizontal compared to other orientations. This cross-species phenomenon is often explained by "efficient coding", whereby more neurons show sharper tuning for the orientations most common in natural vision. However, it is unclear if experience alone can account for such biases. Here, we measured orientation representations in a convolutional neural network, VGG-16, trained on modified versions of ImageNet (rotated by 0{degrees}, 22.5{degrees}, or 45{degrees} counter-clockwise of upright). Discriminability for each model was highest near the orientations that were most common in the networks training set. Furthermore, there was an over-representation of narrowly tuned units selective for the most common orientations. These effects emerged in middle layers and increased with depth in the network. Biases emerged early in training, consistent with the possibility that non-uniform representations may play a functional role in the networks task performance. Together, our results suggest that biased orientation representations can emerge through experience with a non-uniform distribution of orientations, supporting the efficient coding hypothesis.

sentences: Sialoglycan microarray encoding reveals differential sialoglycan binding of phylogenetically-related bacterial AB5 toxin B subunits Vertebrate sialic acids (Sias) display much diversity in modifications, linkages and underlying glycans. Slide microarrays allow high-throughput explorations of sialoglycan-protein interactions. A microarray presenting [~]150 structurally-defined sialyltrisaccharides with various Sias linkages and modifications still poses challenges in planning, data sorting, visualization and analysis. To address these issues, we devised a simple 9-digit code for sialyltrisaccharides with terminal Sias and underlying two monosaccharides assigned from the non-reducing end, with three digits assigning a monosaccharide, its modifications, and linkage. Calculations based on the encoding system reveals >113,000 likely linear sialyltrisaccharides in nature. Notably a biantennary N-glycan with two terminal sialyltrisaccharides could thus have >1010 potential combinations and a triantennary N-glycan with three terminal sequences, >1015 potential combinations. While all possibilities likely do not exist in nature, sialoglycans encode enormous diversity. While glycomic approaches are used to probe such diverse sialomes, naturally-occurring bacterial AB5 toxin B subunits are simpler tools to track the dynamic sialome in biological systems. Sialoglycan microarray was utilized to compare sialoglycan-recognizing bacterial toxin B subunits. Unlike the poor correlation between B subunits and species phylogeny, there is stronger correlation with Sia-epitope preferences. Further supporting this pattern, we report a B subunit (YenB) from Yersinia enterocolitica (broad host range) recognizing almost all sialoglycans in the microarray, including 4-O-acetylated-Sias not recognized by a Y. pestis orthologue (YpeB). Differential Sia-binding patterns were also observed with phylogenetically-related B subunits from Escherichia coli (SubB), Salmonella Typhi (PltB), S. Typhimurium (ArtB), extra-intestinal E.coli (EcPltB), Vibrio cholera (CtxB), and cholera family homologue of E. coli (EcxB).

sentences: Boosting annotation in nutrimetabolomics by Feature-Based Molecular Networking: analytical and computational strategies applied to human urine samples from an untargeted LC-MS/MS based bilberry-blueberry intervention study. Urine represents a challenging metabolite mixture to decipher. Yet, it contains valuable information on dietary intake patterns as typically investigated using randomized, single-blinded, intervention studies. This research demonstrates how the use of Feature-Based Molecular Networking in combination with public spectral libraries, further expanded with an "In-house" library of metabolite spectra, improved the non-trivial annotation of metabolites occurring in human urine samples following bilberry and blueberry intake. Following this approach, 65 berry-related and human endogenous metabolites were annotated, increasing the annotation coverage by 72% compared to conventional annotation approaches. Furthermore, the structures of 15 additional metabolites were hypothesized by spectral analysis. Then, by leveraging the MzMine quantitative information, several molecular families of phase II (e.g., glucuronidated phenolics) and phase I (e.g., phenylpropionic acid and hydroxybenzoic acid molecular scaffolds) metabolism were identified by correlation analysis of postprandial kinetics, and the dietary impact of endogenous and exogenous metabolites following bilberry-blueberry intake was estimated.

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Relevance of Auditory Errors Decreases When Errors Are Introduced Suddenly PurposeWhen the speech motor system encounters errors, it generates adaptive responses to compensate for the errors. We previously showed that adaptive responses to task-irrelevant errors are significantly smaller than responses to task-relevant errors when errors are introduced gradually. The current study aimed to examine responses to task-irrelevant and task-relevant errors when errors are introduced suddenly.

MethodWe used an adaptation paradigm in which participants experienced task-relevant errors (induced by formant-shift perturbations) and task-irrelevant errors (induced by formant-clamp perturbations). For one group of participants (N = 30), we applied the perturbations gradually. The second group of participants (N = 30) received the perturbations suddenly. We designed the perturbations based on participant-specific vowel configurations such that a participants first and second formants of /{varepsilon}/ were perturbed toward their /ae/. To estimate adaptive responses, we measured formant changes (within 0-100 ms of the vowel onset) in response to the formant perturbations.

ResultsWe found that (1) the difference between adaptive responses to formant-shift and formant-clamp perturbations was the smallest for the suddenly introduced perturbations, and (2) responses to formant-shift perturbations positively correlated with responses to formant-clamp perturbations for the suddenly (but not gradually) introduced perturbations.

ConclusionsThese results showed that the speech motor system responds to task-relevant errors and task-irrelevant errors more differently when errors are introduced gradually than suddenly. Overall, the speech motor system evaluates the relevance of errors and uses its evaluation to modulate its adaptive responses to errors.

sentences: Genomic databanks and targeted assays help characterise mosquito incursions within Australia Biosecurity strategies aiming to restrict the spread of invasive pests can benefit from knowing where new incursions have come from. This knowledge can be acquired using genomic databanks, where genetic variation in incursion samples is compared against variation in reference samples. Here, we use genomic databanks to investigate domestic incursions of two mosquito species in Australia. We used a deep learning method to trace a 2021 invasion of Aedes aegypti in Tennant Creek, Northern Territory, to Townsville, Queensland, and to trace Ae. albopictus incursions between several invaded and uninvaded islands in the Torres Strait. We observed high precision of tracing despite 30-70 generations separating incursions and source samples. Targeted assays also improved precision, in this case by comparing Wolbachia infection data to mitochondrial DNA variation in Ae. aegypti. For Ae. albopictus, incursions detected in 2021 were traced to different islands than incursions detected in 2019. Patterns of relatedness and inbreeding indicated that Tennant Creek was likely invaded by Ae. aegypti from one family, whereas Torres Strait incursions involved distinct kinship groups. Our results highlight the value of repeated tracing with genomic databanks that remain informative over years, and demonstrate how additional targeted assays (e.g. Wolbachia) can improve inferences.

sentences: Ser68 phosphoregulation is essential for CENP-A deposition, centromere function and viability in mice Centromere identity is defined by nucleosomes containing CENP-A, a histone H3 variant. The deposition of CENP-A at centromeres is tightly regulated in a cell-cycle-dependent manner. We previously reported that the spatiotemporal control of centromeric CENP-A incorporation is mediated by the phosphorylation of CENP-A Ser68. However, a recent report argued that Ser68 phosphoregulation is dispensable for accurate CENP-A loading. Here, we report that the substitution of Ser68 of endogenous CENP-A with either Gln68 or Glu68 severely impairs CENP-A deposition and cell viability. We also find that mice harboring the corresponding mutations are lethal. Together, these results indicate that the dynamic phosphorylation of Ser68 ensures cell-cycle-dependent CENP-A deposition and cell viability.

sentences: Preliminary report of preclinical efficacy and safety analysis of gamma-irradiated inactivated SARS-CoV-2 vaccine candidates, SK-01 version 1 and OZG-3861 version 1 COVID-19 outbreak caused by SARS-CoV-2 created an unprecedented health crisis since there is no vaccine for this novel virus. Therefore, SARS-CoV-2 vaccines have become crucial for reducing morbidity and mortality. In this study, in vitro and in vivo safety and efficacy analyzes of lyophilized vaccine candidates inactivated by gamma-irradiation were performed. The candidate vaccines in this study were OZG-3861 version 1 (V1), an inactivated SARS-CoV-2 virus vaccine, and SK-01 version 1 (V1), a GM-CSF adjuvant added vaccine. The candidate vaccines were applied intradermally to BALB/c mice to assess toxicity and immunogenicity. Preliminary results in vaccinated mice are reported in this study. Especially, the vaccine models containing GM-CSF caused significant antibody production with neutralization capacity in absence of the antibody-dependent enhancement feature, when considered in terms of T and B cell responses. Another important finding was that the presence of adjuvant was more important in T cell in comparison with B cell response. Vaccinated mice showed T cell response upon restimulation with whole inactivated SARS-CoV-2 or peptide pool. This study shows that the vaccines are effective and leads us to start the challenge test to investigate the gamma-irradiated inactivated vaccine candidates for infective SARS-CoV-2 virus in humanized ACE2+ mice.

sentences: A machine learning approach for detecting vicarious trial and error behaviors Vicarious trial and error behaviors (VTEs) indicate periods of indecision during decision-making, and have been proposed as a behavioral marker of deliberation. In order to understand the neural underpinnings of these putative bridges between behavior and neural dynamics, researchers need the ability to readily distinguish VTEs from non-VTEs. Here we utilize a small set of trajectory-based features and standard machine learning classifiers to identify VTEs from non-VTEs for rats performing a spatial delayed alternation task (SDA) on an elevated plus maze. We also show that previously reported features of the hippocampal field potential oscillation can be used in the same types of classifiers to separate VTEs from non-VTEs with above chance performance. However, we caution that the modest classifier success using hippocampal population dynamics is not sufficient for identifying trials where VTEs occur, and show that combining oscillation-based features with trajectory-based features degrades classifier performance compared to trajectory-based features alone. Overall, we propose a standard set of features useful for trajectory-based VTE classification and support previous suggestions that VTEs are supported by a network including, but likely extending beyond, the hippocampus.

sentences: Sortilin exhibits tumor suppressor-like activity by limiting EGFR transducing function Lung cancer is the leading cause of cancer deaths worldwide and remains one of the most incurable. Tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR), are often aberrantly activated and drive tumor growth. Monotherapy with tyrosine kinase inhibitors to deactivate EGFR has shown initial efficacy, but their benefits tend to decline over time. EGFR acts as a transcriptional factor promoting the expression of co-oncogenic drivers, which, in turn, interact with canonical EGFR mutations to induce therapeutic relapse. This study reports that sortilin, a crucial regulator of cytoplasmic EGFR, attenuates its transducing function. Genome-wide chromatin binding revealed that sortilin interacts with gene regulatory elements occupied by EGFR. These results suggest a model, in which sortilin exhibits potential tumor suppressor-like activity by concurrently binding to regulatory elements of cMYC. Sortilin expression in lung adenocarcinoma may be predictive of the efficacy of anti-EGFR strategies.

sentences: Internet of Things Architecture for Cellular Biology The Internet of Things (IoT) provides a simple framework to easily control online devices. IoT is now a commonplace tool used by technology companies, but it is rarely used in biology experiments. IoT can benefit research through alarm notifications, automation, and the real-time monitoring of experiments. We developed and implemented an IoT architecture to control biological devices used in experiments. We developed our own electrophysiology, microscopy, and microfluidic devices so that may be controlled through a unified IoT architecture. The system allows each device to be monitored and controlled through an online web tool. We present our IoT architecture so other labs may replicate it for their own experiments.

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Genome scale CRISPR Cas9a knockout screen reveals genes that controls glioblastoma susceptibility to the alkylating agent temozolomide Glioblastoma is the most fatal of all primary human brain tumors with 14 months survival, at best. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy and tumor treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impact their overall survival and outcome. However, the lack of progress in this area could be attributed to lack of tools to probe unbiasedly at the genome wide level the coding and non-coding elements contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients.

CRISPR Cas9a has emerged as a functional genomics tool to study at genome level the factors that control cancer resistance to drugs. Recently, we used genome wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understand genes that control glioblastoma response to temozolomide by using genome scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.

sentences: Genetic mapping of sex and self-incompatibility determinants in the androdioecious plant Phillyrea angustifolia AO_SCPLOWBSTRACTC_SCPLOWThe diversity of mating and sexual systems in angiosperms is spectacular, but the factors driving their evolution remain poorly understood. In plants of the Oleaceae family, an unusual self-incompatibility (SI) system has been discovered recently, whereby only two distinct homomorphic SI specificities segregate stably. To understand the role of this peculiar SI system in preventing or promoting the diversity of sexual phenotypes observed across the family, an essential first step is to characterize the genetic architecture of these two traits. Here, we developed a high-density genetic map of the androdioecious shrub P. angustifolia based on a F1 cross between a hermaphrodite and a male parent with distinct SI genotypes. Using a double restriction-site associated digestion (ddRAD) sequencing approach, we obtained reliable genotypes for 196 offspring and their two parents at 10,388 markers. The resulting map comprises 23 linkage groups totaling 1,855.13 cM on the sex-averaged map. We found strong signals of association for the sex and SI phenotypes, that were each associated with a unique set of markers on linkage group 12 and 18 respectively, demonstrating inheritance of these traits as single, independent, mendelian factors. The P. angustifolia linkage map shows robust synteny to the olive tree genome overall. Two of the six markers strictly associated with SI in P. angustifolia have strong similarity with a recently identified 741kb chromosomal region fully linked to the SI phenotype on chromosome 18 of the olive tree genome, providing strong cross-validation support. The SI locus stands out as being markedly rearranged, while the sex locus has remained relatively more collinear between the two species. This P. angustifolia linkage map will be a useful resource to investigate the various ways by which the sex and SI determination systems have co-evolved in the broader phylogenetic context of the Oleaceae family.

sentences: Antennapedia regulates metallic silver wing scale development and cell shape in Bicyclus anynana butterflies Butterfly wing scale cells can develop very intricate cuticular nanostructures that interact with light to produce structural colors including silvery hues, but the genetic basis of such nanostructures is mostly unexplored. Here, we first identified that optix is necessary for metallic scale development in the butterfly Bicyclus anynana. We then characterized different subtypes of wildtype metallic silver scales and addressed the function of five genes - apterous A, Ultrabithorax, doublesex, Antennapedia, and optix - in the differentiation of silver scales, at a single-cell resolution, by leveraging crispants that exhibited either ectopic gains or losses of silver scales. Wildtype silver scales were generally rounded and had low amounts of pigmentation, exhibiting a common ultrastructural modification for metallic broadband reflectance, i.e., an undulatory air layer enclosed by an upper and lower lamina. Our results indicated that the varying air layer thickness was the important parameter of the bilaminate scale for producing a broadband reflectance across visible wavelengths. While a single lamina of the appropriate thickness could also produce broadband colors, the bilaminate structure is advantageous as it increases the overall reflectivity. Crispant brown scales differed from wildtype silver scales via the loss of the continuous upper lamina, increased lower lamina thickness, and increased pigmentation. The reverse was seen when brown scales became silver. We identified Antennapedia and optix as high-level regulators in the network differentiating different silver scale subtypes and determining overall cell shape in both sexes. In addition, Antp exhibits a novel, post-embryonic role in the determination of ridge and crossrib orientation.

sentences: Attractor Decision Network with Selective Inhibition The ability to decide swiftly and accurately in an urgent scenario is crucial for an organisms survival. The neural mechanisms underlying the perceptual decision and trade-off between speed and accuracy have been extensively studied in the past few decades. Among several theoretical models, the attractor neural network model has successfully captured both behavioral and neuronal data observed in many decision experiments. However, a recent experimental study revealed additional details that were not considered in the original attractor model. In particular, the study shows that the inhibitory neurons in the posterior parietal cortex of mice are as selective to decision making results as the excitatory neurons, whereas the previous attractor model assumes the inhibitory neurons to be unselective. In this study, we investigate the attractor model with selective inhibition (selective model), which can be considered as a general case of the previous attractor model (unselective model). Our model reproduces several behavioral and neurophysiological observations. To analyze the dynamics of the selective model, we reduce it and show that selectivity adds a time-varying component to the energy landscape. This time dependence of the energy landscape allows the selective model to integrate information carefully in initial stages, then quickly converge to an attractor once the choice is clear. This results in the selective model having a more efficient speed-accuracy trade-off that is unreachable by unselective models.

sentences: A functional topography within the cholinergic basal forebrain for processing sensory cues associated with reward and punishment Basal forebrain cholinergic neurons (BFCNs) project throughout the cortex to regulate arousal, stimulus salience, plasticity, and learning. Although often treated as a monolithic structure, the basal forebrain features distinct connectivity along its anteroposterior axis that could impart regional differences in BFCN processing. Here, we performed simultaneous bulk calcium imaging from anterior and posterior BFCNs over a one-month period of variable reinforcement learning in mice. BFCNs in both regions showed equivalently weak responses to unconditioned visual stimuli and anticipated rewards. Anterior BFCNs in the horizontal limb of the diagonal band were more responsive to reward omission, more accurately classified behavioral outcomes, and more closely tracked fluctuations in pupil-indexed global brain state. Posterior BFCNs in globus pallidus and substantia innominata were more responsive to unconditioned auditory stimuli, orofacial movements, aversive reinforcement, and showed robust associative plasticity for punishment-predicting cues. These results identify a functional topography that diversifies cholinergic modulatory signals broadcast to downstream brain regions.

sentences: Robust Working Memory through Short-Term Synaptic Plasticity Working memory has long been thought to arise from sustained spiking/attractor dynamics. However, recent work has suggested that short-term synaptic plasticity (STSP) may help maintain attractor states over gaps in time with little or no spiking. To determine if STSP endows additional functional advantages, we trained artificial recurrent neural networks (RNNs) with and without STSP to perform an object working memory task. We found that RNNs with and without STSP were both able to maintain memories over distractors presented in the middle of the memory delay. However, RNNs with STSP showed activity that was similar to that seen in the cortex of monkeys performing the same task. By contrast, RNNs without STSP showed activity that was less brain-like. Further, RNNs with STSP were more robust to noise and network degradation than RNNs without STSP. These results show that STSP can not only help maintain working memories, it also makes neural networks more robust.

sentences: Patient brain organoids identify a link between the 16p11.2 copy number variant and the RBFOX1 gene. Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.2 CNVs lead to disease are unclear. Relevant models, like human cortical organoids (hCOs), are needed to understand the human-specific mechanisms of neurodevelopmental disease. We generated hCOs from 18 patients and controls, profiling 167,958 cells with single cell (sc)RNA-seq. Analysis revealed neuronal-specific differential expression of genes outside of the 16p11.2 region that were related to cell-cell adhesion, neuronal projection growth, and neurodevelopmental disorders. Furthermore, 16p11.2 deletion syndrome organoids exhibited reduced mRNA and protein levels of RBFOX1, a gene which can also harbor CNVs linked to neurodevelopmental phenotypes. We found that many genes previously shown to be regulated by RBFOX1 are also perturbed in organoids from patients with 16p11.2 deletion syndrome, and thus identified a novel link between independent CNVs associated with neuronal development and autism. Overall, this work suggests convergent signaling, which indicates the possibility of a common therapeutic mechanism across multiple rare neuronal diseases.

sentences: mGluR5 Negative Modulators for Fragile X: Resistance and Persistence Fragile X syndrome (FXS) is caused by silencing of the human FMR1 gene and is the leading monogenic cause of intellectual disability and autism. Abundant preclinical data indicated that negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5) might be efficacious in treating FXS in humans. Initial attempts to translate these findings in clinical trials have failed, but these failures provide the opportunity for new discoveries that will improve future trials. The emergence of acquired treatment resistance ("tolerance") after chronic administration of mGluR5 NAMs is a potential factor in the lack of success. Here we confirm that FXS model mice display acquired treatment resistance after chronic treatment with the mGluR5 NAM CTEP in three assays commonly examined in the mouse model of FXS: (1) audiogenic seizure susceptibility, (2) sensory cortex hyperexcitability, and (3) hippocampal protein synthesis. Cross-tolerance experiments suggest that the mechanism of treatment resistance likely occurs at signaling nodes downstream of glycogen synthase kinase 3 (GSK3), but upstream of protein synthesis. The rapid emergence of tolerance to CTEP begs the question of how previous studies showed an improvement in inhibitory avoidance (IA) cognitive performance after chronic treatment. We show here that this observation was likely explained by timely inhibition of mGluR5 during a critical period, as brief CTEP treatment in juvenile mice is sufficient to provide a persistent improvement of IA behavior measured many weeks later. These data will be important to consider when designing future fragile X clinical trials using compounds that target the mGluR5-to-protein synthesis signaling cascade.

sentences: Efficacy of antiviral drugs against the omicron variant of SARS-CoV-2. The Omicron variant of the SARS-CoV-2 virus was first detected in South Africa in November 2021. The analysis of the sequence data in the context of earlier variants suggested that it may show very different characteristics, including immune evasion and increased transmission. These assumptions were partially confirmed, and the reduction in protection in convalescent patients and vaccinated individuals have been confirmed. Here, we have evaluated the efficacy of antivirals against SARS-CoV-2 variants, Omicron, Delta, and the early 2020 isolate.

sentences: Local adaptation of Aedes aegypti mosquitoes to Wolbachia-induced fitness costs Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavorable conditions. Aedes aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We therefore compared the costs of wMel infection for quiescent egg viability in field-derived and laboratory populations. Quiescent egg viability was highly variable in wMel-infected populations, with greater costs of wMel in field-derived populations. In contrast, there was little variation between matched field-derived and long-term laboratory populations lacking wMel, suggesting that laboratory adaptation does not influence this trait and that differences are due to wMel infection. Comparisons of populations collected a year apart show a decline in costs under laboratory rearing conditions involving a rapid turnover of mosquito generations; this pattern was consistent across populations despite their origin, suggesting adaptation of mosquitoes to the wMel infection under laboratory conditions. Reciprocal crossing experiments confirm that differences in quiescent egg viability were mainly due to the genetic background and not Wolbachia alone. wMel-infected mosquitoes hatching from long-term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long-term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and local adaptation. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection.

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Polycystin promotes renal fibrosis through EZH2 Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations.

sentences: Animal-appropriate housing of ball pythons (Python regius)-- Behavior-based evaluation of two types of housing systems Considering animal welfare, animals should be kept in animal-appropriate and stress-free housing conditions in all circumstances. To assure such conditions, not only basic needs must be met, but also possibilities must be provided that allow animals in captive care to express all species-typical behaviors. Rack housing systems for snakes have become increasingly popular and are widely used; however, from an animal welfare perspective, they are no alternative to furnished terrariums. In this study, we therefore evaluated two types of housing systems for ball pythons (Python regius) by considering the welfare aspect animal behavior. In Part 1 of the study, ball pythons (n = 35) were housed individually in a conventional rack system. The pythons were provided with a hiding place and a water bowl, temperature control was automatic, and the lighting in the room served as indirect illumination. In Part 2 of the study, the same ball pythons, after at least 8 weeks, were housed individually in furnished terrariums. The size of each terrarium was correlated with the body length of each python. The terrariums contained substrate, a hiding place, possibilities for climbing, a water basin for bathing, an elevated basking spot, and living plants. The temperature was controlled automatically, and illumination was provided by a fluorescent tube and a UV lamp. The shown behavior spectrum differed significantly between the two housing systems (p < 0.05). The four behaviors basking, climbing, burrowing, and bathing could only be expressed in the terrarium. Abnormal behaviors that could indicate stereotypies were almost exclusively seen in the rack system. The results show that the housing of ball pythons in a rack system leads to a considerable restriction in species-typical behaviors; thus, the rack system does not meet the requirements for animal-appropriate housing.

sentences: Two-photon patterned photostimulation with low-power, high-efficiency and reliable single-cell optogenetic control Two-photon optogenetics enables selectively stimulating individual cells for manipulating neuronal ensembles. As the general photostimulation strategy, the patterned two-photon excitation has enabled millisecond-timescale activation for single or multiple neurons, but its activation efficiency is suffered from high laser power due to low beam-modulation efficiency. Here, we develop a high- efficiency beam-shaping method based on the Gerchberg-Saxton (GS) algorithm with spherical-distribution initial phase (GSSIP) to reduce the patterned two-photon excitation speckles and intensity. It can well control the phase of shaped beams to attain speckle-free accurate patterned illumination with an improvement of 44.21% in the modulation efficiency compared with that of the traditional GS algorithm. A combination of temporal focusing and the GSSIP algorithm (TF-GSSIP) achieves patterned focusing through 500-m-thickness mouse brain slices, which is 2.5 times deeper than the penetration depth of TF-GS with the same signal-to-noise ratio (SNR). With our method, the laser power can be reduced to only 55.56% of that with traditional method (the temporal focusing with GS, TF-GS) to reliably evoke GCaMP6s response in C1V1-expressing cultured neurons with single-cell resolution. Besides, the photostimulation efficiency is remarkably increased by 80.19% at the same excitation density of 0.27 mW/m2. This two-photon stimulation method with low-power, reliable and patterned illumination may pave the way for analyzing neural circuits and neural coding and decoding mechanism.

sentences: Enabling interpretable machine learning for biological data with reliability scores Machine learning has become an important tool across biological disciplines, allowing researchers to draw conclusions from large datasets, and opening up new opportunities for interpreting complex and heterogeneous biological data. Alongside the rapid growth of machine learning, there have also been growing pains: some models that appear to perform well have later been revealed to rely on features of the data that are artifactual or biased; this feeds into the general criticism that machine learning models are designed to optimize model performance over the creation of new biological insights. A natural question thus arises: how do we develop machine learning models that are inherently interpretable or explainable? In this manuscript, we describe reliability scores, a new concept for scientific machine learning studies that assesses the ability of a classifier to produce a reliable classification for a given instance. We develop a specific implementation of a reliability score, based on our work in Sugden et al. 2018 in which we introduced SWIF(r), a generative classifier for detecting selection in genomic data. We call our implementation the SWIF(r) Reliability Score (SRS), and demonstrate the utility of the SRS when faced with common challenges in machine learning including: 1) an unknown class present in testing data that was not present in training data, 2) systemic mismatch between training and testing data, and 3) instances of testing data that are missing values for some attributes. We explore these applications of the SRS using a range of biological datasets, from agricultural data on seed morphology, to 22 quantitative traits in the UK Biobank, and population genetic simulations and 1000 Genomes Project data. With each of these examples, we demonstrate how interpretability tools for machine learning like the SRS can allow researchers to interrogate their data thoroughly, and to pair their domain-specific knowledge with powerful machine-learning frameworks. We hope that this tool, and the surrounding discussion, will aid researchers in the biological machine learning space as they seek to harness the power of machine learning without sacrificing rigor and biological understanding.

sentences: Environmentally sensitive hotspots in the methylome of the early human embryo In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin specific methylation and germline DMRs, supporting a periconceptional environmental influence. Many SoC-sensitive CpGs overlapped enhancers or sites of active transcription in H1 ESCs and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental  hotspots providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease.

sentences: Red backgrounds have different effects on electrophysiological responses to fearful faces in groups with low and high autistic tendency Visual processing differences in the magnocellular pathway have been reported across the autistic spectrum. On the basis that the firing of primate Type IV magnocellular cells is suppressed by diffuse red backgrounds, several groups have used red backgrounds as a means to investigate magnocellular contributions to visual processing in humans. Here, we measured emotional identification accuracy, and compared the P100 and N170 responses from groups with low (n=21; AQ<11) and high (n=22; AQ>22) Autism Spectrum Quotient (AQ) scores, in response to low (LSF) and high (HSF) spatially filtered fearful and neutral face stimuli presented on red and green backgrounds. For the LSF stimuli, the low AQ group correctly identified fearful expressions more often when presented on a red compared to a green background. The low AQ group also showed red backgrounds reduced the effect of LSF fearful expressions on P100 amplitudes. In contrast, the high AQ group showed that background colour did not significantly alter P100 responses to LSF stimuli. Interestingly, red background reduced the effects of HSF stimuli for the high AQ group. The effects of background color on LSF and HSF facial emotion responses were not evident for the N170 component. Our findings suggest that presenting face stimuli on a red background alters both magnocellular and parvocellular contributions to the P100 waveform, and that these effects differ for groups with low and high autistic tendencies. In addition, a theoretical model for explaining the temporal differences in facial emotion processing for low and high AQ groups is proposed.

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A critical test of deep convolutional neural networks ability to capture recurrent processing in the brain using visual masking Recurrent processing is a crucial feature in human visual processing supporting perceptual grouping, figure-ground segmentation, and recognition under challenging conditions. There is a clear need to incorporate recurrent processing in deep convolutional neural networks (DCNNs) but the computations underlying recurrent processing remain unclear. In this paper, we tested a form of recurrence in deep residual networks (ResNets) to capture recurrent processing signals in the human brain. Though ResNets are feedforward networks, they approximate an excitatory additive form of recurrence. Essentially, this form of recurrence consists of repeating excitatory activations in response to a static stimulus. Here, we used ResNets of varying depths (reflecting varying levels of recurrent processing) to explain electroencephalography (EEG) activity within a visual masking paradigm. Sixty-two humans and fifty artificial agents (10 ResNet models of depths - 4, 6, 10, 18 and 34) completed an object categorization task. We show that deeper networks (ResNet-10, 18 and 34) explained more variance in brain activity compared to shallower networks (ResNet-4 and 6). Furthermore, all ResNets captured differences in brain activity between unmasked and masked trials, with differences starting at [~]98ms (from stimulus onset). These early differences indicated that EEG activity reflected  pure feedforward signals only briefly (up to [~]98ms). After [~]98ms, deeper networks showed a significant increase in explained variance which peaks at [~]200ms, but only within unmasked trials, not masked trials. In summary, we provided clear evidence that excitatory additive recurrent processing in ResNets captures some of the recurrent processing in humans.

Significance statementThe challenge of modeling recurrent processes is not trivial and the operationalization of recurrent processing is highly contested. In this paper, we tested the ability of deep residual networks (ResNets) to explain recurrent processes in the human brain. Though ResNets are feedforward networks, they have been shown to equate operations in recurrent neural networks. In this study, we show that deeper networks explained more variance in brain activity than shallower networks. However, all networks still performed far from the noise ceiling. Thus, we conclude that recurrent processing in ResNets captures a form of recurrent processing in humans though other types of recurrent processing (inhibition, multiplicative) that are not present in current regular deep neural networks (alexnet, cornet, resnet) are necessary for building better visual models.

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Sex-Dependent Shared and Non-Shared Genetic Architecture Across Mood and Psychotic Disorders BACKGROUNDSex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk.

METHODSWe conducted the largest to date genome-wide genotype-by-sex (GxS) interaction of risk for these disorders, using 85,735 cases (33,403 SCZ, 19,924 BIP, 32,408 MDD) and 109,946 controls from the Psychiatric Genomics Consortium (PGC) and iPSYCH.

RESULTSAcross disorders, genome-wide significant SNP-by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815; p=3.2x10-8), that interacts with sodium/potassium-transporting ATPase enzymes implicating neuronal excitability. Three additional loci showed evidence (p<1x10-6) for cross-disorder GxS interaction (rs7302529, p=1.6x10-7; rs73033497, p=8.8x10-7; rs7914279, p=6.4x10-7) implicating various functions. Gene-based analyses identified GxS interaction across disorders (p=8.97x10-7) with transcriptional inhibitor SLTM. Most significant in SCZ was a MOCOS gene locus (rs11665282; p=1.5x10-7), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509; p=1.1x10-7) in a locus containing IDO2, a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant GxS of genes regulating vascular endothelial growth factor (VEGF) receptor signaling in MDD (pFDR<0.05).

CONCLUSIONSIn the largest genome-wide GxS analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development, immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway enrichment levels.

sentences: Abortive Intussusceptive Angiogenesis Causes Multi-Cavernous Vascular Malformations Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a over-expression and resultant aberrant flow signaling. Surviving adult fish manifested histologically-typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.

sentences: MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis Atherosclerosis is the underlying cause of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. It is a lipid-triggered chronic inflammatory condition of the arterial vascular wall that is driven by various inflammatory pathways including atherogenic cytokines and chemokines. D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2 (MIF-2), belongs to the MIF protein family, which is best known for its pathogenic role in a variety of inflammatory and immune conditions including CVDs. While MIF is well known as a promoter of atherogenic processes, MIF-2 has not been studied in atherosclerosis. Here, we investigated atherosclerosis in hyperlipidemic Mif-2-/-Apoe-/- mice and studied the role of MIF-2 in various atherogenic assays in vitro. We found that global Mif-2 deficiency as well as its pharmacological blockade by 4-CPPC protected against atherosclerotic lesion formation and vascular inflammation in models of early and advanced atherogenesis. On cellular level, MIF-2 promoted monocyte migration in 2D and 3D and monocyte arrest on aortic endothelial monolayers, promoted B-cell chemotaxis in vitro and B-cell homing in vivo, and increased macrophage foam cell formation. Dose curves and direct comparison in a 3D migration set-up suggest that MIF-2 may be a more potent chemokine than MIF for monocytes and B cells. We identify CXCR4 as a novel receptor for MIF-2. The evidence relies on a CXCR4 inhibitor, CXCR4 internalization experiments, MIF-2/CXCR4 binding studies by yeast-CXCR4 transformants, and fluorescence spectroscopic titrations with a soluble CXCR4 surrogate. Of note, Mif-2-/-Apoe-/- mice exhibited decreased plasma cholesterol and triglyceride levels, lower body weights, smaller livers, and profoundly reduced hepatic lipid accumulation compared to Apoe-/- mice. Mechanistic experiments in Huh-7 hepatocytes suggest that MIF-2 regulates the expression and activation of sterol-regulatory element binding protein-1 and -2 (SREBP-1, SREBP-2) to induce lipogenic downstream genes such as FASN and LDLR, while it attenuated the activation of the SREBP inhibiting AMPK pathway. Studies using receptor Inhibitors showed that SREBP activation and hepatic lipoprotein uptake by MIF-2 is mediated by both CXCR4 and CD74. Lastly and in line with a combined role of MIF-2 in vascular inflammation and hepatic lipid accumulation, MIF-2 was found to be profoundly upregulated in unstable human carotid plaques, underscoring a critical role for MIF-2 in advanced stages of atherosclerosis. Together, these data identify MIF-2 as a novel atherogenic chemokine and CXCR4 ligand that not only promotes lesion formation and vascular inflammation but also strongly affects hepatic lipogenesis in an SREBP-mediated manner, possibly linking atherosclerosis and hepatic steatosis.

sentences: Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane metalloprotease SpoIVFB Intramembrane proteases function in numerous signaling pathways that impact health, but knowledge about regulation of intramembrane proteolysis is limited. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that BofA residues in and near a predicted transmembrane segment are required for SpoIVFB inhibition, and cross-linking experiments indicated that this transmembrane segment occupies the SpoIVFB active site cleft. SpoIVFA is also required for SpoIVFB inhibition. The inhibitory proteins block access of the substrate N-terminal Proregion to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a transmembrane segment and a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of intramembrane protease inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics.

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Complexity of genomic epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Colombia urges the reinforcement of whole genome sequencing based surveillance programs BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging public health problem. This study explores the specifics of CRKP epidemiology in Colombia based on whole genome sequencing (WGS) of the National Reference Laboratory at Instituto Nacional de Salud (INS)s 2013-2017 sample collection.

MethodsA total of 425 CRKP isolates from 21 departments were analyzed by HiSeq-X10(R)Illumina high-throughput sequencing. Bioinformatic analysis was performed, primarily using the pipelines developed collaboratively by the National Institute for Health Research Global Health Research Unit (GHRU) on Genomic Surveillance of AMR, and AGROSAVIA.

ResultsOf the 425 CRKP isolates, 91.5% were carbapenemase-producing strains. The data support a recent expansion and the endemicity of CRKP in Colombia with the circulation of 7 high-risk clones, the most frequent being CG258 (48.39% of isolates). We identified genes encoding carbapenemases blaKPC-3, blaKPC-2, blaNDM-1, blaNDM-9, blaVIM-2, blaVIM-4, and blaVIM-24, and various mobile genetic elements (MGE). The virulence of CRKP isolates was low, but colibactin (clb3) was present in 25.2% of isolates, and a hypervirulent CRKP clone (CG380) was reported for the first time in Colombia. ST258, ST512, and ST4851 were characterized by low levels of diversity in the core genome (ANI> 99.9%).

ConclusionsThe study outlines complex CRKP epidemiology in Colombia. CG258 expanded clonally and carries specific carbapenemases in specific MGEs, while the other high-risk clones (CG147, CG307, and CG152) present a more diverse complement of carbapenemases. The specifics of the Colombian situation stress the importance of WGS-based surveillance to monitor evolutionary trends of STs, MGE, and resistance and virulence genes.

summaryIn Colombia, the dissemination of carbapenemases in carbapenem-resistant Klebsiella pneumoniae is attributed to horizontal gene transfer and successful circulation of CG258, and, to a lesser extent, other clones such as ST307, ST147, and ST152.

sentences: Conditional resampling improves calibration and sensitivity in single cell CRISPR screen analysis Single-cell CRISPR screens are the most promising biotechnology for mapping regulatory elements to their target genes at genome-wide scale. However, the analysis of these screens presents significant statistical challenges. For example, technical factors like sequencing depth impact not only expression measurement but also perturbation detection, creating a confounding effect. We demonstrate on two recent high multiplicity of infection single-cell CRISPR screens how these challenges cause calibration issues among existing analysis methods. To address these challenges, we propose SCEPTRE: analysis of single-cell perturbation screens via conditional re-sampling. This methodology, designed to avoid calibration issues due to technical confounders and expression model misspecification, infers associations between perturbations and expression by resampling the former according to a working model for perturbation detection probability in each cell. SCEPTRE demonstrates excellent calibration and sensitivity on the CRISPR screen data and yields hundreds of new regulatory relationships, supported by orthogonal functional evidence.

sentences: Endogenous but not sensory-driven activity controls migration, morphogenesis and survival of adult-born neurons in the mouse olfactory bulb The development and survival of adult-born neurons is believed to be driven by sensory signaling. By genetically manipulating excitability of adult-born cells (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), longitudinal in vivo monitoring of their Ca2+ signaling and transcriptome analyses, we show that endogenous but not sensory-driven activity governs migration, morphogenesis, survival, and functional integration of adult-born juxtaglomerular neurons in the mouse olfactory bulb. The proper development of these cells required fluctuations of cytosolic Ca2+ levels, phosphorylation of CREB, and pCREB-mediated gene expression. Attenuating Ca2+ fluctuations via K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in the vulnerable and immature state. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as proper Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons.

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Behavioral signatures of Y-like neuronal responses in human vision Retinal ganglion cells (RGCs) initiating the magnocellular/Y-cell visual pathways are distinguished by responding nonlinearly to high spatial frequencies (SFs) and temporal frequencies (TFs). This nonlinearity is implicated in the processing of contrast modulation (CM) stimuli in cats and monkeys, but its contribution to human visual perception is not well understood. Here we formulated an approach to reveal the function of human Y-like RGCs by leveraging the Y-like carrier response properties of cortical neurons to CM stimuli consisting of a high SF contrast-reversing grating carrier whose contrast is modulated by a low SF drifting sinewave envelope. Subjects reported the direction of motion of CM envelopes or luminance modulation (LM) gratings, presented at different eccentricities. The dependence of task performance on SF (for LMs) or carrier SF (for CMs) was measured for different TFs (LMs) or carrier TFs (CMs). The best performance for LM stimuli was at lower TFs and SFs, which decreased systematically with eccentricity. However, performance with CM stimuli was bandpass with carrier SF, largely independent of carrier TF, and at the highest carrier TF (20 Hz) decreased minimally with eccentricity. Since the nonlinear subunits of Y -cells respond better at higher TFs compared to the linear response components, and respond best at higher SFs that are relatively independent of eccentricity, these results suggest that behavioral tasks employing CM stimuli with high spatiotemporal carrier frequencies can reveal the nonlinear contributions of retinal Y-like cells to human perception.

Significance StatementY-like retinal ganglion cells (RGCs), characterized by responding linearly at low spatial and temporal frequencies and nonlinearly at high spatiotemporal frequencies, have been well characterized neurophysiologically in cats and monkeys. However, a clear counterpart of Y-like RGCs has remained elusive in humans. Here, we leveraged these response properties to reveal a behavioral signature of Y-like cells in human observers. To drive linear or nonlinear response mechanisms, we presented drifting luminance modulation and contrast modulation stimuli respectively at multiple spatial and temporal frequencies and eccentricities. Our results suggest that humans exhibit behavioral response patterns to contrast modulation stimuli that are consistent with the nonlinear response properties of Y-like cells.

sentences: Slow phase-locked endogenous modulations support selective attention to sound To make sense of complex soundscapes, listeners must select and attend to task-relevant streams while ignoring uninformative sounds. One possible neural mechanism underlying this process is alignment of endogenous oscillations with the temporal structure of the target sound stream. Such a mechanism has been suggested to mediate attentional modulation of neural phase-locking to the rhythms of attended sounds. However, such modulations are compatible with an alternate framework, where attention acts as a filter that enhances exogenously-driven neural auditory responses. Here we attempted to adjudicate between theoretical accounts by playing two tone steams varying across condition in tone duration and presentation rate; participants attended to one stream or listened passively. Attentional modulation of the evoked waveform was roughly sinusoidal and scaled with rate, while the passive response did not. This suggests that auditory attentional selection is carried out via phase-locking of slow endogenous neural rhythms.

sentences: Understanding SARS-CoV-2 budding through molecular dynamics simulations of M and E protein complexes SARS-CoV-2 and other coronaviruses pose major threats to global health, yet computational efforts to understand them have largely overlooked the process of budding, a key part of the coronavirus life cycle. When expressed together, coronavirus M and E proteins are sufficient to facilitate budding into the ER-Golgi intermediate compartment (ERGIC). To help elucidate budding, we ran atomistic molecular dynamics (MD) simulations using the Feig laboratorys refined structural models of the SARS-CoV-2 M protein dimer and E protein pentamer. Our MD simulations consisted of M protein dimers and E protein pentamers in patches of membrane. By examining where these proteins induced membrane curvature in silico, we obtained insights around how the budding process may occur. Multiple M protein dimers acted together to induce global membrane curvature through protein-lipid interactions while E protein pentamers kept the membrane planar. These results could eventually help guide development of antiviral therapeutics which inhibit coronavirus budding.

sentences: Maternal microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice The gut microbiota plays a central role in regulating host metabolism. However, while substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. The effects of B. breve UCC2003 on feto-placental growth are mediated, in part, by changes in the maternal and placental metabolome (i.e. acetate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. This study provides knowledge towards a novel and safe therapeutic strategy for treating pregnancy disorders via modulation of the maternal gut microbiota.

sentences: Fuzzy set intersection based paired-end short-read alignment Much genomic data comes in the form of paired-end reads: two reads that represent genetic material with a small gap between. We present a new algorithm for aligning both reads in a pair simultaneously by fuzzily intersecting the sets of candidate alignment locations for each read. This algorithm is often much faster and produces alignments that result in variant calls having roughly the same concordance as the best competing aligners.

sentences: Elucidating the structural features of ABCA1 in its heterogeneous membrane environment. ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by a sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular "reservoir". This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within ECDs but not in theTMDs giving it an outward-facing conformation. This structure was hypothesized to substantiate the lateral access theory, whereby ApoA1 obtained lipids from the proximal end laterally. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a large heterogeneous lipid environment and found that along with several large conformational changes, the protein harbours a continuous tunnel that traverses the entire length of the protein. In this study, we have characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.

sentences: Tracking the fates of iron-labeled tumor cells in vivo using Magnetic Particle Imaging The use of imaging to detect and monitor the movement and accumulation of cells in living subjects can provide significant insights that can improve our understanding of metastasis and guide therapeutic development. For cell tracking using Magnetic Resonance Imaging (MRI), cells are labeled with iron oxides and the effects of the iron on water provides contrast. However, due to low specificity and difficulties in quantification with MRI, other modalities and approaches need to be developed. Magnetic Particle Imaging (MPI) is an emerging imaging technique which directly detects magnetic iron, allowing for a specific, quantitative and sensitive readout. Here, we use MPI to image iron-labeled tumor cells longitudinally, from implantation and growth at a primary site to movement to distant anatomic sites. In vivo bioluminescent imaging (BLI) was used to localize tumor metastases and computed tomography (CT) allowed for correlation of these signals to anatomic locations. These three imaging modalities provide information on immune escape and metastasis of iron-labeled, and unlabeled, tumor cells, and the accumulation of cell-free iron contrast over time. We identified iron signals by MPI and tumor cells via BLI, and correlated these positive contrast images with CT scans to reveal the anatomic sites with cancer cells; histologic analysis confirmed the presence of iron-labeled tumor cells in the tissues, suggesting that the metastatic cells retained enough iron for MPI detection. The use of multi-modality cell tracking reveals the movement, accumulation and fates of labeled cells that will be helpful understanding cancer progression and guiding the development of targeted therapies.

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Xenon LFP Analysis Platform is a Novel Graphical User Interface for Analysis of Local Field Potential from Large-Scale MEA Recordings High-density multi-electrode array (HD-MEA) has enabled neuronal measurements at high spatial resolution to record local field potentials (LFP), extracellular spikes, and network activity with ease. Whilst we have advanced recording systems with over 4000 electrodes, capable of recording data at over 20 kHz, it still presents computational challenges to handle, process, extract, and view information from these large recordings. It can be challenging for researchers to extract and view even a single channel that has more than a billion data points, let alone process a group of channels.

We have created a computational method, and an open-source toolkit built on Python, rendered on a web browser using Plotlys Dash for extracting and viewing the data, and creating interactive visualization. In addition to extracting and viewing entire or small chunks of data sampled at lower or higher frequencies, respectively, it provides a framework to collect user inputs, analyze channel groups, generate raster plots, view quick summary measures for LFP activity, detect and isolate noise channels, and generate plots and visualization in both time and frequency domain. Incorporated into our Graphical User Interface (GUI), we also have created a novel seizure detection method, which can be used to detect the onset of seizures in all or a selected group of channels and provide the following measures of seizures: distance, duration, and propagation across the region of interest.

We demonstrate the utility of this toolkit, using datasets collected from the 3Brain BioCAM duplex system. For the current analysis, we demonstrate the toolkit and methods with a low sampling frequency dataset (300 Hz) and a group of approximately 400 channels. Using this toolkit, we present novel data demonstrating increased seizure propagation speed from slices of Scn1aHet mice compared to littermate controls.

With advances in HD-MEA recording systems with high spatial and temporal resolution, limited tools are available for researchers to view and process these big datasets. We now provide a user-friendly toolkit to analyze LFP activity obtained from large-scale MEA recordings with translatable applications to EEG recordings, and demonstrate the utility of this new graphic user interface with novel biological findings.

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The Effects of Posture on the Three-Dimensional Gait Mechanics of Human Walking in Comparison to Bipedal Chimpanzees Humans walk with an upright posture on extended limbs during stance and with a double-peaked vertical ground reaction force. Our closest living relatives, chimpanzees, are facultative bipeds that walk with a crouched posture on flexed, abducted hind limbs and with a single-peaked vertical ground reaction force. Differences in human and bipedal chimpanzee three-dimensional kinematics have been well quantified; however, it is unclear what the independent effects of using a crouched posture are on three-dimensional gait mechanics for humans, and how they compare with chimpanzees. Understanding the relationships between posture and gait mechanics, with known differences in morphology between species, can help researchers better interpret the effects of trait evolution on bipedal walking. We quantified pelvis and lower limb three-dimensional kinematics and ground reaction forces as humans adopted a series of upright and crouched postures and compared them with data from bipedal chimpanzee walking. Human crouched posture gait mechanics were more similar to bipedal chimpanzee gait than normal human walking, especially in sagittal plane hip and knee angles. However, there were persistent differences between species, as humans walked with less transverse plane pelvis rotation, less hip abduction, and greater peak horizontal ground reaction force in late stance than chimpanzees. Our results suggest that human crouched posture walking reproduces only a small subset of the characteristics of three-dimensional kinematics and ground reaction forces of chimpanzee walking, with the remaining differences likely due in large part to the distinct musculoskeletal morphologies of humans and chimpanzees.

Summary StatementDifferences between human crouched posture gait and bipedal chimpanzee gait illustrate the limitations of using modern day humans to infer the evolution of hominin bipedalism.

sentences: Effects of Perinatal Fluoxetine Exposure on Novelty-induced Social and Non-Social Investigation Behaviors in a Seminatural Environment Selective serotonin reuptake inhibitors (SSRIs) are increasingly prescribed as medication for various affective disorders during pregnancy. SSRIs cross the placenta and affect serotonergic neurotransmission in the fetus, but the neurobehavioral consequences for the offspring remain largely unclear. Recent rodent research has linked perinatal SSRI exposure to alterations in both social and non-social aspects of behavior. However, this research has mainly focused on behavior within simplified environments. The current study investigates the effects of perinatal SSRI exposure on social and non-social investigation behaviors of adult rat offspring upon introduction to a novel seminatural environment with unknown conspecifics. During the perinatal period (gestational day 1 until postnatal day 21), rat dams received daily treatment with either an SSRI (fluoxetine, 10 mg/kg) or vehicle. Adult male and female offspring were observed within the first hour after introduction to a seminatural environment. The results showed that perinatal fluoxetine exposure altered aspects of non-social investigation behaviors, while not altering social investigation behaviors. More specifically both fluoxetine exposed males and females spent more total time on locomotor activity than controls. Furthermore, fluoxetine exposed females spent less time exploring objects and specific elements in the environment. The data suggest that perinatal exposure to SSRIs leads to a quicker, less detailed investigation strategy in novel environments, and that the alteration is mostly pronounced in females.

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Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the family Goodeidae, the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trohotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Summary statementHere, we report that an endocytic pathway is a candidate for nutrient absorption in pseudoplacenta of a viviparous teleost. The trait may have developed from common intestinal mechanism among vertebrates.

sentences: A luciferase prosubstrate and a red bioluminescent calcium indicator to image neuronal activity Genetically encoded fluorescent indicators have been broadly used to monitor neuronal activity in live animals, but invasive surgical procedures are required. This study presents a functional bioluminescence imaging (fBLI) method for recording the activity of neuronal ensembles in the brain in awake mice. We developed a luciferase prosubstrate activatable in vivo by nonspecific esterase to enhance the brain delivery of the luciferin. We further engineered a bright, bioluminescent indicator with robust responsiveness to calcium ions (Ca2+) and appreciable emission above 600 nm. Integration of these advantageous components enabled the imaging of Ca2+ dynamics in awake mice minimally invasively with excellent signal- to-background and subsecond temporal resolution. This study thus establishes a new paradigm for studying brain functions in health and disease.

sentences: Powerful, efficient QTL mapping in Drosophila melanogaster using bulked phenotyping and pooled sequencing Despite the value of Recombinant Inbred Lines (RILs) for the dissection of complex traits, large panels can be difficult to maintain, distribute, and phenotype. An attractive alternative to RILs for many traits leverages selecting phenotypically-extreme individuals from a segregating population, and subjecting pools of selected and control individuals to sequencing. Under a bulked or extreme segregant analysis paradigm, genomic regions contributing to trait variation are revealed as frequency differences between pools. Here we describe such an extreme quantitative trait locus, or X-QTL mapping strategy that builds on an existing multiparental population, the DSPR (Drosophila Synthetic Population Resource), and involves phenotyping and genotyping a population derived by mixing hundreds of DSPR RILs. Simulations demonstrate that challenging, yet experimentally tractable X-QTL designs (>=4 replicates, >=5000 individuals/replicate, and a selection intensity of 5-10%) yield at least the same power as traditional RIL-based QTL mapping, and can localize variants with sub-centimorgan resolution. We empirically demonstrate the effectiveness of the approach using a 4-fold replicated X-QTL experiment that identifies 7 QTL for caffeine resistance. Two mapped X-QTL factors replicate loci previously identified in RILs, 6/7 are associated with excellent candidate genes, and RNAi knock-downs support the involvement of 4 genes in the genetic control of trait variation. For many traits of interest to drosophilists a bulked phenotyping/genotyping X-QTL design has considerable advantages.

sentences: Characterization of a vaccine-elicited human antibody with sequence homology to VRC01-class antibodies that binds the C1C2 gp120 domain Broadly HIV-1 neutralizing VRC01-class antibodies bind the CD4-binding site of the HIV-1 envelope (Env) and contain VH1-2*02-derived heavy chains paired with light chains expressing five amino acid long CDRL3s. Their unmutated forms do not recognize Env or neutralize HIV-1. The lack of elicitation of VRC01-class antibodies in human clinical trials could potentially be due to the absence of activation of the corresponding naive B cells by the vaccine Env immunogens. To address this point directly, we examined Env-specific BCR sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed only one displayed sequence homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in both the CDRH3 and CDRL3 were necessary. Our findings support the use of specifically designed immunogens to activate VRC01-class B cells in future human vaccine trials.

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Functional group-dependent responses of forest bird communities to invasive predator control and habitat fragmentation AimMounting global pressure on bird populations from invasive predators and habitat loss has driven a rapid growth in restorative and protective conservation action around the world, yet the efficacy of such actions is still not well understood. We investigated the relative effects of invasive predator control and habitat fragmentation on the abundance of native birds and invasive mammalian predators in native forest fragments.

LocationWaikato region, New Zealand

MethodsWe sampled invasive mammalian predator and native bird abundances using camera traps and bird counts at 26 sites in 15 forest fragments across New Zealands Waikato region. Fragment area, shape complexity, and surrounding land cover of exotic and native forest were determined in ArcMap. We further created two composite gradients reflecting predator control intensity and temporal distribution of control based on seven quantitative variables recorded in each of the five years preceding native bird data collection. Finally, we estimated the relative influence of these drivers on invasive mammals and functional groups of native birds using model averaging.

ResultsOf the two invasive predator control variables, only control intensity significantly affected invasive predator abundance and was also a more important driver than landscape or fragment structure, but responses varied among invasive mammal species. In contrast, both invasive predator control intensity and fragment structure were similarly important drivers of native bird abundance, though bird community responses varied markedly between functional groups.

Main conclusionsOur findings suggest that spatial extent of invasive mammal control is important for controlling mammal numbers and enhancing bird abundance, especially for small insectivorous species, and that habitat fragmentation is less important for invasive mammals but at least as important for native bird communities. Consequently, both drivers should be given strong consideration when undertaking landscape-scale conservation and restoration of bird communities in human-altered landscapes threatened by invasive predators.

sentences: The MLL3/4 complexes and MiDAC act antagonistically as genome-wide regulators of H4K20ac to control a specific gene expression program The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data shows that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, that are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2 and H3K27ac. We find that MiDAC antagonizes the recruitment of the MLL3/4 complexes to negatively regulate H4K20ac, H3K4me2 and H3K27ac resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.

sentences: A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing, and contribute to neuronal hyperexcitability and disease pathophysiology. Here we show, for the first time, that INaR can be reconstituted in a heterologous system by co-expression of sodium channel -subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin-resistant neuronal channels and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.

sentences: Precise topology of adjacent domain-general and sensory-biased regions in the human brain Recent functional MRI studies identified sensory-biased regions across much of the association cortices and cerebellum. However, their anatomical relationship to multiple-demand (MD) regions, characterized as domain-general due to their co-activation during multiple cognitive demands, remains unclear. For a better anatomical delineation, we used multimodal MRI techniques of the Human Connectome Project to scan subjects performing visual and auditory versions of a working memory (WM) task. The contrast between hard and easy WM showed strong domain generality, with essentially identical patterns of cortical, subcortical and cerebellar MD activity for visual and auditory materials. In contrast, modality preferences were shown by contrasting easy WM with baseline; most MD regions showed visual preference while immediately adjacent to cortical MD regions, there were interleaved regions of both visual and auditory preference. The results may exemplify a general motif whereby domain-specific regions feed information into and out of an adjacent, integrative MD core.

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Eliminating accidental deviations to minimize generalization error and maximize replicability: applications in connectomics and genomics Replicability, the ability to replicate scientific findings, is a prerequisite for scientific discovery and clinical utility. Troublingly, we are in the midst of a replicability crisis. A key to replicability is that multiple measurements of the same item (e.g., experimental sample or clinical participant) under fixed experimental constraints are relatively similar to one another. Thus, statistics that quantify the relative contributions of accidental deviations--such as measurement error--as compared to systematic deviations--such as individual differences--are critical. We demonstrate that existing replicability statistics, such as intra-class correlation coefficient and fingerprinting, fail to adequately differentiate between accidental and systematic deviations in very simple settings. We therefore propose a novel statistic, discriminability, which quantifies the degree to which an individuals samples are relatively similar to one another, without restricting the data to be univariate, Gaussian, or even Euclidean. Using this statistic, we introduce the possibility of optimizing experimental design via increasing discriminability and prove that optimizing discriminability improves performance bounds in subsequent inference tasks. In extensive simulated and real datasets (focusing on brain imaging and demonstrating on genomics), only optimizing data discriminability improves performance on all subsequent inference tasks for each dataset. We therefore suggest that designing experiments and analyses to optimize discriminability may be a crucial step in solving the replicability crisis, and more generally, mitigating accidental measurement error.

Author SummaryIn recent decades, the size and complexity of data has grown exponentially. Unfortunately, the increased scale of modern datasets brings many new challenges. At present, we are in the midst of a replicability crisis, in which scientific discoveries fail to replicate to new datasets. Difficulties in the measurement procedure and measurement processing pipelines coupled with the influx of complex high-resolution measurements, we believe, are at the core of the replicability crisis. If measurements themselves are not replicable, what hope can we have that we will be able to use the measurements for replicable scientific findings? We introduce the "discriminability" statistic, which quantifies how discriminable measurements are from one another, without limitations on the structure of the underlying measurements. We prove that discriminable strategies tend to be strategies which provide better accuracy on downstream scientific questions. We demonstrate the utility of discriminability over competing approaches in this context on two disparate datasets from both neuroimaging and genomics. Together, we believe these results suggest the value of designing experimental protocols and analysis procedures which optimize the discriminability.

sentences: Spatially heterogeneous microstructural development within subcortical regions from 9-13 years During late childhood behavioral changes, such as increased risk-taking and emotional reactivity, have been associated with the maturation of cortico-subcortical circuits. Understanding microstructural changes in subcortical regions may aid our understanding of how individual differences in these behaviors emerge. Restriction spectrum imaging (RSI) is a framework for modelling diffusion-weighted imaging that decomposes the diffusion signal from a voxel into hindered and restricted compartments. This yields greater specificity than conventional methods of characterizing intracellular diffusion. Using RSI, we modelled voxelwise restricted isotropic, N0, and anisotropic, ND, diffusion across the brain and measured cross-sectional and longitudinal age associations in a large sample (n=8,039) from the Adolescent Brain and Cognitive Development (ABCD) study aged 9-13 years. Older participants had higher N0 and ND across subcortical regions. The largest associations for N0 were within the basal ganglia and for ND within the ventral diencephalon. Importantly, age associations varied with respect to the internal cytoarchitecture within subcortical structures, for example age associations differed across thalamic nuclei. This suggests that developmental effects may map onto specific cell populations or circuits and highlights the utility of voxelwise compared to ROI-wise analyses. Future analyses will aim to understand the relevance of this subcortical microstructural developmental for behavioral outcomes.

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The edible seaweed Laminaria japonica contains cholesterol analogues that inhibit Lipid Peroxidation and Cyclooxygenase Enzymes In this study, 5 sterols were isolated and purified from Laminaria japonica, commonly known as edible brown seaweed, and their structures were identified based on detailed chemical methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3{beta}-ol, saringosterol (24-vinyl-cholest-5-ene-3{beta},24-diol), 24-methylenecholesterol, fucosterol (stigmasta-5,24-diene-3{beta}-ol), and 24-Hydroperoxy-24-vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the other compounds. In addition, the results of molecular docking revealed that the 5 sterols were located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin methine achieved the highest binding energy (-7.85 and -9.02 kcal/mol) through hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica and its significant anti-inflammatory and antioxidant activity.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/463984v1_ufig1.gif" ALT="Figure 1">
View larger version (21K):
org.highwire.dtl.DTLVardef@16c3f43org.highwire.dtl.DTLVardef@1ad7385org.highwire.dtl.DTLVardef@7b3c4borg.highwire.dtl.DTLVardef@b3675a_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LISterols 29-hydroperoxy-stigmasta-5,24(28)-dien-3{beta}-ol and 24-hydroperoxy-24-vinyl-cholesterol are identified for the first time in L. japonica.
C_LIO_LISaringosterol, 24-methylenecholesterol and fucosterol showed strong LPO inhibitory activity.
C_LIO_LIFucosterol showed highest binding affinity for COX-1 and -2 enzymes through hydrophobic interactions.
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sentences: Linking Population Dynamics to Microbial Kinetics for Hybrid Modeling of Engineered Bioprocesses Mechanistic and data-driven models have been developed to provide predictive insights into the design and optimization of engineered bioprocesses. These two modeling strategies can be combined to form hybrid models to address the issues of parameter identifiability and prediction interpretability. Herein, we developed a novel and robust hybrid modeling strategy by incorporating microbial population dynamics into model construction. The hybrid model was constructed using bioelectrochemical systems (BES) as a platform system. We collected 77 samples from 13 publications, in which the BES were operated under diverse conditions, and performed holistic processing of the 16S rRNA amplicon sequencing data. Community analysis revealed core populations composed of putative electroactive taxa Geobacter, Desulfovibrio, Pseudomonas, and Acinetobacter. Primary Bayesian networks were trained with the core populations and environmental parameters, and directed Bayesian networks were trained by defining the operating parameters to improve the prediction interpretability. Both networks were validated with Bray-Curtis similarly, relative root-mean-square error (RMSE), and a null model. The hybrid model was developed by first building a three-population mechanistic component and subsequently feeding the estimated microbial kinetic parameters into network training. The hybrid model generated a simulated community that shared a Bray-Curtis similarity of 72% with the actual microbial community and an average relative RMSE of 7% for individual taxa. When examined with additional samples that were not included in network training, the hybrid model achieved accurate prediction of current production with a relative error-based RMSE of 0.8 and outperformed the data-driven models. The genomics-enabled hybrid modeling strategy represents a significant step toward robust simulation of a variety of engineered bioprocesses.

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Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo. BackgroundMicroRNAs (miRNA) and other components contained in extracellular vesicles may reflect the presence of a disease. Lung tissue, sputum and sera of individuals with idiopathic pulmonary fibrosis (IPF) show alterations in miRNA expression. We designed this study to test whether urine and/or tissue derived exosomal miRNAs from individuals with IPF carry cargo that can promote fibrosis.

MethodsExosomes were isolated from urine (U-IPFexo), lung tissue myofibroblasts (MF-IPFexo), serum from individuals with IPF (n=16) and age/sex-matched controls without lung disease (n=10). We analyzed microRNA expression of isolated exosomes and their in vivo bio-distribution. We investigated the effect on ex vivo skin wound healing and in in vivo mouse lung models.

ResultsU-IPFexo or MF-IPFexo expressed miR let-7d, miR-29a-5p, miR 181b-3p and miR-199a-3p consistent with previous reports of miRNA expression obtained from lung tissue/sera from patients with IPF. In vivo bio-distribution experiments detected bioluminescent exosomes in the lung of normal C57Bl6 mice within 5 minutes after intravenous infusion, followed by distribution to other organs irrespective of exosome source. Exosomes labeled with gold nanoparticles and imaged by transmission electron microscopy were visualized in alveolar epithelial type I and type II cells. Treatment of human and mouse lung punches obtained from control, non-fibrotic lungs with either U-IPFexo or MF-IPFexo produced a fibrotic phenotype. A fibrotic phenotype was also induced in a human ex vivo skin model and in in vivo lung models.

ConclusionsOur results provide evidence of a systemic feature of IPF whereby exosomes contain pro-fibrotic miRNAs when obtained from a fibrotic source and interfere with response to tissue injury as measured in skin and lung models.

FundingThis work was supported in part by Lester and Sue Smith Foundation and The Samrick Family Foundation and NIH grants R21 AG060338 (SE and MKG), U01 DK119085 (IP, RS, MTC).

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Inferring Population Size Histories using Coalescent Hidden Markov Models with TMRCA and Total Branch Length as Hidden States Unraveling the complex demographic histories of natural populations is a central problem in population genetics. Understanding past demographic events is of general anthropological interest and is also an important step in establishing accurate null models when identifying adaptive or disease-associated genetic variation. An important class of tools for inferring past population size changes are Coalescent Hidden Markov Models (CHMMs). These models make efficient use of the linkage information in population genomic datasets by using the local genealogies relating sampled individuals as latent states that evolve across the chromosome in an HMM framework. Extending these models to large sample sizes is challenging, since the number of possible latent states increases rapidly.

Here, we present our method CHIMP (CHMM History-Inference ML Procedure), a novel CHMM method for inferring the size history of a population. It can be applied to large samples (hundreds of haplotypes) and only requires unphased genomes as input. The two implementations of CHIMP that we present here use either the height of the genealogical tree (TMRCA) or the total branch length, respectively, as the latent variable at each position in the genome. The requisite transition and emission probabilities are obtained by numerically solving certain systems of differential equations derived from the ancestral process with recombination. The parameters of the population size history are subsequently inferred using an Expectation-Maximization algorithm. In addition, we implement a composite likelihood scheme to allow the method to scale to large sample sizes.

We demonstrate the efficiency and accuracy of our method in a variety of benchmark tests using simulated data and present comparisons to other state-of-the-art methods. Specifically, our implementation using TMRCA as the latent variable shows comparable performance and provides accurate estimates of effective population sizes in intermediate and ancient times. Our method is agnostic to the phasing of the data, which makes it a promising alternative in scenarios where high quality data is not available, and has potential applications for pseudo-haploid data.

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Cavitation Induced Fracture of Intact Brain Tissue Nonpenetrating traumatic brain injuries (TBI) are linked to cavitation. The structural organization of the brain makes it particularly susceptible to tears and fractures from these cavitation events, but limitations in existing characterization methods make it difficult to understand the relationship between fracture and cavitation in this tissue. More broadly, fracture energy is an important, yet often overlooked, mechanical property of all soft tissues. We combined needle-induced cavitation (NIC) with hydraulic fracture models to induce and quantify fracture in intact brains at precise locations. We report here the first measurements of the fracture energy of intact brain tissue that range from 1.5 to 8.9 J/m2, depending on the location in the brain and the model applied. We observed that fracture consistently occurs along interfaces between regions of brain tissue. These fractures along interfaces allow cavitation-related damage to propagate several millimeters away from the initial injury site. Quantifying the forces necessary to fracture brain and other soft tissues is critical for understanding how impact and blast waves damage tissue in vivo and has implications for the design of protective gear and tissue engineering.

SignificanceMild injuries associated with concussion and blast waves cause tearing of brain tissue, which leads to traumatic brain injury (TBI). TBI is a leading cause of death and disability among children and young adults in the U.S., with 1.5 million Americans reporting a TBI each year. We introduce a novel approach to visualize these tears in intact brain tissue, and report the energies associated with brain fracture. Quantifying the fracture energy of brain, as we have done here, is critical to understand the forces from injury that lead to TBI.

sentences: The compound of baicalein, wogonin, and oroxylin-A inhibits EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC.

sentences: GENE DUPLICATION TO THE Y CHROMOSOME IN TRINIDADIAN GUPPIES Differences in allele frequencies at autosomal genes between males and females in a population can result from two scenarios. Unresolved sexual conflict over survival produces allelic differentiation between the sexes. However, given the substantial mortality costs required to produce allelic differences between males and females at each generation, it remains unclear how many loci within the genome experience significant sexual conflict over survival. Alternatively, recent studies have shown that similarity between autosomal and Y sequence, arising from duplication onto the Y, can create perceived allelic differences, and this represents potentially resolved sexual conflict. However, Y duplications are most likely in species with large non-recombining regions, in part because they simply represent larger targets for duplications. We assessed the genomes of 120 wild-caught guppies, which experience extensive predation- and pathogen-induced mortality and have a relatively small ancestral Y chromosome. We identified seven autosomal genes that show allelic differences between male and female adults. Five of these genes show clear evidence of whole or partial gene duplication to the Y chromosome, suggesting that the male-specific region of the guppy Y chromosome, although relatively small, may nonetheless act as a hotspot for the resolution of sexual conflict. The remaining two genes show evidence of partial homology to the Y. Overall, our findings suggest that the guppy genome experiences a very low level of unresolved sexual conflict over survival, and instead the Y chromosome, despite its small ancestral size and recent origin, acts as a major mechanism of conflict resolution.

sentences: Mixed matrix factorization: a novel algorithm for the extraction of kinematic-muscular synergies Synergistic models have been employed to investigate motor coordination separately in the muscular and kinematic domains. However, the relationship between muscle synergies, constrained to be non-negative, and kinematic synergies, whose elements can be positive and negative, has received limited attention. Existing algorithms for extracting synergies from combined kinematic and muscular data either do not enforce non-negativity constraints or separate non-negative variables into positive and negative components. We propose a mixed matrix factorization (MMF) algorithm based on a gradient descent update rule which overcomes these limitations. It allows to directly assess the relationship between kinematic and muscle activity variables, by enforcing the non-negativity constrain on a subset of variables. We validated the algorithm on simulated kinematic-muscular data generated from known spatial synergies and temporal coefficients, by evaluating the similarity between extracted and ground truth synergies and temporal coefficients when the data are corrupted by different noise levels. We also compared the performance of MMF to that of non-negative matrix factorization applied to separate positive and negative components (NMFpn). Finally, we factorized kinematic and EMG data collected during upper-limb movements to demonstrate the potential of the algorithm. MMF achieved almost perfect reconstruction on noiseless simulated data. It performed better than NMFpn in recovering the correct spatial synergies and temporal coefficients with noisy simulated data. It also allowed to correctly select the original number of ground truth synergies. We showed meaningful applicability to real data; MMF can also be applied to any multivariate data that contains both non-negative and unconstrained variables.

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Pickering stabilization of a dynamic intracellular emulsion Biomolecular condensates are cellular compartments that form by phase separation in the absence of limiting membranes. Studying the P granules of C. elegans, we find that condensate dynamics are regulated by protein clusters that adsorb to the condensate interface. Using in vitro reconstitution, live observations and theory, we demonstrate that localized assembly of P granules is controlled by MEG-3, an intrinsically disordered protein that forms low dynamic assemblies on P granules. Following classic Pickering emulsion theory, MEG-3 clusters lower surface tension and slow down coarsening. During zygote polarization, MEG-3 recruits DYRK/MBK-2 kinase to accelerate localized growth of the P granule emulsion. By tuning condensate-cytoplasm exchange, interfacial clusters regulate the structural integrity of biomolecular condensates, reminiscent of the role of lipid bilayers in membrane-bound organelles.

One Sentence SummaryBiomolecular condensates are stabilized by interfacial nanoscale protein clusters.

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The novel, recurrent mutation in the TOP2A gene results in the enhanced topoisomerase activity and transcription deregulation in glioblastoma BackgroundHigh grade gliomas (HGGs) are aggressive, primary brain tumors with poor clinical outcomes. We aim to better understand glioma pathobiology and find potential therapeutic susceptibilities.

MethodsWe designed a custom panel of 664 cancer- and epigenetics-related genes, and employed targeted next generation sequencing to study the genomic landscape of somatic and germline variants in 182 gliomas of different malignancy grades. mRNA sequencing was performed to detect transcriptomic abnormalities.

ResultsIn addition to known alterations in TP53, IDH1, ATRX, EGFR genes found in this cohort, we identified a novel, recurrent mutation in the TOP2A gene coding for Topoisomerase 2A occurring only in glioblastomas (GBM, WHO grade IV gliomas). Biochemical assays with recombinant proteins demonstrated stronger DNA binding and DNA supercoil relaxation activities of the variant proteins. GBM patients carrying the mutated TOP2A had shorter overall survival than those with the wild type TOP2A. Computational analyses of transcriptomic data showed that GBMs with the mutated TOP2A have different transcriptomic patterns suggesting higher transcriptomic activity.

ConclusionWe identified a novel TOP2A E948Q variant that strongly binds to DNA and is more active than the wild type protein. Our findings suggest that the discovered TOP2A variant is gain-of-function mutation.

Key pointsO_LIThe most frequent genetic alterations in high grade gliomas are reported.
C_LIO_LIA new mutation in the TOP2A gene was found in 4 patients from Polish population.
C_LIO_LIA E948Q substitution changes TOP2A activities towards DNA.
C_LIO_LIThe recurrent TOP2A variant is a gain-of-function mutation.
C_LI

Importance of the studyGlioblastoma is a deadly disease. Despite recent advancements in genomics and innovative targeted therapies, glioblastoma therapy has not shown improvements. Insights into glioblastoma biology may improve diagnosis, prognosis, and treatment prediction, directing to a better outcome. We performed targeted sequencing of 664 cancer genes, and identified a new variant of the TOP2A gene encoding topoisomerase 2A in glioblastomas. The TOP2A protein variant shows a higher affinity towards DNA and causes transcriptional alterations, suggesting a higher de novo transcription rate.

sentences: A parametric logistic equation with light flux and medium concentration for cultivation planning of microalgae Microalgae are considered to be promising producers of bioactive chemicals, feeds, and fuels from carbon dioxide by photosynthesis. Thus, the prediction of microalgal growth profiles is important for the planning of cost-effective and sustainable cultivation-harvest cycles. This paper proposes a mathematical model capable of predicting the effect of light flux into culture and medium concentration on the growth profiles of microalgae by incorporating these growth-limiting factors into the logistic equation. The specific form of the equation is derived based on the experimentally measured growth profiles of Monoraphidium sp., a microalgal strain isolated by the authors, under 16 conditions consisting of the combinations of incident light fluxes into culture and initial medium concentrations. Using a cross-validation method, it is shown that the proposed model has the ability to predict necessary incident light flux into culture and initial medium concentration for harvesting target biomass at target time. Finally, the model-guided cultivation planning is performed and is evaluated by comparing the result with the experimental data.

sentences: Parallel evolution of phage resistance - virulence trade - offs during in vitro and nasal Pseudomonas aeruginosa phage treatment With rising antibiotic resistance, there has been increasing interest in treating pathogenic bacteria with bacteriophages (phage therapy). One limitation of phage therapy is the ease at which bacteria can evolve resistance. Negative effects of resistance may be mitigated when resistance results in reduced bacterial growth and virulence, or when phage coevolve to overcome resistance. Resistance evolution and its consequences are contingent on the bacteria-phage combination and their environmental context, making therapeutic outcomes hard to predict. One solution might be to conduct "in vitro evolutionary simulations" using bacteria-phage combinations from the therapeutic context. Overall, our aim was to investigate parallels between in vitro experiments and in vivo dynamics in a human participant. Evolutionary dynamics were similar, with high levels of resistance evolving quickly with limited evidence of phage evolution. Resistant bacteria - evolved in vitro and in vivo - had lower virulence. In vivo, this was linked to lower growth rates of resistant isolates, whereas in vitro phage resistant isolates evolved greater biofilm production. Population sequencing suggests resistance resulted from selection on de novo mutations rather than sorting of existing variants. These results highlight the speed at which phage resistance can evolve in vivo, and in vitro experiments may give useful insights for clinical evolutionary outcomes.

sentences: Invariant Differential Expression Analysis Reveals Mechanism of Cancer Resistance to Cell Cycle Inhibitors Retinoblastoma (RB) is a good model to study drug resistance to cell-cycle inhibitors because it is driven by mutations in the core components of cell-cycle, i.e, Rb gene. However, there is limited gene expression dataset in RB which has major reproducibility issues. We have developed invariant differential expression analysis (iDEA) that improves the state of the art in differential expression analysis (DEA). iDEA uses strong Boolean implication relationships in a large diverse human dataset GSE119087 (n = 25,955) to filter the noisy differentially expressed genes (DEGs). iDEA was applied to RB datasets and a gene signature was computed that led to prediction and mechanism of drug sensitivity. The prediction was confirmed using drugs-sensitive/resistant RB cell-lines and mouse xenograft models using CDC25 inhibitor NSC663284. iDEA improved reproducibility of differential expression across diverse retina/RB cohorts and RB cell-lines with different drug sensitivity (Y79/Weri vs NCC). Pathway analysis revealed WNT/{beta}-catenin involved in distinguishing drug sensitivity to CDC25 inhibitor NSC663284. NSC663284 inhibited tumour cell proliferation in mouse xenograft model containing Y79 cells indicating novel therapeutic option in RB. Invariant differentially expressed genes (iDEGs) are robustly associated with outcome in diverse cancer datasets and supports for a fundamental mechanism of drug resistance.

sentences: Potent macrocycle inhibitors of the human SAGA deubiquitinating module The SAGA transcriptional coactivator contains a four-protein subcomplex called the DUB module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit, USP22, which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 as compared to a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for development.

sentences: A structural atlas of the developing zebrafish telencephalon based on spatially-restricted transgene expression 1.Zebrafish telencephalon acquires an everted morphology by a two-step process that occurs from 1 to 5 days post-fertilization (dpf). Little is known about how this process affects the positioning of discrete telencephalic cell populations, hindering our understanding of how eversion impacts telencephalic structural organisation. In this study, we characterise the neurochemistry, cycle state and morphology of an EGFP positive (+) cell population in the telencephalon of Et(gata2:EGFP)bi105 transgenic fish during eversion and up to 20dpf. We map the transgene insertion to the early-growth-response-gene-3 (egr3) locus and show that EGFP expression recapitulates endogenous egr3 expression throughout much of the pallial telencephalon. Using the gata2:EGFPbi105 transgene, in combination with other well-characterised transgenes and structural markers, we track the development of various cell populations in the zebrafish telencephalon as it undergoes the morphological changes underlying eversion. These datasets were registered to reference brains to form an atlas of telencephalic development at key stages of the eversion process (1dpf, 2dpf and 5dpf) and compared to expression in adulthood. Finally, we registered gata2:EGFPbi105 expression to the Zebrafish Brain Browser 6dpf reference brain (ZBB, see Marquart et al., 2015, 2017; Tabor et al. 2019), to allow comparison of this expression pattern with anatomical data already in ZBB.

sentences: Transcriptional interference in toehold switch-based RNA circuits Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5 untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.

sentences: NOTCH1 drives immune-escape mechanisms in B cell malignancies NOTCH1 is a recurrently mutated gene in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Functional studies to investigate its role have been hampered by the inability to genetically manipulate primary human lymphoma cells, attributed to low transduction-efficacy and procedure-associated toxicity. To overcome these limitations, we have developed a novel method to retrovirally transfer genes into malignant human B cells. We generated isogenic human tumor cells from patients with CLL and MCL, differing only in their expression of NOTCH1. Our data demonstrate that NOTCH1 facilitates immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-{gamma} signaling. In addition, NOTCH1 causes silencing of the entire HLA-class II locus via suppression of the transcriptional co-activator CIITA. These NOTCH1-mediated immune escape mechanisms are associated with the expansion of CD4+ T cells in vivo, further contributing to the poor clinical outcome of NOTCH1-mutated CLL and MCL.

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A method for the efficient iron-labeling of patient-derived xenograft cells and cellular imaging validation There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumour behavior, and metastatic properties observed in the original tumour. These models are more predictive of clinical outcomes and are superior to cell lines for preclinical drug evaluation and therapeutic strategies. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metastases and validate this method with imaging during tumour development.

Luciferase expressing human breast cancer PDX cells (F2-7) were successfully labeled after incubation with micron-sized iron oxide particles (MPIO; 25 g Fe/mL). NOD/SCID/ILIIrg-/- (n=5) mice received injections of 1x106 iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. In vivo BLI revealed that signal increased over time with tumour development. MPI revealed decreasing signal in the tumours and increasing signal in the liver region over time.

Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumour. To accomplish this, we also demonstrate the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.

sentences: Multi-omics Analyses Provide Insight into the Biosynthesis Pathways of Fucoxanthin in Isochrysis galbana Isochrysis galbana is considered an ideal bait for functional foods and nutraceuticals in humans because of its high fucoxanthin (Fx) content. However, multi-omics analysis of the regulation networks for Fx biosynthesis in I. galbana has not been reported. In this study, we report a high-quality genome sequence of I. galbana LG007, which has a 92.73 Mb genome size, with a contig N50 of 6.99 Mb and 14,900 protein-coding genes. Phylogenomic inferences confirmed the monophyly of Haptophyta, with I. galbana sister to Emiliania huxleyi and Chrysochromulina tobinii. Evolutionary analysis revealed an estimated divergence time between I. galbana and E. huxleyi of ~ 133 million years ago (Mya). Gene family analysis indicated that lipid metabolism-related genes exhibited significant expansion, including IgPLMT, IgOAR1 and {Delta}-4 desaturase. Metabolome analysis showed that the content of carotenoid in I. galbana cultured under green light for 7 days was higher than that of white light, and {beta}-carotene was the main carotenoids, accounting for 79.09% of the total carotenoids. Comprehensive analysis of multi-omics analysis revealed that {beta}-carotene, antheraxanthin, zeaxanthin, and Fx content was increased by green light induction, which was significantly correlated with the expression of IgMYB98, IgZDS, IgPDS, IgLHCX2, IgZEP, IgLCYb, and IgNSY. These findings contribute to understanding Fx biosynthesis and its regulation, providing a valuable reference for food and pharmaceutical applications.

sentences: Pre-Clinical Application of Functional Human Induced Pluripotent Stem Cell-Derived Airway Epithelial Grafts Airway pathologies including cancer, trauma and stenosis lack effective treatments, meanwhile airway transplantation and available tissue engineering approaches fail due to epithelial dysfunction. Autologous progenitors do not meet the clinical need for regeneration due to their insufficient expansion and differentiation, for which human induced pluripotent stem cells (hiPSCs) are promising alternatives. Airway epithelial grafts are engineered by differentiating hiPSC-derived airway progenitors into physiological proportions of ciliated (73.9{+/-}5.5%) and goblet (2.1{+/-}1.4%) cells on a Silk Fibroin-Collagen Vitrigel Membrane (SF-CVM) composite biomaterial for transplantation in porcine tracheal defects ex vivo and in vivo. Evaluation of ex vivo tracheal repair using hiPSC-derived SF-CVM grafts demonstrate native-like tracheal epithelial metabolism and maintenance of mucociliary epithelium to day 3. In vivo studies reveal SF-CVM integration, maintenance of airway patency, showing 80.8{+/-}3.6% graft coverage with an hiPSC-derived pseudostratified epithelium and 70.7{+/-}2.3% coverage with viable cells, 3 days post-operatively. We demonstrate the utility of bioengineered, hiPSC-derived epithelial grafts for airway repair in a pre-clinical survival model, providing a significant leap for airway reconstruction approaches.

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Alcohol Use Disrupts Age-Appropriate Cortical Thinning in Adolescence: A Data Driven Approach ObjectiveCortical thickness changes dramatically during development and is influenced by adolescent drinking. However, previous findings have been inconsistent and limited by region-of-interest approaches that are underpowered because they do not conform to the underlying heterogeneity from the effects of alcohol.

MethodsAdolescents (n=657; 12-22 years at baseline) from the National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA) who endorsed little to no alcohol use at baseline were assessed with structural MRI and followed longitudinally at four yearly intervals. Seven unique spatially covarying patterns of cortical thickness were obtained from the baseline scans by applying a novel data-driven method called non-negative matrix factorization (NMF). The cortical thickness maps of all participants longitudinal scans were projected onto vertex-level cortical patterns to obtain participant-specific coefficients for each pattern. Linear mixed-effects models were fit to each pattern to investigate longitudinal effects of alcohol consumption on cortical thickness.

ResultsIn most NMF-derived cortical thickness patterns, the longitudinal rate of decline in no/low drinkers was similar for all age cohorts, among moderate drinkers the decline was faster in the younger cohort and slower in the older cohort, among heavy drinkers the decline was fastest in the younger cohort and slowest in the older cohort (FDR corrected p-values < 0.01).

ConclusionsThe NMF method can delineate spatially coordinated patterns of cortical thickness at the vertex level that are unconstrained by anatomical features. Age-appropriate cortical thinning is more rapid in younger adolescent drinkers and slower in older adolescent drinkers.

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Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reactions cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the birds-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes.

Graphical Abstract

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Age-related differences in functional network segregation in the context of sex and reproductive stage Age is accompanied by differences in the organization of functional brain networks, which impact behavior in adulthood. Functional networks tend to become less segregated and more integrated with age. However, sex differences in network segregation declines with age are not well-understood. Further, network segregation in the context of female reproductive stage is relatively understudied, though unmasking such relationships would be informative for elucidating biological mechanisms that contribute to sex-specific differences in aging. In the current work, we used data from the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) repository to evaluate differences in resting-state network segregation as a product of sex and reproductive stage. Reproductive stage was categorized using the Stages of Reproductive Aging Workshop (STRAW+10) criteria. Replicating prior work, we investigated the following functional networks: auditory, cerebellar-basal ganglia, cingulo-opercular task control, default mode, dorsal attention, fronto-parietal task control, salience, sensory somatomotor mouth, sensory somatomotor hand, ventral attention, and visual. First, our results mirror findings from previous work indicating that network segregation is lower with increasing age. Second, when analyzing associations between network segregation and age within each sex separately, we find differences between females and males. Finally, we report significant effects of reproductive stage on network segregation, though these findings are likely driven by age. Broadly, our results suggest that impacts of sex are important to evaluate when investigating network segregation differences across adulthood, though further work is needed to determine the unique role of menopause and sex hormones on the organization of functional brain networks within aging females.

Key PointsO_LISegregation of functional brain networks declines with increasing age
C_LIO_LIAge-segregation relationships are modified by biological sex
C_LIO_LIReproductive stage may impact sex differences in brain network organization
C_LI

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Enabling multiscale variation analysis with genome graphs BackgroundStandard approaches to characterising genetic variation revolve around mapping reads to a reference genome and describing variants in terms of differences from the reference; this is based on the assumption that these differences will be small and provides a simple coordinate system. However this fails, and the coordinates break down, when there are diverged haplotypes at a locus (e.g. one haplotype contains a multi-kilobase deletion, a second contains a few SNPs, and a third is highly diverged with hundreds of SNPs). To handle these, we need to model genetic variation that occurs at different length-scales (SNPs to large structural variants) and that occurs on alternate backgrounds. We refer to these together as multiscale variation.

ResultsWe model the genome as a directed acyclic graph consisting of successive hierarchical subgraphs ("sites") that naturally incorporate multiscale variation, and introduce an algorithm for genotyping, implemented in the software gramtools. This enables variant calling on different sequence backgrounds. In addition to producing regular VCF files, we introduce a JSON file format based on VCF, which records variant site relationships and alternate sequence backgrounds.

We show two applications. First, we benchmark gramtools against existing state-of-the-art methods in joint-genotyping 17 M. tuberculosis samples at long deletions and the overlapping small variants that segregate in a cohort of 1,017 genomes. Second, in 706 African and SE Asian P. falciparum genomes, we analyse a dimorphic surface antigen gene which possesses variation on two diverged backgrounds which appeared to not recombine. This generates the first map of variation on both backgrounds, revealing patterns of recombination that were previously unknown.

ConclusionsWe need new approaches to be able to jointly analyse SNP and structural variation in cohorts, and even more to handle variants on different genetic backgrounds. We have demonstrated that by modelling with a directed, acyclic and locally hierarchical genome graph, we can apply new algorithms to accurately genotype dense variation at multiple scales. We also propose a generalisation of VCF for accessing multiscale variation in genome graphs, which we hope will be of wide utility.

sentences: Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in nonhuman primates is coincident with an anamnestic antibody response in the lower airway mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined [~]3-log10 compared to control animals. In nasal swabs, sgRNA declined 1-log10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

sentences: Population receptive fields in non-human primates from whole-brain fMRI and large-scale neurophysiology in visual cortex Population receptive field (pRF) modeling is a popular fMRI method to map the retinotopic organization of the human brain. While fMRI-based pRF-maps are qualitatively similar to invasively recorded single-cell receptive fields in animals, it remains unclear what neuronal signal they represent. We addressed this question in awake non-human primates comparing whole-brain fMRI and large-scale neurophysiological recordings in areas V1 and V4 of the visual cortex. We examined the fits of several pRF-models based on the fMRI BOLD-signal, multi-unit spiking activity (MUA) and local field potential (LFP) power in different frequency bands. We found that pRFs derived from BOLD-fMRI were most similar to MUA-pRFs in V1 and V4, while pRFs based on LFP gamma power also gave a good approximation. FMRI-based pRFs thus reliably reflect neuronal receptive field properties in the primate brain. In addition to our results in V1 and V4, the whole-brain fMRI measurements revealed retinotopic tuning in many other cortical and subcortical areas with a consistent increase in pRF-size with increasing eccentricity, as well as a retinotopically specific deactivation of default-mode network nodes similar to previous observations in humans.

sentences: PME-1 suppresses anoikis, and is associated with therapy relapse of PTEN-deficient prostate cancers While organ-confined PCa is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumors with concomitantly compromised function of two tumor suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having less than 50% 5-year secondary-therapy free patient survival. Functionally, overexpression of PME-1, a PP2A inhibitor protein, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumor xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1 deficient PCa cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype corresponding to increased apoptosis sensitivity. In summary, we discover that PME-1 overexpression supports anoikis resistance in PTEN-deficient PCa cells. Clinically, the results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa.

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Improved SNV discovery in barcode-stratified scRNA-seq alignments Single cell SNV analysis is an emerging and promising strategy to connect cell-level genetic variation to cell phenotypes. At the present, SNV detection from 10x Genomics scRNA-seq data is typically performed on the pooled sequencing reads across all cells in a sample. Here, we assess the gain of information of SNV assessments from individual cell scRNA-seq data, where the alignments are split by barcode prior to the variant call. For our analyses we use publicly available sequencing data on the human breast cancer cell line MCF7 cell line generated at consequent time-points during anti-cancer treatment. We analyzed SNV calls by three popular variant callers - GATK, Strelka2 and Mu-tect2, in combination with a method for cell-level tabulation of the sequencing read counts bearing SNV alleles - SCReadCounts. Our analysis shows that variant calls on individual cell alignments identify at least two-fold higher number of SNVs as compared to the pooled scRNA-seq. We demonstrate that scSNVs exclusively called in the single cell alignments (scSNVs) are substantially enriched in novel genetic variants and in coding functional annotations, in particular, stop-codon and missense substitutions. Furthermore, we find that the expression of some scSNVs correlates with the expression of their harbouring gene (cis-scReQTLs).

Overall, our study indicates an immense potential of SNV calls from individual cell scRNA-seq data and emphasizes on the need of cell-level variant detection approaches and tools. Given the growing accumulation of scRNA-seq datasets, cell-level variant assessments are likely to significantly contribute to the understanding of the cellular heterogeneity and the relationship between genetics variants and functional phenotypes. In addition, cell-level variant assessments from scRNA-seq can be highly informative in cancer where they can help elucidate somatic mutations evolution and functionality.

sentences: STEREOLOGICAL EVALUATION OF TISSUE PRESERVATION AFTERNEUROPROTECTIVE TREATMENTS FOR TRAUMATIC SPINAL CORDINJURY Spinal cord injury (SCI) is a major cause of permanent disability and its causes and pathophysiological effects are very variables between patients. The assessment of tissue damage extent and neurodegeneration degree correlated with the functional evaluation are the most accepted tools to diagnose and prognose the trauma severity. Animal models of SCI have been used for treatment development and in the present work we evaluate the potency of stereological tools to estimate damage degree for a diagnostic of neural degeneration and locomotor and sensorial disability after SCI and the efficacy of different types of therapeutic strategies.

sentences: Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India. Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

sentences: Joint evolution of mycorrhizal type, pollination, and seed dispersal mode in trees Although tree diversity is fundamental to terrestrial ecosystems, the processes that generate it remain uncertain. Mycorrhizal type, pollination mode, and seed dispersal mode may be key drivers of tree diversity. We predicted that mycorrhizal symbiosis drove evolution of pollination and seed dispersal modes because arbuscular mycorrhizal (AM) associations would favour long-range seed or pollen dispersal owing to their negative plant-soil-feedback effects on conspecific individuals, and ectomycorrhizal (EcM) associations would favour short-range dispersal owing to their positive effects. We analysed evolutionary relationships among mycorrhizal type, seed dispersal mode, and pollination mode in 704 tree species and conducted a meta-analysis of the dispersal distances of the various seed dispersal and pollination modes. We found evidence of joint evolution of all three features. Most AM-associated trees had endozoochorous seed dispersal and biotic pollination and these dispersal modes had long dispersal distances, whereas most EcM-associated trees had anemochorous seed dispersal and wind pollination and these dispersal modes had relatively shorter dispersal distances. Overall, evolution of mycorrhizal type, seed dispersal mode, and pollination mode were linked, strongly suggesting that mycorrhizal symbiosis drives the evolution of seed and pollination modes and contributes to tree diversification.

sentences: Differential impact of TNFRSF co-stimulation on CD8+ T cell cytokine production by feedback control of surface receptor expression T cell responses towards infections and cancers are regulated by a host of co-signalling receptors that are largely grouped into the binary categories of co-stimulation and co-inhibition. The TNF receptor superfamily (TNFRSF) members 4-1BB, CD27, GITR, and OX40 are well-established co-stimulation receptors with largely shared molecular pathways raising the question of whether they also have a similar impact on quantitative T cell responses, such as the efficacy, sensitivity, and duration of T cell responses. Here, we systematically stimulated primary human CD8+ T cell blasts with dose ranges of antigen and ligands for TNFRSF members to screen for their quantitative effects on cytokine production. Although both 4-1BB and CD27 increased efficacy, only 4-1BB was able to prolong the duration of cytokine production, and both had only a modest impact on antigen sensitivity. An operational model could explain these divergent quantitative phenotypes using a shared signalling mechanism based on the surface expression of 4-1BB, but not CD27, being regulated through a signalling feedback. The model predicted that CD27 co-stimulation would increase 4-1BB expression and subsequent 4-1BB co-stimulation, which we confirmed experimentally. Although GITR and OX40 produced only minor changes in cytokine production on their own, we found that like 4-1BB, CD27 could enhance GITR expression and subsequent GITR co-stimulation. Thus, feedback control of induced TNFRSF surface expression explains both synergy and differential impact on cytokine production. The work highlights that different co-stimulation receptors can have different quantitative phenotypes on the same output allowing for highly regulated control of T cell responses.

sentences: New Segmentation and Feature Extraction Algorithm for the Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate.

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Rapid homeostatic modulation of transsynaptic nanocolumn rings Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization, and how synaptic nano-architecture is transsynaptically regulated to achieve stable synaptic transmission remains enigmatic. Using time-gated stimulated emission depletion (gSTED) microscopy at the Drosophila neuromuscular junction, we here find that presynaptic nano-rings formed by the active-zone scaffold Bruchpilot (Brp) precisely align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor ~5 transsynaptically-aligned Brp-GluR  nanocolumns. Intriguingly, acute GluR impairment rapidly triggers the formation of new transsynaptic nanocolumns on the minute time scale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic reorganization preceding presynaptic modulation. Finally, the auxiliary GluR subunit Neto-{beta} promotes structural and functional homeostatic plasticity. Thus, transsynaptic nanocolumns arrange in stereotypic rings that are rapidly modulated during homeostatic plasticity to stabilize synaptic efficacy.

One Sentence SummaryRapid changes of transsynaptic nanocolumn rings during homeostatic plasticity.

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Targeted in silico characterization of fusion transcripts in tumor and normal tissues via FusionInspector BackgroundGene fusions play a key role as driving oncogenes in tumors, and their reliable discovery and detection is important for cancer research, diagnostics, prognostics and guiding personalized therapy. While discovering gene fusions from genome sequencing can be laborious and costly, the resulting "fusion transcripts" can be recovered from RNA-seq data of tumor and normal samples. However, alleged and putative fusion transcript can arise from multiple sources in addition to the chromosomal rearrangements yielding fusion genes, including cis- or trans-splicing events, experimental artifacts during RNA-seq or computational errors of transcriptome reconstruction methods. Understanding how to discern, interpret, categorize, and verify predicted fusion transcripts is essential for consideration in clinical settings and prioritization for further research. Here, we present FusionInspector for in silico characterization and interpretation of candidate fusion transcripts from RNA-seq, enabling exploration of sequence and expression characteristics of fusions and their partner genes.

ResultsWe applied FusionInspector to thousands of tumor and normal transcriptomes, and identified statistical and experimental features enriched among biologically impactful fusions. Through clustering and machine learning, we identified large collections of fusions potentially relevant to tumor and normal biological processes. We show that biologically relevant fusions are enriched for relatively high expression of the fusion transcript, imbalanced fusion allelic ratios, and canonical splicing patterns, and are deficient in sequence microhomologies detected between partner genes.

ConclusionWe demonstrate FusionInspector to accurately in silico validate fusion transcripts, and to help identify numerous understudied fusions in tumor and normal tissues samples. FusionInspector is freely available as open source for screening, characterization, and visualization of candidate fusions via RNA-seq. We believe that this work will continue driving the discipline of transparent explanation and interpretation of machine learning predictions and tracing the predictions to their experimental sources.

sentences: A Synthesis of Game Theory and Quantitative Genetic Models of Social Evolution Two popular approaches for modeling social evolution, evolutionary game theory and quantitative genetics, ask complementary questions but are rarely integrated. Game theory focuses on evolutionary outcomes, with models solving for evolutionarily stable equilibria, whereas quantitative genetics provides insight into evolutionary processes, with models predicting short-term responses to selection. Here we draw parallels between evolutionary game theory and interacting phenotypes theory, which is a quantitative genetic framework for understanding social evolution. First, we show how any evolutionary game may be translated into two quantitative genetic selection gradients, nonsocial and social selection, which may be used to predict evolutionary change from a single round of the game. We show that synergistic fitness effects may alter predicted selection gradients, causing changes in magnitude and sign as the population mean evolves. Second, we show how evolutionary games involving plastic behavioral responses to partners can be modeled using indirect genetic effects, which describe how trait expression changes in response to genes in the social environment. We demonstrate that repeated social interactions in models of reciprocity generate indirect effects and conversely, that estimates of parameters from indirect genetic effect models may be used to predict the evolution of reciprocity. We argue that a pluralistic view incorporating both theoretical approaches will benefit empiricists and theorists studying social evolution. We advocate the measurement of social selection and indirect genetic effects in natural populations to test the predictions from game theory, and in turn, the use of game theory models to aid in the interpretation of quantitative genetic estimates.

sentences: Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

sentences: Computational design of highly signaling active membrane receptors through de novo solvent-mediated allosteric networks Protein catalysis and allostery require the atomic-level orchestration and motion of residues, ligand, solvent and protein effector molecules, but the ability to design protein activity through precise protein-solvent cooperative interactions has not been demonstrated. Here, we report the design of a dozen novel membrane receptors catalyzing G-protein nucleotide exchange through diverse de novo engineered allosteric pathways mediated by cooperative networks of intra-protein, protein-ligand and solvent molecule interactions. Consistent with the predictions, designed protein activities strongly correlated with the level of solvent-mediated interaction network plasticity at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared to related natural receptors. The most stable and active variant crystallized in an unforeseen signaling active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal a space of allosteric interactions larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein catalytic and signaling activities.

sentences: BayesDeBulk: A Flexible Bayesian Algorithm for the Deconvolution of Bulk Tumor Data Characterizing the tumor microenvironment is crucial in order to improve responsiveness to immunotherapy and develop new therapeutic strategies. The fraction of different cell-types in the tumor microenvironment can be estimated based on transcriptomic profiling of bulk tumor data via deconvolution algorithms. One class of such algorithms, known as reference-based, rely on a reference signature containing gene expression data for various cell-types. The limitation of these methods is that such a signature is derived from the gene expression of pure cell-types, which might not be consistent with the transcriptomic profiling in solid tumors. On the other hand, reference-free methods usually require only a set of cell-specific markers to perform deconvolution; however, once the different components have been estimated from the data, their labeling can be problematic. To overcome these limitations, we propose BayesDeBulk - a new reference-free Bayesian method for bulk deconvolution based on gene expression data. Given a list of markers expressed in each cell-type (cell-specific markers), a repulsive prior is placed on the mean of gene expression in different cell-types to ensure that cell-specific markers are upregulated in a particular component. Contrary to existing reference-free methods, the labeling of different components is decided a priori through a repulsive prior. Furthermore, the advantage over reference-based algorithms is that the cell fractions as well as the gene expression of different cells are estimated from the data, simultaneously. Given its flexibility, BayesDeBulk can be utilized to perform bulk deconvolution beyond transcriptomic data, based on other data types such as proteomic profiles or the integration of both transcriptomic and proteomic profiles.

sentences: Coherent activity at three major lateral hypothalamic neural outputs controls the onset of motivated behavior responses The lateral hypothalamus (LH) plays an important role in motivated behavior. However, it is not known how LH neural outputs dynamically signal to major downstream targets to organize behavior. We used multi-fiber photometry to show that three major LH neural outputs projecting to the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and lateral habenula (LHb) exhibit significant coherent activity in mice engaging motivated responses, which decrease during immobility. Mice engaging active coping responses exhibit increased activity at LH axon terminals that precedes an increase in the activity of serotonin neurons and dopamine neurons, indicating that they may play a role in initiating active responses stemming from LH signal transmissions. The optogenetic activation of LH axon terminals in either the DRN, VTA, or LHb was sufficient to increase mobility but had different effects on passive avoidance and sucrose consumption, suggesting that LH outputs use complementary mechanisms to control behavioral responses. Our results support the notion that the three LH neural outputs play complementary roles in initiating motivated behaviors.

sentences: Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available - the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, long-read data. To demonstrate the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs with >90% accuracy. These results demonstrate that nanopore sequencing platforms are capable of revealing additional variation in and around STR loci depending on read coverage. As the first long-read platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

sentences: Improved production and expanded application of CVS-N2c-ΔG virus for retrograde tracing Neurotropic virus tracers, particularly those with low toxicity and high efficient tracing, are powerful tools for structural and functional dissections of neural circuits. The retrograde trans-mono-synaptic technology based on rabies virus CVS-N2c strain has reduced cytotoxicity and enhanced efficiency, attains long-term gene manipulation for functional studies, but suffers from difficult preparation and low yield. To overcome these shortcomings, an improved production system was established for rapid rescue and preparation of CVS-N2c-{Delta}G virus, CVS-N2c-{Delta}G with the same titer as SAD-B19-{Delta}G can be prepared within a short time. Meanwhile, we found that N2cG coated CVS-N2c-{Delta}G allows efficient retrograde access to projection neurons, and further expand its application in VTA/SNc to DLS pathway that unaddressed by rAAV9-Retro, and the efficiency is 6 folds higher than that of rAAV9-Retro. Then the trans-synaptic efficiency of CVS-N2c-{Delta}G virus was evaluated. Results showed that the trans-mono-synaptic efficiency of oG-mediated CVS-N2c-{Delta}G was 2-3 folds higher than that of oG-mediated SAD-B19-{Delta}G, but there was no difference between oG-mediated and N2cG-mediated CVS-N2c-{Delta}G system. In addition, codon modified N2cG (optiG) did not increase the efficiency of CVS-N2c-{Delta}G tracing. Finally, we found that the CVS-N2c-{Delta}G produced by the improved method can be used for monitoring neural activity of projection neurons, and the time window can be maintained for 3 weeks, and it can also express sufficient recombinases for efficient transgene recombination. That is, the virus produced by the improved production system does not affect its own function, paving the way for its further optimization, popularization and application in structural and functional studies of neural circuits.

sentences: Inka2, a novel Pak4 inhibitor, regulates actin dynamics in dendritic spine development The actin filament is a fundamental part of the cytoskeleton defining cell morphology and regulating various physiological processes, including filopodia formation and dendritic spinogenesis of neurons. Serine/threonine-protein kinase Pak4, an essential effector, links Rho GTPases to control actin polymerization. Previously, we identified the Inka2 gene, a novel mammalian protein exhibiting sequence similarity to Inka1, which serves as a possible inhibitor for Pak4. Although Inka2 is dominantly expressed in the nervous system and involved in focal-adhesion dynamics, its molecular role remains unclear. Here, we found that Inka2-iBox directly binds to Pak4 catalytic domain to suppress actin polymerization. Inka2 promoted actin depolymerization and inhibited the formation of cellular protrusion caused by Pak4 activation. We further generated the conditional knockout mice of the Inka2 gene. The beta-galactosidase reporter indicated the preferential Inka2 expression in the dorsal forebrain neurons. Cortical pyramidal neurons of Inka2-/- mice exhibited decreased density and aberrant morphology of dendritic spines with marked activation/phosphorylation of downstream molecules of Pak4 signal cascade, including LIMK and cofilin. These results uncovered the unexpected function of endogenous Pak4 inhibitor in neurons. Unlike Inka1, Inka2 is a critical mediator for actin reorganization required for dendritic spine development.

sentences: Merlin Tumor Suppressor Function is Regulated by PIP2-Mediated Dimerization Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central -helical region and a C-terminal domain that binds to the FERM domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. These conformational transitions are regulated by both phosphorylation and phosphoinositide binding. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay and found that Merlin dimerizes via a FERM-FERM interaction in a parallel orientation that requires an uncovered N-terminus and the first 18 amino acids of the FERM domain. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Dimerization requires an open conformation, is inhibited by phosphorylation at serine 518 and is enhanced by PIP2 binding. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with significant implications for the development of therapies designed to compensate for Merlin loss.

sentences: SARS -CoV-2 T-cell immunity to variants of concern following vaccination Recently, two mRNA vaccines to severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) have become available, but there is also an emergence of SARS-CoV-2 variants with increased transmissibility and virulence1-6. A major concern is whether the available vaccines will be equally effective against these variants. The vaccines are designed to induce an immune response against the SARS-CoV-2 spike protein7, 8, which is required for viral entry to host cells9. Immunity to SARS-CoV-2 is often evaluated by antibody production, while less is known about the T-cell response. Here we developed, characterized, and implemented two standardized, functional assays to measure T-cell immunity to SARS-CoV-2 in uninfected, convalescent, and vaccinated individuals. We found that vaccinated individuals had robust T-cell responses to the wild type spike and nucleocapsid proteins, even more so than convalescent patients. We also found detectable but diminished T-cell responses to spike variants (B.1.1.7, B.1.351, and B.1.1.248) among vaccinated but otherwise healthy donors. Since decreases in antibody neutralization have also been observed with some variants10-12, investigation into the T-cell response to these variants as an alternative means of viral control is imperative. Standardized measurements of T-cell responses to SARS-CoV-2 are feasible and can be easily adjusted to determine changes in response to variants.

sentences: Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphogenesis Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogeneous substrates, in collagen gels, and on micropatterned substrates. We find that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A scale-bridging mathematical model explains our observations by relating actin fiber stability to the molecular rates of the myosin crossbridge cycle. A dynamic cell stretch/release experiment confirms these predictions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

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Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias.

sentences: Cryo-EM of the yeast VO complex reveals distinct binding sites for macrolide V-ATPase inhibitors Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.

sentences: The OPAQUE1/DISCORIDA2 myosin XI is required for phragmoplast guidance during asymmetric cell division in maize Asymmetric divisions produce cells with different fates and are critical for development. Here we show that a maize myosin XI protein, OPAQUE1 (O1), is necessary for asymmetric divisions during maize stomatal development. We analyzed stomatal precursor cells prior to and during asymmetric division to determine why o1 mutants have abnormal division planes. Cell polarization and nuclear positioning occur normally in the o1 mutant, and the future site of division is correctly specified. The defect in o1 occurs during late cytokinesis, when the plant-specific phragmoplast -- made of microtubules, actin and other proteins - forms the nascent cell plate. The phragmoplast becomes misguided and does not meet the previously established division site. Initial phragmoplast guidance is correct in o1. However, as phragmoplast expansion continues, phragmoplasts in o1 stomatal precursor cells become misguided and do not meet the cortex at the established division site. To understand how this myosin protein contributes to phragmoplast guidance, we identified O1-interacting proteins. Other myosins, specific actin-binding proteins, and maize kinesins related to the Arabidopsis thaliana division site markers PHRAGMOPLAST ORIENTING KINESINs (POKs) interact with O1. We propose that different myosins are important at multiple steps of phragmoplast expansion, and the O1 actin motor and POK-like microtubule motors work together to ensure correct late-stage phragmoplast guidance.

sentences: Streptococcus pneumoniae, S. pyogenes, and S. agalactiae membrane phospholipid remodeling in response to human serum Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS), and S. agalactiae (Group B Streptococcus; GBS) are major etiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization mass spectrometry, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS, and GBS in routine undefined laboratory medium, streptococcal defined medium, and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodeling by the major pathogenic streptococci in response to metabolites present in human serum.

sentences: Comprehensive mouse gut metagenomecatalog reveals major difference to thehuman counterpart Mouse is the most used model for studying the impact of microbiota on its host, but the repertoire of species from the mouse gut microbiome remains largely unknown. Here, we construct a Comprehensive Mouse Gut Metagenome (CMGM) catalog by assembling all currently available mouse gut metagenomes and combining them with published reference and metagenome-assembled genomes. The 50011 genomes cluster into 1699 species, of which 78.1% are uncultured, and we discovered 226 new genera, 7 new families, and 1 new order. Rarefaction analysis indicates comprehensive sampling of the species from the mouse gut. CMGM enables an unprecedented coverage of the mouse gut microbiome exceeding 90%. Comparing CMGM to the human gut microbiota shows an overlap 64% at the genus, but only 16% at the species level, demonstrating that human and mouse gut microbiota are largely distinct.

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Molecular Drivers of Tumor Progression in Microsatellite Stable APC Mutation-Negative Colorectal Cancers BackgroundThe tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut-) CRCs.

MethodsWe analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs.

ResultsTranscriptionally, APCmut- CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3.

ConclusionsAPCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

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A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27 Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimethylarsenite (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9- based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus {Delta}TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

ImportanceWe here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator, controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65{degrees}C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

sentences: Learning cell communication from spatial graphs of cells Tissue niches are sources of cellular variation and key to understanding both single-cell and tissue phenotypes. The interaction of a cell with its niche can be described through cell communication events. These events cannot be directly observed in molecular profiling assays of single cells and have to be inferred. However, computational models of cell communication and variance attribution defined on data from dissociated tissues suffer from multiple limitations with respect to their ability to define and to identify communication events. We address these limitations using spatial molecular profiling data with node-centric expression modeling (NCEM), a computational method based on graph neural networks which reconciles variance attribution and communication modeling in a single model of tissue niches. We use these models in varying complexity across spatial assays, such as immunohistochemistry and MERFISH, and biological systems to demonstrate that the statistical cell-cell dependencies discovered by NCEM are plausible signatures of known molecular processes underlying cell communication. We identify principles of tissue organisation as cell communication events across multiple datasets using interpretation mechanisms. In the primary motor cortex, we found gene expression variation that is due to niche composition variation across cortical depth. Using the same approach, we also identified niche-dependent cell state variation in CD8 T cells from inflamed colon and colorectal cancer. Finally, we show that NCEMs can be extended to mixed models of explicit cell communication events and latent intrinsic sources of variation in conditional variational autoencoders to yield holistic models of cellular variation in spatial molecular profiling data. Altogether, this graphical model of cellular niches is a step towards understanding emergent tissue phenotypes.

sentences: Network- and Enrichment-based Inference of Phenotypes and Targets from large-scale Disease Maps Disease maps have emerged as computational knowledge bases for exploring and modeling diseasespecific molecular processes. By capturing molecular interactions, disease-associated processes, and phenotypes in standardized representations, disease maps provide a platform for applying bioinformatics and systems biology approaches. Applications range from simple map exploration to algorithm-driven target discovery and network perturbation. The web-based MINERVA environment for disease maps provides a platform to develop tools not only for mapping experimental data but also to identify, analyze and simulate disease-specific regulatory networks. We have developed a MINERVA plugin suite based on network topology and enrichment analyses that facilitate multi-omics data integration and enable in silico perturbation experiments on disease maps. We demonstrate workflows by analyzing two RNA-seq datasets on the Atlas of Inflammation Resolution (AIR). Our approach improves usability and increases the functionality of disease maps by providing easy access to available data and integration of selfgenerated data. It supports efficient and intuitive analysis of omics data, with a focus on disease maps.

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A Mechanistic Modeling Framework Reveals the Key Principles Underlying Tumor Metabolism While aerobic glycolysis, or the Warburg effect, has for a long time been considered a hallmark of tumor metabolism, recent studies have revealed a far more complex picture. Tumor cells exhibit widespread metabolic heterogeneity, not only in their presentation of the Warburg effect but also in the nutrients and the metabolic pathways they are dependent on. Moreover, tumor cells can switch between different metabolic phenotypes in response to environmental cues and therapeutic interventions. A framework to analyze the observed metabolic heterogeneity and plasticity is, however, lacking. Using a mechanistic model that includes the key metabolic pathways active in tumor cells, we show that the inhibition of phosphofructokinase by excess ATP in the cytoplasm can drive a preference for aerobic glycolysis in fast-proliferating tumor cells. The differing rates of ATP utilization by tumor cells can therefore drive heterogeneity with respect to the presentation of the Warburg effect. Building upon this idea, we couple the metabolic phenotype of tumor cells to their migratory phenotype, and show that our model predictions are in agreement with previous experiments. Next, we report that the reliance of proliferating cells on different anaplerotic pathways depends on the relative availability of glucose and glutamine, and can further drive metabolic heterogeneity. Finally, using treatment of melanoma cells with a BRAF inhibitor as an example, we show that our model can be used to predict the metabolic and gene expression changes in cancer cells in response to drug treatment. By making predictions that are far more generalizable and interpretable as compared to previous tumor metabolism modeling approaches, our framework identifies key principles that govern tumor cell metabolism, and the reported heterogeneity and plasticity. These principles could be key to targeting the metabolic vulnerabilities of cancer.

Author SummaryTumor cells exhibit heterogeneity and plasticity in their metabolic behavior, relying on distinct nutrients and metabolic pathways, and switching to reliance on different pathways when challenged by an environmental change or a drug. While multiple previous studies have focused on identifying metabolic signatures that can distinguish tumor cells from non-tumorigenic ones, frameworks to analyze the metabolic heterogeneity in tumors have been lacking. Here, we present a mechanistic mathematical model of some of the key metabolic pathways active in tumor cells and analyze the steady state behaviors the model can exhibit. We find that the rate of ATP use by tumor cells can be a key determinant of the metabolic pathway via which tumor cells utilize glucose. We further show that tumor cells can utilize different pathways for satisfying the same metabolic requirements, and explore the implications of such behavior for the response of tumor cells to drugs targeting tumor metabolism. At each step, we discuss how our model predictions fit within the context of experimental observations made across tumor types. The present modeling framework represents an important step towards reconciling the wide array of experimental observations concerning tumor metabolism, and towards a more methodical approach to targeting tumors metabolic vulnerabilities.

sentences: Regulators of proteostasis are translationally repressed in fibroblasts from sporadic and LRRK2-G2019S Parkinson's patients Parkinsons disease (PD) is characterized by heterogeneous motor and non-motor symptoms. The majority of PD cases are sporadic, whereas the leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common monogenic cause of familial PD and symptomatically indistinguishable from the sporadic form. Deficits in protein synthesis have been reported in PD. However, it is not known which proteins are affected or if there are synthesis differences between patients with sporadic and LRRK2 G2019S PD. Here we used non-canonical amino acid labeling and mass-spectrometry based proteomics to perform a quantitative comparison of nascent proteins in fibroblasts from sporadic and G2019S mutation PD patients compared to healthy individuals. We found that several proteins were under-represented among nascent proteins in cells from PD patients. Among these were regulators endo-lysosomal sorting, mRNA processing and translation itself. We further validated which of these proteins were dysregulated at the static proteome level using targeted proteomics. Key downregulated proteins included the autophagy regulator ATG9A and translational stability regulator YTHDF3. Notably, 68% of the identified proteins were downregulated in both sporadic and LRRK2-G2019S forms of PD. We identify translational repression of key regulators of proteostasis in patient fibroblasts that are common to both sporadic and LRRK2-G2019S PD.

sentences: Sea anemone genomes reveal ancestral metazoan chromosomal macrosynteny Cnidaria (sea anemones, jellyfish, corals and hydra) form a close sister group to Bilateria. Within this clade, the sea anemone Nematostella vectensis has emerged as a slow evolving model for investigating characteristics of the cnidarian-bilaterian common ancestor, which diverged near the Cambrian explosion. Here, using long read sequencing and high throughput chromosome conformation capture, we generate high quality chromosome-level genome assemblies for N. vectensis and the closely related edwardsiid sea anemone, Scolanthus callimorphus. In both cases we find a robust set of 15 chromosomes comprising a stable linkage group detectable within all major clades of sequenced cnidarian genomes. Further, both genomes show remarkable chromosomal conservation with chordates. In contrast with Bilateria, we report that extended Hox and NK gene clusters are chromosomally linked but do not retain a tight spatial conservation. Accordingly, there is a lack of evidence for topologically associated domains, which have been implicated in the evolutionary pressure to retain tight microsyntenic gene clusters. We also uncover ultra-conserved noncoding elements at levels previously undetected in non-chordate lineages. Both genomes are accessible through an actively updated genome browser and database at https://simrbase.stowers.org

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The neurogenic fate of the hindbrain boundaries: a Notch-dependent behavioral switch triggers asymmetric division of boundary stem cells The generation of cell diversity in the central nervous system occurs during embryogenesis and requires a precise balance between cell proliferation, commitment to specific fates, and further neuronal differentiation. The cellular and molecular mechanisms regulating this balance in the embryonic brain are still poorly understood. Here we study how the neurogenic capacity in the embryonic hindbrain is spatiotemporally allocated, and how the fate and the growth of the hindbrain boundary cells are regulated. By generating a CRISPR-based knock-in zebrafish transgenic line to specifically label the hindbrain boundaries, we unveiled that boundary cells undergo a functional transition to become neurogenic during hindbrain segmentation concurrently as they maintain the progenitor cell pool. Boundary cells engaged in neurogenesis coinciding with the onset of Notch signaling, which triggered their asymmetrical cell division. Our findings reveal that distinct neurogenic phases take place during hindbrain growth and suggest that boundary cells contribute to refine the final number, identity, and proportion of neurons in the brain.

SUMMARYHindbrain boundary cells undergo a functional transition to become neurogenic concurrently as they are maintained as a progenitor cell pool. This involves a behavioral switch from symmetrically dividing progenitor cells to asymmetrically dividing progenitors, which depends on Notch-activity.

sentences: Seeing the future: predictive control in neural models of ocular accommodation Ocular accommodation is the process of adjusting the eyes crystalline lens so as to bring the retinal image into sharp focus. The major stimulus to accommodation is therefore retinal defocus, and in essence, the job of accommodative control is to send a signal to the ciliary muscle which will minimize the magnitude of defocus. In this paper, we first provide a tutorial introduction to control theory to aid vision scientists without this background. We then present a unified model of accommodative control that explains properties of the accommodative response for a wide range of accommodative stimuli. Following previous work, we conclude that most aspects of accommodation are well explained by dual integral control, with a "fast" or "phasic" integrator enabling response to rapid changes in demand, which hands over control to a "slow" or "tonic" integrator which maintains the response to steady demand. Control is complicated by the sensorimotor latencies within the system, which delay both information about defocus and the accommodation changes made in response, and by the sluggish response of the motor plant. These can be overcome by incorporating a Smith predictor, whereby the system predicts the delayed sensory consequences of its own motor actions. For the first time, we show that critically-damped dual integral control with a Smith predictor accounts for adaptation effects as well as for the gain and phase for sinusoidal oscillations in demand. In addition, we propose a novel proportional-control signal to account for the power spectrum of accommodative microfluctuations during steady fixation, which may be important in hunting for optimal focus, and for the nonlinear resonance observed for low-amplitude, high-frequency input. Complete Matlab/Simulink code implementing the model is provided at https://doi.org/10.25405/data.ncl.14945550.

sentences: Self-confocal NIR-II fluorescence microscopy for in vivo imaging Benefiting from low scatter of NIR-II light in biological tissues and high spatial resolution of confocal microscopy, NIR-II fluorescence confocal microscopy has been developed recently and achieve deep imaging in vivo. However, independence of excitation point and detection point makes this system difficult to be adjusted. New, improved, self-confocal NIR-II fluorescence confocal systems are created in this work. Based on a shared pinhole for excitation light and fluorescence, the system is easy and controlled to be adjusted. The fiber-pinhole confocal system is constructed for cerebrovascular and hepatocellular NIR-II fluorescence intensity imaging. The air-pinhole confocal system is constructed for cerebrovascular NIR-II fluorescence intensity imaging, hepatic NIR-II fluorescence lifetime imaging, and hepatic multiphoton imaging.

sentences: Extraordinarily precise nematode sex ratios: Adaptive responses to vanishingly rare mating options Sex ratio theory predicts both mean sex ratio and variance under a range of population structures. Here, we compare two genera of phoretic nematodes (Parasitodiplogaster and Ficophagus spp.) associated with twelve fig-pollinating wasp species in Panama. The host wasps exhibit classic Local Mate Competition: only inseminated females disperse from natal figs, and their offspring form mating pools that consist of scores of the adult offspring contributed by one or a few foundress mothers. In contrast, in both nematode genera, only sexually undifferentiated juveniles disperse, and their mating pools routinely consist of eight or fewer adults. Across all mating pool sizes, the sex ratios observed in both nematode genera are consistently female-biased (~0.34 males), which is markedly less female-biased than is often observed in the host wasps (~0.10 males). In further contrast with their hosts, variances in nematode sex ratios are also consistently precise (significantly less than binomial). The constraints associated with predictably small mating pools within highly subdivided populations appear to select for precise sex ratios that contribute both to the reproductive success of individual nematodes, and to the evolutionary persistence of nematode species. We suggest that some form of environmental sex determination underlies these precise sex ratios.

sentences: Cell- and non-cell-autonomous ARF3 coordinates meristem proliferation and organ patterning in Arabidopsis In cell-cell communication, non-cell-autonomous transcription factors play vital roles in controlling plant stem cell fate. We previously reported that AUXIN RESPONSE FACTOR 3 (ARF3), a member of the ARF family with critical roles in floral meristem maintenance and determinacy, has a distinct accumulation pattern that differs from the expression domain of its encoding gene in the shoot apical meristem (SAM). However, the biological meaning of this difference is obscure. Here, we demonstrate that ARF3 expression is mainly activated at the periphery of the SAM by auxin, where ARF3 cell-autonomously regulates the expression of meristem-organ boundary-specific genes, such as CUP-SHAPED COTYLEDON1-3 (CUC1-3), BLADE ON PETIOLE1-2 (BOP1-2) and TARGETS UNDER ETTIN CONTROL3 (TEC3) to determine organ patterning. We also show that ARF3 is translocated into the organizing center, where it represses cytokinin activity and WUSCHEL expression to regulate meristem activity non-cell-autonomously. Therefore, ARF3 acts as a molecular link that mediates the interaction of auxin and cytokinin signaling in the SAM while coordinating the balance between meristem maintenance and organogenesis. Our findings reveal an ARF3-mediated coordination mechanism through cell-cell communication in dynamic SAM maintenance.

sentences: In-phase and in-antiphase connectivity in EEG Zero-lag synchrony is generally discarded from functional connectivity studies to eliminate the confounding effect of volume conduction. Demonstrating genuine and significant unlagged synchronization between distant brain regions would indicate that most electroencephalography (EEG) connectivity studies neglect an important mechanism for neuronal communication. We previously demonstrated that local field potentials recorded intracranially tend to synchronize with no lag between homotopic brain regions. This synchrony occurs most frequently in antiphase, potentially supporting corpus callosal inhibition and interhemispheric rivalry. We are now extending our investigation to EEG. By comparing the coherency in a recorded and a surrogate dataset, we confirm the presence of a significant proportion of genuine zero-lag synchrony unlikely to be due to volume conduction or to recording reference artifacts. These results stress the necessity for integrating zero-lag synchrony in our understanding of neural communication and for disentangling volume conduction and zero-lag synchrony when estimating EEG sources and their functional connectivity.

sentences: Stability and dynamics of a spectral graph model of brain oscillations We explore the stability and dynamic properties of a hierarchical, linearized, and analytic spectral graph model for neural oscillations that integrates the structuring wiring of the brain. Previously we have shown that this model can accurately capture the frequency spectra and the spatial patterns of the alpha and beta frequency bands obtained from magnetoencephalography recordings without regionally varying parameters. Here, we show that this macroscopic model based on long-range excitatory connections exhibits dynamic oscillations with a frequency in the alpha band even without any oscillations implemented at the mesoscopic level. We show that depending on the parameters, the model can exhibit combinations of damped oscillations, limit cycles, or unstable oscillations. We determined bounds on model parameters that ensure stability of the oscillations simulated by the model. Finally, we estimated time-varying model parameters to capture the temporal fluctuations in magnetoencephalography activity. We show that a dynamic spectral graph modeling framework with a parsimonious set of biophysically interpretable model parameters can thereby be employed to capture oscillatory fluctuations observed in electrophysiological data in various brain states and diseases.

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A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants BackgroundThe high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects.

ResultsIn this study, we performed a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, revealed that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring the CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicated that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the plant genome. Furthermore, we detected off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tended to cluster, while off-target A>G DNA mutations rarely clustered.

ConclusionOur findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.

sentences: Mapping the dynamic transfer functions of epigenome editing Biological information can be encoded in the dynamics of signaling components which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions--modulating the frequency, intensity, and pulse width of light--regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed highly tunable gene expression and filtering behaviors and provided the most comprehensive quantification to date of the maximum amount of information that can be reliably transferred across a promoter as [~]1.7 bits. Using a library of over 100 orthogonal epigenome editors, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic epigenome editing.

sentences: Optimising expression quantitative trait locus mapping workflows for single-cell studies Single-cell RNA-sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states, and promises to improve our understanding of genetic regulation across tissues in both health and disease. While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimise sc-eQTL mapping. Here, we evaluate the role of different normalisation and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches and provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

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Complement Regulator Factor H is a Cofactor for Thrombin in both Pro- and Anticoagulant Roles BackgroundComplement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin.

MethodsTail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR).

ResultsTail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed.

ConclusionComplement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis.

Key pointsO_LIAbsence of FH prolongs tail bleeding time in CFH-/- mice and activated partial thromboplastin time (aPTT) is elevated in human FH-depleted plasma.
C_LIO_LIFH acts a cofactor for thrombin by enhancing fibrin generation, altering fibrin clot structure and enhancing TM-thrombin mediated protein C activation
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sentences: Elucidating design principles for engineering cell-derived vesicles to inhibit SARS-CoV-2 infection The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles--cell-mimicking particles that bind and inhibit virions--are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

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Gradual development of non-adjacent dependency learning during early childhood In order to become proficient native speakers, children have to learn the morpho-syntactic relations between distant elements in a sentence, so-called non-adjacent dependencies (NADs). Previous research suggests that NAD learning in children comprises different developmental stages, where until 2 years of age children are able to learn NADs associatively under passive listening conditions, while starting around the age of 3 to 4 years children fail to learn NADs during passive listening. To test whether the transition between these developmental stages occurs gradually, we tested childrens NAD learning in a foreign language using event-related potentials (ERPs). We found ERP evidence of NAD learning across the ages of 1, 2 and 3 years. The amplitude of the ERP effect indexing NAD learning, however, decreased with age. These findings might indicate a gradual transition in childrens ability to learn NADs associatively. Cognitively, this transition might be driven by childrens increasing knowledge of their native language, hindering NAD learning in novel contexts. Neuroanatomically, maturation of the prefrontal cortex might play a crucial role, promoting top-down learning, affecting bottom-up, associative learning. In sum, our study suggests that NAD learning under passive listening conditions undergoes a gradual transition between different developmental stages during early childhood.

Research HighlightsO_LITransition between developmental stages of non-adjacent dependency (NAD) learning
C_LIO_LIChildren between 1 and 3 years of age showed learning of NADs in a foreign language
C_LIO_LIBrain responses revealed associative NAD learning, triggered by passive listening
C_LIO_LIGradual decrease of the strength of associative non-adjacent dependency learning
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Two tales of one neural link predict blind individual's Braille reading proficiency Natural Braille reading, a demanding cognitive skill, poses a huge challenge for the brain network of the blind. Here, with behavioral measurement and functional MRI imaging data, we pinpointed the neural pathway and investigated the neural mechanisms of individual differences in Braille reading in late blindness. Using resting state fMRI, we identified a distinct neural link between the higher-tier  visual cortex--the lateral occipital cortex (LOC), and the inferior frontal cortex (IFC) in the late blind brain, which is significantly stronger than sighted controls. Individual Braille reading proficiency positively correlated with the left-lateralized LOC-IFC functional connectivity. In a natural Braille reading task, we found an enhanced bidirectional information flow with a stronger top-down modulation of the IFC-to-LOC effective connectivity. Greater top-down modulation contributed to higher Braille reading proficiency via a broader area of task-engaged LOC. Together, we established a model to predict Braille reading proficiency, considering both functional and effective connectivity of the LOC-IFC pathway. This  two-tale model suggests that developing the underpinning neural circuit and the top-down cognitive strategy contributes uniquely to superior Braille reading performance.

SIGNIFICANCE STATEMENTFor late blind humans, one of the most challenging cognitive skills is natural Braille reading. However, little is known about the neural mechanisms of significant differences in individual Braille reading performance. Using functional imaging data, we identified a distinct neural link between the left lateral occipital cortex (LOC) and the left inferior frontal cortex (IFC) for natural Braille reading in the late blind brain. To better predict individual Braille reading proficiency, we proposed a linear model with two variables of the LOC-IFC link: the resting-state functional connectivity and the task-engaged top-down effective connectivity. These findings suggest that developing the underpinning neural pathway and the top-down cognitive strategy contributes uniquely to superior Braille reading performance.

sentences: The response network of HSP70 defines vulnerabilities in cancer cells with the inhibited proteasome Human neoplasias are often addicted to the proteasome machinery. However, cancers have evolved efficient response mechanisms to overcome proteasome inhibition with bortezomib and carfilzomib - drugs approved for multiple myeloma treatment. To understand these responses we investigated proteome changes upon the proteasome inhibition with carfilzomib - in multiple myeloma, normal fibroblasts, and cancers of lung, colon, and pancreas. A pathway-oriented siRNA screen based on the proteomics results showed that molecular chaperones, autophagy- and endocytosis-related proteins are cancer-specific vulnerabilities combined with carfilzomib. Targeting of HSPA1A/B (HSP70 family chaperones) most specifically sensitized cancer cells and patient-derived organoids to the proteasome inhibition. A high level of HSPA1A/B mRNA correlated with a low proteasome activity in cancer patient tissues and is a risk factor in cancer patients with a low proteasome expression. Mechanistically, HSPA1A/B governed autophagy, unfolded protein response, endocytic trafficking, and chaperoned the proteasome machinery, suppressing the effect of the proteasome inhibition, but did not control the NRF1/2-driven proteasome subunit transcriptional bounce-back. Consequently, downregulation of NRF1 most specifically decreased the viability of cancer cells with the inhibited proteasome and HSP70.

sentences: CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

sentences: Reading instruction causes changes in category-selective visual cortex Education sculpts specialized neural circuits for skills like reading that are critical to success in modern society but were not anticipated by the selective pressures of evolution. Does the emergence of brain regions that selectively process novel visual stimuli like words occur at the expense of cortical representations of other stimulus categories like faces and objects? To answer this question we designed a randomized controlled trial where pre-literate children (five years of age) were randomly assigned to intervention programs that either taught reading skills (Letter Intervention) or oral language comprehension skills (Language Intervention). Magnetoencephalography (MEG) data collected before and after the interventions revealed that being assigned to the Letter versus Language Intervention induced different patterns of changes in category selective responses in high-level visual cortex. We found moderate support for the notion that words compete with other objects for cortical territory as children become attuned to this new class of visual stimuli. How these changes play out over a longer timescale is still unclear but, based on these data, we can surmise that high-level visual cortex undergoes rapid changes as children enter school and begin establishing new skills like literacy.

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Basal forebrain parvalbumin neurons modulate vigilant attention. Attention is impaired in many neuropsychiatric disorders1 and by sleep disruption, leading to decreased workplace productivity and increased risk of accidents2-4. Thus, understanding the underlying neural substrates is important for developing treatments. The basal forebrain (BF) is a brain region which degenerates in dementia5-7 and is implicated in the negative effects of sleep disruption on vigilance and cognition8,9. Previous studies demonstrated that the BF controls cortical fast oscillations that underlie attention10-12 and revealed the important role of cholinergic neurons13-15. However, the role of other neurochemically defined BF subtypes is unknown. Recent work has shown that one population of BF GABAergic neurons containing the calcium-binding protein parvalbumin (PV) control cortical fast oscillations and arousals from sleep16-19 but their role in awake behavior is unclear. Thus, here we test the hypothesis that BF-PV neurons modulate vigilant attention in mice. A lever release version of the rodent psychomotor vigilance test (rPVT) was used to assess vigilant attention as measured by reaction time. Brief and continuous low power optogenetic excitation of BF-PV neurons (1s,473nm@5mW) that preceded the cue light signal by 0.5s improved vigilant attention as indicated by quicker reaction times. In contrast, both sleep deprivation (8h) and optogenetic inhibition of BF-PV neurons (1s,530nm@10mW) slowed reaction times. Importantly, BF-PV excitation rescued the reaction time deficits in sleep deprived mice. These findings reveal for the first time a role for BF-PV neurons in attention.

HIGHLIGHTSO_LIOptogenetic methods tested the neural circuitry of vigilant attention in mice
C_LIO_LIExcitation of basal forebrain parvalbumin neurons quickened reaction times
C_LIO_LISleep deprivation or inhibition of parvalbumin neurons slowed reaction times
C_LIO_LIExcitation of parvalbumin neurons rescued deficits produced by sleep deprivation
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sentences: Welfare concerns for mounted load carrying by working donkeys in Pakistan Working donkeys (Equus asinus) are vital to peoples livelihoods. They are essential for carrying goods, however globally, overloading is one of the primary welfare concerns of working donkeys. We studied mounted load carrying by donkeys and associated factors in Pakistan. A cross-sectional study of donkey owners (n = 332) was conducted, and interviews were undertaken based on a questionnaire. Owners estimated that the median weight of their donkeys was 110kg (interquartile range (IQR) 100-120kg), and that they carried a median mounted load of 81.5kg (IQR 63-99kg). We found that 87.4% of donkeys carried a load above 50% of their bodyweight ratio (BWR), the median BWR carried was 77.1% (IQR 54.5-90.7%), and 25.3% of donkeys carried above 90% BWR. Donkeys that were loaded at more than 50% BWR were more likely to sit, compared to donkeys loaded with less weight (p=0.01). Donkeys working in peri-urban and urban areas were more likely to carry a greater BWR than donkeys working in rural areas (P<0.001), as were those carrying construction materials or bricks, compared to agricultural materials (p=0.004). Age (p=0.03) and breed (p=0.01) were also associated with carrying a higher weight. Overloading based on current recommendations (50% BWR) was common, with the majority (87.4%) of donkeys reported to carry more than the recommended 50% limit. This survey provides evidence of on-the-ground working practices and factors associated with mounted load carrying, which is critical for developing evidence-based recommendations for loading, in order to improve the welfare of working donkeys.

sentences: Complementary Phenotyping of Maize Root Architecture by Root Pulling Force and X-Ray Computed Tomography The root system is critical for the survival of nearly all land plants and a key target for improving abiotic stress tolerance, nutrient accumulation, and yield in crop species. Although many methods of root phenotyping exist, within field studies one of the most popular methods is the extraction and measurement of the upper portion of the root system, known as the root crown, followed by trait quantification based on manual measurements or 2D imaging. However, 2D techniques are inherently limited by the information available from single points of view. Here, we used X-ray computed tomography to generate highly accurate 3D models of maize root crowns and created computational pipelines capable of measuring 71 features from each sample. This approach improves estimates of the genetic contribution to root system architecture, and is refined enough to detect various changes in global root system architecture over developmental time as well as more subtle changes in root distributions as a result of environmental differences. We demonstrate that root pulling force, a high-throughput method of root extraction that provides an estimate of root biomass, is associated with multiple 3D traits from our pipeline. Our combined methodology can therefore be used to calibrate and interpret root pulling force measurements across a range of experimental contexts, or scaled up as a stand-alone approach in large genetic studies of root system architecture.

sentences: Critical role of DNA unwinding and Cas9 conformational changes in modelling off-target effects CRISPR-Cas9 off-target effects interfere with the ability to accurately perform genetic edits. To predict off-target effects CRISPR-Cas9 researchers perform high throughput guide RNA mismatch and bulge experiments and then use the data to fit thermodynamic binding models. While impactful from an engineering perspective such models are not based on the experimentally observed target interrogation process and thus incorrectly measure the energetic effects mismatches have on the system. In this work we convert an experimentally deduced qualitive model of target interrogation to a linear ODE model and demonstrate that the mismatch tolerance patterns observed in experiments do not need to be caused by differences in energetic penalties of mismatches but rather are emergent effects of the timing and coordination of target DNA unwinding and Cas9 conformational changes.

sentences: Age-related differences in the neural network dynamics underlying the predictability gain Speech comprehension is often challenged by increased background noise, but can be facilitated via the semantic context of a sentence. This predictability gain relies on an interplay of language-specific semantic and domain-general brain regions. However, age-related differences in the interactions within and between semantic and domain-general networks remain poorly understood. Using functional neuroimaging, we investigated commonalities and differences in network interactions enabling processing of degraded speech in healthy young and old participants. Participants performed a sentence repetition task while listening to sentences with high and low predictable endings and varying intelligibility. Stimulus intelligibility was adjusted to individual hearing abilities. Older adults showed an undiminished behavioural predictability gain. Likewise, both groups recruited a similar set of semantic and cingulo-opercular brain regions. However, we observed age-related differences in effective connectivity for high predictable speech of increasing intelligibility. Young adults exhibited stronger connectivity between regions of the cingulo-opercular network and between left insula and the posterior middle temporal gyrus. Moreover, these interactions were excitatory in young adults but inhibitory in old adults. Finally, the degree of the inhibitory influence between cingulo-opercular regions was predictive of the behavioural sensitivity towards changes in intelligibility for high predictable sentences in older adults only. Our results demonstrate that the predictability gain is relatively preserved in older adults when stimulus intelligibility is individually adjusted. While young and old participants recruit similar brain regions, differences manifest in underlying network interactions. Together, these results suggest that ageing affects the network configuration rather than regional activity during successful speech comprehension under challenging listening conditions.

sentences: mTOR inhibition enhances delivery and activity of antisense oligonucleotides in uveal melanoma cells Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Due to a lack of effective treatments, patients with metastatic disease have a median survival time of 6-12 months. We recently demonstrated that the SAMMSON long non-coding RNA (lncRNA) is essential for uveal melanoma cell survival and that antisense oligonucleotide (ASO)-mediated silencing of SAMMSON impaired cell viability and tumor growth in vitro and in vivo. By screening a library of 2911 clinical stage compounds, we identified the mTOR inhibitor GDC-0349 to synergize with SAMMSON inhibition in UM. Mechanistic studies revealed that mTOR inhibition enhanced uptake and reduced lysosomal accumulation of lipid complexed SAMMSON ASOs, improving SAMMSON knockdown and further decreasing UM cell viability. We found mTOR inhibition to also enhance target knockdown in other cancer cell lines as well as normal cells when combined with lipid nanoparticle complexed or encapsulated ASOs or small interfering RNAs (siRNAs). Our results are relevant to nucleic acid treatment in general and highlight the potential of mTOR inhibition to enhance ASO and siRNA mediated target knockdown.

sentences: Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration. Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

sentences: Fractal dimension of EEG signal senses complexity of fractal animations We are surrounded with many fractal and self-similar patterns which has been area of many researches in the recent years. We can perceive self-similarities in various spatial and temporal scales; however, the underlying neural mechanism needs to be well understood. In this study, we hypothesized that complexity of visual stimuli directly influence complexity of information processing in the brain. Therefore, changes in fractal pattern of EEG signal must follow change in fractal dimension of animation. To investigate this hypothesis, we recorded EEG signal of fifteen healthy participants while they were exposed to several 2D fractal animations. Fractal dimension of each frame of the animation was estimated by box counting method. Subsequently, fractal dimensions of 32 EEG channels were estimated in a frequency specific manner. Then, association between pattern of fractal dimensions of the animations and pattern of fractal dimensions of EEG signals were calculated using the Pearsons correlation algorithm. The results indicated that fractal animation complexity is mainly sensed by changes in fractal dimension of EEG signals at the centro-parietal and parietal regions. It may indicate that when the complexity of visual stimuli increases the mechanism of information processing in the brain also enhances its complexity to better attend and comprehend the stimuli.

sentences: Motor usage imprints microtubule stability in the shaft Tubulin dimers assemble into dynamic microtubules which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network is commonly thought to be regulated at the polymer ends, where tubulin-dimers can be added or removed. Here we show that molecular motors running on microtubules cause exchange of dimers along the shaft. These sites of dimer exchange act as rescue sites where depolymerising microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft serving as traces of microtubule usage to organize the polarity landscape of the cell.

sentences: Mammal virus diversity estimates are unstable due to accelerating discovery effort Host-virus association data form the backbone of research into eco-evolutionary drivers of viral diversity and host-level zoonotic risk. However, knowledge of the wildlife virome is inherently constrained by historical discovery effort, and there are concerns that the reliability of ecological inference from host-virus data may be undermined by taxonomic and geographical sampling biases. Here, we evaluate whether current estimates of host-level viral diversity in wild mammals are stable enough to be considered biologically meaningful, by analysing a comprehensive dataset of discovery dates of 6,571 unique mammal host-virus associations between 1930 and 2018. We show that virus discovery rates in mammal hosts are still either constant or accelerating, with little evidence of declines towards viral richness asymptotes in even highly-sampled hosts. Consequently, inference of relative viral richness across host species has been unstable over time, particularly in bats, where intensified surveillance since the early 2000s caused a rapid rearrangement of species ranked viral richness. Our results show that comparative inference of host-level virus diversity across mammals is highly sensitive to even short-term changes in sampling effort. We advise caution to avoid overinterpreting patterns in current data, since our findings suggest that an analysis conducted today could feasibly draw quite different conclusions than one conducted only a decade ago.

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Super-doses of dietary vitamin D3 intake in aged laying hens illustrates limitation of 24,25-dihydroxycholecalciferol conversion ABSRACTO_ST_ABSBackgroundC_ST_ABSHumans take vitamin D supplements to reduce risk of vitamin D deficiency and reduce the risk of osteoporosis. However, it is unclear how dietary super-doses (10,000x greater than requirement) can affect vitamin D status in aged animals. Aged laying hens could be a model to compare with women in peri- or postmenopausal stages of life. The hens bone health is physiologically taxed from egg production and they are highly susceptible to osteoporosis.

ObjectiveWe investigated dietary super-dose impacts of cholecalciferol (vitamin D3) on vitamin D status in aged laying hens in production.

MethodsForty-eight 68 wk old Hy-Line Brown laying hens were individually housed in cages with eight hens per dietary treatment for eleven weeks. Hens were randomly assigned to one of six groups of dietary vitamin D3 supplementation and fed ad libitum. Supplementation levels were 400, 800, 7,400, 14,000, 20,000, and 36,000 IU D3/kg of feed. At termination of the study, all hens were euthanized and we collected tissue samples and their feces. Tissue calcium, plasma and egg yolk vitamin D metabolites, and gene expression levels were measured.

ResultsWe observed that increasing dietary vitamin D3 increased plasma vitamin D3, 25-hydroxycholecalciferol, and 24,25-dihydroxycholecalciferol concentrations (p < 0.0001 for all 3 metabolites). Also, egg yolk vitamin D3 and 25-hydroxycholecalciferol showed a similar effect like plasma vitamin D metabolites (p < 0.0001 for both metabolites). We also observed super-dose fed hens had decreased kidney 24-hydroxylase expression (p = 0.0006).

ConclusionsDietary super-doses of vitamin D3 led to greater plasma and egg yolk vitamin D levels to show that aged laying hens can deposit excess vitamin D in egg yolk. We suggest future research should explore how 24-hydroxylation mechanisms are affected by vitamin D supplementation. Further understanding of 24-hydroxylation can help ascertain ways to reduce risk of vitamin D toxicity.

sentences: Emergence of Probabilistic Representation in the Neural Network of Primary Visual Cortex During the early development of mammalian visual system, the distribution of neuronal preferred orientations in the primary visual cortex (V1) gradually shifts to match the major orientation features of an environment, achieving optimal representation of the environment. By combining the computational modeling and experimental electrophysiological recording, we provide a circuitry plasticity mechanism that underlies the developmental emergence of such matched representation in the visual cortical network. Specifically, in a canonical circuit of densely interconnected pyramidal cells and inhibitory parvalbumin-expressing (PV+) fast-spiking interneurons in the V1 layer 2/3, our model successfully simulates the experimental observations and further reveals that the non-uniform inhibition, mediated by local interneurons, exerts a key role in shaping the network representation through spike timing-dependent synaptic modifications. The experimental results confirm that PV+ interneurons in the V1 are capable of providing such non-uniform inhibition during a short period after the vision onset. Thus, our study elucidates a circuitry mechanism for acquisition of the prior knowledge of environment for optimal inference in sensory neural system.

sentences: Failure of the brain glucagon-like peptide-1-mediated control of intestinal redox homeostasis in a rat model of sporadic Alzheimer's disease The gastrointestinal system may be involved in the etiopathogenesis of the insulin-resistant brain state (IRBS) and Alzheimers disease (AD). Gastrointestinal hormone glucagon-like peptide-1 (GLP-1) is being explored as a potential therapy as activation of brain GLP-1 receptors (GLP-1R) exerts neuroprotection and controls peripheral metabolism. Intracerebroventricular administration of streptozotocin (STZ-icv) is used to model IRBS and GLP-1 dyshomeostasis seems to be involved in the development of neuropathological changes. The aim was to explore i) gastrointestinal homeostasis in the STZ-icv model ii) assess whether the brain GLP-1 is involved in the regulation of gastrointestinal redox homeostasis and iii) analyze whether brain-gut GLP-1 axis is functional in the STZ-icv animals. Acute intracerebroventricular treatment with exendin-3(9-39)amide was used for pharmacological inhibition of brain GLP-1R in the control and STZ-icv rats, and oxidative stress was assessed in plasma, duodenum and ileum. Acute inhibition of brain GLP-1R increased plasma oxidative stress. TBARS were increased, and LMWT, SH, and superoxide dismutase (SOD) were decreased in the duodenum, but not in the ileum of the controls. In the STZ-icv, TBARS and CAT were increased, LMWT and SH were decreased at baseline, and no further increment of oxidative stress was observed upon central GLP-1R inhibition. The presented results indicate that i) oxidative stress is increased in the duodenum of the STZ-icv rat model of AD, ii) brain GLP-1R signaling is involved in systemic redox regulation, iii) brain-gut GLP-1 axis regulates duodenal, but not ileal redox homeostasis, and iv) brain-gut GLP-1 axis is dysfunctional in the STZ-icv model.

sentences: Experimental sexual selection reveals rapid divergence in male and female reproductive transcriptomes and their interactions Postcopulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these processes to rapid transcriptomic evolution in sex-specific tissues and substantial transcriptomic postmating responses in females, patterns of which are altered when mating between reproductively isolated species. Here we test multiple predictions arising from sexual selection and conflict theory about the evolution of sex-specific and tissue-specific gene expression and the postmating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the postmating response, particularly in the female reproductive tract - the main arena for sexual conflict - compared to ovaries. This affect was asymmetric with monandry females typically showing more postmating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the postmating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.

sentences: Tau forms dynamic hot spots that are resistant to microtubule perturbations and cholesterol depletion Accumulation of Tau aggregates is a pathological hallmark of Alzheimers disease, but little is known about where these aggregates assemble and how stable these assemblies are. Using quantitative, single-molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane, resulting in the formation of nanometer-sized hot spots smaller than the diffraction limit. The hot spots lasted tens of seconds, much longer than the short dwell time (~40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not affect hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.

sentences: Can Stephen Curry really know? - Conscious access to outcome prediction of motor actions The NBA player Stephen Curry has a habit of turning away from the basket right after taking three-point shots, presumably because he can predict the success of his shot. For such a consciously accessible prediction to be possible, Stephen Curry needs access to internal processes of outcome prediction and valuation. Computational simulations and empirical data suggest that the quality of internal prediction processes is related to motor expertise. Whether the results of internal predictions can reliably be consciously accessed is less clear. In the current study, 30 participants each practiced a virtual goal-oriented throwing task for 1000 trials. Every second trial, they were required to verbally predict the success of the current throw. Results showed that on average, conscious prediction accuracy was above an individually computed chance level, taking into account individual success rates and response strategies. Furthermore, prediction accuracy was related to throwing performance. Participants with better performance predicted the success of their throws more accurately than participants with poorer performance. Moreover, for the poorer performing individuals, movement execution was negatively affected by the verbalized predictions required, and they did not show variation in speech characteristics (response latency) between correct and incorrect predictions. This indicates reduced quality of conscious access to internal processes of outcome prediction.

sentences: Deep Heterogeneous Dilation of LSTM for Transient-phase Gesture Prediction through High-density Electromyography: Towards Application in Neurorobotics Deep networks have been recently proposed to estimate motor intention using conventional bipolar surface electromyography (sEMG) signals for myoelectric control of neurorobots. In this regard, Deepnets are generally challenged by long training times (affecting practicality and calibration), complex model architectures (affecting the predictability of the outcomes), and a large number of trainable parameters (increasing the need for big data). Capitalizing on our recent work on homogeneous temporal dilation in a Recurrent Neural Network (RNN) model, this paper proposes, for the first time, heterogeneous temporal dilation in an LSTM model and applies that to high-density surface electromyography (HD-sEMG), allowing for the decoding of dynamic temporal dependencies with tunable temporal foci. In this paper, a 128-channel HD-sEMG signal space is considered due to the potential for enhancing the spatiotemporal resolution of human-robot interfaces. Accordingly, this paper addresses a challenging motor intention decoding problem of neurorobots, namely, transient intention identification. Our approach uses only the dynamic and transient phase of gesture movements when the signals are not stabilized or plateaued, which can significantly enhance the temporal resolution of human-robot interfaces. This would eventually enhance seamless real-time implementations. Additionally, this paper introduces the concept of "dilation foci" to modulate the modeling of temporal variation in transient phases. In this work a high number (e.g., 65) of gestures is included, which adds to the complexity and significance of the understudied problem. Our results show state-of-the-art performance for gesture prediction in terms of accuracy, training time, and model convergence.

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Cortical network structure mediates response to stimulation: an optogenetic study in non-human primates As aberrant network-level functional connectivity underlies a variety of neural disorders, the ability to induce targeted functional reorganization would be a profound development towards therapies for neural disorders. Brain stimulation has been shown to alter large-scale network-wide functional connectivity, but the mapping from stimulation to the modification is unclear. Here, we leverage advances in neural interfaces, interpretable machine learning, and graph theory to arrive at a model which accurately predicts stimulation-induced network-wide functional reorganization. The model jointly considers the stimulation protocol and the cortical network structure, departing from the standard approach which only considers the stimulation protocol. We validate our approach in the primary sensorimotor cortex of non-human primates using paired optogenetic stimulation through a large-scale optogenetic interface. We observe that the stimulation protocol only predicts a small portion of the induced functional connectivity changes while the network structure predicts much more, indicating that the underlying network is the primary mediator of the response to stimulation. We extract the relationships linking the stimulation and network characteristics to the functional connectivity changes and observe that the mappings diverge over frequency bands and successive stimulations. Finally, we uncover shared processes governing real-time and longer-term effects of stimulation. Our framework represents a paradigm shift for targeted neural stimulation and can be used to interrogate, improve, and develop stimulation-based interventions for neural disorders.

TeaserBrain stimulation rewires the brain, but the pre-existing network structure of the brain controls the rewiring.

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Identification of new Dickeya dadantii virulence factors secreted by the type 2 secretion system Dickeya are plant pathogenic bacteria able to provoke disease on a wide range of plants. A type 2 secretion system (T2SS) named Out is necessary for bacterial virulence. Its study in D. dadantii showed that it secretes a wide range of plant cell wall degrading enzymes, including pectinases and a cellulase. However, the full repertoire of exoproteins it can secrete has probably not yet been identified. Secreted proteins are first addressed to the periplasm before their secretion by Out. No secretion signal present on the protein allows the identification of substrates of a T2SS. To identify new Out substrates, we analyzed D. dadantii transcriptome data obtained in plant infection condition and searched for genes strongly induced encoding a protein with a signal sequence. We identified four new Out-secreted proteins: the expansin YoaJ, the putative virulence factor VirK and two proteins of the DUF 4879 family, SvfA and SvfB. We showed that SvfA and SvfB are required for full virulence of D. dadantii and showed that svf genes are present in a variable number of copies in other Pectobacteriaceae, up to three in D. fanghzongdai. This work opens the way to the study of the role of non-pectinolytic proteins secreted by the Out pathway in Pectobacteriaceae.

IMPORTANCEThe plant pathogen Dickeya rely on a type 2 secretion system named Out for their pathogenicity. Importance of plant cell wall degrading enzymes secreted by this system has been well studied. However, existence and role of other Out-secreted proteins has barely been investigated. By mining D. dadantii transcriptome data, we identified four new Out-secreted proteins. We showed that two of them, SvfA and SvfB, are necessary for the full virulence of the bacteria. These findings show that identification of all the proteins secreted by the Dickeya Out system is necessary for a better knowledge of the virulence of these bacteria.

sentences: LINE-1 Retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5'-single cell RNA-Seq LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method - 5 scL1seq - that makes use of a widely available library preparation method (10x Genomics 5 single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors - present in cancer cells, absent from immune cells - and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases.

sentences: Iterative community-driven development of a SARS-CoV-2 tissue simulator The 2019 novel coronavirus, SARS-CoV-2, is a pathogen of critical significance to international public health. Knowledge of the interplay between molecular-scale virus-receptor interactions, single-cell viral replication, intracellular-scale viral transport, and emergent tissue-scale viral propagation is limited. Moreover, little is known about immune system-virus-tissue interactions and how these can result in low-level (asymptomatic) infections in some cases and acute respiratory distress syndrome (ARDS) in others, particularly with respect to presentation in different age groups or pre-existing inflammatory risk factors. Given the nonlinear interactions within and among each of these processes, multiscale simulation models can shed light on the emergent dynamics that lead to divergent outcomes, identify actionable "choke points" for pharmacologic interventions, screen potential therapies, and identify potential biomarkers that differentiate patient outcomes. Given the complexity of the problem and the acute need for an actionable model to guide therapy discovery and optimization, we introduce and iteratively refine a prototype of a multiscale model of SARS-CoV-2 dynamics in lung tissue. The first prototype model was built and shared internationally as open source code and an online interactive model in under 12 hours, and community domain expertise is driving regular refinements. In a sustained community effort, this consortium is integrating data and expertise across virology, immunology, mathematical biology, quantitative systems physiology, cloud and high performance computing, and other domains to accelerate our response to this critical threat to international health. More broadly, this effort is creating a reusable, modular framework for studying viral replication and immune response in tissues, which can also potentially be adapted to related problems in immunology and immunotherapy.

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Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD.

Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination.

sentences: TRAK proteins encode distinct MIRO-dependent and MIRO-independent mechanisms for associating with the mitochondrial outer membrane I.Mitochondria are essential to proper cell function, mislocalization of mitochondria leads to disease. Previous research has indicated that MYO19, an unconventional myosin, localizes to the mitochondria outer membrane (MOM) and enables actin-based mitochondrial movement along the cytoskeleton. MOM insertion of MYO19 is assisted by the small GTPase MIRO, a "molecular switch" that facilitates MYO19/mitochondria interactions. MIRO serves as a "recruiter" of MYO19 to the MOM, rather than the tether/receptor that mediates attachment, as MYO19 contains a second, MIRO-independent mitochondrial association domain. MIRO proteins have previously been reported to serve as attachment points for the microtubule-based motors through interactions with the adaptor protein, TRAK. Past research has identified a MIRO-binding domain of TRAK (TRAKMBD) that directly participates in the interaction with MIRO. We chose to investigate whether the interactions between TRAK and MOM paralleled our hypothesized mechanism for MYO19/MOM interactions by examining the MIRO-mediated enhancement of TRAK protein localization to mitochondria, by identifying the location of a MIRO-independent mitochondrial association domain in the c-terminus of TRAK proteins, and by examining the steady-state binding kinetics of various TRAK constructs to mitochondria. We interpret these data to indicate that MIRO proteins serve in the initial recruitment of TRAK proteins to mitochondria, but that the MIRO-independent domain plays a significant role in long-term association between TRAK and MOM.

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

sentences: T cell-intrinsic vitamin A metabolism and its signaling are targets for memory T cell-based cancer immunotherapy Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T- cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh10 deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

sentences: Strategy for detecting off-target sites in genome-edited rice Genome-editing using the CRISPR-Cas9 system can substantially accelerate crop breeding. Because off-target editing is the main problem with this system, a reliable method for comprehensively detecting off-target sites is required for the editing of food crop genomes. However, a method that accurately predicts off-target sites has not been established. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq is an unbiased method applicable for the in vitro detection of double-strand breaks (DSBs). To analyze SITE-Seq data, we developed a novel Galaxy system, which can perform simple and reproducible analyses without a command line operation. We conducted a SITE-Seq analysis of a rice genome modified by OsFH15 gRNA-Cas9, and identified 41 DSB sites in the annotated regions. Amplicon-sequencing revealed mutations at one off-target site in the genome-edited rice. The presence of an uncommon protospacer adjacent motif (NTG PAM) likely makes this off-target site difficult to identify using in silico methods. Of the six tested programs, only CRISPRdirect predicted this off-target site, but it also predicted 6,080 off-target sites in total. These results suggest the SITE-Seq method presented herein can efficiently predict off-target sites and is useful for assessing the safety of genome-edited food.

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Characterization of gut microbiome and metabolome in Helicobacter pylori patients in an underprivileged community in the United States BackgroundHelicobacter pylori, a bacterium that infects approximately half of the worlds population, is associated with various gastrointestinal diseases, including peptic ulcers, non-ulcer dyspepsia, gastric adenocarcinoma, and gastric lymphoma. To combat the increasing antibiotic resistance of H. pylori, the need for new therapeutic strategies has become more pressing. Characterization of the interactions between H. pylori and the fecal microbiome, as well as the mechanisms underlying these interactions, may offer new therapeutic approaches. Exploration of changes in fatty acid metabolism associated with H. pylori-mediated alterations of the fecal microbiome may also reveal strategies to help prevent progression to neoplasia.

AimTo characterize the gut microbiome and metabolome in H. pylori patients in a socioeconomically challenged and underprivileged inner-city community.

MethodsStool samples from 19 H. pylori patients and 16 control subjects were analyzed. 16S rRNA gene sequencing was performed on normalized pooled amplicons using the Illumina MiSeq System using a MiSeq reagent kit v2. Alpha and beta diversity analyses were performed in QIIME 2. Non-targeted fatty acid analysis of the samples was carried out using gas chromatography-mass spectrometry (GC-MS), which measures the total content of 30 fatty acids in stool after conversion into their corresponding fatty acid methyl esters. Multi-dimensional scaling (MDS) was performed on Bray-Curtis distance matrices created from both the metabolomics and microbiome datasets and a Procrustes test was performed on the metabolomics and microbiome MDS coordinates.

ResultsFecal microbiome analysis showed that alpha diversity was lowest in H. pylori patients over 40 years of age compared to control subjects of similar age group. Beta diversity analysis of the samples revealed significant differences in microbial community structure between H. pylori patients and control subjects. Thirty-eight and six taxa had lower and higher relative abundance in H. pylori patients, respectively. Taxa that were enriched in H. pylori patients included Atopobium, Gemellaceae, Micrococcaceae, Gemellales and Rothia (R. mucilaginosa). Notably, relative abundance of the phylum Verrucomicrobia was decreased in H. pylori patients compared to control subjects, suggesting disruption of the gut mucosal environment by H. pylori. Procrustes analysis showed a significant relationship between the microbiome and metabolome datasets. Stool samples from H. pylori patients showed increases in several fatty acids including the polyunsaturated fatty acids (PUFAs) 22:4n6, 22:5n3, 20:3n6 and 22:2n6, while decreases were noted in other fatty acids including the PUFA 18:3n6. The pattern of changes in fatty acid concentration correlated to the Bacteroidetes:Firmicutes ratio determined by 16S rRNA gene analysis.

ConclusionAn individualized understanding of gut microbiome features among H. pylori patients will pave the way for improved community impact, reduced healthcare burdens of repeated treatment, and decreased mounting resistance.

sentences: A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, we mapped the developmental origin of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and identify them in a synapse-scale TEM reconstruction of the Drosophila larval CNS. We find that lineages concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, we show that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, we demonstrate that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system.

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Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive distal myopathy with rimmed vacuoles that typically manifests in late adolescence/early adulthood. GNE encodes an enzyme that is the rate-limiting step in sialic acid biosynthesis which is necessary for proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study we aimed to address this knowledge gap by querying underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem cell (iPSC) model. Muscle and skin biopsies were acquired from two patients with GNE myopathy that presented with distinct histopathological features. Control and patient-specific iPSCs were derived from skin fibroblasts and differentiated down a skeletal muscle lineage in a three-stage process analogous to muscle development and muscle regeneration. Initial studies revealed: 1) the ability of patient-derived GNE iPSC clones to recapitulate key characteristics of the human pathology including TDP-43 accumulation and evidence of dysregulated autophagy, and 2) a striking defect in myogenic progression of the more severe GNE iPSC clone. Single-cell RNA sequencing time course studies were then performed to more rigorously explore myogenesis defects. Cluster-based bioinformatics analyses revealed clear differences between control and GNE iPSC-derived muscle precursor cells (iMPCs). On a transcriptional level, late stage GNE iMPCs resembled that of early stage control iMPCs, confirming stalled myogenic progression on a molecular level. Comparative expression and pathway studies revealed EIF2 signaling as a top signaling pathway altered in GNE iMPCs. In summary, we report a novel in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a likely novel contributing mechanism to the etiology of GNE myopathy.

SUMMARY STATEMENTDevelopment of a novel cell-based model of GNE myopathy, utilizing GNE patient-derived samples, which recapitulates human disease characteristics, uncovered myogenic differentiation defects, and can elucidate possible mechanistic contributors to the disease.

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Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.

Author summaryMiltefosine was developed as an anticancer drug but is commonly used to treat infections with the protozoan parasites Leishmania and Trypanosoma cruzi. More recently, it has been used to treat fungal infections, and in 2021 it was designated as an orphan drug by the US Food and Drug Administration for treatment of invasive candidiasis. Miltefosine is a derivative of phosphatidylcholine (PC), a major constituent of the cell membrane. PC and other phospholipids are asymmetrically distributed across the cell membrane. The mechanism of action of miltefosine is unknown. Here, we show that either increasing the activity of a putative floppase, which controls outward "flop" movement of phospholipids, or decreasing the activity of flippases, which control inward "flip" movement, results in increased resistance of the fungal pathogen Candida parapsilosis to miltefosine. This result suggests that miltefosine acts by controlling the localisation of PC or other phospholipids in the membrane. Importantly, we find that many C. parapsilosis isolates carry mutations in one flippase gene, which renders them partially resistant to miltefosine, and prone to easily acquiring increased resistance.

sentences: Protein farnesylation is upregulated in Alzheimer's human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer's model mice The pathogenic mechanisms underlying the development of Alzheimers disease (AD) remain elusive and to date there are no effective prevention or treatment for AD. Farnesyltransferase (FT) catalyzes a key posttranslational modification process called farnesylation, in which the isoprenoid farnesyl pyrophosphate is attached to target proteins, facilitating their membrane localization and their interactions with downstream effectors. Farnesylated proteins, including the Ras superfamily of small GTPases, are involved in regulating diverse physiological and pathological processes. Emerging evidence suggests that isoprenoids and farnesylated proteins may play an important role in the pathogenesis of AD. However, the dynamics of FT and protein farnesylation in human brains and the specific role of neuronal FT in the pathogenic progression of AD are not known. Here, using postmortem brain tissue from individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimers dementia, we found that the levels of FT and membrane-associated H-Ras, an exclusively farnesylated protein, and its downstream effector ERK were markedly increased in AD and MCI compared with NCI. To elucidate the specific role of neuronal FT in AD pathogenesis, we generated the transgenic AD model APP/PS1 mice with forebrain neuron-specific FT knockout, followed by a battery of behavioral assessments, biochemical assays, and unbiased transcriptomic analysis. Our results showed that the neuronal FT deletion mitigates memory impairment and amyloid neuropathology in APP/PS1 mice through suppressing amyloid generation and reversing the pathogenic hyperactivation of mTORC1 signaling. These findings suggest that aberrant upregulation of protein farnesylation is an early driving force in the pathogenic cascade of AD and that targeting FT or its downstream signaling pathways presents a viable therapeutic strategy against AD.

sentences: OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

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Ecosystem size and complexity dictate riverine biodiversity Larger ecosystems support more species; this ubiquitous pattern is the foundation of current conservation schemes. However, many ecosystems possess a complex spatial structure that cannot be represented by area, and the role of such complexity in scaling biodiversity is largely unknown. Here, we use theory and extensive fish community data from two distinct geographic regions (Japan and United States) to show that ecosystem size and complexity dictate riverine biodiversity. We found that larger and more branched  complex river networks harbored greater species richness due to increased space and environmental heterogeneity. The complexity effect was comparable to the size effect, and this pattern has emerged regardless of ecological contexts. The dual control of biodiversity may be a pervasive feature that has far-reaching implications for biodiversity conservation.

One sentence summaryThis study provides the first evidence that ecosystem size and complexity play comparable roles in regulating biodiversity.

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Microglia Detect Externalized Phosphatidylserine on Synapses for Elimination via TREM2 in Alzheimer's Disease Models Genetic studies implicate phagocytosis pathways in microglia to be a major Alzheimers disease (AD)-associated process. Microglia phagocytose synapses in AD mouse models, suggesting a role for microglia in region-specific synapse loss, a pathological hallmark of AD. However, whether specific synapses are targeted for elimination, and if so, how, remains to be elucidated. Here, we show that synapses externalize phosphatidylserine (PtdSer) upon challenge by {beta}-amyloid oligomers, which are then selectively engulfed by microglia. Mechanistically, we find that Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is critical for microglia to sense and preferentially engulf AD synapses. In brains of mice and humans, TREM2 dysfunction leads to exacerbation of apoptotic synapses. Our work altogether suggests a fundamental role for microglia as brain-resident macrophages to remove damaged synapses in AD. We provide mechanistic insight into how TREM2 variants associated with increased risk of developing AD may contribute to defective microglia-synapse function.

One-Sentence summaryMicroglia selectively engulf synapses in Alzheimer-like mouse brains via PtdSer-TREM2 signaling.

sentences: Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice. The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

sentences: Alpha power and stimulus-evoked activity dissociate metacognitive reports of attention, visibility and confidence in a visual detection task Variability in the detection and discrimination of weak visual stimuli has been linked to oscillatory neural activity. In particular, the amplitude of activity in the alpha-band (8-12 Hz) has been shown to impact upon the objective likelihood of stimulus detection, as well as measures of subjective visibility, attention, and decision confidence. We aimed to clarify how preparatory alpha influences performance and phenomenology, by recording simultaneous subjective measures of attention and confidence (Experiment 1), or attention and visibility (Experiment 2) on a trial-by-trial basis in a visual detection task. Across both experiments, alpha amplitude was negatively and linearly correlated with the intensity of subjective attention. In contrast to this linear relationship, we observed a quadratic relationship between the strength of alpha oscillations and subjective ratings of confidence and visibility. We find that this same quadratic relationship links alpha amplitude to the strength of stimulus evoked responses. Visibility and confidence judgements corresponded to the strength of evoked responses, but confidence, uniquely, incorporated information about attentional state. As such, our findings reveal distinct psychological and neural correlates of metacognitive judgements of attentional state, stimulus visibility, and decision confidence.

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

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

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

sentences: Connectivity and Synaptic Physiology in the Mouse and Human Cortex To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

sentences: Le Petit Prince: A multilingual fMRI corpus using ecological stimuli Neuroimaging using more ecologically valid stimuli such as audiobooks has advanced our understanding of natural language comprehension in the brain. However, prior naturalistic stimuli have typically been restricted to a single language, which limited generalizability beyond small typological domains. Here we present the Le Petit Prince fMRI Corpus (LPPC-fMRI), a multilingual resource for research in the cognitive neuroscience of speech and language during naturalistic listening (Open-Neuro: ds003643). 49 English speakers, 35 Chinese speakers and 28 French speakers listened to the same audiobook The Little Prince in their native language while multi-echo functional magnetic resonance imaging was acquired. We also provide time-aligned speech annotation and word-by-word predictors obtained using natural language processing tools. The resulting timeseries data are shown to be of high quality with good temporal signal-to-noise ratio and high inter-subject correlation. Data-driven functional analyses provide further evidence of data quality. This annotated, multilingual fMRI dataset facilitates future re-analysis that addresses cross-linguistic commonalities and differences in the neural substrate of language processing on multiple perceptual and linguistic levels.

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Biclique extension as an effective approach to predict novel interaction partners in metabolic compound-protein interaction networks. MotivationMetabolic networks are complex systems of connected chemical reactions with physical interactions between metabolites and proteins playing a critical role for both metabolic conversion and regulation. In this study, we aimed to predict previously unknown compound-protein interactions (CPI) by transferring the concept of biclique extension, which was developed in the context of drug-target interaction prediction and that is based on the rationale that interactions that readily extend an existing biclique are real, to metabolic CPI networks.

ResultsWe developed and tested a workflow to predict CPIs based on the concept of extending existing bicliques and applied it to E. coli and human using their respective known CPI network as input. Depending on the chosen biclique size, for the E. coli network we reached a sensitivity of 39% with an associated precision of 59%. For the larger human CPI network, a sensitivity of 78% with a false-positive rate of less than 5% and an associated precision of 75% was obtained. At more stringent settings, a precision as high as 95% was attainable at the expense of a lowered recall. Prediction performance significantly exceeded that obtained using randomized networks as input. Predicted novel interactions were tested for biomolecular function involvement, with TCA-cycle and ribosomal processes found associated with particularly pronounced statistical significance. As we demonstrate, our approach holds great potential to increase efficiency of experimental testing of CPIs and can readily be transferred to other species of interest.

Availability and implementationThe R code and datasets are available at https://github.com/SandraThieme/BiPredict.

sentences: A comparative analysis of computational tools for the prediction of epigenetic DNA methylation from long-read sequencing data Recent development of Oxford Nanopore long-read sequencing has opened new avenues of identifying epigenetic DNA methylation. Among the different epigenetic DNA methylations, N6-methyladenosine is the most prevalent DNA modification in prokaryotes and 5-methylcytosine is common in higher eukaryotes. Here we investigated if N6-methyladenosine and 5-methylcytosine modifications could be predicted from the nanopore sequencing data. Using publicly available genome sequencing data of Saccharomyces cerevisiae, we compared the open-access computational tools, including Tombo, mCaller, Nanopolish and DeepSignal for predicting 6mA and 5mC. Our results suggest that Tombo and mCaller can predict DNA N6-methyladenosine modifications at a specific location, whereas, Tombo dampened fraction, Nanopolish methylation likelihood and DeepSignal methylation probability have comparable efficiency for 5-methylcytosine prediction from Oxford Nanopore sequencing data.

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A local insulin reservoir ensures developmental progression in condition of nutrient shortage in Drosophila Insulin/IGF signalling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides (Ilp) is produced by brain neurosecretory cells and exerts systemic functions. Here, we describe the local uptake and storage of Ilps in the Corpora Cardiaca (CC), a group of alpha cell homolog that produces the glucagon-like hormone AKH. Dilp uptake relies on the expression of Impl2, an IGF-BP that accumulates in the CCs. During nutrient shortage, this specific reserve of Ilps is released and activates IIS in a paracrine manner in the prothoracic gland, securing accelerated entry into pupal development through the production of the steroid hormone ecdysone. We therefore uncover a sparing mechanism whereby local Ilp storage and release activates the production of steroids and ensures early developmental progression in adverse food conditions.

Highlights- Dilps are uptaken by CC cells through the IGF-BP Imp-L2
- the CC-Dilp store is released upon nutrient shortage and activates IIS through CC projections on the PG
- upon nutrient shortage, IIS activation in the PG ensures an accelerated transition from larval feeding stage to metamorphosis.

sentences: Fluorescence-Activated Droplet Sorting of Polyethylene terephthalate-degrading Enzymes Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. However, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of PETases using synthetic biology.

sentences: HSP90 function is required for T2R bitter taste receptor nitric oxide production and innate immune responses in human airway epithelial cells and macrophages Bitter taste receptors (T2Rs) are G protein-coupled receptors (GPCRs) expressed in various cell types including ciliated airway epithelial cells and macrophages. T2Rs in these two airway innate immune cell types are activated by bitter products, including some secreted by Pseudomonas aeruginosa, leading to Ca2+-dependent activation of endothelial nitric oxide (NO) synthase (eNOS). NO enhances mucociliary clearance and has direct antibacterial effects in ciliated epithelial cells and increases phagocytosis by macrophages. Using biochemistry and live cell imaging, we explored the role of heat shock protein 90 (HSP90) in regulating T2R-dependent NO pathways in primary sinonasal epithelial cells, primary monocyte-derived macrophages, and a human bronchiolar cell line (H441). We used immunofluorescence to show that H441 cells express eNOS and certain T2Rs and that the bitterant denatonium benzoate activates NO production in an HSP90-dependent manner in cells grown either as submerged cultures and at air liquid interface. In primary sinonasal epithelial cells, we determined that HSP-90 inhibition reduces T2R-stimulated NO production and ciliary beating which are crucial for pathogen clearance. In primary monocyte-derived macrophages, we found that HSP-90 is integral to T2R-stimulated NO production and phagocytosis of FITC-labeled Escherichia coli and pHrodo-Staphylococcus aureus. Our study demonstrates that HSP90 serves an innate immune role by regulating NO production downstream of T2R signaling by augmenting eNOS activation without impairing upstream calcium signaling. These findings suggest that HSP90 plays an important role in airway antibacterial innate immunity and may be an important target in airway diseases like chronic rhinosinusitis, asthma, or cystic fibrosis.

sentences: Distinct phases within large RNP granules of C. elegans oocytes are associated with differential stress responses of RNA binding proteins One emerging paradigm of cellular organization of RNA and RNA binding proteins is the formation of membraneless organelles (MLOs). Examples of MLOs include several types of RNP (ribonucleoprotein) granules that form via phase separation. Proper regulation of the phase transitions of RNA binding proteins is critical as dysregulation can lead to disease states. Germ granules are small RNP granules conserved across metazoa. In C. elegans, when oogenesis undergoes an extended meiotic arrest, germ granules assemble into much larger, more complex RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. As a step towards gaining insight into the function of RNP granules, in this report we characterize distinct phases for four RNA binding proteins in arrested oocytes. We find that PGL-1 is dynamic and has liquid-like properties, while MEG-3 has gel-like properties, both similar to their properties in small germ granules of embryos. We also show that MEX-3 exhibits gel-like properties in many regards but is more dynamic than MEG-3. We find CGH-1 is dynamic but does not consistently behave liquid-like, and may be an intermediate phase within RNP granules. We also show that the distinct phases of the RNA binding proteins are associated with differential responses to stress.

sentences: The Gradient Clusteron: A model neuron that learnsvia dendritic nonlinearities, structural plasticity, and gradient descent Synaptic clustering on neuronal dendrites has been hypothesized to play an important role in implementing pattern recognition. Neighboring synapses on a dendritic branch can interact in a synergistic, cooperative manner via the nonlinear voltage-dependence of NMDA receptors. Inspired by the NMDA receptor, the single-branch clusteron learning algorithm (Mel 1991) takes advantage of location-dependent multiplicative nonlinearities to solve classification tasks by randomly shuffling the locations of "under-performing" synapses on a model dendrite during learning ("structural plasticity"), eventually resulting in synapses with correlated activity being placed next to each other on the dendrite. We propose an alternative model, the gradient clusteron, or G-clusteron, which uses an analytically-derived gradient descent rule where synapses are "attracted to" or "repelled from" each other in an input- and location- dependent manner. We demonstrate the classification ability of this algorithm by testing it on the MNIST handwritten digit dataset and show that, when using a softmax activation function, the accuracy of the G-clusteron on the All-vs-All MNIST task ([~]85%) approaches that of logistic regression ([~]93%). In addition to the location update rule, we also derive a learning rule for the synaptic weights of the G-clusteron ("functional plasticity") and show that a G-clusteron that utilizes the weight update rule can achieve [~]89% accuracy on the MNIST task. We also show that a G-clusteron with both the weight and location update rules can learn to solve the XOR problem from arbitrary initial conditions.

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Metagenomic sequencing reveals a lack of virus exchange between native and invasive freshwater fish across the Murray-Darling Basin, Australia Biological invasions are among the biggest threats to freshwater biodiversity. This is increasingly relevant in the Murray-Darling Basin, Australia, particularly since the introduction of the common carp (Cyprinus carpio). This invasive species now occupies up to 90% of fish biomass, with hugely detrimental impacts on native fauna and flora. To address the ongoing impacts of carp, cyprinid herpesvirus 3 (CyHV-3) has been proposed as a potentially effective biological control. Crucially, however, it is unknown whether CyHV-3 and other cyprinid herpesviruses already exist in the Murray-Darling. Further, little is known about those viruses that naturally occur in wild freshwater fauna, and the frequency with which these viruses jump species boundaries. To document the evolution and diversity of freshwater fish viromes and better understand the ecological context to the proposed introduction of CyHV-3, we performed a meta-transcriptomic viral survey of invasive and native fish across the Murray-Darling Basin, covering over 2,200 km of the river system. Across a total of 36 RNA libraries representing 10 species, we failed to detect CyHV-3 nor any closely related viruses. Rather, meta-transcriptomic analysis identified 18 vertebrate-associated viruses that could be assigned to the Arenaviridae, Astroviridae, Bornaviridae, Caliciviridae, Coronaviridae, Chuviridae, Flaviviridae, Hantaviridae, Hepeviridae, Paramyxoviridae, Picornaviridae, Poxviridae, Reoviridae and Rhabdoviridae families, and a further 27 that were deemed to be associated with non-vertebrate hosts. Notably, we revealed a marked lack of viruses that are shared among invasive and native fishes sampled here, suggesting that there is little virus transmission from common carp to native fish species. Overall, this study provides the first data on the viruses naturally circulating in a major river system and supports the notion that fish harbour a large diversity of viruses with often deep evolutionary histories.

Author SummaryThe ongoing invasion of the common carp in the Murray-Darling Basin, Australia, has wreaked havoc on native freshwater ecosystems. This has stimulated research into the possible biological control of invasive carp through the deliberate release of the virus cyprinid herpesvirus 3 (CyHV-3). However, little is known on the diversity of viruses that naturally circulate in wild freshwater fauna, whether these viruses are transmitted between invasive and native species, nor if CyHV-3 or other cyprinid herpesviruses are already present in the basin. To address these fundamental questions we employed meta-transcriptomic next-generation sequencing to characterise the total assemblage of viruses (i.e. the viromes) in three invasive and seven native fish species cohabiting at 10 sites across 2,200 km of the river system. From this analysis we identified 18 vertebrate-associated viruses across 14 viral families, yet a marked lack of virus transmission between invasive and native species. Importantly, no CyHV-3 was detected. This study shows that freshwater fish harbour a high diversity and abundance of viruses, that viruses have likely been associated with fish for millennia, and that there is likely little direct virus transmission between introduced and native species.

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In-shoe plantar pressure depends on walking speed and type of weight-bearing activity in people with diabetes at high risk of ulceration BackgroundIn evaluating the biomechanical properties of therapeutic footwear, most often in-shoe plantar pressure is obtained during mid-gait steps at self-selected speed in a laboratory setting. However, this may not represent plantar pressures or indicate the cumulative stress experienced in daily life, where people adopt different walking speeds and weight-bearing activities.

Research questionIn people with diabetes at high risk of ulceration, 1) what is the effect of walking speed on plantar pressure measures, and 2) what is the difference in plantar pressure measures between walking at self-selected speed and other weight-bearing activities?

MethodsIn a cross-sectional study, we included 59 feet of 30 participants (5 females, mean age: 63.8 (SD 9.2) years). We assessed in-shoe plantar pressure with the Pedar-X system during three standardized walking speeds (0.8, 0.6 and 0.4 m/s) and eight types of activities versus walking at self-selected speed (3 components of the Timed Up and Go test (TUG), standing, accelerating, decelerating, stair ascending and descending and standing). Peak plantar pressure (PPP) and pressure-time integral (PTI) were determined for the hallux, metatarsal 1, metatarsal 2-3 and metatarsal 4-5. For statistical comparisons we used linear mixed models (<0.05) with Holm-Bonferroni correction.

ResultsWith increasing walking speed, PPP increased and PTI decreased for all regions (p[≤]0.001). Standing, decelerating, stair ascending and TUG showed lower PPP than walking at self-selected speed for most regions (p[≤]0.004), whereas accelerating and stair descending showed similar PPP. Stair ascending and descending showed higher PTI than walking at self-selected speed (p[≤]0.002), standing showed lower PTI (p[≤]0.001), while the other activities showed similar PTI for most regions.

SignificanceTo best evaluate the biomechanical properties of therapeutic footwear, and to assess cumulative plantar tissue stress of people with diabetes at high risk of ulceration, plantar pressures during different walking speeds and activities of daily living should be considered.

sentences: Rapid evolution of sex role specialization in a hermaphrodite under sex-limited selection The evolution of separate sexes from hermaphroditism is thought to have occurred independently many times, and is linked to the evolution of sex chromosomes. Even though we have a good understanding of the theoretical steps in the evolution of sex chromosomes from a hermaphrodite ancestor, the initial stages are still hard to study because many sex chromosome systems are old. We addressed this problem by experimentally selecting a hermaphrodite via sex-limited experimental evolution for several generations, simulating the early stages in the evolution of a sex chromosome. More specifically, we used a GFP (green fluorescent protein) marker as a proxy for a sex-determining locus, and selected replicate populations of the simultaneously hermaphroditic flatworm Macrostomum lignano for fitness via the male sex role, female sex role, or both (i.e. a control). After 14 generations, a fitness assay revealed clear evidence for incipient sex role specialization, presumably reflecting the release from constraints usually imposed by selection on the other sex role. Importantly, however, this was not simply explained by differential sex allocation in the different selection regimes - insofar as morphological traits reflect the underlying trade-off over resource allocation to the male and female sex functions - because testis and ovary sizes did not diverge among treatments. Our study shows that sex role specialization can occur rapidly as a result of sex-limited selection, which is consistent with genetic constraints between sex-roles, and in line with the first predicted steps towards the evolution of a new sex chromosome system.

sentences: A novel DNA-binding protein regulates sexual differentiation in malaria parasites and is essential for transmission. Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary for transmission to the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their dearth of transcriptional and chromatin regulators and the molecular mediators of these changes remain largely unknown. We recently identified HomeoDomain Protein 1 (HDP1) as a DNA-binding protein that enhances the expression of key genes that are critical for early sexual differentiation. The discovery of a homeodomain-like DNA-binding protein marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. In this study, we show that HDP1 facilitates the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes they characteristic shape and is required for gametocyte maturation and parasite transmission.

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COVID-19 lung disease shares driver AT2 cytopathic features with Idiopathic pulmonary fibrosis BackgroundIn the aftermath of Covid-19, some patients develop a fibrotic lung disease, i.e., post-COVID-19 lung disease (PCLD), for which we currently lack insights into pathogenesis, disease models, or treatment options.

MethodUsing an AI-guided approach, we analyzed > 1000 human lung transcriptomic datasets associated with various lung conditions using two viral pandemic signatures (ViP and sViP) and one covid lung-derived signature. Upon identifying similarities between COVID-19 and idiopathic pulmonary fibrosis (IPF), we subsequently dissected the basis for such similarity from molecular, cytopathic, and immunologic perspectives using a panel of IPF-specific gene signatures, alongside signatures of alveolar type II (AT2) cytopathies and of prognostic monocyte-driven processes that are known drivers of IPF. Transcriptome-derived findings were used to construct protein-protein interaction (PPI) network to identify the major triggers of AT2 dysfunction. Key findings were validated in hamster and human adult lung organoid (ALO) pre-clinical models of COVID-19 using immunohistochemistry and qPCR.

FindingsCOVID-19 resembles IPF at a fundamental level; it recapitulates the gene expression patterns (ViP and IPF signatures), cytokine storm (IL15-centric) and the AT2 cytopathic changes, e.g., injury, DNA damage, arrest in a transient, damage-induced progenitor state, and senescence-associated secretory phenotype (SASP). These immunocytopathic features were induced in pre-clinical COVID models (ALO and hamster) and reversed with effective anti-CoV-2 therapeutics in hamsters. PPI-network analyses pinpointed ER stress as one of the shared early triggers of both diseases, and IHC studies validated the same in the lungs of deceased subjects with COVID-19 and SARS-CoV-2-challenged hamster lungs. Lungs from tg-mice, in which ER stress is induced specifically in the AT2 cells, faithfully recapitulate the host immune response and alveolar cytopathic changes that are induced by SARS-CoV-2.

InterpretationLike IPF, COVID-19 may be driven by injury-induced ER stress that culminates into progenitor state arrest and SASP in AT2 cells. The ViP signatures in monocytes may be key determinants of prognosis. The insights, signatures, disease models identified here are likely to spur the development of therapies for patients with IPF and other fibrotic interstitial lung disease.

FundingThis work was supported by the National Institutes for Health grants R01-GM138385 and AI155696 and funding from the Tobacco-Related disease Research Program (R01RG3780).

One Sentence SummarySevere COVID-19 triggers cellular processes seen in fibrosing Interstitial Lung Disease

sentences: Epigenetic alterations at distal enhancers are linked to proliferation in human breast cancer Breast cancer is a highly heterogeneous disease driven by multiple factors including genetic and epigenetic alterations. DNA methylation patterns have been shown to be altered on a genome-wide scale and previous studies have highlighted the critical role of aberrant DNA methylation on gene expression and breast cancer pathogenesis. Here, we perform genome-wide expression-methylation Quantitative Trait Loci (emQTL), a method for integration of CpG methylation and gene expression to identify disease-driving genes under epigenetic control. By grouping these emQTLs by biclustering we identify associations representing important biological processes associated with breast cancer pathogenesis such as proliferation and tumor infiltrating fibroblasts. We report hypomethylation at enhancers carrying transcription factor binding sites of key proliferation-driving transcription factors such as CEBP-{beta}, FOSL1, and FOSL2, with concomitant high expression of cell cycle- and proliferation-related genes in aggressive breast tumors. The identified CpGs and genes were found to be connected through chromatin loops, together indicating that proliferation in aggressive breast tumors is under epigenetic regulation by DNA methylation. Interestingly, there was a significant correlation between proliferation-related DNA methylation and gene expression also within subtypes of breast cancer, thereby showing that varying proliferation may be explained by epigenetic profiles across breast cancer subtypes. Indeed, the identified proliferation gene signature was prognostic both in the Luminal A and Luminal B subtypes. Taken together, we show that proliferation in breast cancer is linked to hypomethylation at specific enhancers and transcription factor binding mediated through chromatin loops.

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Identification of diverse viruses associated with grasshoppers unveils phylogenetic congruence between hosts and viruses Locusts and grasshoppers are one of the most dangerous agricultural pests. Environmentally benign microbial pesticides are increasingly desirable for controlling locust outbreaks in fragile ecosystems. Here we use metagenomic sequencing to profile the rich viral communities in 34 grasshopper species and report 322 viruses, including 202 novel species. Most of the identified viruses are related to other insect viruses and some are targeted by antiviral RNAi pathway, indicating they infect grasshoppers. Some plant/fungi/vertebrate associated viruses are also abundant in our samples. Our analysis of relationships between host and virus phylogenies suggests that the composition of viromes is closely allied with host evolution, and there is significant phylogenetic relatedness between grasshoppers and viruses from Lispiviridae, Partitiviridae, Orthomyxoviridae, Virgaviridae and Flaviviridae. Overall, this study is a thorough exploration of viruses in grasshoppers and provide an essential evolutionary and ecological context for host-virus interaction in Acridoidea.

Author SummaryLocusts are the most destructive migratory pest in the world and continue to cause massive damages that endanger food security and threaten millions of people in 21st century. While chemical pesticides are still heavily relied on, biological pesticides developed from natural pathogens offer a reliable, less harmful alternative for controlling locust outbreaks in fragile ecosystems. Unfortunately, little is known about natural pathogens infecting this pest. In this study, we profile the viral communities in 34 grasshopper species include some major swarming species. While we identified as many as 202 novel viral species associated with grasshoppers, some of them are of potential to be developed as biocontrol agents. Our analysis of relatedness of phylogenies of grasshoppers and associated viruses helps to shed light on the eco-evolutionary interactions between insects and viruses. This work provides a valuable dataset of both academic and applied interest.

sentences: Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during algal blooms Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global nutrient cycling. When blooms collapse, organic carbon is transferred to higher trophic levels, microbial respiration or sinking in proportions that depend on the dominant mortality agent. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains an open question. Here, we characterized the consequences of viral infection on the microbiome composition and biogeochemical landscape of marine ecosystems by conducting a large-scale mesocosm experiment. Moniroting of seven induced coccolithophore blooms, which showed different degrees of viral infection, revealed that only high levels of viral infection caused significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, viral infection favored the growth of eukaryotic heterotrophs (thraustochytrids) over bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection can increase per-cell rates of extracellular carbon release by 2-4.5 fold. This happened via production of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms.

sentences: Linguistic modulation of the neural encoding of phonemes Speech comprehension entails the neural mapping of the acoustic speech signal onto learned linguistic units. This acousto-linguistic transformation is bi-directional, whereby higher-level linguistic processes (e.g., semantics) modulate the acoustic analysis of individual linguistic units. Here, we investigated the cortical topography and linguistic modulation of the most fundamental linguistic unit, the phoneme. We presented natural speech and  phoneme quilts (pseudo-randomly shuffled phonemes) in either a familiar (English) or unfamiliar (Korean) language to native English speakers while recording fMRI. This design dissociates the contribution of acoustic and linguistic processes towards phoneme analysis. We show that (1) the four main phoneme classes (vowels, nasals, plosives, fricatives) are differentially and topographically encoded in human auditory cortex, and that (2) their acoustic analysis is modulated by linguistic analysis. These results suggest that the linguistic modulation of cortical sensitivity to phoneme classes minimizes prediction error during natural speech perception, thereby aiding speech comprehension in challenging listening situations.

sentences: The influence of the extracellular domains on the dynamic behavior of membrane proteins The dynamic behavior of the plasma membrane proteins mediates various cellular processes, such as cell-cell interactions, transmembrane transport and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interaction of the intracellular domain with cytosolic components such as cortical actin. However, the impact of the extracellular domains (ECDs) on the dynamics of membrane proteins is rather unexplored. Here, we investigate how the ECD size influences protein dynamics in lipid bilayer. We reconstitute ECDs of different molecular weights and heights in model membrane systems and analyze ECD-driven protein sorting in lipid domains as well as protein mobility. We observe that increasing the ECD size leads to a decrease in ordered domain partitioning as well as diffusivity. Our data suggests a critical role of the ECDs on membrane protein behavior in the plasma membrane and paves the way to a more complete understanding of membrane protein dynamics that includes interaction with the extracellular matrix and glycocalyx in health and disease.

sentences: Metacommunity stability and persistence for predation turnoff in selective patches Predation as an important trophic interaction of ecological communities controls the large-scale patterns of species distribution, population abundance and community structure. Numerous studies address that predation can mediate diversity and regulate the ecological community and food web stability through changes in the behaviour, morphology, development, and abundance of prey. Since predation has large effects on persistence and diversity, the local loss or removal of predation in a community can trigger a cascade of extinctions. In ecological theory, the effect of predation removal has been well studied in foodwebs, but it remains unclear in the case of a spatially distributed community connected by dispersal. In this study, the interaction between local and spatial processes is taken into account, we present how a predation turnoff in selective patches affects the stability and persistence of a metacommunity. Using a simple predator-prey metacommunity with a diffusive dispersal, we show the impact of predation on synchronized, asynchronized and source-sink dynamics. Our results reveal that predation turnoff in very few patches alters a perfectly synchronized oscillatory state into multicluster states consisting of various patterns. In a source-sink behaviour, predation turnoff in a source patch reduces the number of sink patches and changes the clusters. In general, predation turnoff in a finite number of patches increases the number of clusters through asynchronized (inhomogeneous) states, whereas predation turnoff in a larger number of patches can lead to the complete extinction of predators. Typically, there exists a critical number of patches below which the predation turnoff results in asynchronized states and above that predation turnoff leads to a synchronized state in prey population with complete extinction of predators. Further, our results identify the network configurations that exhibit a unique number of clusters. Moreover, prey density from the patches where predation is absent goes to a saturating state near the carrying capacity. Thus, this study stresses that predation turnoff in selective patches acts as a stabilizing mechanism that can promote metacommunity persistence.

sentences: Montage electron tomography of vitrified specimens Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 15 [A] were observed in the montaged tilt-series, and diverse cellular features were evident in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

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Fitting deterministic population models tostochastic data: from trajectory matching tostate-space models O_LIPopulation and community ecology traditionally has a very strong theoretical foundation with well-known models, such as the logistic and its many variations, and many modification of the classical Lotka-Volterra predator-prey and interspecific competition models. More and more, these classical models are confronted to data via fitting to empirical time-series for purposes of projections or for estimating model parameters of interest. However, the interface between mathematical population or community models and data, provided by a statistical model, is far from trivial.
C_LIO_LIIn order to help empiricists, especially researchers working with experimental laboratory populations in micro- and mesocosms, make informed decisions, we here ask which error structure one should use when fitting classical deterministic ODE models to empirical data, from single species to community dynamics and trophic interactions. We use both realistically simulated data and empirical data from microcosms to answer this question in a Bayesian framework.
C_LIO_LIWe find that many model parameters can be estimated precisely with an appropriate choice of error structure using pure observation error or state-space models, if observation errors are not too high. However, Allee effect models are typically hard to identify and state-space models should be preferred with when model complexity increases.
C_LIO_LIOur work shows that, at least in the context of experimental laboratory populations, deterministic models can be used to describe stochastic population dynamics that include process variability and observation error. Also, many scenarios do not require a complex state-space model formulation and simpler trajectory matching is sufficient for accurate parameter estimates. Finally, we provide a comprehensive tutorial for fitting these models in R.
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A folder mechanism ensures size uniformity among C. elegans individuals by coupling growth and development Animals increase by orders of magnitude in their volume during development. Hence, even small differences in the growth rates between individuals could generate large differences in their adult body size. Yet, such volume divergence among individuals is usually not observed in nature.

We combined theory and experiment to understand the mechanisms of body size uniformity. Using live imaging, we measured the volume growth of hundreds of individuals of C. elegans over the entire span of their postembryonic development. We find that C. elegans grows exponentially in volume with a coefficient of variation of the growth rate of [~]7%, but that individuals diverge much less in volume than expected from this heterogeneity. The mechanism counteracting size divergence does not involve size thresholds for developmental milestones. Instead, an inverse coupling of the growth rate and the duration of development produces a constant volume fold change per larval stage.

The duration of larval stages of C. elegans is determined by the period of a developmental oscillator. Using mathematical modelling, we show that an anti-correlation between the growth rate and the oscillatory period emerges as an intrinsic property of a genetic oscillator. We propose that the robustness of body volume fold change is a hard-wired characteristic of the oscillatory circuit and does not require elaborate mechanisms of size control by cellular signalling. Indeed, the coupling of growth and development was unaltered by mutation of canonical pathways of growth control. This novel concept of size homeostasis may broadly apply to other multicellular systems controlled by genetic oscillators.

sentences: Probabilistic Modeling Confirms Delayed, But Non-Deficient Locomotor Development in Low Birth Weight Piglets 1In the study of vertebrate locomotion, kinematic measures of gait, dynamic posture, and coordination often change when comparing subjects of different body mass, size, and age. Is it, conversely, possible to infer subject characteristics from the kinematic measures? For this study, piglets (Sus domesticus) were filmed from lateral perspective during their first ten hours of life, an age at which body mass and size have major consequences for survival. We then apply deep learning methods for point digitization (DeepLabCut), calculate joint angle profiles, and apply information-preserving transfromations (Fourier Series and PCA) to retrieve a multivariate kinematic data set. We train a probabilistic model to predict subject characteristics from kinematics. The model infers subject characteristics accurately for strides from piglets of normal birth weight (i.e. the category it was trained on), but surprisingly predicts the body mass and size of low birth weight piglets (which were left out of training, out-of-sample prediction) to be "normal". The discrepancies between prediction and observation confirm that dynamic posture and coordination are unaffected by lower birth weight and lower size, which is evidence that low birth weight does not imply locomotor deficits. However, the age of some (but not all) low birth weight individuals is underestimated, supporting the hypothesis that these piglets can experience a delay in locomotor maturation.

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Integration of the Salmonella Typhimurium methylome and transcriptome reveals DNA methylation and transcriptional regulation are largely decoupled under virulence-related conditions Despite being in a golden age of bacterial epigenomics, little work has systematically examined the plasticity and functional impacts of the bacterial DNA methylome. Here, we leveraged SMRT sequencing to examine the m6A DNA methylome of two Salmonella enterica ser. Typhimurium strains: 14028s and a {Delta}metJ mutant with derepressed methionine metabolism, grown in Luria Broth or a media that simulates the intracellular environment. We find that the methylome is remarkably static--over 95% of adenosine bases retain their methylation status across conditions. Integration of methylation with transcriptomic data revealed limited correlation between changes in methylation and gene expression. Further, examining the transcriptome in {Delta}yhdJ bacteria, lacking the m6A methylase with the most dynamic methylation pattern in our dataset, revealed little evidence of YhdJ-mediated gene regulation. Curiously, despite G(m6A)TC motifs being particularly resistant to change across conditions, incorporating dam mutants into our analyses revealed two examples where changes in methylation and transcription may be linked across conditions. This includes the novel finding that the {Delta}metJ motility defect may be partially driven by hypermethylation of the chemotaxis gene tsr. Together, these data redefine the S. Typhimurium epigenome as a highly stable system that has rare, but important, roles in transcriptional regulation. Incorporating these lessons into future studies will be critical as we progress through the epigenomic era.

ImportanceWhile recent breakthroughs have enabled intense study of bacterial DNA modifications, limitations in current work have potentiated a surprisingly untested narrative that DNA methylation is a common mechanism of the bacterial response to environmental conditions. Essentially, whether epigenetic regulation of bacterial transcription is a common, generalizable phenomenon is a critical unanswered question that we address here. We find that most DNA methylation is static in Salmonella enterica serovar Typhimurium, even when the bacteria are grown under dramatically different conditions that cause broad changes in the transcriptome. Further, even when the methylation of individual bases change, these changes generally do not correlate with changes in gene expression. Finally, we demonstrate methods by which data can be stratified in order to identify coupled changes in methylation and gene expression.

sentences: Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains. Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

sentences: Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

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Genes with Specificity for Expression in the Round Cell Layer of the Growth Plate are Enriched in GWAS of Human Height Human adult height reflects the outcome of childhood skeletal growth. Growth plate (epiphyseal) chondrocytes are key determinants of height. As epiphyseal chondrocytes mature and proliferate, they pass through three developmental stages, which are organized into three distinct layers in the growth plate: 1) resting (round), 2) proliferative (flat), and 3) hypertrophic. Recent genome-wide association studies (GWAS) of human height identified numerous associated loci, which are enriched for genes expressed in growth plate chondrocytes. However, it remains unclear which specific genes expressed in which layers of the growth plate regulate skeletal growth and human height.

To connect the genetics of height and growth plate biology, we analyzed GWAS data through the lens of gene expression in the three dissected layers of murine newborn tibial growth plate. For each gene, we derived a specificity score for each growth plate layer and regressed these scores against gene-level p-values from recent height GWAS data. We found that specificity for expression in the round cell layer, which contains chondrocytes early in maturation, is significantly associated with height GWAS p-values (p=8.5x10-9); this association remains after conditioning on specificity for the other cell layers. The association also remains after conditioning on membership in an "OMIM gene set" (genes known to cause monogenic skeletal growth disorders, p<9.7x10-6). We replicated the association in RNA-seq data from maturing chondrocytes sampled at early and late time points during differentiation in vitro: we found that expression early in differentiation is significantly associated with p-values from height GWAS (p=6.1x10-10) and that this association remains after conditioning on expression at 10 days in culture and on the OMIM gene set (p<0.006). These findings newly implicate genes highlighted by GWAS of height and specifically expressed in the round cell layer as being potentially important regulators of skeletal biology.

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Learning torus PCA based classification for multiscale RNA backbone structure correction with application to SARS-CoV-2 MotivationReconstructions of structure of biomolecules, for instance via X-ray crystallography or cryo-EM frequently contain clashes of atomic centers. Correction methods are usually based on simulations approximating biophysical chemistry, making them computationally expensive and often not correcting all clashes.

ResultsWe propose a computationally fast data-driven statistical method yielding suites free from within-suite clashes: From such a clash free training data set, devising mode hunting after torus PCA on adaptive cutting average linkage tree clustering (MINTAGE), we learn RNA suite shapes. With classification based on multiscale structure enhancement (CLEAN), for a given clash suite we determine its neighborhood on a mesoscopic scale involving several suites. As corrected suite we propose the Frechet mean on a torus of the largest classes in this neighborhood. We validate CLEAN MINTAGE on a benchmark data set, compare it to a state of the art correction method and apply it, as proof of concept, to two exemplary suites adjacent to helical pieces of the frameshift stimulation element of SARS-CoV-2 which are difficult to reconstruct. In contrast to a recent reconstruction proposing several different structure models, CLEAN MINTAGE unanimously proposes structure corrections within the same clash free class for all suites.

Code Availabilityhttps://gitlab.gwdg.de/henrik.wiechers1/clean-mintage-code

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Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents.

sentences: Extremely bright, near-IR emitting spontaneously blinking fluorophores enable ratiometric multicolor nanoscopy in live cells New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multi-color, live-cell, single-molecule localization microscopy (SMLM), especially for experiments that demand ultra-high-resolution live imaging. Here we report the design, synthesis, and biological evaluation of Yale676sb, a photostable, near-IR emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.

sentences: DNA motif analysis of shear stress responsive endothelial enhancers reveals differential association of KLF and ETV/ETS binding sites with gained and lost enhancers Endothelial cells (EC) lining blood vessels are exposed to mechanical forces, such as shear stress exerted by the flowing blood. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation in addition to cell size and shape. Despite a good understanding of the genes and signaling pathways responding to shear stress, our insights into the transcriptional regulation of these responses is much more limited. In particular, we do not know the different sets of regulatory elements (enhancers) that might control increases or decreases in gene expression. Here, we set out to study changes in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 hours of laminar shear stress. Our results show a correlation of gained and lost enhancers with up- and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized shear stress responsive genes in ECs of zebrafish embryos using RNA-Seq. Together, our results reveal the presence of shear stress responsive DNA regulatory elements and lay the groundwork for the future exploration of these elements and the TFs binding to them in controlling EC biology.

sentences: Dissecting the effects of adaptive encoding and predictive inference on a single perceptual estimation The human visual system exploits continuity in the visual environment, which induces two contrasting perceptual phenomena: repulsive aftereffects and attractive serial dependences. Recent studies have theorized that these two effects concurrently arise in perceptual processing, but empirical evidence that directly supports this hypothesis lacks. Here we show that the two effects concurrently modulate estimation behavior in a typical sequence of perceptual tasks. We first demonstrate that observers estimation error as a function of both the previous stimulus and response cannot be fully described by either attractive or repulsive bias but is instead well captured by a summation of repulsion from the previous stimulus and attraction toward the previous response. We then reveal that the repulsive bias is centered on the observers sensory encoding of the previous stimulus, which is again repelled away from the preceding trials, whereas the attractive bias is centered precisely on the previous response, which is the observers best prediction about the incoming stimuli. Our findings provide strong evidence that sensory coding is shaped by dynamic tuning of the system to the past stimuli, inducing repulsive aftereffects, and followed by inference incorporating the prediction from the past estimation, leading to attractive serial dependence.

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Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

Impact statementLacham-Hartman et al. demonstrate enhancement of binding affinity through avidity in a complex between a bivalent ligand and a soluble monomeric target with a single binding site. Avidity effects have previously been demonstrated only for clustered receptor molecules presenting multiple binding sites. Our model may explain how polyvalent ligands can agonize or antagonize biological interactions involving nonclustered target molecules that are crucial for intra- and extracellular structural, metabolic, signaling, and regulatory pathways.

sentences: ChAdOx1 nCoV-19 (AZD1222) protects hamsters against SARS-CoV-2 B.1.351 and B.1.1.7 1 disease We investigated ChAdOx1 nCoV-19 (AZD1222) vaccine efficacy against SARS-CoV-2 variants of concern (VOCs) B.1.1.7 and B.1.351 in Syrian hamsters. We previously showed protection against SARS-CoV-2 disease and pneumonia in hamsters vaccinated with a single dose of ChAdOx1 nCoV-19. Here, we observed a 9.5-fold reduction of virus neutralizing antibody titer in vaccinated hamster sera against B.1.351 compared to B.1.1.7. Vaccinated hamsters challenged with B.1.1.7 or B.1.351 did not lose weight compared to control animals. In contrast to control animals, the lungs of vaccinated animals did not show any gross lesions. Minimal to no viral subgenomic RNA (sgRNA) and no infectious virus was detected in lungs of vaccinated animals. Histopathological evaluation showed extensive pulmonary pathology caused by B.1.1.7 or B.1.351 replication in the control animals, but none in the vaccinated animals. These data demonstrate the effectiveness of the ChAdOx1 nCoV-19 vaccine against clinical disease caused by B.1.1.7 or B.1.351 VOCs.

sentences: Diverse classes of constraints enable broader applicability of a linear programming-based dynamic metabolic modeling framework Current metabolic modeling tools suffer from a variety of limitations, from scalability to simplifying assumptions, that preclude their use in many applications. We recently created a modeling framework, LK-DFBA, that addresses a key gap: capturing metabolite dynamics and regulation while retaining a potentially scalable linear programming structure. Key to this frameworks success are the linear kinetics and regulatory constraints imposed on the system. However, while the linearity of these constraints reduces computational complexity, it may not accurately capture the behavior of many biochemical systems. Here, we developed three new classes of LK-DFBA constraints to better model interactions between metabolites and the reactions they regulate. We tested these new approaches on several synthetic and biological systems, and also performed the first-ever comparison of LK-DFBA predictions to experimental data. We found that no single constraint approach was optimal across all systems examined, and systems with the same topological structure but different parameters were often best modeled by different types of constraints. However, we did find that the optimal constraint approach was generally robust to local perturbations of the system, indicating that just a single wild-type dataset could allow identification of the ideal constraint for a given system. These results suggest that the availability of multiple constraint approaches will allow LK-DFBA to model a wider range of metabolic systems.

sentences: Replacement of microglia by brain-engrafted macrophages provide protection against concussive injury Brain resident microglia have a distinct origin compared to macrophages in other organs. Under physiological conditions, microglia are maintained by self-renewal from the local pool, independent of hematopoietic progenitors. Pharmacological depletion of microglia during therapeutic whole-brain irradiation prevents synaptic loss and long-term recognition memory deficits but the mechanisms behind these protective effects are unknown. Here we demonstrate that after a combination of therapeutic whole-brain irradiation and microglia depletion, macrophages originating from circulating monocytes engraft into the brain and replace the microglia pool. Comparisons of transcriptomes reveal that brain-engrafted macrophages have an intermediate phenotype that resembles both monocytes and embryonic microglia. Brain-engrafted macrophages display reduced phagocytic activity for synaptic compartments compared to microglia from normal brains in response to a secondary concussive brain injury. In addition to sparing mice from brain radiotherapy-induced long-term cognitive deficits, replacement of microglia by brain-engrafted macrophages can prevent concussive injury-induced memory loss. These results demonstrate the long-term functional role of brain-engrafted macrophages as a possible therapeutic tool against radiation-induced cognitive deficits.

sentences: Intracellular transport of electrotransferred DNA cargo is governed by coexisting ergodic and non ergodic anomalous diffusion The ability of exogenous DNA cargo to overcome the active and viscoelastic eukaryotic cytoplasm is a principal determinant for the gene delivery efficacy. During DNA electrotransfer, DNA forms complexes with the membrane (DNA cargo) which is transported through the cytoplasm through a combination of passive diffusion and active transport. However, this process is poorly understood limiting rational optimization of DNA cargo to be delivered to different cell types. We have investigated the intracellular transport of DNA cargo (of sizes 100 bp, 250 bp and 500 bp) delivered by electrotransfer to non-cancerous and cancerous mammalian cells. We demonstrate that intracellular DNA cargo transport is governed by coexisting ergodic and non ergodic anomalous diffusion for all the tested DNA sizes and cell types. The apparent diffusion coefficient of the electrotransferred DNA cargo in the cytoplasm decreases when the DNA size is increased from 100 bp to 500 bp. Interestingly, the electrotransferred DNA cargo (500 bp) transport is strongly dependent on the cells cancer state. Intracellular electrotransferred DNA cargo transport has a higher probability of superdiffusive transport and lower probability of caging in metastatic cells compared to malignant cells followed by benign cells.

sentences: Evolution avoids a pathological stabilizing interaction in the immune protein S100A9 Protein stability constrains protein evolution. While much is known about evolutionary constraints on destabilizing mutations, much less is known about evolutionary constraints on mutations that increase protein stability. We recently found that the M63F mutation in the innate immune protein S100A9 increases its stability but disrupts its natural ability to activate inflammation. Here we show, through careful biophysical and functional studies, that this mutation stabilizes a nonfunctional form of the protein through a direct interaction with another amino acid, Phe37. Phe can be tolerated at position 37 or 63, but not at both sites simultaneously. We find that this pattern has been conserved over millions of years of evolution. Our work highlights an underappreciated evolutionary constraint on stabilizing mutations: they must avoid inappropriately stabilizing non-functional protein conformations.

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ColabFold - Making protein folding accessible to all ColabFold offers accelerated protein structure and complex predictions by combining the fast homology search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFolds 40 - 60x faster search and optimized model use allows predicting close to a thousand structures per day on a server with one GPU. Coupled with Google Colaboratory, ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software available at github.com/sokrypton/ColabFold. Its novel environmental databases are available at colabfold.mmseqs.com

Contactmilot.mirdita@mpibpc.mpg.de, so@fas.harvard.edu, martin.steinegger@snu.ac.kr

sentences: A bacterial genome and culture collection of gut microbial in weanling piglet The microbiota hosted in the pig gastrointestinal tract are important for productivity of livestock. However, the individual species and functional repertoires that make up the pig gut microbiome remain largely undefined. Here we comprehensively investigated the genomes and functions of the piglet gut microbiome using culture-based and metagenomics approaches. A collection included 266 cultured genomes and 482 metagenome-assembled genomes (MAGs) that were clustered to 428 species across 10 phyla was established. Among these clustered species, 333 genomes represent potential new species. Less matches between cultured genomes and MAGs revealed a substantial bias for the acquisition of reference genomes by the two strategies. Glycoside hydrolases was the dominant category of carbohydrate-active enzymes. 445 secondary metabolite biosynthetic genes were predicted from 292 genomes with bacteriocin being the most. Pan genome analysis of Limosilactobacillus reuteri uncover the biosynthesis of reuterin was strain-specific and the production was experimentally determined. These genomic resources will enable a comprehensive characterization of the microbiome composition and function of pig gut.

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RNA decay defines the therapeutic response to transcriptional perturbation in cancer Transcriptionally dysregulated cancers are sensitive to the inhibition of RNA Polymerase II (RNAPII) - driven gene expression. The therapeutic effect is attributed to selective inhibition of discrete oncogenes regulated at the chromatin level, however the role of RNA stability remains largely unexplored. Using integrated transcriptomic technologies, we discovered that RNA decay is a key determinant in defining gene expression responses to transcriptional perturbation, where total RNA signatures are dominated with genes that have short transcript half-lives, including oncogenic drivers such as c-MYC. Experimentally increasing c-MYC RNA stability maintained total c-MYC RNA levels following RNAPII perturbation, despite a concordant decrease in nascent RNA. Taken together, these data demonstrate that RNA decay shapes the molecular and therapeutic response to transcriptional perturbation in cancer.

HIGHLIGHTSO_LISelective inhibition of oncogenic transcription in response to epigenetic and transcriptional inhibitors in cancer under-estimates the role of RNA decay
C_LIO_LIGene intrinsic RNA decay rates are a key determinant in shaping the total mRNA response to transcriptional perturbation in cancer
C_LIO_LISelective disruption of core-transcription factor networks is a result of short transcript half-lives
C_LIO_LIModulation of c-MYC decay rates can render it insensitive to transcriptional targeting
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sentences: Progressive alterations in polysomal architecture in a mouse model of Huntington's disease Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntingtons disease (HD). We examined the architecture of polysomes in their native brain context by performing 3D electron tomography of striatal tissue from the zQ175 knock-in mouse model of HD. Results revealed progressive remodelling towards a more compacted polysomal architecture, an effect that coincided with the emergence and progression of HD related symptoms. This aberrant architecture is compatible with ribosome stalling phenomena. In fact, we also detected an increase in the striatal expression of the stalling relief factor EIF5A2. Polysomal sedimentation gradients showed a significant increase in the accumulation of free 40S ribosomal subunits in zQ175 striatal samples. Together, these findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, thus opening new avenues for understanding the progression of this disease.

sentences: Total Biosynthesis of Triacsin Featuring an N-hydroxytriazene Pharmacophore Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing hydrazine-forming enzyme, Tri28, and a nitrous acid-utilizing N-nitrosating enzyme, Tri17. This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.

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

sentences: Identification of Piperlongumine (PL) as an new E3 ligase ligand to induce targeted protein degradation PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. Through competitive activity-based protein profiling, we found that piperlongumine (PL), a natural product, binds multiple E3 ligases. To evaluate whether PL can be used as an E3 ligase ligand, we generated a series of PL and SNS-032 (a selective CDK9 inhibitor) conjugates and found that the lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome dependent manner. In addition, 955 is more potent than SNS-032 against various tumor cells in vitro. Through TurboID-based proteomics and mechanistic studies, we identified KEAP1 as the E3 ligase recruited by PL to degrade CDK9. These findings demonstrate that PL is a novel E3 ligase ligand that can be used to generate potent anticancer PROTACs.

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From zero to infinity: minimum to maximum diversity of the planet by spatio-parametric Rao's quadratic entropy AimThe majority of work done to gather information on Earth diversity has been carried out by in-situ data, with known issues related to epistemology (e.g., species determination and taxonomy), spatial uncertainty, logistics (time and costs), among others. An alternative way to gather information about spatial ecosystem variability is the use of satellite remote sensing. It works as a powerful tool for attaining rapid and standardized information. Several metrics used to calculate remotely sensed diversity of ecosystems are based on Shannons Information Theory, namely on the differences in relative abundance of pixel reflectances in a certain area. Additional metrics like the Raos quadratic entropy allow the use of spectral distance beside abundance, but they are point descriptors of diversity, namely they can account only for a part of the whole diversity continuum. The aim of this paper is thus to generalize the Raos quadratic entropy by proposing its parameterization for the first time.

InnovationThe parametric Raos quadratic entropy, coded in R, i) allows to represent the whole continuum of potential diversity indices in one formula, and ii) starting from the Raos quadratic entropy, allows to explicitly make use of distances among pixel reflectance values, together with relative abundances.

Main conclusionsThe proposed unifying measure is an integration between abundance- and distance-based algorithms to map the continuum of diversity given a satellite image at any spatial scale.

sentences: Pressure for rapid and accurate mate recognition promotes avian-perceived plumage sexual dichromatism in true thrushes (genus: Turdus) Ecological conditions limiting the time to find a compatible mate or increasing the difficulty in doing so likely promote the evolution of traits used for species and mate recognition. Conspicuous traits that signal an individuals species, sex, and breeding status reduce the challenge of identifying a compatible conspecific mate, and should be more common in migratory rather than sedentary species, species with shorter breeding seasons, and species breeding under high sympatry with many closely-related heterospecifics. Here, we tested this recognition hypothesis for promoting plumage sexual dichromatism in the true thrushes (Turdus spp.), a large and diverse genus of passerine birds. We used receptor-noise limited models of avian vision to quantify avian-perceived chromatic and achromatic visual contrasts between male and female plumage patches and tested the influence of breeding season length, spatial distribution, and sympatry with other Turdus species on plumage dichromatism. As predicted, we found that 1) true thrush species with migratory behaviour have greater plumage sexual dichromatism than non-migratory species, 2) species with longer breeding seasons have less plumage sexual dichromatism, and 3) greater numbers of Turdus thrush species breeding in sympatry is associated with more plumage sexual dichromatism. These results suggest that social recognition systems, including species and mate recognition, play a prominent role in the evolution of plumage sexual dichromatism in true thrushes.

sentences: Functional analysis of Mmd2 and related PAQR genes during sex determination in mice Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

sentences: Homolog of ELAC2 is a central regulator of the mitochondrial unfolded protein response The mitochondrial unfolded protein response (UPRmt) has emerged as a predominant mechanism that preserves mitochondrial function. Consequently, multiple pathways likely exist to modulate UPRmt. We unexpectedly discovered that the tRNA processing enzyme, homolog of ELAC2 (HOE-1), is central to UPRmt regulation in Caenorhabditis elegans. We find that nuclear HOE-1 is necessary and sufficient to robustly activate UPRmt. We show that HOE-1 acts via transcription factors ATFS-1 and DVE-1 that are crucial for UPRmt. Mechanistically, we show that HOE-1 likely mediates its effects via tRNAs, as blocking tRNA export prevents HOE-1-induced UPRmt. Interestingly, we find that HOE-1 does not act via the integrated stress response, which can be activated by uncharged tRNAs, pointing towards its reliance on a new mechanism. Finally, we show that the subcellular localization of HOE-1 is responsive to mitochondrial stress and is subject to negative regulation via ATFS-1. Together, we have discovered a novel RNA-based cellular pathway that modulates UPRmt.

sentences: One mucosal administration of a live attenuated recombinant COVID-19 vaccine protects non-human primates from SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein, spike, for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live attenuated, recombinant human respiratory syncytial virus (RSV) expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200- fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunity in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against two virus variants of concern. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in phase 1 clinical trials as a single-dose intranasal COVID-19 vaccine.

sentences: Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

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Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Mimiviridae, and prokaryotes in a coastal ecosystem Coastal seawater is the habitat of different microbial communities. These communities are affected by seasonal environmental changes and fluctuating nutrient availability, as well as competitive and cooperative interspecific interactions. In this work, we investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses infecting eukaryotes (i.e. Mimiviridae), as well as prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and the Mimiviridae polB gene as marker genes in 43 seawater samples collected during 20 months. Communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles for eukaryotes, Mimiviridae, and prokaryotes. However, the community dynamics showed intriguing differences in several aspects such as the recovery rate after a year. We further show that the differences in the community dynamics can be explained by differences in the recurrence/persistence levels of individual ASVs among eukaryotes, Mimiviridae, and prokaryotes. Mimiviridae ASVs were less persistent than eukaryotic ASVs, and prokaryotic ASVs were the most persistent. We argue that the differences in the specificity of interactions (i.e. virus-eukaryote vs prokaryote-eukaryote) as well as the survival strategies are at the origin of the distinct community dynamics among eukaryotes, Mimiviridae, and prokaryotes.

One sentence summaryA one year observation of coastal microbial communities revealed similar but different community dynamics for eukaryotes, a group of large viruses, and prokaryotes.

sentences: Using Graph Convolutional Neural Networks to Learn a Representation for Glycans As the only nonlinear and most diverse biological sequence, glycans offer substantial challenges for computational biology. These complex carbohydrates participate in nearly all biological processes - from protein folding to the cellular entry of viruses - yet are still not well understood. There are few computational methods to link glycan sequences to functions and those that do exist do not take full advantage of all the available information of glycans. SweetNet is a graph convolutional neural network model that uses graph representation learning to facilitate a computational understanding of glycobiology. SweetNet explicitly incorporates the nonlinear nature of glycans and establishes a framework to map any glycan sequence to a representation. We show that SweetNet outperforms other computational methods in predicting glycan properties on all reported tasks. More importantly, we show that glycan representations, learned by SweetNet, are predictive of organismal phenotypic and environmental properties. Finally, we present a new application for glycan-focused machine learning, the prediction of viral glycan-binding, that can be used to discover new viral receptors and monitor rapidly mutating viruses.

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A different type of tolerance: selection for and phenotypic characteristics of antibiotic tolerance evaluated at the single cell level under recurring antibiotic exposure in Escherichia coli. Non-heritable drug resistance, such as tolerance and persistence towards antibiotics, is little characterized compared to genetic resistance. Tolerance and persistence allow cells to survive application of antibiotics that are bactericidal to non-tolerant cells. Non-heritable drug resistance challenges antibiotic treatment, particularly of recurrent infections, and have implications towards heritable resistance evolution. Tolerant cells have commonly been characterized as growth arrested cells prior and during antibiotic application that quickly resume growth post-application. Here, we explore characteristic of tolerant and susceptible E. coli single bacteria cells to different levels of recurrent antibiotic exposure and quantify their occurrence. In using a high throughput single-cell microfluidic device, we find that tolerant cells reduce their growth rate by about 50%, but contrary to previous findings do not go into growth arrest or near growth arrest. The growth reduction is induced by antibiotic exposure and not caused by a stochastic switch or predetermined state as previously described. Cells exhibiting constant intermediate growth survived best under antibiotic exposure and selection did not primarily act on fast growing cells, as expected for a {beta}-lactam antibiotic. Control experiments on population cultures confirmed and challenged scaling of single cell findings to population level processes. Our findings suggest a prevalent type of tolerance that differs from previously described tolerance and persister characteristics. Our described characteristics and its high frequency of occurrence supports acclaims of an underappreciated role of tolerant cells towards resistance evolution.

Author SummaryResistance to antibiotics is a major cause of treatment failure in modern medicine. In addition to genetic resistance, bacterial cells can sustain periods of bactericidal application in a so-called tolerant state. Previously tolerant cells were characterized by growth arrest prior and during exposure to antibiotics. Here, we show a distinctive type of tolerance that arises out of normally growing cells, where during antibiotic exposure, cell growth is slowed but does not go into growth arrest. Cells with an intermediate growth have the best survival, which is contrary to the assumptions of exposure to a {beta}-lactam antibiotic where selection is assumed to act against the fastest growing cells and the least against non-growing cells. The novel characteristics of tolerance that we have uncovered provides bacteria with another route to evade antibiotics and that could be a considerable threat to the efficiency of antibiotic treatment.

sentences: Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity Satellite DNA spans megabases of eukaryotic sequence and evolves rapidly. Paradoxically, satellite-rich genomic regions mediate strictly conserved, essential processes like chromosome segregation and nuclear structure. A leading resolution to this paradox posits that satellite DNA and satellite-associated chromosomal proteins coevolve to preserve these essential functions. We experimentally test this model of intra-genomic coevolution by conducting the first evolution-guided manipulation of both chromosomal protein and DNA satellite. The 359bp satellite spans an 11Mb array in D. melanogaster that is absent from its sister species, D. simulans. This species-specific DNA satellite colocalizes with the adaptively evolving, ovary-enriched protein, Maternal Haploid (MH)-the Drosophila homolog of Spartan. To determine if MH and 359 coevolve, we swapped the D. simulans version of MH ("MH[sim]") into D. melanogaster. MH[sim] triggers ovarian cell death, reduced ovary size, and loss of mature eggs. Surprisingly, the D. melanogaster mh null mutant has no such ovary phenotypes, suggesting that MH[sim] is toxic in a D. melanogaster background. Using both cell biology and genetics, we discovered that MH[sim] poisons oogenesis through a DNA damage pathway. Remarkably, deleting the D. melanogaster-specific 359 satellite array completely restores mh[sim] germline genome integrity and fertility, consistent with a history of coevolution between these two fast-evolving loci. Germline genome integrity and fertility are also restored by overexpressing Topoisomerase II (Top2), suggesting that MH[sim] interferes with Top2-mediated processing of 359. The observed 359-MH[sim] cross-species incompatibility supports a model under which ostensibly inert repetitive DNA and essential chromosomal proteins must coevolve to preserve germline genome integrity.

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Using a Homeogram to Detect Sleep in Free-living Animals O_LISleep is appreciated as a behavior critical to homeostasis, performance, and fitness. Yet, most of what we know about sleep comes from humans or controlled laboratory experiments. Assessing sleep in wild animals is challenging, as it is often hidden from view, and electrophysiological recordings that define sleep states are difficult to obtain. Accelerometers have offered great insight regarding gross movement, although ambiguous quiescent states like sleep have been largely ignored, limiting our understanding of this ubiquitous behavior.
C_LIO_LIWe developed a broadly applicable sleep detection method called a homeogram that can be applied to accelerometer data collected from wild animals. We applied our methodology to detect sleep in free-ranging North American red squirrels (Tamiasciurus hudsonicus) in a region that experiences drastic seasonal shifts in light, temperature, and behavioral demands.
C_LIO_LIOur method characterized sleep in a manner consistent with limited existing studies and expanded those observations to provide evidence that red squirrels apply unique sleep strategies to cope with changing environments.
C_LIO_LIApplying our analytical strategy to accelerometer data from other species may open new possibilities to investigate sleep patterns for researchers studying wild animals.
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Machine learning-based approach KEVOLVE efficiently identifies SARS-CoV-2 variant-specific genomic signatures Machine learning has proven to be a powerful tool for the identification of distinctive genomic signatures among viral sequences. Such signatures are motifs present in the viral genome that differentiate species or variants. In the context of SARS-CoV-2, the identification of such signatures can contribute to taxonomic and phylogenetic studies, help in recognizing and defining distinct emerging variants, and focus the characterization of functional properties of polymorphic gene products. Here, we study KEVOLVE, an approach based on a genetic algorithm with a machine learning kernel, to identify several genomic signatures based on minimal sets of k-mers. In a comparative study, in which we analyzed large SARS-CoV-2 genome dataset, KEVOLVE performed better in identifying variant-discriminative signatures than several gold-standard reference statistical tools. Subsequently, these signatures were characterized to highlight potential biological functions. The majority were associated with known mutations among the different variants, with respect to functional and pathological impact based on available literature. Notably, we found show evidence of new motifs, specifically in the Omicron variant, some of which include silent mutations, indicating potentially novel, variant-specific virulence determinants. The source code of the method and additional resources are available at: https://github.com/bioinfoUQAM/KEVOLVE.

Author summaryAdvances in cloning and sequencing technologies have yielded a vast repository of viral genomic sequence data. To analyze this complex and massive data, Machine learning, which refers to the development and application of computer algorithms that improve with experience, has proven to be efficient. Although many methods have been developed to classify viruses into different characteristic groups, it is often difficult to explain the predictions of these methods. To overcome this, we are working in our laboratory on the design of machine learning based methods for discriminative signatures identification within viral genomic sequences. These signatures which are a specific motifs to groups of viruses known to be pervasive in their genome, are used to 1) build accurate and explainable prediction tools for pathogens and 2) highlight mutations potentially associated with functional changes. In this paper we present the potential of our latest approach KEVOLVE. We first compare it to three discriminating motif identification tools with data sets covering several SARS-CoV-2 variant genomes. We then focus on the identified motifs by KEVOLVE to analyze the mutations associated with the different variants and the potential changes in biological functions that they may involve.

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Macrophage proliferation machinery leads to PDAC progression, but susceptibility to innate immunotherapy. Tumor-associated macrophages (TAMs) are involved in many aspects of cancer progression and correlate with poor clinical outcomes in many cancer types, including pancreatic ductal adenocarcinomas (PDACs). Previous studies have shown that TAMs can populate PDAC tumors not only by monocyte recruitment but also by local proliferation. However, the impact local proliferation might have on macrophage phenotype and cancer progression is unknown. Here, we utilized genetically engineered cancer models, single-cell RNA-sequencing data, and in vitro systems to show that proliferation of TAMs was driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. The p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression, also drive response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

SummaryTAMs are indicative of poor clinical outcomes and in PDAC their number is sustained in part by local proliferation. This study shows that stromal desmoplasia drives local proliferation of TAMs, and induces their immunosuppressive ability through altering cell cycle machinery, including p21 expression. Serendipitously, these changes in p21 in TAMs also potentially render tumors more sensitive to CD40 agonist therapy.

sentences: Influence of Response Criterion on Nociceptive Detection Thresholds and Evoked Potentials Pain scientists and clinicians search for objective measures of altered nociceptive processing to study and stratify chronic pain patients. Nociceptive processing can be studied by observing a combination of nociceptive detection thresholds and evoked potentials. However, it is unknown whether the nociceptive detection threshold measured using a Go-/No-Go (GN) procedure can be biased by a response criterion. In this study, we compared nociceptive detection thresholds, psychometric slopes and central evoked potentials obtained during a GN procedure with those obtained during a 2-interval forced choice (2IFC) procedure to determine 1) if the nociceptive detection threshold during a GN procedure is biased by a criterion and 2) to determine if nociceptive evoked potentials observed in response to stimuli around the detection threshold are biased by a criterion. We found that the detection threshold can be higher when assessed using a GN procedure in comparison with the 2IFC procedure. The average P2 component in the central evoked potential showed on-off behavior with respect to stimulus detection and increased proportionally with the detection probability during a GN procedure. These data suggest that nociceptive detection thresholds estimated using a GN procedure are subject to a response criterion.

sentences: Distribution of the National Science Foundation's Advancing Informal STEM Learning Awards (AISL) between 2006-21 The COVID-19 pandemic tested many fundamental connections between science and society. A growing field working to strengthen those connections exists within the informal STEM learning (ISL) community which provides diverse learning and engagement environments outside the formal classroom. One of the largest funders of ISL initiatives is the National Science Foundation (NSF) which runs the Advanced Informal STEM Learning (AISL) program in the United States. The AISL program supports initiatives through six categories that include pilots and feasibility studies, research in service to practice, innovations in development, broad implementation, literature reviews, syntheses, or meta-analyses, and conferences. However, a number of questions remain unanswered with respect to the distribution and ultimately the broad impact of the awards. In this study, we analyzed publicly available awardee information across a 15-year period (2006-2021) to provide a preliminary analysis of how the AISL grants are distributed across individual states, organizations, and the principal investigators. Several states, organizations, and principal investigators stood out as prolific awardees. Massachusetts and California represented the largest share of awards at 14% and 13% respectively during that time. WGBH Educational Foundation located in Massachusetts and the Exploratorium located in California, received the largest number of awards during the 15 year period. Notably, 67% of the AISL awards list at least one co-principal investigator. Our report brings to light a number of new questions and charts new paths of exploration for future studies.

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Epigenetic reprogramming of human macrophages by an intestinal pathogen Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61 % of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.

Author SummaryHuman pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages.

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Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory.

We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ, with no manipulation of spheroids.

As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX(R), mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies.

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sentences: Unique role and vulnerability of EP300 KIX domain in small-cell lung cancer EP300 (E1A binding protein p300) is a versatile transcription co-activator important in cell proliferation and differentiation. The gene EP300 is frequently mutated in diverse cancer types, including small-cell lung cancer (SCLC). While it is widely believed that these mutations result in loss of EP300 function, the impact on SCLC pathogenesis remains largely unknown. Here we demonstrate that mutant EP300 variants lacking histone acetyltransferase (HAT) domain accelerate tumor development in autochthonous mouse models of SCLC. However, unexpectedly, complete knockout of Ep300 suppresses tumor development and inhibits proliferation of both human and mouse SCLC cells. Genetic dissection of EP300 domains identifies kinase-inducible domain (KID)-interacting (KIX) domain, specifically its interaction with transcription factors such as CREB1 and MYB, as the determinant of pro-tumorigenic activity. Blockade of the KIX-mediated protein interactions using a small molecule and a recombinant peptide mimicking the KIX-binding sequences of EP300-interacting partners inhibits the growth of SCLC cells. These findings identify domain-specific roles of EP300 in SCLC and unique vulnerability of the EP300 KIX domain to potential therapeutics.

sentences: Purification of age-distinct insulin secretory granules through antigen restriction Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. The competence of secretory granules for exocytosis depends on spatio-temporal variables such as proximity to the plasma membrane and age, with newly-generated granules being preferentially released. Despite recent advances, we still lack a comprehensive view about the molecular composition of insulin granules and how this may change over the lifetime of the organelles. Here we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtained intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time along with the physical properties of the membrane.

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Phosphorylation of the selective autophagy receptor TAX1BP1 by canonical and noncanonical IκB kinases promotes its lysosomal localization and clearance of MAVS aggregates TAX1BP1 is a selective autophagy receptor which inhibits NF-{kappa}B and RIG-I-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as p62/SQSTM1 and OPTN are phosphorylated by the noncanonical I{kappa}B kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that the noncanonical I{kappa}B kinases TBK1 and IKKi phosphorylate TAX1BP1 to regulate its basal turnover, whereas the canonical I{kappa}B kinase IKK and the core autophagy factor ATG9 play essential roles in RNA virus-mediated TAX1BP1 autophagosomal degradation. TAX1BP1 phosphorylation by canonical and noncanonical I{kappa}B kinases promotes its localization to lysosomes resulting in its degradation. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 augments its MAVS aggrephagy function. Together, our data support a model whereby I{kappa}B kinases license TAX1BP1 selective autophagy function to inhibit MAVS and RLR signaling.

Author SummaryThe RIG-I-like receptor (RLR) pathway induces type I interferon (IFN) and proinflammatory cytokines in response to RNA virus infection. MAVS is a mitochondrial adaptor protein in the RLR pathway that forms prion-like aggregates upon activation; however, how MAVS aggregates are cleared to restore homeostasis is unclear. Autophagy is a lysosomal degradation pathway important for the clearance of potentially cytotoxic protein aggregates that could induce inflammation and/or cell death. TAX1BP1 is a selective autophagy receptor that inhibits RLR signaling, but the precise mechanisms remain unknown. Here, we found that TAX1BP1 is a substrate for multi-site phosphorylation by canonical and noncanonical I{kappa}B kinases which triggered its lysosomal localization and degradation. We also found that TAX1BP1 was critical for the clearance of MAVS aggregates in a phosphorylation-dependent manner. Overall, our data suggest that phosphorylation serves a key regulatory function for TAX1BP1 to inhibit RLR signaling.

sentences: Unsupervised clustering analysis reveals global population structure of SARS-CoV-2 Identifying the population structure of the newly emerged coronavirus SARS-CoV-2 has significant potential to inform public health management and diagnosis. As SARS-CoV-2 sequencing data accrued, grouping them into clusters is important for organizing the landscape of the population structure of the virus. Due to the limited prior information on the newly emerged coronavirus, we utilized four different clustering algorithms to group 16,873 SARS-CoV-2 strains, which automatically enables the identification of spatial structure for SARS-CoV-2. A total of six distinct genomic clusters were identified using mutation profiles as input features. Comparison of the clustering results reveals that the four algorithms produced highly consistent results, but the state-of-the-art unsupervised deep learning clustering algorithm performed best and produced the smallest intra-cluster pairwise genetic distances. The varied proportions of the six clusters within different continents revealed specific geographical distributions. In particular, our analysis found that Oceania was the only continent on which the strains were dispersively distributed into six clusters. In summary, this study provides a concrete framework for the use of clustering methods to study the global population structure of SARS-CoV-2. In addition, clustering methods can be used for future studies of variant population structures in specific regions of these fast-growing viruses.

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Cerebellum controls timing of periaqueductal grey encoding of fear memory and expression of fear conditioned behaviour The pivotal role of the periaqueductal grey (PAG) in fear learning is reinforced by the identification of neurons in rat ventral PAG (vPAG) that encode fear memory through signalling the onset and offset of an auditory conditioned stimulus during presentation of the unreinforced conditioned tone during retrieval. Some units only display CS+ onset or offset responses and the two signals differ in extinction sensitivity, suggesting they are independent of each other. In addition, understanding cerebellar contributions to survival circuits is advanced by the discovery that: (i) reversible inactivation of the medial cerebellar nucleus (MCN) during fear consolidation leads in subsequent retrieval to: (a) disruption of the temporal precision of vPAG offset, but not onset responses to the unreinforced conditioned tone, and (b) an increase in duration of freezing behaviour. And (ii) chemogenetic manipulation of the MCN-vPAG projection during fear acquisition: (a) reduces the occurrence of fear- related ultrasonic vocalisations and (b) during subsequent retrieval, slows the extinction rate of fear- related freezing. These findings show that the cerebellum is part of the survival network that regulates fear memory processes at multiple timescales and in multiple ways; raising the possibility that dysfunctional interactions in the cerebellar-survival network may underlie fear-related disorders and comorbidities.

Impact StatementCerebellar-periaqueductal grey interactions contribute to fear conditioned processes and, as such, provide a novel target for treating psychological conditions including post-traumatic stress disorder.

sentences: A within-host infection model to explore tolerance andresistance How are some individuals surviving infections while others die? The answer lies in how infected individuals invest into controlling pathogen proliferation and mitigating damage, two strategies respectively called resistance and disease tolerance. Pathogen within-host dynamics (WHD), influenced by resistance, and its connection to host survival, determined by tolerance, decide the infection outcome. To grasp these intricate effects of resistance and tolerance, we used a deterministic theoretical model where pathogens interact with the immune system of a host. The model describes the positive and negative regulation of the immune response, consider the way damage accumulate during the infection and predicts WHD. When chronic, infections stabilize at a Set-Point Pathogen Load (SPPL). Our model predicts that this situation can be transient, the SPPL being then a predictor of life span which depends on initial condition (e.g. inoculum). When stable, the SPPL is rather diagnostic of non lethal chronic infections. In lethal infections, hosts die at a Pathogen Load Upon Death (PLUD) which is almost independent from the initial conditions. As the SPPL, the PLUD is affected by both resistance and tolerance but we demonstrate that it can be used in conjunction with mortality measurement to distinguish the effect of disease tolerance from that of resistance. We validate empirically this new approach, using Drosophila, melanogaster and the pathogen Providencia rettgeri.

sentences: Genetic and thermal variation influences adaptation to fluctuating temperature in the seed beetle, Callosobruchus maculatus The impacts of climate change on biological systems are notoriously difficult to measure, and laboratory studies often do not realistically represent natural fluctuations in environmental conditions. To date, most experimental studies of thermal adaptation test populations at constant temperatures, or they make incremental changes to an otherwise constant mean background state. To address this, we examined the long-term effects of stressful fluctuating daily temperature on several key life history traits in two laboratory populations of the seed beetle, Callosobruchus maculatus. These populations were kept for 19 generations at either a constant control temperature, T=29{degrees}C, or at a fluctuating daily cycle with Tmean = 33{degrees}C, Tmax=40, and Tmin=26. The latter being a simple representation of daily temperatures in southern central India in May, where this species originally evolved. We found that beetles that had evolved in stressful environments were smaller in body size when switched to a constant 29{degrees}C and had far greater reproductive fitness in comparison to beetles from both the constant control and continuously stressful 33{degrees}C environments. This suggests that beetles raised in environments with stressful fluctuating temperatures were more phenotypically plastic and had greater genetic variability than control treatment beetles and indicates that populations that experience fluctuations in temperature may be better able to respond to short-term changes in environmental conditions.

sentences: Elevated HLA-E and NKG2A as a consequence of chronic immune activation defines resistance to M. bovis BCG immunotherapy in Non-Muscle-Invasive Bladder Cancer Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the first-line treatment for non-muscle invasive bladder cancer (NMIBC), promotes the production of inflammatory cytokines, particularly interferon (IFN)-{gamma}. Prolonged inflammation and IFN-{gamma} exposure are known to cause an adaptive immune response, enabling immune escape and proliferation by tumor cells. We investigated HLA-E and NKG2A, a novel T and NK cell checkpoint pathway, as a driver of adaptive resistance in BCG unresponsive NMIBC. We observed ubiquitous inflammation in all patients after BCG immunotherapy, regardless of recurrence status. IFN-{gamma} was shown to drive tumor expression of HLA-E and PD-L1. Further, NKG2A-expressing NK and CD8 T cells were enriched in BCG unresponsive tumors and with enhanced capacity for cytolytic functions. Strikingly, in situ spatial analyses revealed that HLA-EHIGH tumors are activated to recruit NK and T cells via chemokine production, potentially sparing HLA-ELOW tumors that would otherwise be susceptible to lysis. Finally, blood-derived NK cells retained anti-tumor functions at the time of tumor recurrence. These data support combined NKG2A and PD-L1 blockade for BCG unresponsive disease.

sentences: Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.

sentences: Dual effects of presynaptic membrane mimetics on α-synuclein amyloid aggregation Aggregation of intrinsically disordered -synuclein (SN) under various conditions is closely related to synucleinopathies. Although various biological membranes have shown to alter the structure and aggregation propensity of SN, a thorough understanding of the molecular and mechanical mechanism of amyloidogenesis in membranes remains unanswered. Herein, we examined the structural changes, binding properties, and amyloidogenicity of three variations of SN mutants under two types of liposomes, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and presynaptic vesicle mimetic (Mimic) membranes. While neutrally charged DOPC membranes elicited marginal changes in the structure and amyloid fibrillation of SNs, negatively charged Mimic membranes induced dramatic helical folding and biphasic amyloid generation. At low concentration of Mimic membranes, the amyloid fibrillation of SNs was promoted in a dose-dependent manner. However, further increases in the concentration constrained the fibrillation process. These results suggest the dual effect of Mimic membranes on regulating the amyloidogenesis of SN, which is rationalized by the amyloidogenic structure of SN and condensation-dilution of local SN concentration. Finally, we propose physicochemical properties of SN and membrane surfaces, and their propensity to drive electrostatic interactions as decisive factors of amyloidogenesis.

sentences: Reciprocity and interaction effectiveness in generalised mutualisms among free-living species Mutualistic interactions among free-living species generally involve weak links and highly asymmetric dependence among partners, yet our understanding of factors beyond their emergence is still limited. Using individual-based interactions of a super-generalist fleshy-fruited plant with its frugivore assemblage we estimate the Resource Provisioning Effectiveness (RPE) and Seed Dispersal Effectiveness (SDE) to assess the balance in the exchange of resources. Plants were highly dependent on a few super-generalist frugivore species, while these interacted with most individual plants, resulting in strong asymmetries in mutual dependence. Both RPE and SDE were mainly driven by interaction frequency. Despite highly asymmetric dependences, the strong reliance on quantity largely determined high reciprocity in rewards between partners (i.e., higher energy provided, more seedlings recruited), not obscured by minor variations in the quality of animal or plant service. We anticipate reciprocity will emerge in low-intimacy mutualisms where the mutualistic outcome largely relies upon interaction frequency.

sentences: A pleiotropic flowering time QTL exhibits gene-by-environmental interaction for fitness in a perennial grass Flowering time is crucial for wild plant populations to adapt to their local environments. Although the genetic basis of flowering variation has been studied in many plant species, its mechanisms in non-model organisms and its adaptive value in the field are still poorly understood. Here, we report new insights into the genetic basis of flowering time and its effect on fitness in Panicum hallii, a native perennial grass. We conducted genetic mapping in populations derived from representative inland and coastal ecotypes to identify flowering time QTL and loci exhibited extensive QTL-by-environment interactions. Patterns of segregation within recombinant hybrids provide strong support for directional selection driving ecotypic divergence in flowering time. A major QTL on chromosome 5 (q-FT5) was detected in all experiments and is a key locus controlling flowering variation. Fine-mapping and expression studies identified a FLOWERING LOCUS T orthologue, FT-like 9 (PhFTL9), as the candidate underlying q-FT5. We used reciprocal transplant experiment to test for global local adaptation and the specific impact of q-FT5 on performance. We did not observe local adaptation in terms of fitness tradeoffs when contrasting ecotypes in home versus away habitats. However, we observed that the coastal allele of q-FT5 conferred a fitness advantage only in its local habitat but not at the inland site. Sequence analysis of the PhFTL9 promoter identified ecotypic specific cis-element variation associated with environmental responsiveness. Together, our findings demonstrate the genetic basis of flowering variation in a perennial grass and provide evidence for conditional neutrality underlying flowering divergence.

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RNA-silencing induces target gene relocalization toward a specialized nuage domain The authors wish to withdraw this manuscript and apologize for errors in the initial submission. All the original experiments were performed by YY. Unfortunately, JP and members of the Mello lab have not been able to replicate some aspects of the study. JP has failed to independently reproduce the specific results showing RNAi-triggered relocalization of target RNA, and P granule specific accumulation of (the P granule component GLH-1) as reported. The conditions/strains analyzed by JP were as follows: (1) oma-1 FISH on WT worms [control, 6 hr and 12 hr oma-1(RNAi)]. (2) oma-1 FISH on OMA-1:GFP worms [control, 6 hr oma-1(RNAi), or 6 hr gfp(RNAi)]. (3) oma-1 FISH on WT worms [control, 4hr, 6hr, 8 hr, and 10 hr oma-1(RNAi)]. 10-23 gonads were analyzed per experiment. Fixation conditions were essentially as described, with the only known difference being that gonads were not exposed to detergent prior to fixation. Using YYs reagents and protocol the Mello lab has not observed an obvious relocalization of target RNA to P granules (marked by GFP::GLH-1) after 6 hrs oma-1(RNAi); n=92 gonads. CM, JP and DG consider that the published images accurately represent the image stacks provided by YY as representative, raw data, but JP and CM note configurations of FISH signals in germ nuclei and gonad anatomy that they consider unusual. CM, JP and DG have not detected any evidence of image manipulation.

YY states that none of the raw image data were manipulated beyond standard adjustments for brightness and contrast prior to processing images for publication as described. However, YY reports that the images were not representative of the majority of sample gonads, and instead were pre-selected under low magnification for rare examples with asymmetrical, expanded P granules.

Efforts to identify conditions that explain the rare gonads imaged by YY continue in the Mello lab, as do efforts to reproduce independently each of the other reported results; we plan to provide an update in the near future.

sentences: Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin Near-infrared fluorescent protein (iRFP) is a bright and stable fluorescent protein with excitation and emission maxima at 690 nm and 713 nm, respectively. Unlike the other conventional fluorescent proteins such as GFP, iRFP requires biliverdin (BV) as a chromophore because iRFP originates from bacteriophytochrome. Here, we report that phycocyanobilin (PCB) functions as a brighter chromophore for iRFP than BV, and biosynthesis of PCB allows live-cell imaging with iRFP in the fission yeast Schizosaccharomyces pombe. We initially found that fission yeast cells did not produce BV, and therefore did not show any iRFP fluorescence. The brightness of iRFP attached to PCB was higher than that of iRFP attached to BV in vitro and in fission yeast. We introduced SynPCB, a previously reported PCB biosynthesis system, into fission yeast, resulting in the brightest iRFP fluorescence. To make iRFP readily available in fission yeast, we developed an endogenous gene tagging system with iRFP and all-in-one integration plasmids, which contain genes required for the SynPCB system and the iRFP-fused marker proteins. These tools not only enable the easy use of iRFP in fission yeast and the multiplexed live-cell imaging in fission yeast with a broader color palette, but also open the door to new opportunities for near-infrared fluorescence imaging in a wider range of living organisms.

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Dynamic distortion of orientationrepresentation after learning in the mouseprimary visual cortex Learning is an essential cognitive mechanism allowing behavioral adaptation through adjustments in neuronal processing. It is associated with changes in the activity of sensory cortical neurons evoked by task-relevant stimuli. However, the exact nature of those modifications and the computational advantages they may confer are still debated. Here, we investigated how learning an orientation discrimination task alters the neuronal representations of the cues orientations in the primary visual cortex (V1) of male and female mice. When comparing the activity evoked by the task stimuli in naive mice and mice performing the task, we found that the representations of the orientation of the rewarded and non-rewarded cues were more accurate and stable in trained mice. This better cue representation in trained mice was associated with a distortion of the orientation representation space such that stimuli flanking the task-relevant orientations were represented as the task stimuli themselves, suggesting that those stimuli were generalized as the task cues. This distortion was context dependent as it was absent in trained mice passively viewing the task cues and enhanced in the behavioral sessions where mice performed best. Those modifications of the V1 population orientation representation in performing mice were supported by a suppression of the activity of neurons tuned for orientations neighboring the orientations of the task cues. Thus, visual processing in V1 is dynamically adapted to enhance the reliability of the representation of the learned cues and favor generalization in the task-relevant computational space.

Significance statementPerformance improvement in a task often requires facilitating the extraction of the information necessary to its execution. Here, we demonstrate the existence of a suppression mechanism that improves the representation of the orientations of the task stimuli in the V1 of mice performing an orientation discrimination task. We also show that this mechanism distorts the V1 orientation representation space, leading stimuli flanking the task stimuli orientations to be generalized as the task stimuli themselves.

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InvL, an invasin-like adhesin, is a type II secretion system substrate required for Acinetobacter baumannii uropathogenesis. Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Herein, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the {beta}-barrel domain associated with T5SSs, but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis.

IMPORTANCEWhile pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Herein, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Though InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacters urinary tract virulence, and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.

sentences: The neuropeptide receptor NMUR-1 regulates the specificity of C. elegans innate immunity against pathogen infection A key question in current immunology is how the innate immune system generates high levels of specificity. Using the Caenorhabditis elegans model system, we demonstrate that functional loss of NMUR-1, a neuronal G protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U, has diverse effects on C. elegans innate immunity against various bacterial pathogens. Transcriptomic analyses and functional assays revealed that NMUR-1 modulates C. elegans transcription activity by regulating the expression of transcription factors involved in binding to RNA polymerase II regulatory regions, which, in turn, controls the expression of distinct immune genes in response to different pathogens. These results uncovered a molecular basis for the specificity of C. elegans innate immunity. Given the evolutionary conservation of NMUR-1 signaling in immune regulation across multicellular organisms, our study could provide mechanistic insights into understanding the specificity of innate immunity in other animals, including mammals.

sentences: A Computational Dissection of Spike protein of SARS-CoV-2 Omicron Variant The emergence of SARS-CoV-2 omicron variant in late November, 2021 and its rapid spread to different countries, warns the health authorities to take initiative to work on containing its spread. The omicron SARS-CoV-2 variant is unusual from the other variants of concerns reported earlier as it harbors many novel mutations in its genome particularly with >30 mutations in the spike glycoprotein alone. The current study investigated the variation in binding mechanism which it carries compared to the wild type. The study also explored the interaction profile of spike-omicron with human ACE2 receptor. The structure of omicron spike glycoprotein was determined though homology modeling. The interaction analysis was performed through docking using HADDOCK followed by binding affinity calculation. Finally, the comparison of interactions were performed among spike-ACE2 complex of wild type, delta and omicron variants. The interaction analysis has revealed the involvement of highly charged and polar residues (H505, Arg498, Ser446, Arg493, and Tyr501) in the interactions. The important novel interactions in the spike-ACE2-omicron complex was observed as S494:H34, S496:D38, R498:Y41, Y501:K353, and H505:R393 and R493:D38. Moreover, the binding affinity of spike-ACE2-omicron complex (-17.6Kcal/mol) is much higher than wild type-ACE2 (-13.2Kcal/mol) and delta-ACE2 complex (-13.3Kcal/mol). These results indicate that the involvement of polar and charged residues in the interactions with ACE2 may have an impact on increased transmissibility of omicron variant.

sentences: An integral genomic signature approach for tailored cancer targeted therapy using genome-wide sequencing data With the advent of low-cost sequencing, transcriptome and genome sequencing is expected to become clinical routine and transform precision oncology within next decade. However, viable genome-wide modeling methods that can facilitate rational selection of patients for tailored intervention while tolerating sequencing biases are far lacking. Here we propose an integral genomic signature (iGenSig) analysis as a new class of transparent, interpretable, and resilient methods for precision oncology based on multiple types of genome-wide sequencing data. We postulate that the redundant high-dimensional genomic features, which are typically eliminated during multi-omics modeling, may help overcome the sequencing biases. We thus conceive a novel method that models the therapeutic response using the high-dimensional transcriptional and mutational features predictive of tumor response, which we termed as an integral genomic signature (iGenSig), and then algorithmically resolve the feature redundancy tailored for each patient subject. Using genomic dataset of chemical perturbations, we developed the iGenSig models for predicting targeted therapy responses, and applied selected models to independent datasets for cancer cell lines, patient-derived xenografts, and patient subjects. iGenSig models exhibit outstanding cross-dataset performance compared to artificial intelligence methods, with exceptional resilience against simulated errors in genomic features. In particular, the iGenSig model for the EGFR inhibitor Erlotinib significantly predicted the responses of patient-derived xenografts and patients from a clinical trial, biological interpretation of which led to new insights into the predictive signature pathways with clinical relevance. Together, iGenSig will provide a computational infrastructure to empower tailored cancer intervention based on genome-wide sequencing data.

sentences: Functional maps of the primate cortex revealed by through-skull wide-field optical imaging The primate cerebral cortex is organized into specialized areas representing different functional modalities (e.g., vision, audition, touch) and their associations along a continuous surface. The functional maps of these areas, however, are often investigated in a single modality at a time. Here, we developed and applied to awake primates a polarization-enhanced wide-field optical imaging method for measuring cortical hemodynamics through the intact skull. Adjacent somatosensory, auditory, and visual cortices were noninvasively localized and rapidly parcellated in awake marmosets (Callithrix jacchus), a primate model featuring a smooth cortex. Detailed somatotopy, tonotopy, and retinotopy were also mapped out on an individual-subject basis, with a new pure-tone-responsive tonotopic gradient discovered outside the auditory core. Moreover, the motion-sensitive extent surrounding the primate-specific MT/V5 and the location of a face-sensitive patch were both revealed with respect to retinotopy. This approach provides a powerful tool for mapping the functional landscape across modalities in a single non-human primate subject, and thus opens new opportunities for probing how primate cortical system is organized to enable real-world behaviors.

sentences: Integrating transcriptomes to investigate genes associated with adaptation to aquatic environments, and assess phylogenetic conflict and whole-genome duplications in Alismatales Land plants first evolved from freshwater algae, and flowering plants returned to water as early as the Cretaceous and multiple times subsequently. Alismatales is the largest clade of aquatic angiosperms including all marine angiosperms, as well as terrestrial plants. We used Alismatales to explore plant adaptation to aquatic environments by analyzing a data set that included 95 samples (89 Alismatales species) covering four genomes and 91 transcriptomes (59 generated in this study). To provide a basis for investigating adaptation, we assessed phylogenetic conflict and whole-genome duplication (WGD) events in Alismatales. We recovered a relationship for the three main clades in Alismatales as (Tofieldiaceae, Araceae) + core Alismatids. We also found phylogenetic conflict among the backbone of the three main clades that could be explained by incomplete lineage sorting and introgression. Overall, we identified 18 putative WGD events across Alismatales. One of them occurred at the most recent common ancestor of core Alismatids, and three occurred at seagrass lineages. We also found that lineage and life-form were both important for different evolutionary patterns for the genes related to freshwater and marine adaptation. For example, several light or ethylene-related genes were lost in the seagrass Zosteraceae, but are present in other seagrasses and freshwater species. Stomata-related genes were lost in both submersed freshwater species and seagrasses. Nicotianamine synthase genes, which are important in iron intake, expanded in both submersed freshwater species and seagrasses. Our results advance the understanding of the adaptation to aquatic environments and whole-genome duplications using phylogenomics.

sentences: Human macrophage polarization determines bacterial persistence of Staphylococcus aureus in a liver-on-chip-based infection model Infections with Staphylococcus aureus (S. aureus) have been reported from various organs ranging from asymptomatic colonization to severe infections and sepsis associated with multiple organ dysfunction. Although considered an extracellular pathogen, S. aureus can invade and persist in professional phagocytes such as monocytes and macrophages. Its capability to persist and manipulate phagocytes is considered a critical step to evade host antimicrobial reactions. For the first time we leveraged a human liver-on-chip model and tailored image analysis algorithms to demonstrate that S. aureus (USA300) specifically targets macrophages in the liver models as essential niche facilitating bacterial persistence and phenotype switching to small colony variants (SCVs). In vitro M2 polarization was found to favor SCV-formation and was associated with increased intracellular bacterial loads in macrophages, increased cell death, and impaired recruitment of circulating monocytes to sites of infection. These findings expand the knowledge about the role of liver macrophages in the course of systemic infection. Further, the results might be relevant for understanding infection mechanisms in patients with chronic liver disease such as fibrosis that display increased frequencies of M2 polarized liver macrophages and have a higher risk for developing chronic infections and relapsing bacteremia.

sentences: Computationally scalable regression modeling for ultrahigh-dimensional omics data with ParProx Statistical analysis of ultrahigh-dimensional omics scale data has long depended on univariate hypothesis testing. With growing data features and samples, the obvious next step is to establish multivariable association analysis as a routine method for understanding genotype-phenotype associations. Here we present ParProx, a state-of-the-art implementation to optimize overlapping group lasso regression models for time-to-event and classification analysis, guided by biological priors through coordinated variable selection. ParProx not only enables model fitting for ultrahigh-dimensional data within the architecture for parallel or distributed computing, but also allows users to obtain interpretable regression models consistent with known biological relationships among the independent variables, a feature long neglected in statistical modeling of omics data. We demonstrate ParProx using three different omics data sets of moderate to large numbers of variables, where we use genomic regions and pathways to arrive at sparse regression models comprised of biologically related independent variables. ParProx is naturally applicable to a wide range of studies using ultrahigh-dimensional omics data, ranging from genome-wide association analysis to single cell sequencing studies where multivariable modeling is computationally intractable.

sentences: The epigenetic landscape in purified myonuclei from fast and slow muscles Muscle cells have different phenotypes adapted to different usage and can be grossly divided into fast/glycolytic and slow/oxidative types. While most muscles contain a mixture of such fiber types, we aimed at providing a genome-wide analysis of chromatin environment by ChIP-Seq in two muscle extremes, the almost completely fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus muscles. Muscle is a heterogeneous tissue where less than 60% of the nuclei are inside muscle fibers. Since cellular homogeneity is critical in epigenome-wide association studies we devised a new method for purifying skeletal muscle nuclei from whole tissue based on the nuclear envelope protein Pericentriolar material 1 (PCM1) being a specific marker for myonuclei. Using antibody labeling and a magnetic-assisted sorting approach we were able to sort out myonuclei with 95% purity. The sorting eliminated influence from other cell types in the tissue and improved the myo-specific signal. A genome-wide comparison of the epigenetic landscape in EDL and soleus reflected the functional properties of the two muscles each with a distinct regulatory program involving distal enhancers, including a glycolytic super-enhancer in the EDL. The two muscles are also regulated by different sets of transcription factors; e.g. in soleus binding sites for MEF2C, NFATC2 and PPARA were enriched, while in EDL MYOD1 and SOX1 binding sites were found to be overrepresented. In addition, novel factors for muscle regulation such as MAF, ZFX and ZBTB14 were identified.

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BIN2 phosphorylates the Thr280 of CO to restrict its function in promoting Arabidopsis flowering CONSTANS (CO) is a central regulator of floral initiation in response to photoperiod. In this study, we show that the GSK3 kinase BIN2 physically interacts with CO and the gain-of-function mutant bin2-1 displays late flowering phenotype through down-regulation of FT transcription. Genetic analyses show that BIN2 genetically acts upstream of CO in regulating flowering time. Further, we illustrate that BIN2 phosphorylates the Thr280 residue of CO. Importantly, the BIN2 phosphorylation of Thr280 residue restricts the function of CO in promoting flowering. Moreover, we reveal that the N-terminal part of CO harboring the B-Box domain mediates the interaction of both CO-CO and BIN2-CO. We find that BIN2 inhibits the formation of CO dimer/oligomer. Taken together, this study reveals that BIN2 regulates flowering time through phosphorylating the Thr280 of CO and inhibiting the CO-CO interaction in Arabidopsis.

HighlightBIN2 regulates flowering time through phosphorylating the Thr280 of CO in Arabidopsis.

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The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferons by engaging cytosolic DNA sensing in macrophages Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics, demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA surveillance pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this we found that a population of ~12% of R. equi phagosomes recruited the galectin-3, -8 and -9 danger receptors. Interesting, neither phagosomal damage nor induction of type I IFN required the R. equis virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulated ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

IMPORTANCERhodococcus equi is a facultative intracellular bacterial pathogen of horses and other domestic animals, as well as an opportunistic pathogen of immunocompromised and rarely immunocompetent humans. In human patients, Rhodococcus pneumonia bears some pathological similarities to pulmonary tuberculosis, and poses a risk for misdiagnosis. In horses, R. equi infection has a major detrimental impact on the equine breeding industry due to a lack of an efficacious vaccine and its ubiquitous distribution in soil. Given the prevalence of subclinical infection and high false positive rate in current screening methods, there exists a critical need to identify factors contributing to positive patient outcomes. Our research identifies innate immune sensing events and immune transcriptional signatures that may lead to biomarkers for clinical disease, more accurate screening methods, and insight into susceptibility to infection.

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Identification of the Rigid Core for Aged Liquid Droplets of the TDP-43 Low Complexity Domain The biomolecular condensation of proteins with low complexity sequences plays a functional role in RNA metabolism and a pathogenic role in neurodegenerative diseases. The formation of dynamic liquid droplets brings biomolecules together to achieve complex cellular functions. The rigidification of liquid droplets into {beta}-strand-rich hydrogel structures composed of protein fibrils is thought to be purely pathological in nature. However, low complexity sequences often harbor multiple fibril-prone regions with delicately balanced functional and pathological interactions. Here, we investigate the maturation of liquid droplets formed by the low complexity domain of the TAR DNA-binding protein 43 (TDP-43). Solid state nuclear magnetic resonance measurements on the aged liquid droplets identify a structured core region distinct from the region thought to be most important for pathological fibril formation and aggregation. The results of this study show that multiple segments of this low complexity domain are prone to form fibrils, and that stabilization of {beta}-strand-rich structure in one segment precludes the other region from adopting rigid fibril structure.

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sentences: Fractions of traditionally brewed rice beverage ameliorate anxiety and improve spatial memory in mice Rice beverages are traditionally prepared and consumed popularly by the different ethnic groups of North East India. To investigate its effects on behavior, mice were treated with different fractions of rice beverage that included the beverage as a whole, insoluble and soluble fractions. Intragastric treatments of these fractions were given to the mice (n=6 per group) for 30 days and behavioral studies were performed on elevated plus and Y maze to evaluate anxiety and spatial memory, respectively. Next generation sequencing of metagenomic DNA of the beverage indicated presence of 157 OTUs and 26 bacterial genera were dominant with an abundance of 0.1%. The insoluble fraction treated animals showed lowest anxiety like symptoms. Spatial memory improved in all the treatments compared to the control, of which the rice beverage treatment showed the highest levels (p<0.05). Gas chromatography and mass spectroscopy-based metabolite profiling of the beverage revealed 10 alcohols, 29 sachharides, 43 acids and 13 amino acids. Findings of this study suggest a positive effect of rice beverage components on anxiety and spatial memory of mice.

sentences: Snowball Earths, population bottlenecks, and the evolution of marine photosynthetic bacteria Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean. A massive DNA loss event occurred in their early evolutionary history, leading to highly reduced genomes in nearly all lineages, as well as enhanced efficiency in both nutrient uptake and light absorption. The environmental landscape that shaped this ancient genome reduction, however, remained unknown. Through careful molecular clock analyses, we established that this Prochlorococcus genome reduction occurred during the Neoproterozoic Snowball Earth climate catastrophe. The lethally low temperature and exceedingly dim light during the Snowball Earth event would have inhibited Prochlorococcus growth and proliferation and caused severe population bottlenecks. These bottlenecks are recorded as an excess of deleterious mutations that accumulated across genomic regions in the descendant lineages. Prochlorococcus adaptation to extreme environmental conditions during Snowball Earth intervals can be inferred by tracing the evolutionary paths of genes that encode key metabolic potential. This metabolic potential includes modified lipopolysaccharide structure, strengthened peptidoglycan biosynthesis, the replacement of a sophisticated circadian clock with an hourglass-like mechanism that resets daily for dim light adaption, and the adoption of ammonia diffusion as an efficient membrane transporter-independent mode of nitrogen acquisition. In this way, the Neoproterozoic Snowball Earth event altered the physiological characters of Prochlorococcus, shaping their ecologically vital role as the most abundant primary producers in the modern oceans.

sentences: Inducible Yeast Two-Hybrid with Quantitative Measures The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.

sentences: Sequence determinants of the aggregation of proteins within condensates generated by liquid-liquid phase separation The transition between the native and amyloid states of proteins can proceed via a deposition pathway via oligomeric intermediates or via a condensation pathway via liquid droplet intermediates generated through liquid-liquid phase separation. Here we investigate the sequence determinants of aggregation from within the droplet state based on generic interactions. We describe a model in which these sequence determinants can be captured by three features, the droplet-promoting propensity, the aggregation-promoting propensity and the binding mode entropy. By using this approach, we propose a formula to identify aggregation-promoting mutations in droplet-forming proteins. This analysis provides insights into the amino acid code for the conversion of proteins between liquid-like and solid-like condensates.

sentences: Synaptosome microRNAs regulate synapse functions in Alzheimer's disease MicroRNAs (miRNAs) are found in nerve terminals, synaptic vesicles, and synaptosomes, but it is unclear whether synaptic and cytosolic miRNA populations differ in Alzheimers disease (AD) or if synaptosomal miRNAs affect AD synapse activity. To address these questions, we generated synaptosomes and cytosolic fractions from postmortem brains of AD and unaffected control (UC) samples and analyzed them using a global Affymetrix miRNAs microarray platform. A group of miRNAs significantly differed (p<0.0001) with high fold changes variance (+/- >200-fold) in their expressions in different comparisons- 1) UC synaptosome vs UC cytosol, 2) AD synaptosomes vs AD cytosol, 3) AD cytosol vs UC cytosol, and 4) AD synaptosomes vs UC synaptosomes. MiRNAs data analysis revealed that some potential miRNAs were consistently different across sample groups. These differentially expressed miRNAs were further validated using AD postmortem brains, brains of APP transgenic (Tg2576), Tau transgenic (P301L), and wild type mice. The miR-501-3p, miR-502-3p and miR-877-5p were identified as potential synaptosomal miRNAs upregulated with disease progression based on AD Braak stages. Gene Ontology Enrichment and Ingenuity Pathway Analysis of synaptosomal miRNAs showed the involvement of miRNAs in nervous system development, cell junction organization, synapse assembly formation, and function of GABAergic synapse. This is the first description of synaptic versus cytosolic miRNAs in AD and their significance in synapse function.

sentences: Single cell quantification of ribosome occupancy in early mouse development Technological limitations precluded transcriptome-wide analyses of translation at single cell resolution. To solve this challenge, we developed a novel microfluidic isotachophoresis approach, named RIBOsome profiling via IsoTachoPhoresis (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key regulatory mechanism of genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high coverage measurements enabled the first analysis of allele-specific ribosome engagement in early development and led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes. Finally, by integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle stage oocytes is the predominant determinant of protein abundance in the zygote. Taken together, these findings resolve the long-standing paradox of low correlation between RNA expression and protein abundance in early embryonic development. The novel Ribo-ITP approach will enable numerous applications by providing high coverage and high resolution ribosome occupancy measurements from ultra-low input samples including single cells.

sentences: Over-expression of wild-type ACVR1 in fibrodysplasia ossificans progressiva mice rescues perinatal lethality and inhibits heterotopic ossification Fibrodysplasia ossificans progressiva (FOP) is a devastating disease of progressive heterotopic bone formation for which effective treatments are currently unavailable. FOP is caused by dominant gain-of-function mutations in the receptor ACVR1 (also known as ALK2), which render the receptor inappropriately responsive to activin ligands. In previous studies, we developed a genetic mouse model of FOP that recapitulates most clinical aspects of the disease. In this model, genetic loss of the wild-type Acvr1 allele profoundly exacerbated heterotopic ossification, suggesting the hypothesis that the stoichiometry of wild-type and mutant receptors dictates disease severity. Here, we tested this model by producing FOP mice that conditionally over-express human wild-type ACVR1. Injury-induced heterotopic ossification (HO) was completely blocked in FOP mice when expression of both the mutant and wild-type receptor were targeted to Tie2-positive cells, which includes fibro/adipogenic progenitors (FAPs). Perinatal lethality of Acvr1R206H/+ mice was rescued by constitutive ACVR1 over-expression and these mice survived to adulthood at predicted Mendelian frequencies. Constitutive over-expression of ACVR1 also provided protection from spontaneous HO, and the incidence and severity of injury-induced HO in these mice was dramatically reduced. Analysis of pSMAD1/5/8 signaling both in cultured cells and in vivo indicates that ACVR1 over-expression functions cell-autonomously by reducing osteogenic signaling in response to activin A. Manipulating the stoichiometry of FOP-causing and wild-type ACVR1 receptors may provide the foundation for novel therapeutic strategies to treat this devastating disease.

sentences: Sub-diffraction error mapping for localization microscopy images Assessing the quality of localization microscopy images is highly challenging due to difficulty in reliably detecting errors in experimental data, with artificial sharpening being a particularly common failure mode of the technique. Here we use Haar wavelet kernel analysis (HAWK), a localization microscopy data analysis method which is known to give results without artificial sharpening, to generate a reference image. This enables the mapping and quantification of this common artefact. By suppressing intensity information, we are able to map sharpening errors in a way which is not influenced by nonlinearity in the localisation imaging process. The HAWK Method for the Assessment of Nanoscopy (HAWKMAN) is a general approach which allows the reliability of localization information to be assessed.

sentences: Dynamic and stationary brain connectivity during movie watching as revealed by functional MRI Spatially remote brain regions show synchronized activity as typically revealed by correlated functional MRI (fMRI) signals. An emerging line of research has focused on the temporal fluctuations of connectivity; however, its relationships with stationary connectivity have not been clearly illustrated. We examined dynamic and stationary connectivity when the participants watched four different movie clips. We calculated point-by-point multiplication between two regional time series to estimate the time-resolved dynamic connectivity, and estimated the inter-individual consistency of the dynamic connectivity time series. Widespread consistent dynamic connectivity was observed for each movie clip, which also showed differences between the clips. For example, a cartoon movie clip, Wall-E, showed more consistent of dynamic connectivity with the posterior cingulate cortex and supramarginal gyrus, while a court drama clip, A Few Good Men, showed more consistent of dynamic connectivity with the auditory cortex and temporoparietal junction, which might suggest the involvement of specific brain processing for different movie contents. In contrast, the stationary connectivity as measured by the correlations between regional time series was highly similar among the movie clips, and showed fewer statistically significant differences. The patterns of consistent dynamic connectivity could be used to classify different movie clips with higher accuracy than the stationary connectivity and regional activity. These results support the functional significance of dynamic connectivity in reflecting functional brain changes, which could provide more functionally relevant information than stationary connectivity.

sentences: CSNet: Estimating cell-type-specific gene co-expression networks from bulk gene expression data Inferring and characterizing gene co-expression networks has led to important insights on the molecular mechanisms of complex diseases. Most co-expression analyses to date have been performed on gene expression data collected from bulk tissues with different cell type compositions across samples. As a result, the co-expression estimates only offer an aggregate view of the underlying gene regulations and can be confounded by heterogeneity in cell type compositions, failing to reveal gene coordination that may be distinct across different cell types. In this paper, we describe a flexible framework for estimating cell-type-specific gene co-expression networks from bulk sample data, without making specific assumptions on the distributions of gene expression profiles in different cell types. We develop a novel sparse least squares estimator, referred to as CSNet, that is efficient to implement and has good theoretical properties. Using CSNet, we analyzed the bulk gene expression data from a cohort study on Alzheimers disease and identified previously unknown cell-type-specific co-expressions among Alzheimers disease risk genes, suggesting cell-type-specific disease pathology for Alzheimers disease.

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Chronic effects of short-term aerobic and anaerobic physical training on the ventral prostate of adult rats AimsTo analyze the chronic effects of short-term aerobic and anaerobic physical training on prostate compartments, extracellular matrix, microvascularization, TGF{beta}, cyclooxygenase-2, inducible nitric oxide synthase (iNOS) and macrophage immunostaining, and ventral prostate histopathology in adult rats.

Main methodsFifteen male rats (90 days old) were divided into three groups (n = 5/group): sedentary, aerobic (AE) (swimming), and anaerobic physical training (AN) (jumping), performed three days per week, for 8-weeks. The animals were sacrificed within 21 weeks of age. The ventral prostate was collected, weighed, and processed for histological and immunohistochemical analyses.

Key findingsOur results show that AE increases blood capillaries and reduces the percentage and increases the thickness of smooth muscle. AN promotes increased iNOS levels in the prostatic tissue, and both modalities reduce TGF{beta} and elastic fibers, in addition to being protective against benign prostatic hyperplasia and atrophy.

SignificanceDifferent physical training modalities can activate specific mechanisms that modify the prostate environment.

sentences: Experimental and computational analysis of calcium dynamics in 22q11.2 deletion model astrocytes Intracellular calcium dynamics in spontaneously active cells such as neurons or astrocytes is an information-rich readout of the physiological state of the cell. Methods for deriving mechanistic information from biological time courses, as well as for algorithmically extracting cellular activity time courses from imaging data, have significantly advanced in recent years but been mostly applied to neuronal data. At the same time, the role for astrocytes, a type of glial brain cells, in cognition and psychiatric diseases remains poorly understood. Using calcium imaging, computer vision, and Bayesian kinetic inference, we analyze calcium dynamics in primary astrocytes derived from control or Df1/+ mice, a model of 22q11.2 deletion syndrome (DiGeorge syndrome). Inference of highest-likelihood molecular kinetic characteristics from the intracellular calcium time courses pinpoints a significant change in the activity of the sarcoendoplasmic reticulum calcium ATPase (SERCA). Applying a SERCA inhibitor to the control cells reproduces the differences detected in the deletion-bearing cells. Our work identifies for the first time the molecular changes driving the calcium kinetics in 22q11.2 deletion model astrocytes. We conclude that Bayesian kinetic inference is a useful tool for mechanistic dissection of a complex cellular phenotype, calcium dynamics, in glial cells. This method has the potential to facilitate formulation of specific hypotheses concerning the underlying molecular mechanisms, prioritization of experiments testing such hypotheses, and, in the future, individualized functional molecular diagnostics.

sentences: A microRNA Signature of Metastatic Colorectal Cancer Although microRNAs (miRNA) are involved in all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood and contradictory results have been published. The aim of this work was to identify miRNAs associated with metastatic progression of colorectal cancer (CRC). Novel and previously published next generation sequencing (NGS) datasets generated from 268 samples with primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues were analyzed. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, utilizing miRNA-tailored quality control and processing, and applying a physiologically meaningful cut-off value (100 reads per million). The results were adjusted for host tissue background expression and samples from the different metastatic sites were independently analyzed. A metastatic signature containing five miRNAs up-regulated at multiple metastatic sites was identified (Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p (mir-141-3p), Mir-1307_5p, and Mir-155_5p) along with a number of miRNAs that were differentially expressed at individual metastatic sites. Several of these have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The identified differentially expressed miRNAs confirm known associations and contribute novel insights into miRNA involvement in the metastatic process. The use of open science practices facilitates reproducibility, and new datasets may easily be added to the publicly available pipeline to continuously improve the knowledge in the field. The identified set of miRNAs provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.

sentences: Pango lineage designation and assignment using SARS-CoV-2 spike gene nucleotide sequences More than 2 million SARS-CoV-2 genome sequences have been generated and shared since the start of the COVID-19 pandemic and constitute a vital information source that informs outbreak control, disease surveillance, and public health policy. The Pango dynamic nomenclature is a popular system for classifying and naming genetically-distinct lineages of SARS-CoV-2, including variants of concern, and is based on the analysis of complete or near-complete virus genomes. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. It is therefore important to understand how much information about Pango lineage status is contained in spike-only nucleotide sequences. Here we explore how Pango lineages might be reliably designated and assigned to spike-only nucleotide sequences. We survey the genetic diversity of such sequences, and investigate the information they contain about Pango lineage status. Although many lineages, including the main variants of concern, can be identified clearly using spike-only sequences, some spike-only sequences are shared among tens or hundreds of Pango lineages. To facilitate the classification of SARS-CoV-2 lineages using subgenomic sequences we introduce the notion of designating such sequences to a "lineage set", which represents the range of Pango lineages that are consistent with the observed mutations in a given spike sequence. These data provide a foundation for the development of software tools that can assign newly-generated spike nucleotide sequences to Pango lineage sets.

sentences: Active Inference as a Framework for Brain-Computer Interfaces As Karl Friston explained during the International Symposium on Artificial Intelligence and Brain Science 2020, active inference provides a way of using abstract rule-learning and approximate Bayesian inference to show how minimizing (expected) free energy leads to active sampling of novel contingencies. Friston elaborated how there were ways of making an optimal decision using active inference that can offer perspectives to advances in artificial intelligence. These methods of optimization within the context of active inference can also be used as a framework for improving brain-computer interfaces (BCI). This way, BCIs can give rise to artificial curiosity in the way Friston had described during his session. Using Fristons free energy principle, we can optimize the criterion a BCI uses to infer the intentions of the user from EEG observations. Under Fristons criteria for making an optimal decision, BCIs can expand their framework of optimal decision-making using active inference.

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Loss of muscarinic M5 receptor function manifests disparate impairments in exploratory behavior in male and female mice despite common dopamine regulation There are five cloned muscarinic acetylcholine receptors (M1-M5). Of these, the muscarinic type 5 receptor (M5) is the only one localized to dopamine neurons in the ventral tegmental area and substantia nigra. Unlike M1-M4, the M5 receptor has relatively restricted expression in the brain, making it an attractive therapeutic target. Here we performed an in-depth characterization of M5-dependent potentiation of dopamine transmission in the nucleus accumbens and accompanying exploratory behaviors in male and female mice. We show that M5 receptors potentiate dopamine transmission by acting directly on the terminals within the nucleus accumbens. Using the agonist oxotremorine, we revealed a unique concentration response curve and a sensitivity to repeated stressor exposure. We found that constitutive deletion of M5 receptors reduced exploration of the center of an open field while at the same time impairing normal habituation only in male mice. In addition, M5 deletion reduced exploration of salient stimuli, especially under conditions of high novelty, yet had no effect on hedonia. We conclude that M5 receptors are critical for both engaging with the environment and updating behavioral output in responses to the environment cues, specifically in male mice. A cardinal feature of mood and anxiety disorders is a withdrawal from the environment. These data indicate that boosting M5 receptor activity may be a useful therapeutic target for ameliorating these symptoms of depression and anxiety.

Significance StatementThe basic physiological and behavioral functions of the muscarinic M5 receptor remain understudied. Furthermore, its presence on dopamine neurons, relatively restricted expression in the brain, and recent crystallization make it an attractive target for therapeutic development. Yet, most preclinical studies of M5 receptor function have primarily focused on substance use disorders in male rodents. Here we characterized the role of M5 receptors in potentiating dopamine transmission in the nucleus accumbens, finding impaired functioning after stress exposure. Furthermore, we show that M5 receptors can modulate exploratory behavior in a sex-specific manner, without impacting hedonic behavior. These findings further illustrate the therapeutic potential of the M5 receptor, warranting further research in the context of treating mood disorders.

sentences: A global timing mechanism regulates cell-type specific wiring programs The assembly of neural circuits is dependent upon precise spatiotemporal expression of cell recognition molecules1-6. Factors controlling cell-type specificity have been identified7-9, but how timing is determined remains unknown. Here we describe the induction of a cascade of transcription factors by a steroid hormone (Ecdysone) in all fly visual system neurons spanning target recognition and synaptogenesis. We demonstrate through single cell sequencing that the Ecdysone pathway regulates the expression of a common set of targets required for synaptic maturation and cell-type specific targets enriched for cell surface proteins regulating wiring specificity. Transcription factors in the cascade regulate the expression of the same wiring genes in complex ways, including activation in one cell-type and repression in another. We show that disruption of the Ecdysone-pathway generates specific defects in dendritic and axonal processes and synaptic connectivity, with the order of transcription factor expression correlating with sequential steps in wiring. We also identify shared targets of a cell-type specific transcription factor and the Ecdysone pathway which regulate specificity. We propose neurons integrate a global temporal transcriptional module with cell-type specific transcription factors to generate different cell-type specific patterns of cell recognition molecules regulating wiring.

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Contribution of cytoplasm viscoelastic properties to mitotic spindle positioning Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original position. These forces are independent of cytoskeletal force generators, yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

Significance StatementThe regulation of mitotic spindle positioning is a key process for tissue architecture, embryo development and stem cells. To date, most models have assumed that spindles are positioned by forces exerted by polar cytoskeleton networks, like microtubule asters or acto-myosin bundles. Here, using in situ magnetic tweezers to apply calibrated forces and torques to mitotic spindles in live dividing sea urchin cells, we found that the viscoelastic properties of the cytoplasm medium in which spindles are embedded can hold spindles in place, and move them back if their original position is perturbed. These viscoelastic forces are large and may significantly participate in the force balance that position and orient mitotic spindles in many cell types.

sentences: Tissue fixation effects on human retinal lipid analysis by MALDI imaging and LC-MS/MS technologies Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge cross-links between formaldehyde and amine/ thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artefactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared to fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.

sentences: A novel 5' DNA binding site in RFC facilitates PCNA loading for gap DNA repair RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {gamma}. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures revealing a novel 2nd DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located in the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. It appears 5' DNA binds after 3' DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. We propose that the 5' site facilitates RFCs PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5' DNA site in longer gaps including during lagging strand DNA synthesis.

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Gut regulatory T cells mediate immunological tolerance in Salmonella Typhimurium-infected superspreader hosts by suppressing cytotoxic activity of T cells. Superspreader hosts carry out most pathogen transmission events and are often disease tolerant since they remain asymptomatic despite high pathogen burdens. Here we describe the superspreader immune state that allows for disease tolerance. In a model of Salmonella infection, superspreader mice develop colitis with robust CD4+ and CD8+ T-cell responses, however, they remain asymptomatic. We found that superspreaders have significantly more regulatory T cells (Tregs) in the distal gut compared to non-superspreader infected hosts. Surprisingly, the depletion of Tregs did not induce pathogen clearance but rather exacerbated weight loss, increased gut inflammation, and compromised epithelial intestinal barrier. This loss of tolerance correlated with dramatic increases in cytotoxic CD4+ and CD8+ T cells. Interestingly, CD4 neutralization in Tregs-depleted superspreaders was sufficient to rescue tolerance. Our results indicate that Tregs play a crucial role in maintaining immunologic tolerance in the guts of superspreader mice by suppressing cytotoxic CD4+ and CD8+ T-cell activities.

AUTHOR SUMMARYSuperspreader hosts are the main cause of disease transmission and a very important public health concern. Here, we evaluated the immunological tolerance of the Salmonella infected superspreaders in a mouse model. By manipulating Tregs, we demonstrated the immunological mechanism from the host to maintain health status and high pathogen burden. Tregs depletion in the superspreaders led to severe disease, with damage of the intestinal epithelia, and high morbidity without having any effect on shedding and systemic Salmonella burden. Furthermore, we demonstrated that the damage of the intestinal epithelia was related to cytotoxic activity of T cells. When Tregs were depleted, CD8+ T cells produced high levels of granzyme B and perforin. CD8+ T cells neutralization in Tregs depleted mice led to increased cytotoxic CD4+ T cells. Interestingly, neutralization of CD4+ T cells in the Tregs depleted mice led to a reduction in the CD8+ T cells producing granzyme B and it was sufficient to rescue host tolerance in this model. We demonstrate for the first time that cytotoxic CD4+ T cells damage the epithelial intestinal barrier and contribute to loss of tolerance in the context of a superspreader host. These findings open new perspectives to understand mechanisms of tolerance in the intestine of a superspreader host.

sentences: Intra- and inter-specific density dependence of body condition, growth, and habitat temperature in chub mackerel (Scomber japonicus) The density dependence of growth and body condition have important impacts on fish population dynamics and fisheries management. Although population density is known to affect the temperature of the habitat selected, how this affects the density dependence of growth and body condition remains unclear. Here, we investigated annual changes in body condition, habitat temperature, and cohort-specific growth of chub mackerel (Scomber japonicus) in the western North Pacific and examined quarterly changes in the density dependence of body condition. We hypothesized that chub mackerel body condition is affected both directly (e.g., through competition for food) and indirectly (through changes in habitat temperature) by the abundance of both conspecifics (i.e., chub mackerel) and heterospecifics (the Japanese sardine Sardinops melanostictus). Indeed, chub mackerel body condition, habitat temperature, and growth all decreased with increasing conspecific and heterospecific abundance. Mean annual growth rates in chub mackerel were positively corelated with body condition. The best model showed that conspecific and/or heterospecific abundance had strong negative effects on chub mackerel body condition in all seasons, and influenced habitat temperature in some seasons. By contrast, temperature effects on body condition were weak. Therefore, direct effects likely have more impact than indirect effects on density-dependent body condition and growth.

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Multiple levels of triggered factors and positive effect of cell-to-cell bottleneck in mutation repair of RNA viruses Debilitating mutation in RNA viruses can be repaired via different mechanisms, while triggering factors of mutation repair were poorly understood. In this study, multiple levels of triggering factors of mutation repair was identified based on genetic damage of tRNA-like structure (TLS) in cucumber mosaic virus (CMV). TLS mutation in different RNAs of CMV distinctively impacted the pathogenicity and mutation repair. Relative quantity defect of RNA2 or quality defect of RNA3 resulting from TLS mutation was correlated with high rate of mutation repair, and TLS mutation of RNA1 failed to be repaired. However, TLS mutation of RNA1 can be repaired in the mixed inoculation with RNA2 having pre-termination mutation of 2b or at the low dose of original inoculation, especially around dilution end-point. Taken together, TLS mutation resulting into quality or quantity defect of viral genome or TLS mutation at low dose around dilution end-point was inclined to be repaired. In addition, different levels of mutation repair of TLS necessarily required the cell-to-cell movement, which implied the positive effect of cell-to-cell bottleneck on evolution of low-fitness virus, a phenomenon opposite to the Muller ratchet. This study provided important revelations on virus evolution and application of viral mild vaccine.

Author summaryDue to the low-fidelity of replicase, debilitating RNA viruses can be repaired through different mechanisms, which implied the resilience of RNA viruses. In this study, we identified multiple levels of triggered factors and occurrence occasion of mutation repair using the divided genome of CMV, which contained conserved cis-element tRNA-like structure (TLS) at the 3end. TLS mutation of different RNA in CMV presented different rate of mutation repair from 0-80%. TLS mutation resulting into genomic quality or quantity defect or at low dose around dilution end-point was inclined to be repaired. However, all above types of mutation repair necessarily required cell-to-cell movement, which presented the positive effect of cell-to-cell bottleneck on virus evolution and increased fitness of low-fitness RNA viruses. It is an opposite phenomenon to the Muller ratchet, in which bottleneck always decreased the fitness of viruses. Except to identify the triggering factors of mutation repair in RNA viruses, this study also provided important revelations on creation and application of mild vaccine based on RNA viruses.

sentences: Prevalence of Bourbon and Heartland viruses in field collected ticks at an environmental field station in St. Louis County, Missouri, USA Heartland and Bourbon viruses are pathogenic tick-borne viruses putatively transmitted by Amblyomma americanum, an abundant tick species in Missouri. To assess the prevalence of these viruses in ticks, we collected 2778 ticks from 8 sampling sites at Tyson Research Center, an environmental field station within St. Louis County and close to the City of St. Louis, from May - July in 2019 and 2021. Ticks were pooled according to life stage and sex, grouped by year and sampling site to create 355 pools and screened by RT-qPCR for Bourbon and Heartland viruses. Overall, 14 (3.9%) and 27 (7.6%) of the pools were positive for Bourbon virus and Heartland virus respectively. In 2019, 11 and 23 pools were positive for Bourbon and Heartland viruses respectively. These positives pools were of males, females and nymphs. In 2021, there were 4 virus positive pools out of which 3 were positive for both viruses and were comprised of females and nymphs. Five out of the 8 sampling sites were positive for at least one virus. This included a site that was positive for both viruses in both years. Detection of these viruses in an area close to a relatively large metropolis presents a greater public health threat than previously thought.

sentences: A Comparison of Macaque Hair Hormone Concentration Following Enhanced Cognitive Experiences or Standard Nonhuman Primate Environmental Enrichment Experience with enriched environments positively impacts the health and wellbeing of nonhuman animals ranging from rodents to primates. Little is known, however, about the specific effects of enhanced cognitive enrichment (ECE) on nonhuman primates. The study reported here used archival samples to provide preliminary analysis of ECE on hormones associated with stress and wellbeing, as well as evaluation of persistent effects of infant social rearing. Hair samples from 24 adult male rhesus macaques were analyzed via LC-MS/MS technique for the main stress response hormones: cortisol, cortisone, and dehydroepiandrosterone. The ECE and care-as-usual (CAU) groups did not significantly differ in any of the mean analyte values. Cortisol and dehydroepiandrosterone were strongly and positively correlated in the ECE group. By contrast, for the CAU group the two analytes were weakly correlated. Within the ECE group, cortisol and dehydroepiandrosterone were significantly and positively correlated in the mother-reared group, but not in the nursery-reared group. Overall, these results provide preliminary evidence that experience with ECE could help to maintain a balanced cortisol:dehydroepiandrosterone ratio, possibly indicative of a healthy stress response. Further examination of this ratio in hair is needed to support this hypothesis. These observations may also suggest that nursery rearing could have persistent effects, including dysregulation of the hypothalamic-pituitary-adrenal axis, apparent in the unbalanced cortisol:dehydroepiandrosterone ratio. Together these findings are consistent with the growing literature that supports the use of ECE to promote nonhuman primate wellbeing and healthy development.

sentences: Duplication of NRAMP3 gene in poplars generated two homologous transporters with distinct functions Transition metals are essential for a wealth of metabolic reactions, but their concentrations need to be tightly controlled across cells and cell compartments, as metal excess or imbalance has deleterious effects. Metal homeostasis is achieved by a combination of metal transport across membranes and metal binding to a variety of molecules. Gene duplication is a key process in evolution, as emergence of advantageous mutations on one of the copies can confer a new function. Here, we report that the poplar genome contains two paralogues encoding NRAMP3 metal transporters localized in tandem. All Populus species analyzed had two copies of NRAMP3, whereas only one could be identified in Salix species indicating that duplication occurred when the two genera separated. Both copies are under purifying selection and encode functional transporters, as shown by expression in the yeast heterologous expression system. However, genetic complementation revealed that only one of the paralogues has retained the original function in release of metals stored in the vacuole previously characterized in A. thaliana. Confocal imaging showed that the other copy has acquired a distinct localization to the Trans Golgi Network (TGN). Expression in poplar suggested that the copy of NRAMP3 localized on the TGN has a novel function in the control of cell-to-cell transport of manganese. This work provides a clear case of neo-functionalization through change in the subcellular localization of a metal transporter as well as evidence for the involvement of the secretory pathway in cell-to-cell transport of manganese.

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

sentences: Microglia-secreted TNF-α affects differentiation efficiency and viability of pluripotent stem cell-derived human dopaminergic precursors Parkinsons Disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic cells of the substantia nigra pars compacta. Even though successful transplantation of dopamine-producing cells into the striatum exhibits favourable effects in animal models and clinical trials; transplanted cell survival is low. Since every transplant elicits an inflammatory response which can affect cell survival and differentiation, we aimed to study in vivo and in vitro the impact of the pro-inflammatory environment on human dopaminergic precursors. We first observed that transplanted human dopaminergic precursors into the striatum of immunosuppressed rats elicited an early and sustained activation of astroglial and microglial cells after 15 days post-transplant. This long-lasting response was associated with Tumor necrosis factor alpha expression in microglial cells. In vitro conditioned media from activated BV2 microglial cells increased cell death, decreased Tyrosine hydroxylase -positive cells and induced morphological alterations on human neural stem cells-derived dopaminergic precursors at two differentiation stages: 19 days and 28 days. Those effects were ameliorated by inhibition of Tumor necrosis factor alpha, a cytokine which was previously detected in vivo and in conditioned media from activated BV-2 cells. Our results suggest that a pro-inflammatory environment is sustained after transplantation under immunosuppression, providing a window of opportunity to modify this response to increase transplant survival and differentiation. In addition, our data show that the microglia-derived pro-inflammatory microenvironment has a negative impact on survival and differentiation of dopaminergic precursors. Finally, Tumor necrosis factor alpha plays a key role in these effects, suggesting that this cytokine could be an interesting target to increase the efficacy of human dopaminergic precursors transplantation in Parkinsons Disease.

sentences: A behavioral paradigm for cortical control of a robotic arm by freely moving rats in a one-dimensional two-target reaching task Controlling the trajectory of a neuroprosthetic device to reach multiple targets is a commonly used brain-machine interface (BMI) task in primates and has not been available for rodents yet. Here, we describe a novel experimental paradigm which enables this task for rats in one-dimensional space for reaching two distant targets depending on their limited cognitive and visual capabilities compared to primates. An online transform was used to convert the activity of a pair of primary motor cortex (M1) units into two robotic actions. The rats were shaped to adapt to the transform and direct the robotic actuator toward the selected target by modulating the activity of the M1 neurons. All three rats involved in the study were capable of achieving randomly selected targets with at least 78% accuracy. A total of 9 out of 16 pairs of units examined were eligible for exceeding this success criterion. Two out of three rats were capable of reversal learning, where the mapping between the activity of the unit pairs and the robotic actions were reversed. The present work is the first demonstration of trajectory-based control of a neuroprosthetic device by rodents to reach two distant targets using visual feedback. The paradigm introduced here may be used as a cost-effective platform for elucidating the information processing principles in the neural circuits related to neuroprosthetic control and for studying the performance of novel BMI technologies using freely moving rats.

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Differential impact of transdiagnostic, dimensional psychopathology on multiple scales of functional connectivity IntroductionDimensional psychopathology strives to associate different domains of cognitive dysfunction with brain circuitry. Connectivity patterns as measured by functional magnetic resonance imaging (fMRI) exist at multiple scales, with global networks of connectivity composed of microscale interactions between individual nodes. It remains unclear how separate dimensions of psychopathology might differentially impact these different scales of organization.

MethodsPatients experiencing anxious misery symptomology (depression, anxiety and trauma; n = 192) were assessed for symptomology and received resting-state fMRI scans. Three modeling approaches (seed-based correlation analysis [SCA], support vector regression [SVR] and Brain Basis Set Modeling [BSS]), each relying on increasingly dense representations of functional connectivity patterns, were used to associate connectivity patterns with six different dimensions of psychopathology: anxiety sensitivity, anxious arousal, rumination, anhedonia, insomnia and negative affect. Importantly, a full 50 patients were held-out in a testing dataset, leaving 142 patients as training data.

ResultsDifferent symptom dimensions were best modeled by different scales of brain connectivity: anhedonia and anxiety sensitivity were best modeled with single connections (SCA), insomnia and anxious arousal by mesoscale patterns (SVR) and negative affect and ruminative thought by broad, cortex-spanning patterns (BBS). Dysfunction within the default mode network was implicated in all symptom dimensions that were best modeled by multivariate models.

ConclusionThese results suggest that symptom dimensions differ in the degree to which they impact different scales of brain organization. In addition to advancing our basic understanding of transdiagnostic psychopathology, this has implications for the translation of basic research paradigms to human disorders.

sentences: Early postnatal activation of the hypoxia pathway disrupts β-cell function Hypoxic insults in the perinatal period can lead to persistent hyperinsulinism and profound hypoglycemia in newborns. We studied the impact of the hypoxia-inducible factor 1A (HIF1A) pathway on postnatal {beta}-cell function. Rat pups were treated daily between postnatal day (P)7 to P10 with adaptaquin (AQ), an inhibitor of prolyl hydroxylases, which stabilizes HIF1A. AQ-treated pups were hypoglycemic and had higher plasma insulin concentrations. Their islets had a decreased glucose threshold for insulin secretion, indicative of a delay in {beta}-cell postnatal functional maturation. Histology analyses revealed that AQ-treated pups had increased pancreatic insulin-positive area but no changes in the number of islets or number of {beta}-cells per islet, suggesting larger average {beta}-cell size. AQ-treated rat pups had decreased expression of cell cycle genes and decreased numbers of proliferating {beta}-cells. In conclusion, pharmacologic activation of the HIF1A pathway in the early postnatal period leads to hyperinsulinism, due to the persistence of a low glucose threshold for insulin secretion, and to decreased early postnatal {beta}-cell proliferation, suggesting it can impact adult {beta}-cell mass and diabetes risk.

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Neural Basis of Acoustic Species Recognition in a Cryptic Species Complex Sexual traits that promote species recognition are important drivers of reproductive isolation, especially among closely related species. Identifying neural processes that shape species differences in recognition is crucial for understanding the causal mechanisms of reproductive isolation. Temporal patterns are salient features of sexual signals that are widely used in species recognition by several taxa, including anurans. Recent advances in our understanding of temporal processing by the anuran auditory system provide an excellent opportunity to investigate the neural basis of species-specific recognition. The anuran inferior colliculus (IC) consists of neurons that are selective for temporal features of calls. Of potential relevance are auditory neurons known as interval-counting neurons (ICNs) that are often selective for the pulse rate of conspecific advertisement calls. Here, we took advantage of a species differences in temporal selectivity for pulsatile advertisement calls exhibited by two cryptic species of gray treefrog (Hyla chrysoscelis and Hyla versicolor) to test the hypothesis that ICNs mediate acoustic species recognition. We tested this hypothesis by examining the extent to which the threshold number of pulses required to elicit behavioral responses from females and neural responses from ICNs was similar within each species but potentially different between the two species. In support of our hypothesis, we found that a species difference in behavioral pulse number thresholds corresponded closely to a parallel species difference in neural pulse number thresholds. However, this relationship held only for ICNs that exhibited band-pass tuning for conspecific pulse rates. Together, these findings suggest that differences in temporal processing of a subset of ICNs provide a mechanistic explanation for reproductive isolation between two cryptic and syntopically breeding treefrog species.

Summary StatementTemporal processing by a subset of midbrain auditory neurons plays key roles in decoding information about species identity in anurans.

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Intratumoral Heterogeneity Promotes Collective Cancer Invasion Through NOTCH1 Variation Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a multidisciplinary approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

Summary statementThis study reveals a mechanism that Notch1 variation, instead of the average value, promotes the invasiveness of microtumor, providing a link between intratumoral heterogeneity and collective cancer invasion.

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The genomic basis of host and vector specificity in non-pathogenic trypanosomatids The ability of trypanosome parasites to survive and sustain infections is dependent on diverse and intricate immune evasion mechanisms. Pathogenic trypanosomes often have broad host niches that preclude identification of host specific adaptations. In contrast, some non-pathogenic species of the genus Trypanosoma have highly specific hosts and vectors. Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host, distinct from the dominant variant surface glycoprotein coat of pathogenic African trypanosomes. In both species, their surface proteins are encoded by genes which account for [~]10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these closely related species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genome nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. The genome of T. melophagium contains core genes required for development, glycolysis, RNA interference, and meiotic exchange, each being shared with T. theileri. Comparisons between T. melophagium and T. theileri provide insight into the specific adaptations of these related trypanosomatids to their distinct mammalian hosts and arthropod vectors.

Author summaryNon-pathogenic trypanosomes can have narrow host niches, with closely related trypanosome species expanding into distinct mammalian host and insect vectors. T. theileri, a non-pathogenic trypanosome of bovines, is predicted to have an intricate cell surface which allows it to evade the immune response of its mammalian host. In contrast, T. melophagium is closely related to T. theileri but infects sheep and is transmitted by the sheep ked rather than tabanid flies that transmit T. theileri. Here, we sequence and assemble the T. melophagium genome to identify the genomic basis of host and vector specificity in these non-pathogenic trypanosomes. We confirm the two species are closely related, however, T. melophagium has a smaller genome than T. theileri. Most of the discrepancy in genome size is due to an expansion of putative cell surface genes in T. theileri. The differential investment in cell surface proteins could be due to a focus on adaptation to the mammalian host in T. theileri and the insect host in T. melophagium.

Data summaryThe genomes, transcriptomes and proteomes used in this study were accessed from the TriTrypDB repository or NCBI. T. theileri genome sequencing data was downloaded from NCBI SRA (SRR13482812). T. melophagium data generated during this study is available from the NCBI BioProject PRJNA786535.

RepositoriesT. melophagium DNA and RNA sequencing data, along with the draft genome assembly and its annotation, can be found under the NCBI BioProject PRJNA786535.

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Smaller Hippocampal Volume among Black and Latinx Youth Living in High-Stigma Contexts BackgroundTo determine whether being raised in a stigmatizing context influences neurodevelopment in children.

MethodsWe drew data from one of the first national, multi-site neuroimaging studies with substantial variability in sociopolitical contexts: the Adolescent Brain and Cognitive Development study (N=11,534; M=9.9 years). We measured structural stigma surrounding gender, race, and Latinx ethnicity using objective state-level indicators of social policies and prejudicial attitudes and individual-level experiences of perceived discrimination, and examined associations with two neural markers: hippocampal volume and amygdala reactivity to threat.

ResultsIn a pre-registered analysis, we find that Black (B=-57.27, p=0.025) and Latinx (B=-41.02, p=0.037) youths raised in higher-stigma environments have smaller hippocampal volume than youths raised in low-stigma contexts, controlling for demographics and family socioeconomic status. This association was marginal among girls (p=0.082). The magnitude of the observed difference in hippocampal volume between high and low structural stigma contexts was equivalent to a $20,000 difference in annual family income. Stigmatizing environments were unrelated to hippocampal volume in non-stigmatized youth, providing evidence of specificity. Perceived discrimination was unrelated to hippocampal volume among any of the stigmatized groups. No associations between individual or structural forms of stigma and amygdala reactivity to threat were observed in the stigmatized groups.

ConclusionsWe provide novel evidence that an objective measure of stigma at the contextual level (structural stigma) may have a stronger influence on neurodevelopment than subjective perceptions of stigma measured at the individual level, suggesting that contextual approaches may yield new insights into neurodevelopment.

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Environment symmetry drives a multidirectional code in rat retrosplenial cortex A class of neurons showing bidirectional tuning in a two-compartment environment was recently discovered in dysgranular retrosplenial cortex (dRSC). We investigated here whether these neurons possess a more general environmental symmetry-encoding property, potentially useful in representing complex spatial structure. We report that directional tuning of dRSC neurons reflected environment symmetry in onefold, twofold and fourfold-symmetric environments: this was the case not just globally, but also locally within each sub-compartment. Thus, these cells use environmental cues to organize multiple directional tuning curves, which perhaps sometimes combine via interaction with classic head direction cells. A consequence is that both local and global environmental symmetry are simultaneously encoded even within local sub-compartments, which may be important for cognitive mapping of the space beyond immediate perceptual reach.

One Sentence SummaryRetrosplenial directionally tuned neurons encode global environment symmetry even in local sub-compartments.

sentences: Brain Laterality Dynamics Support Human Cognition Hemispheric lateralization constitutes a core architectural principle of human brain organization underlying cognition, often argued to represent a stable, trait-like feature. However, emerging evidence underlines the inherently dynamic nature of brain networks, in which time-resolved alterations in functional lateralization remain uncharted. Integrating dynamic network approaches with the concept of hemispheric laterality, we map the spatiotemporal architecture of whole-brain lateralization in a large sample of high-quality resting-state fMRI data (N=991, Human Connectome Project). We reveal distinct laterality dynamics across lower-order sensorimotor systems and higher-order associative networks. Specifically, we expose two aspects of the laterality dynamics: laterality fluctuations, defined as the standard deviation of laterality time series, and laterality reversal, referring to the number of zero-crossings in laterality time series. These two measures are associated with moderate and extreme changes in laterality over time, respectively. While laterality fluctuations depict positive association with language function and cognitive flexibility, laterality reversal shows a negative association with the same neurocognitive factors. These opposing interactions indicate a dynamic balance between intra- and inter-hemispheric communication, i.e., segregation and integration of information across hemispheres. Furthermore, in their time-resolved laterality index, the default-mode and language networks correlate negatively with visual/sensorimotor and attention networks, indicating flexible while parallel processing capabilities that are linked to better out-of-scanner cognitive performance. Finally, the laterality dynamics correlate with regional metabolism and structural connectivity and showed significant heritability. Our results provide insights into the adaptive nature of the lateralized brain and new perspectives for future studies of human cognition, genetics and brain disorders.

sentences: Drop, Swap, and Generate: A Self-Supervised Approach for Generating Neural Activity Meaningful and simplified representations of neural activity can yield insights into how and what information is being processed within a neural circuit. However, without labels, finding representations that reveal the link between the brain and behavior can be challenging. Here, we introduce a novel unsupervised approach for learning disentangled representations of neural activity called Swap-VAE. Our approach combines a generative modeling framework with an instance-specific alignment loss that tries to maximize the representational similarity between transformed views of the input (brain state). These transformed (or augmented) views are created by dropping out neurons and jittering samples in time, which intuitively should lead the network to a representation that maintains both temporal consistency and invariance to the specific neurons used to represent the neural state. Through evaluations on both synthetic data and neural recordings from hundreds of neurons in different primate brains, we show that it is possible to build representations that disentangle neural datasets along relevant latent dimensions linked tso behavior.

sentences: Reduced visitation to the buzz-pollinated Cyanella hyacinthoides in the presence of other pollen sources in the hyperdiverse Cape Floristic Region Many plant species have floral morphologies that restrict access to floral resources, such as pollen or nectar, and only a subset of floral visitors can perform the complex handling behaviours required to extract restricted resources. Due to the time and energy required to extract resources from morphologically complex flowers, these plant species potentially compete for pollinators with co-flowering plants that have more easily accessible resources. A widespread floral mechanism restricting access to pollen is the presence of tubular anthers that open through small pores or slits (poricidal anthers). Some bees have evolved the capacity to remove pollen from poricidal anthers using vibrations, giving rise to the phenomenon of buzz-pollination. These bee vibrations that are produced for pollen extraction are presumably energetically costly, and to date, few studies have investigated whether buzz-pollinated flowers may be at a disadvantage when competing for pollinators attention with plant species that present unrestricted pollen resources. Here, we studied Cyanella hyacinthoides (Tecophilaeaceae), a geophyte with poricidal anthers in the hyperdiverse Cape Floristic Region of South Africa, to assess how the composition and relative abundance of flowers with easily accessible pollen affect bee visitation to a buzz-pollinated plant. We found that the number of pollinator species was not influenced by community composition. However, visitation rates to C. hyacinthoides were negatively related to the abundance of flowers with more accessible resources. Visitation rates were strongly associated with petal colour, showing that flower colour is important in mediating these interactions. We conclude that buzz-pollinated plants might be at a competitive disadvantage when many easily accessible pollen sources are available, particularly when competitor species share its floral signals.

sentences: Evaluation of Antibiotic Tolerance in Pseudomonas aeruginosa for Aminoglycosides and its Prediction of Resistance Development Through In-silico Transcriptomic Analysis Pseudomonas aeruginosa causes chronic infections like cystic fibrosis, endocarditis, bacteremia and sepsis, which are life-threatening and difficult to treat. The lack of antibiotic response in P. aeruginosa is due to adaptive resistance mechanism, which prevents the entry of antibiotics into cytosol of the cell to achieve tolerance. Among different groups of antibiotics, aminoglycosides are used as a parental antibiotic for treatment of P. aeruginosa. This study aims to determine the kinetics of antibiotic tolerance and gene expression changes in P. aeruginosa exposed to amikacin, gentamicin, and tobramycin. These antibiotics were exposed to P. aeruginosa at their MICs and the experimental setup was monitored till 72 hours, followed by the measurement of optical density in the interval of every 12 hours. The growth of P. aeruginosa in MICs of antibiotics represents the kinetics of antibiotic tolerance in amikacin, gentamicin, and tobramycin. Transcriptomic profile of antibiotic exposed P. aeruginosa PA14 was taken from Gene Expression Omnibus (GEO), NCBI as microarray datasets. The gene expressions of two datasets were compared by test versus control. Tobramycin exposed P. aeruginosa failed to develop tolerance in MICs 0.5{micro}g/mL, 1{micro}g/mL and 1.5{micro}g/mL. Whereas amikacin and gentamicin treated P. aeruginosa developed tolerance in MICs. This depicts the superior in vitro response of tobramycin over the gentamicin and amikacin. Further, in silico transcriptomic analysis of tobramycin treated P. aeruginosa resulted in low expression of 16s rRNA Methyltransferase E, B & L, alginate biosynthesis genes and several proteins of Type 2 Secretory System (T2SS) and Type 3 Secretory System (T3SS). The Differentially Expressed Genes (DEGs) of alginate biosynthesis, and RNA Methyltransferases suggests increased antibiotic response and low probability of developing resistance. The use of tobramycin as a parental antibiotic with its synergistic combination might combat P. aeruginosa with increased response.

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Topology and contribution to the pore channel lining of plasma membrane-embedded S. flexneri type 3 secretion translocase IpaB Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled crosslinking by membrane permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on post translational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection.

ImportanceType 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the proteins IpaB and IpaC. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by S. flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.

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

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

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

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

sentences: Coordination of genome replication and anaphase entry by rDNA copy number in S. cerevisiae Eukaryotes maintain hundreds of copies of ribosomal DNA (rDNA), many more than required for ribosome biogenesis, suggesting a yet undefined role for large rDNA arrays outside of ribosomal RNA synthesis. We demonstrate that reducing the Saccharomyces cerevisiae rDNA array to 35 copies, which is sufficient for ribosome function, shifts rDNA from being the latest replicating region in the genome to one of the earliest. This change in replication timing results in delayed genome-wide replication and classic replication defects. We present evidence that the requirement for rDNA to replicate late, which is conserved among eukaryotes, also coordinates the completion of genome replication with anaphase entry through the proper sequestration of the mitotic exit regulator Cdc14p in the rDNA-containing nucleolus. Our findings suggest that, instead of being a passive repetitive element, the large late-replicating rDNA array plays an active role in genome replication and cell cycle control.

sentences: The chromosome-scale genome assembly of Jasminum sambac var. unifoliatum provide insights into the formation of floral fragrance The high-quality genome assembly of the JSU-FSP provides valuable information on how jasmine produces the abundant VTs and VPBs that synergistically contribute to the pleasant aroma of jasmine flower.

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Screening of antimicrobial activity of aqueous and ethanolic extracts of some medicinal plants from Cameroon and assessment of their synergy with common antibiotics against multidrug-resistant uropathogenic bacteria. Background and aimThe Cameroonian flora abounds in plants with multidimensional therapeutic virtues which can play an important role in the fight against resistance to antibiotics and the search for new antimicrobials. This study aimed to screen the antimicrobial potential of aqueous and ethanolic extracts of thirteen (13) samples (Bark, leaf, seed) of eight (8) plants from Cameroon against 3 reference pathogens and to evaluate their synergy with conventional antibiotics against eleven (11) multiresistant uropathogenic (MRU) bacteria.

MethodBioactive compounds were extracted from leaves of Leucanthemum vulgare, Cymbopogon citratus (DC.) Stapf, Moringa oleifera Lam and Vernonia amygdalina Delile; barks of Cinchona officinalis and Enantia chlorantha Oliv; barks and seeds of Garcinia lucida Vesque and leaves and seeds of Azadirachta indica (Neem) using distilled water and ethanol as solvents. The extracts were tested against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538 and Candida albicans 10231 using the well diffusion method and the microdilution method. The synergistic effect was assessed (using disc diffusion method and the checkerboard method) against MRU bacteria namely Achromobacter xylosoxidans 4892, Citrobacter freundii 426, Enterococcus avium 1669, Escherichia coli 1449, Klebsiella oxytoca 3003, Kocuria rizophilia. 1542, Moraxella catarrhalis 4222, Morganella morganii 1543, Pseudomonas aeruginosa 3057, Staphylococcus aureus 1449 and Streptococcus agalactiae 3984.

ResultsWe found that distilled water extracted a more important mass of phytochemical compounds (7.9-21.2%) compared to ethanol (5.8-12.4%). Except C. officinalis and G. lucida leaves, the rest of extracts were active with inhibition diameters (ID) ranging from 5 to 36 mm. Both ethanolic (EE) and aqueous extract (AE) of E. chloranta bark (ECB) were the most active against all pathogens with the mean ID of 17 and 36 mm vs S. aureus ATCC 6538, 23 and 14 mm vs E. coli ATCC 25922 and 36 and 19 mm vs C. albicans ATCC 10231. Only the EE of E. chloranta bark (ECB) had a strong activity against all the microorganisms tested (MIC <2 mg / ml); L. vulgare leaves (LVL) and G. lucida seed (GLS) had moderate (average MIC of 8 mg/ml) while all other extracts showed very weak antimicrobial activity. In addition, the fractional inhibitory concentration (FIC) ranged from 0.125 to 0.750. No antagonism (FIC> 4) or indifference (1[≤] FIC[≤]4) was noted between the extracts and the antibiotics, but the best synergies were found with ECB which well-modulated Kanamycin (FIC = 0.125 against S. aureus and 0.250 against E. coli), nitrofurantoin (FIC = 0.250 against S. aureus and 0.188 against E. coli) and ampicillin (FIC = 0.125 against E. coli). Similarly, compared to other extracts, ECB, LVL and GLS also well-modulated ampicillin, ceftazidime, tetracycline, nitrofurantoin, and trimethoprim against all the above-mentioned resistant uropathogenic bacteria with important increase in fold area (IFA).

ConclusionThis study show that E. chlorantha bark, L. vulgare leaves G. lucida seed, have good antimicrobial activity against both bacteria (Gram positive and Gram negative) and fungi (C. albicans); and should be more investigated for their possible use to the fight against MDR and MRU microorganisms.

sentences: The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.

sentences: Constraints and Opportunities for Detecting Land Surface Phenology in Drylands Land surface phenology (LSP) enables global scale tracking of ecosystem processes, but its utility is limited in drylands due to low vegetation cover and resulting low annual amplitudes of vegetation indices (VIs). Due to the importance of drylands for biodiversity, food security, and the carbon cycle it is necessary to understand limitations in measuring dryland dynamics. Here, using simulated data and multi-temporal unmanned aerial vehicle (UAV) imagery of a desert shrubland, we explore the feasibility of detecting LSP with respect to fractional vegetation cover, plant functional types, VI uncertainty, and two different detection algorithms. Using simulated data we found that plants with distinct VI signals, such as deciduous shrubs, can require up to 60% fractional cover to consistently detect LSP. Evergreen plants, with lower seasonal VI amplitude, require considerably higher cover and can have undetectable phenology even with 100% vegetation cover. Our evaluation of two algorithms showed that neither performed the best in all cases. Even with adequate cover, biases in phenological metrics can still exceed 20 days, and can never be 100% accurate due to VI uncertainty from shadows, sensor view angle, and atmospheric interference. We showed how high-resolution UAV imagery enables LSP studies in drylands, and highlighted important scale effects driven by within canopy VI variation. With high-resolution imagery the open canopies of drylands are beneficial as they allow for straightforward identification of individual plants, enabling the tracking of phenology at the individual level. Drylands thus have the potential to become an exemplary environment for future LSP research.

sentences: Using herbarium samples for NGS methods - a methodological comparison Herbaria harbor a tremendous amount of plant specimens that are rarely used for plant systematic studies. The main reason is the difficulty to extract a decent quantity of good quality DNA from the preserved plant material. While the extraction of ancient DNA in animals is well established, studies including old plant material are still underrepresented. In our study we compared the standard Qiagen DNeasy Plant Mini Kit and a specific PTB-DTT protocol on two different plant genera (Xanthium L. and Salix L.). The included herbarium material covered about two centuries of plant collections. A selected subset of samples was used for a standard library preparation as well as a target enrichment approach. The results revealed that PTB-PTT resulted in higher quantity and quality regarding DNA yield. For relatively recent herbarium specimens, and despite the lower overall yield of DNA, the Qiagen Kit resulted in better sequencing results regarding the number of filtered and mapped reads. We were able to successfully sequence a sample from 1820 and conclude that it is possible to include old herbarium specimens in NGS approaches. This opens a treasure box for phylogenomic research.

sentences: Brace root phenotypes predict root lodging susceptibility and the contribution to anchorage in maize Plant mechanical failure (lodging) causes global yield losses of 7-66% in cereal crops. We have previously shown that the above-ground nodal roots (brace roots) in maize are critical for anchorage. However, it is unknown how brace root phenotypes vary across genotypes and the functional consequence of this variation. This study quantifies the contribution of brace roots to anchorage, brace root traits, plant height, and root lodging susceptibility in 52 maize inbred lines. We show that the contribution of brace roots to anchorage and root lodging susceptibility varies among genotypes and this contribution can be explained by plant architectural variation. Additionally, supervised machine learning models were developed and show that multiple plant architectural phenotypes can predict the contribution of brace roots to anchorage and root lodging susceptibility. Together these data define the plant architectures that are important in lodging resistance and show that the contribution of brace roots to anchorage is a good proxy for root lodging susceptibility.

sentences: Moderate-intensity exercise might be a competitive protocol to prevent obesity and obesity-related metabolic diseases To determine effects of exercise intensity on expression levels of cytokines and neurotransmitters beneficial for the prevention and improvement of obesity and obesity-related metabolic diseases. Expression levels of irisin, fibroblast growth factor-21 (FGF21), adiponectin, free fatty acid (FFA), dopamine (DA), and serotonin (5-HT) levels after moderate-intensity (50% of maximal oxygen uptake (VO2max)) and high-intensity treadmill running (80% VO2max) for 30 min in 20 healthy men were compared. Blood samples were collected at three time points: before treadmill running (pre-EX), immediately after treadmill running (post-EX), and at 60 min after treadmill running (60 min post-EX). Expression levels of irisin, FGF21, adiponectin, FFA, DA, and 5-HT were increased after 30 min of treadmill running exercise regardless of exercise intensity. Their levels were increased at 60 min post-EX. They showed no significant difference immediately after exercise regardless of exercise intensity. Only irisin, FGF21, FFA, and DA levels showed significant differences between moderate-intensity (50% VO2max) group and high-intensity group (80% VO2max) at 60 min post-EX. Considering that FFA level at 80% VO2max was significantly lower than that at 50% VO2max during post-EX and side effects of high-intensity exercise, moderate-intensity exercise would be a more competitive method compared to high-intensity for the prevention and improvement of obesity and obesity-related metabolic diseases.

sentences: Characterizing the conformational free-energy landscape of RNA using single-molecule field-effect transistors We have developed and used high-time-resolution, single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are some of the most common RNA structural motifs and serve as building blocks for the formation of more complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been recently interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or on the application of mechanical (un)folding forces, they provide a unique and complementary approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 s time resolution. Our results show that under our experimental conditions, stem-loops fold and unfold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which relatively extended unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or, alternatively, the natively folded conformation. Our findings demonstrate how the combination of single-molecule sensitivity and high time resolution makes smFETs unique and powerful tools for characterizing the conformational free-energy landscape of RNA and highlight the extremely rugged landscape on which even the simplest RNA structural elements fold.

sentences: Synergistic collagen-condiment: Streptococcal collagen-like (Scl) protein in cell-adhesion and diabetic wound-closure matrix Group A streptococcus (GAS), Streptococcus pyogenes manifests plethora of diseases through its explicit virulence factors. Among these, the recently deciphered MSCRAMMs, Streptococcal collagen-like (Scls) adhesins are most studied proteins in context of their biophysically stable collagenous-sequence (Gly-X-Y) despite the difference from analogous mammalian-collagen. Based on recent evidence on collagen-mimetic Scls, we elucidated biomaterial-potential of the unmodified, recombinant Scl1 (rScl1). Initially, rScl1 trimeric- assembly yielded its stability in silico than the monomeric-unit. Thereby, rScl1 matrix characterization was confirmed in vitro. rScl1 exhibited high A549 and HepG2 cell- viability--rScl1 dose incremented to 20.0 {micro}g/ml at time points up to 24 hr, and on 24 hr stored-dishes--deliberating it non-cytotoxic. Imploring cell-adhesion potential, we observed increased cell-counts tangential to rScl1-gradient. This affirmative prelude on rScl1 as a supporting-matrix cued its synergy to collagen; we discerned it through rScl1-augmented, full-thickness diabetic wound-closure in vivo and as a first, we studied > 18-month rabbit alloxan-models. We have ascertained re-epithelialization with higher type III collagen in absence of inflammation evidenced morphometrically and histologically. Finally, we correlated our observations through atomistic-evaluation of rScl1-2{beta}1-integrin interaction, surprisingly, with augmented binding-energy compared to collagen. Hence, connoting recombinant-streptococcal collagen as an  alternate; with further characterization, rScl1 can potentiate important revelations conceding homogeneous and safe, bio-available, biomaterial.

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Automatic determination of the handedness of Single-Particle maps of macromolecules solved by CryoEM Single-Particle Analysis by Cryo-Electron Microscopy is a well-established technique to elucidate the three-dimensional (3D) structure of biological macromolecules. The orientation of the acquired projection images must be initially estimated without any reference to the final structure. In this step, algorithms may find a mirrored version of all the orientations resulting in a mirrored 3D map. It is as compatible with the acquired images as its unmirrored version from the image processing point of view, only that it is not biologically plausible.

In this article, we introduce HaPi (Handedness Pipeline), the first method to automatically determine the hand of electron density maps of macromolecules solved by CryoEM. HaPi is built by training two 3D convolutional neural networks. The first determines -helices in a map, and the second determines whether the -helix is left-handed or right-handed. A consensus strategy defines the overall map hand. The pipeline is trained on simulated and experimental data. The handedness can be detected only for maps whose resolution is better than 5[A]. HaPi can identify the hand in 89% of new simulated maps correctly. Moreover, we evaluated all the maps deposited at the Electron Microscopy Data Bank and 11 structures uploaded with the incorrect hand were identified.

sentences: Architecture of the chikungunya virus replication organelle Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

sentences: Making Common Fund data more Findable: Catalyzing a Data Ecosystem The Common Fund Data Ecosystem (CFDE) has created a flexible system of data federation that enables users to discover datasets from across the U.S. National Institutes of Health Common Fund without requiring that data owners move, reformat, or rehost those data. The CFDEs federation system is centered on a catalog that ingests metadata from individual Common Fund Programs Data Coordination Centers (DCCs) into a uniform metadata model that can then be indexed and searched from a centralized portal. This uniform Crosscut Metadata Model (C2M2) supports the wide variety of data types and metadata terms used by the individual DCCs and is designed to enable easy expansion to accommodate new data types. We describe its use to ingest and index data from ten DCCs.

sentences: Non-canonical Wnt/Ror2 signaling status regulates cell-matrix crosstalk to prompt directional tumor cell invasion and dissemination in breast cancer Bidirectional cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression. We demonstrate the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling in models of breast cancer. Wnt/Ror2 loss-of-function triggers tumor cell invasion, accompanied by changes in actin cytoskeleton, adhesion, and collagen crosslinking gene expression programs. E-cadherin levels decline upon Ror2 depletion, and spatially, we pinpoint the upregulation and redistribution of 5 and {beta}3 integrins together with the production of fibronectin in areas of invasion. Wnt/{beta}-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of reciprocal communication between tumor cells and ECM during cancer invasion and metastasis.

sentences: Transposon-induced inversions activate gene expression in Maize pericarp Chromosomal inversions can have considerable biological and agronomic impacts including disrupted gene function, change in gene expression and inhibited recombination. Here we describe the molecular structure and functional impact of six inversions caused by Alternative Transpositions between p1 and p2 genes responsible for floral pigmentation in maize. In maize line p1-wwB54, the p2 gene is expressed in anther and silk but not in pericarp, making the kernels white. We identified inversions in this region caused by transposition of Ac and fractured Ac (fAc) transposable elements. These inversions change the position of a p1 enhancer and activate the expression of p2 in the kernel pericarp, resulting in red kernel color. We hypothesize that these inversions place the p2 gene promoter near a p1 gene enhancer, thereby activating p2 expression in kernel pericarp.

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CD49a Identifies Polyfunctional Memory CD8 T cell Subsets that Persist in the Lungs after Influenza Infection CD8 T cell memory offers critical antiviral protection, even in the absence of neutralizing antibodies. The paradigm is that CD8 T cell memory within the lung tissue consists of a mix of circulating TEM cells and non-circulating TRM cells. However, based on our analysis, the heterogeneity within the tissue is much higher, identifying TCM, TEM, TRM, and a multitude of populations which do not perfectly fit these classifications. Further interrogation of the populations shows that TRM cells that express CD49a, both with and without CD103, have increased and diverse effector potential compared with CD49a negative populations. These populations function as a one-man band, displaying antiviral activity, chemokine production, release of GM-CSF, and the ability to kill specific targets in vitro with delayed kinetics compared with effector CD8 T cells. Together, this study establishes that CD49a defines multiple polyfunctional CD8 memory subsets after clearance of influenza infection, which act to eliminate virus in the absence of direct killing, recruit and mature innate immune cells, and destroy infected cells if the virus persists.

Contribution to the fieldProtection from previously seen infections requires specialized immune memory cells properly positioned throughout the body to combat the newly invading pathogen. In the case of re-exposure to influenza virus, CD8 T cells resident within the respiratory tract (TRM) are critical for eliminating the virus. Previously, TRM were viewed as mostly homogenous, with a limited range of immune functions. In this study, lung TRM were compared with circulating memory CD8 T cells transiently present within the lung, to define the breadth of their effector capabilities. Using TRM defining surface proteins CD49a and CD103 to identify different memory CD8 T cell subsets, gene and protein expression were evaluated. In addition to demonstrating higher levels of diversity than previously reported, multiple polyfunctional subsets were identified. This polyfunctionality was primarily associated with cell populations expressing CD49a, and these cells produced multiple antiviral factors, chemokines to recruit other immune cells, a growth factor associated with improved antigen presenting cell function, and cytolytic granules. Functional assays further demonstrated killing of target cells by TRM. This study paints a more holistic, complete picture of the phenotype and functions of lung CD8 T cells after viral infection, revealing CD49a as a marker of cells with high effector capacity.

sentences: Effects of High Magnetic Fields on Diffusion of Biologically Active Molecules The diffusion of biologically active molecules is a ubiquitous process, controlling many mechanisms and the characteristic time scales for pivotal processes in living cells. Here, we show how a high static magnetic field (MF) affects the diffusion of paramagnetic and diamagnetic species, including oxygen, hemoglobin, ROS and drugs. We derive and solve the equation describing diffusion of such biologically active molecules in the presence of a MF as well as reveal the underlying mechanism of the MF effect on diffusion. We find that a high MF accelerates diffusion of diamagnetic species while slowing the diffusion of paramagnetic molecules in cell cytoplasm. When applied to oxygen and hemoglobin diffusion in red blood cells, our results suggest that a MF may significantly alter the gas exchange in an erythrocyte and cause swelling. Our prediction that the diffusion rate and characteristic time can be controlled by a MF opens new avenues for experimental studies foreseeing numerous biomedical applications.

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Surfaceome CRISPR Screen Identifies OLFML3 as a Rhinovirus-inducible IFN Antagonist BackgroundRhinoviruses (RVs) cause more than half of common cold and, in some cases, more severe diseases. Functional genomics analyses of RVs using siRNA or genome-wide CRISPR screen uncovered a limited set of host factors, few of which has proven clinical relevance.

ResultsHerein, we systematically compared genome-wide CRISPR screen and surface protein-focused CRISPR screen, referred to as surfaceome CRISPR screen, for their efficiencies in identifying RV host factors. It was found that surfaceome screen outperformed genome-wide screen in the success rate of hit identification. Importantly, using surfaceome screen we have identified olfactomedin like 3 (OLFML3) as a novel host factor of RV serotypes A and B including a clinical isolate. We found that OLFML3 was a RV-inducible suppressor of the innate immune response and that OLFML3 antagonized type I interferon (IFN) signaling in a SOCS3-dependent manner.

ConclusionOur study has suggested that RV-induced OLFML3expression is an important mechanism for RV to hijack the immune system and underscored surfaceome CRISPR screen in identifying viral host factors.

sentences: Sensory suppression and increased neuromodulation during actions disrupt memory encoding of unpredictable self-initiated stimuli Actions modulate sensory processing by attenuating responses to self- compared to externally-generated inputs, which is traditionally attributed to stimulus-specific motor predictions. Yet, suppression has been also found for stimuli merely coinciding with actions, pointing to unspecific processes that may be driven by neuromodulatory systems. Meanwhile, the differential processing for self-generated stimuli raises the possibility of producing effects also on memory for these stimuli, however, evidence remains mixed as to the direction of the effects. Here, we assessed the effects of actions on sensory processing and memory encoding of concomitant, but unpredictable sounds, using a combination of self-generation and memory recognition task concurrently with EEG and pupil recordings. At encoding, subjects performed button presses that half of the time generated a sound (motor-auditory; MA) and listened to passively presented sounds (auditory-only; A). At retrieval, two sounds were presented and participants had to respond which one was present before. We measured memory bias and memory performance by having sequences where either both or only one of the test sounds were presented at encoding, respectively. Results showed worse memory performance - but no differences in memory bias - and attenuated responses and larger pupil diameter for MA compared to A sounds. Critically, the larger the sensory attenuation and pupil diameter, the worse the memory performance for MA sounds. Nevertheless, sensory attenuation did not correlate with pupil dilation. Collectively, our findings suggest that sensory attenuation and neuromodulatory processes coexist during actions, and both relate to disrupted memory for concurrent, albeit unpredictable sounds.

sentences: BAI-Net: Individualized Anatomical Human Cerebral Cartography using Graph Convolutional Network Brain atlas is an important tool in the diagnosis and treatment of neurological disorders. However, due to large variations in the organizational principles of individual brains, many challenges remain in clinical applications. Brain atlas individualization network (BAI-Net) is an algorithm that subdivides individual cerebral cortex into segregated areas using brain morphology and connectomes. BAI-Net integrates topological priors derived from a group atlas, adjusts the areal probability using the connectivity context derived from diffusion tractography, and provides reliable and explainable individualized brain parcels across multiple sessions and scanners. We demonstrate that BAI-Net outperforms the conventional iterative clustering approach by capturing significantly heritable topographic variations in individualized cartographies. The topographic variability of BAI-Net cartographies shows strong associations with individual variability in brain morphology, connectivity fingerprints and cognitive behaviors. This study provides a new framework for individualized brain cartography and paves the way of atlas-based precision medicine in clinical practice.

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Interaction with TopBP1 mediates human papillomavirus 16 E2 plasmid segregation/retention function and stability during the viral life cycle Human papillomavirus 16 (HPV16) E2 is a DNA binding protein that regulates transcription, replication and potentially, segregation of the HPV16 genome during the viral life cycle. In the segregation model, E2 simultaneously binds to viral and host chromatin, acting as a bridge to ensure that viral genomes reside in daughter nuclei following cell division. The host chromatin receptor for E2 mediating this function is unknown. Recently, we demonstrated that CK2 phosphorylation of E2 on serine 23 (S23) is required for interaction with TopBP1, and that this interaction promotes E2 and TopBP1 recruitment to mitotic chromatin. Here, we demonstrate that in U2OS and N/Tert-1 cells expressing wild type E2 and a non-TopBP1 binding mutant (S23A, serine 23 mutated to alanine), interaction with TopBP1 is essential for E2 recruitment of plasmids to mitotic chromatin. Using a novel quantitative segregation assay, we demonstrate that interaction with TopBP1 is required for E2 plasmid segregation. The interaction of E2 with TopBP1 promotes increased levels of E2 protein during mitosis in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by the HPV16 genome. Additionally, interaction with TopBP1 is required for expression of the E2 protein during the viral life cycle. Overall, our results demonstrate that E2 has plasmid segregation activity, and that the E2-TopBP1 interaction is essential for this E2 function and for E2 expression during the viral life cycle.

ImportanceHPV16 causes 3-4% of all human cancers. During the viral life cycle, it is proposed that the viral genome is actively segregated into daughter nuclei, ensuring viral replication in the subsequent S phase. The E2 protein potentially bridges the viral and host genomes during mitosis to mediate segregation of the circular viral plasmid. Here, we demonstrate that E2 has the ability to mediate plasmid segregation, and that this function is dependent upon interaction with the host protein TopBP1. Additionally, we demonstrate that the E2-TopBP1 interaction promotes enhanced E2 expression during mitosis which likely promotes the plasmid segregation function of E2. Overall, our results present a mechanism of how HPV16 can segregate its viral genome during an active infection, a critical aspect of the viral life cycle.

sentences: Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived fromhuman iPSCs With the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

sentences: Influenza A (N1-N9) and Influenza B (B/Vic and B/Yam) Neuraminidase Pseudotypes as Tools for Pandemic Preparedness and Improved Influenza Vaccine design To better understand how inhibition of the influenza neuraminidase (NA) protein contributes to protection against influenza, and to investigate its breadth and cross-neutralizing activity, we have produced lentiviral vectors pseudotyped with an avian H11 hemagglutinin (HA) and the NA (N1-N9) of all influenza A and (B/Victoria and B/Yamagata) influenza B subtypes. These NA viral pseudotypes (PV) possess stable NA activity and can be utilized as target antigens in in vitro assays to assess vaccine immunogenicity. Employing these NA PV, we have developed an enzyme-linked lectin assay (pELLA) for routine serology to measure neuraminidase inhibition (NI) titers of reference antisera, monoclonal antibodies, and post-vaccination sera with various influenza antigens. We have also shown that pELLA is more sensitive than the commercially available NA-Fluor in detecting NA inhibition in these samples. Our studies may lead to establishing the protective NA titer that contributes to NA-based immunity. This will aid in the design of superior, longer lasting, and more broadly protective vaccines that can be employed together with HA-targeted vaccines in a pre-pandemic approach.

sentences: Variation in fine scale recombination rate in temperature-evolved Drosophila melanogaster populations in response to selection. Meiotic recombination plays a critical evolutionary role in maintaining fitness in response to selective pressures due to changing environments. Variation in recombination rate has been observed amongst and between species and populations and within genomes across numerous taxa. Studies have demonstrated a link between changes in recombination rate and selection but the extent to which fine scale recombination rate varies between evolved populations during the evolutionary period in response to selection is under active research. Here we utilize a set of three temperature-evolved Drosophila melanogaster populations that were shown to have diverged in several phenotypes including recombination rate based on the temperature regime in which they evolved. Using whole genome sequencing data of these populations, we generated fine scale recombination maps of the three populations. We compare recombination rates and patterns among the three populations and show that they have diverged at fine scales but are conserved at broader scales. We further demonstrate a correlation between recombination rates and genomic variation in the three populations and observe variation in putative warm-spots between the populations with these enhanced areas and associated genes overlapping areas previously shown to have diverged in the three populations due to selection. These data support the existence of recombination modifiers in these populations which are subject to selection during evolutionary change.

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Replicative Aging Remodels Cell Wall and is Associated with Increased Intracellular Trafficking in Human Pathogenic Yeasts Replicative aging is an underexplored field of research in medical mycology. Cryptococcus neoformans (Cn) and Candida glabrata (Cg) are dreaded fungal pathogens that cause fatal invasive infections. The fungal cell wall is essential for yeast viability and pathogenesis. In this study, we provide data characterizing age-associated modifications to the cell wall of Cn and Cg. Here, we report that old yeast cells upregulate genes of cell wall biosynthesis, leading to cell wall reorganization, and increased levels of all major components, including glucan, chitin and its derivatives, as well as mannan. This results in a significant thickening of the cell wall in aged cells. Old generation yeast cells exhibited drastic ultrastructural changes, including the presence of abundant vesicle-like particles in the cytoplasm, and enlarged vacuoles with altered pH homeostasis. Our findings suggest that the cell wall modifications could be enabled by augmented intracellular trafficking. This work furthers our understanding of the cell phenotype that emerges during aging. It highlights differences in these two fungal pathogens and elucidates mechanisms that explain the enhanced resistance of old cells to antifungals and phagocytic attacks.

IMPORTANCECryptococcus neoformans and Candida glabrata are two opportunistic human fungal pathogens that cause life-threatening diseases. During infection, both microorganisms have the ability to persist for long periods, and treatment failure can occur even if standard testing identifies the yeasts to be sensitive to antifungals. Replicative lifespan is a trait that is measured by the number of divisions a cell undergoes before death. Aging in fungi is associated with enhanced tolerance to antifungals and resistance to phagocytosis, and characterization of old cells may help identify novel antifungal targets. The cell wall remains an attractive target for new therapies because it is essential for fungi and is not present in humans. This study shows that the organization of the fungal cell wall changes remarkably during aging and becomes thicker and is associated with increased intracellular trafficking as well as the alteration of vacuole morphology and pH homeostasis.

sentences: Novel microRNAs targeting NMDA receptor subunits in animal models of schizophrenia N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3-UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia-pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKII, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

sentences: Munc13 supports vesicle fusogenicity after disrupting active zone scaffolds and synaptic vesicle docking Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to Ca2+ entry, and target neurotransmitter release to postsynaptic receptor domains. Simultaneous RIM+ELKS ablation disrupts these scaffolds, abolishes vesicle docking and removes active zone-targeted Munc13, but some vesicles remain releasable. This enduring vesicular fusogenicity may be Munc13-independent or be mediated by non-active zone-anchored Munc13. We tested its Munc13-dependence by ablating Munc13-1 and Munc13-2 on top of RIM+ELKS in cultured hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM+ELKS further impaired action potential-evoked release by decreasing the remaining pool of releasable vesicles. We conclude that Munc13 can support some fusogenicity without RIM and ELKS, and that presynaptic recruitment of Munc13, even without active zone-anchoring, suffices to generate some fusion-competent vesicles.

sentences: Effects of treatment with three antibiotics, vancomycin, neomycin, and AVNM on gut microbiome in C57BL/6 mice Higher organisms, especially mammals, harbor diverse microbiota in the gut that plays a major role in maintaining health and physiological homeostasis. Perturbation of gut flora helps identifying their roles. Antibiotics are potent perturbing agents of microbiome. Select antibiotics like vancomycin, neomycin, and AVNM (an antibiotic cocktail containing ampicillin, vancomycin, neomycin, and metronidazole) were used to perturb the gut microbiota of C57BL/6 male mice to understand their roles in host immunity and metabolism. The current study revealed that the resulting gut microbial composition was different, and diversity (at the phylum and genus level) was reduced differentially following each antibiotic treatment. Vancomycin treatment caused a significant increase in Verrucomicrobia and Proteobacteria phyla. The treatment with neomycin yielded an increase in the Bacteroidetes phylum, while the treatment with AVNM led to an increase in Proteobacteria phylum with lowest diversity of microbiome in the gut. The current results also revealed that the different antibiotics treatment caused variation in the cecal index, expression of immune genes (TNF-, IL-10, IFN-{gamma}) in the colon, and short-chain fatty acids (SCFA) level in the blood of mice. A strong correlation was observed for antibiotic-induced differential dysbiosis patterns of gut microbiota and the altered immune and SCFA profile of the host. The outcome of the present study could be clinically important.

sentences: Annelids win again: the first evidence of Hox antisense transcription in Spiralia. To date it is becoming more and more obvious that multiple non-coding RNAs, once considered to be transcriptional noise, play a huge role in gene regulation during animal ontogenesis. Hox genes are key regulators of embryonic development, growth and regeneration of all bilaterian animals. It was shown that mammalian Hox loci are transcribed in both directions and noncoding RNAs maintain and control the normal functioning of Hox clusters. We revealed antisense transcripts of most of Hox genes in two lophotrochozoans, errant annelids Alitta virens and Platynereis dumerilii. It is for the first time when non-coding RNAs associated with Hox genes are found in spiralian animals. All these asRNAs can be referred to as natural antisense transcripts (NATs). We analyzed the expression of all detected NATs using sense probes to their Hox mRNAs during larval and postlarval development and regeneration by whole mount in situ hybridization (WMISH). We managed to clone several asRNAs (Avi-antiHox4-1, Avi-antiHox4-2 and Avi-antiHox5) of these annelids and analyzed their expression patterns as well. Our data indicate variable and complicated interplay between sense and antisense Hox transcripts during development and growth of two annelids. The presence of Hox antisense transcription in the representatives of different bilaterian clades (mammals, myriapods and annelids) and similar expression relationships in sense-antisense pairs suggest that this can be the ancestral feature of Hox cluster regulation.

sentences: Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads To investigate the role of mechanical constraints in morphogenesis and development, we develop a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane (PDMS) mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the Zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue, and its time evolution.

sentences: Analysis of visual processing capabilities and neural coding strategies of a detailed model for laminar cortical microcircuits in mouse V1 Neural networks of the brain that process visual information have structural properties that differ significantly from those of neural networks which are commonly used for visual processing in AI, such as Convolutional Neural Networks (CNNs). But it has remained unknown how these structural differences are related to network function. We analyze visual processing capabilities of a large-scale model for area V1 that arguably provides the most comprehensive accumulation of anatomical and neurophysiological data that is currently available. Its network structure turns out to induce a number of characteristic visual processing capabilities of the brain, in particular the capability to multiplex different visual processing tasks, also on temporally dispersed visual information, with remarkable robustness to noise. This V1 model also exhibits a number of characteristic neural coding properties of the brain, which provide explanations for its superior noise robustness. Since visual processing in the brain is substantially more energy-efficient than implementations of CNNs in common computer hardware, such brain-like neural network models are likely to have also an impact on technology: As blueprints for visual processing in more energy-efficient neuromorphic hardware.

sentences: Biological integrity enhances the qualitative effectiveness of conditional mice-oak mutualisms Scatter-hoarding decisions by rodents are key for the long-term maintenance of scattered tree populations. Decisions are determined by seed value, competition and predation risk, so that they can be influenced by the integrity of the biological system composed by trees, rodents, ungulate competitors, and rodent predators. We manipulate and model the oak-mice interaction in a Spanish dehesa, an anthropogenic savanna system suffering chronic tree regeneration failure, and quantify the joint effect of intrinsic and extrinsic factors on acorn dispersal effectiveness. First, we conducted a large-scale cafeteria field experiment, where we modified ungulate presence and predation risk, and followed mouse scatter-hoarding decisions under contrasting levels of moonlight and acorn availability. Then, we estimated the net effects of competition and risk by means of transition probability models that simulated mouse scatter-hoarding decisions according to the environmental context. Our results show that suboptimal conditions for mice balance the interaction towards the mutualism as they force mice to forage less efficiently. Under stressful conditions (predation risks and presence of ungulates), lack of antipredatory cover around dehesa trees limited transportation of acorns, but also precluded mice activities outside tree canopies. As a result, post-dispersal predation rates were reduced and large acorns had a higher probability to survive. Our work shows that inter-specific interactions preventing efficient foraging by scatter-hoarders benefitted seed dispersal. Therefore, the maintenance of the full set of producers, consumers, dispersers and predators in ecosystems is key for promoting seed dispersal effectiveness in conditional mutualisms.

sentences: Mathematical model of a cytokine storm Cytokine storm is a life-threatening inflammatory response that is characterized by hyperactivation of the immune system, and which can be caused by various therapies, auto-immune conditions, or pathogens, such as respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease COVID-19. While initial causes of cytokine storms can vary, late-stage clinical manifestations of cytokine storm converge and often overlap, and therefore a better understanding of how normal immune response turns pathological is warranted. Here we propose a theoretical framework, where cytokine storm phenomenology is captured using a conceptual mathematical model, where cytokines can both activate and regulate the immune system. We simulate normal immune response to infection, and through variation of system parameters identify conditions where, within the frameworks of this model, cytokine storm can arise. We demonstrate that cytokine storm is a transitional regime, and identify three main factors that must converge to result in storm-like dynamics, two of which represent individual-specific characteristics, thereby providing a possible explanation for why some people develop CRS, while others may not. We also discuss possible ecological insights into cytokine-immune interactions and provide mathematical analysis for the underlying regimes. We conclude with a discussion of how results of this analysis can be used in future research.

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Recombinant Group A Carbohydrate backbone embedded into Outer Membrane Vesicles is a potent vaccine candidate targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis BackgroundGroup A Streptococcus (GAS) are responsible for a wide range of human-exclusive infections, annually killing more than 500,000 people. Antibiotic resistance incidence of invasive GAS tripled in the past decade and emphasises the need to develop a universal GAS vaccine. We have produced, for the first time, a recombinant polyrhamnose backbone (pRha), a validated universal GAS vaccine candidate. E. coli outer membrane vesicles (OMVs) carrying pRha were investigated for their immunogenicity and efficacy in an animal model.

MethodsOMVs decorated with pRha were administered to C57BL/6J mouse and rabbit models. Flow cytometry, ELISA, Luminex, immunofluorescence microscopy and serum bactericidal assay assays were conducted to investigate the ability of pRha-specific antibodies to recognise and kill clinical (hypervirulent) GAS strains.

ResultsOur results suggest that pRha-OMVs induce specific antibodies which recognise Group A Carbohydrate (GAC) from S pyogenes and S. dysgalactiae subsp. equisimilis. Increased IgG levels correlate with increased bactericidal killing of the hypervirulent GAS M89 strain. Elevated IL-17a from pRha-OMV-immunised splenocytes indicates possible stimulation of long-term memory immune cells.

ConclusionWe are the first to report efficacy and potency of this unique, exogenously produced polysaccharide, pRha, in the induction of humoral-mediated immune responses to GAS.

TopicStreptococcus pyogenes, immunoglobulins, polysaccharides, opsonophagocytosis, acute rheumatic fever, M protein, invasive Group A Streptococcus, hyaluronic acid

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Chloride ion evokes taste sensation by binding to the extracellular ligand-binding domain of sweet/umami taste receptors The taste sensation of salt is multifaceted: table salt (NaCl) at low concentrations is perceived as a preferable taste through the salty taste receptor, while that at higher concentrations is perceived as an aversive taste through distinct pathways. In addition, Cl- is also thought to participate in taste sensation through a currently unknown mechanism. Here we describe a Cl- -ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from medaka fish, an amino-acid taste receptor and the sole T1r member whose structure has been solved, exhibited a specific Cl--binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3. Notably, the Cl--binding site is likely conserved among T1r3 from other species, including humans. The Cl--binding at the site is considered to organize the structures of the amino-acid binding site and heterodimer interface. Indeed, the Cl--binding was found to induce a conformational change of T1r2a/T1r3LBD at sub-mM to low-mM concentrations in a similar manner to canonical taste substances. In order to address the physiological significance of the Cl- action on T1r, single fiber responses from mouse taste nerves connected to T1r-expressing taste cells were investigated. As a result, a Cl- application increased impulse frequencies, which were inhibited by a lingual application of a T1r-specific blocker. These results suggest that the Cl- evokes taste sensations by binding to T1r, underlying a reported "sweet" taste sensation by table salt at a low concentration.

Significance statementSalt taste sensation is essential for regulating salt intake and maintaining body fluid volume and concentration. Therefore, in concentrations close to that of body fluid, the sodium ion is perceived as an appealing taste through a specific taste transduction pathway. In this study, we found that a low concentration of chloride ion, a sodium ion counter anion in table salt, is perceived via the sweet taste transduction pathway mediated by the sweet taste receptor, one of the attractive taste qualities in animals. The chloride perception system discovered in this study could be another attractive pathway for promoting salt intake in an appropriate concentration range.

sentences: A Phylogenetic Independent Contrast Method under the Ornstein-Uhlenbeck Model and its Applications in Correlated Evolution AO_SCPLOWBSTRACTC_SCPLOWPhylogenetic comparative methods are essential in studying the evolution of traits across a phylogeny. Felsensteins phylogenetic independent contrast (PIC) method and the generalized least squares (GLS) regression were often utilized to study whether evolutionary changes between traits were correlated. However, a neutral Brownian model is assumed in the PIC method, which impacts the performance of the PIC method when the trait is subject to adaptation. In recent years, the Ornstein-Uhlenbeck (OU) model has attracted increasing attention in studying the evolution of traits with stabilizing selection. In this study, we extended Felsensteins PIC method under the OU model, which we termed OU-PIC. We simulated trait evolution under the OU model on phylogenetic trees with 8, 10, and 55 species. Compared to the PIC method, the OU-PIC method with correct stabilizing selection parameters achieved an appropriate type I error rate, the highest test power, and the lowest mean squared error. We presented a concise proof of the intrinsic connection between the OU-PIC and the generalized least squares (GLS) regression method in evaluating correlated evolution under the OU model. The OU-PIC method has a broad range of applications when trait evolution could be sufficiently modeled by the OU process. Compared with other phylogenetic comparative methods, OU-PIC avoids the inverse of the covariance matrix and would facilitate the analysis of correlated evolution on large phylogenies.

sentences: Sperm cryopreservation impacts the early development of equine embryos by downregulating specific transcription factors Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8, 10 and 12 h post spontaneous ovulation, maintaining the pairs mare-stallion for the type of semen used. Next generation sequencing (NGS) was performed in all embryos and bioinformatic and enrichment analysis performed on the 21,058 identified transcripts. A total of 165 transcripts were downregulated in embryos obtained with cryopreserved spermatozoa respect embryos resulting from an insemination with fresh spermatozoa (p=0.021, q=0.1). The enrichment analysis using human orthologs using g:profiler on the downregulated transcripts marked an enrichment in transcription factors (TFs) in mRNAs downregulated in embryos obtained after insemination with cryopreserved spermatozoa. The 12 mRNAs (discriminant variables) most significantly downregulated in these embryos included among others, the chromatin-remodeling ATPase INO80, Lipase maturation factor 1 LMF1, the mitochondrial mRNA pseudouridine synthase RPUSD3, LIM and cysteine-rich domains protein 1, LMCD1. Sperm cryopreservation also caused a significant impact on the embryos at 8 to 10 days of development, but especially in the transition from 10 to 12 days. Overall, our findings provide strong evidence that insemination with cryopreserved spermatozoa poses a major impact in embryo development that may compromise its growth and viability, probably due to modifications in sperm proteins induced by cryopreservation. Identification of specific factors in the spermatozoa causing these changes may improve cryopreservation.

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Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. m/lEVs isolated from rotenone (mitochondrial complex I inhibitor) exposed BECs (RTN-m/lEVs) did not increase BECs ATP levels compared to naive m/lEVs. In contrast, RTN-sEVs functionality was minimally affected compared to naive sEVs. Compared to EV/HSP27 mixtures, PEG-DET/EV/HSP27 ternary mixtures increased HSP27 protein loading. (PEG-DET/HSP27)/EV ternary and EV/HSP27 binary mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Highlights[tpltrtarr] Medium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria
[tpltrtarr]m/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs)
[tpltrtarr]Donor m/lEV mitochondria colocalized with host mitochondria in recipient BECs
[tpltrtarr]m/lEVs from rotenone-exposed BECs did not increase recipient BEC ATP levels
[tpltrtarr]PEG-DET/EV/HSP27 ternary mixtures increase exogenous HSP27 protein loading

Graphical Abstract

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Emergent spatiotemporal population dynamics with cell-length control of synthetic microbial consortia AO_SCPLOWBSTRACTC_SCPLOWIncreased complexity of engineered microbial biocircuits highlights the need for distributed cell functionality due to concomitant increases of metabolic and regulatory burdens imposed on single-strain topologies. Distributed systems, however, introduce additional challenges since consortium composition and spatiotemporal dynamics of constituent strains must be robustly controlled to achieve desired circuit behaviors. Here, we address these challenges with a modeling-based investigation of emergent spatiotemporal population dynamics that result from cell-length control of monolayer, two-strain bacterial consortia. We demonstrate that with dynamic control of a strains division length, nematic cell alignment in close-packed monolayers can be destabilized. We found this destabilization conferred an emergent, competitive advantage on smaller-length strains--but by mechanisms that differed depending on the spatial patterns of the population. We used complementary modeling approaches to elucidate underlying mechanisms: an agent-based model to simulate detailed mechanical and signaling interactions between the competing strains and a reductive, stochastic lattice model to represent cell-cell interactions with a single rotational parameter. Our modeling suggests that spatial strain-fraction oscillations can be generated when cell-length control is coupled to quorum-sensing signaling in negative feedback topologies. Our research employs novel methods of population control and points the way to programming strain fraction dynamics in consortial synthetic biology.

Engineered microbial collectives are more versatile and robust than single strain populations. However, the function of such collectives is sensitive to their spatiotemporal organization. Here, we demonstrate control of the spatiotemporal composition of synthetic microbial consortia by dynamically modulating the average cell length of constituent strains. Such modulation confers an emergent "mechanical fitness" advantage upon the shorter length strain. We used both a biophysically realistic agent-based model to test the impact of cell shape on spatiotemporal dynamics and a conceptually simpler stochastic lattice model to explain the essential mechanisms driving the dynamics.

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Rapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch BackgroundKlebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting.

MethodsWe developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16,537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1,636 genomes from four low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR.

ResultsUsing Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (e.g. ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that three O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse.

ConclusionsPathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.

summaryPathogenwatch is a free web-application for analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We improved the representation of genomes from middle-income countries through the Global Health Research Unit on AMR, further facilitating ongoing surveillance.

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The WalRK Two-component system is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile The WalR-WalK two-component regulatory system (TCS) is found in all Firmicutes, where it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. Here we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used RNA-seq to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer and we found WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported in other Firmicutes. For example, down-regulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, down-regulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus.

ImportanceThe WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. Here we investigate the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported in other Firmicutes. We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.

sentences: TME induced TRAIL secretion from engineered macrophages for anti-tumor therapy Capacity of tumor niche chemotaxis and long-term persistency making macrophage as great vehicles for anti-tumor factor delivery. Macrophages-based delivery of chemokines or cytokines have been tested for tissue homeostasis and cancer repression. TRAIL as a promising anti-tumor cytokine, made little clinical progress due to limited stability and off-target toxicity. Here we engineered macrophages with tumor micro-environment (TME)-induced trimerized CP1-TRAIL secretion under the TME specific promoter Arg1 (Tri-TRAIL-iM). The Tri-TRAIL-iM cells displayed high specific inducible activity in both cell-based co-culture assay and in tumor baring mice models. Compared to normal TRAIL over-expressed macrophages under none-inducible promoter, Tri-TRAIL-iM infiltrated to tumor sites and showed superior apoptosis induction of cancer cells and tumor growth repression as well as less systemic side effect. This inducible delivery TRAIL strategy can be effectively further applied in clinical studies and can be coupled with other engineered methods to maximize the therapeutic outcomes for solid tumors.

sentences: Propofol Anesthesia Concentration Rather Than Abrupt Behavioral Unresponsiveness Linearly Degrades Responses in the Rat Primary Auditory Cortex Despite extensive knowledge of its molecular and cellular effects, how anesthesia affects sensory processing remains poorly understood. In particular, it remains unclear whether anesthesia modestly or robustly degrades activity in primary sensory regions, and whether such changes are linked to anesthesia drug concentration vs. behavioral unresponsiveness, since these are typically confounded. Here, we employed slow gradual intravenous propofol anesthesia induction together with auditory stimulation and intermittent assessment of behavioral responsiveness while recording epidural EEG, and neuronal spiking activity in the primary auditory cortex (PAC) of eight rats. We found that all main components of neuronal activity including spontaneous firing rates, onset response magnitudes, onset response latencies, post-onset neuronal silence duration, and late-locking to 40Hz click-trains, gradually deteriorated in a dose- dependent manner with increasing anesthesia levels without showing abrupt changes around loss of righting reflex or other time-points. Thus, the dominant factor affecting PAC responses is the anesthesia drug concentration rather than any sudden, dichotomous behavioral state changes. Our findings explain a wide array of seemingly conflicting results in the literature that, depending on the precise definition of wakefulness (vigilant vs. drowsy) and anesthesia (light vs. deep/surgical), report a spectrum of effects in primary regions ranging from minimal to dramatic differences.

sentences: GLOBAL LAND-USE AND LAND-COVER DATA FOR ECOLOGISTS: HISTORICAL, CURRENT AND FUTURE SCENARIOS Land-use land-cover (LULC) data are important predictors of species occurrence and biodiversity threat. Although there are LULC datasets available under current conditions, there is a lack of such data under historical and future climatic conditions. This hinders, for example, projecting niche and distribution models under global change scenarios at different time scenarios. The Land Use Harmonization Project (LUH2) is a global terrestrial dataset at 0.25{degrees} spatial resolution that provides LULC data from 850 to 2300 for 12 LULC state classes. The dataset, however, is compressed in a file format (NetCDF) that is incompatible for many analyses and requires layer extractions and transformations that are intractable for most researchers. Here we selected and transformed the LUH2 in a standard GIS format data to make it more user-friendly. We provide LULC for every year from 850 to 2100, and from 2015 on, the LULC dataset is provided under two Shared Socioeconomic Pathways (SSP2-4.5 and SSP5-8.5). We provide two types of files for each year: separate files with continuous values for each of the 12 LULC state classes, and a single categorical file with all state classes combined. To create the categorical layer, we assigned the state with the highest value in a given pixel among the 12 continuous data. LUH2 predicts a pronounced decrease in primary forest, particularly noticeable in the Amazon, the Brazilian Atlantic Forest, the Congo Basin and the boreal forests, an equally pronounced increase in secondary forest and non-forest lands, and in croplands in the Brazilian Atlantic Forest and sub-Saharan Africa. The final dataset provides LULC data for 1251 years that will be of interest for macroecology, ecological niche modeling, global change analysis, and other applications in ecology and conservation.

sentences: Water droplet-in-oil digestion method for single-cell proteomics Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

sentences: Crosstalk between enterocytes and innate lymphoid cells drives early IFN-g-mediated control of Cryptosporidium The intestinal parasite, Cryptosporidium, is a major contributor to global child mortality and causes opportunistic infection in immune deficient individuals. Innate resistance to Cryptosporidium, which specifically invades enterocytes, is dependent on the production of IFN-{gamma}, yet whether enterocytes contribute to parasite control is poorly understood. In this study, utilizing the natural mouse pathogen, Cryptosporidium tyzzeri, we show that epithelial-derived IL-18 synergized with IL-12 to stimulate innate lymphoid cell (ILC) production of IFN-{gamma}. This innate IFN-{gamma} was required for early parasite control. Loss of STAT1 in enterocytes, but not dendritic cells or macrophages, antagonized early parasite control. Transcriptional profiling of enterocytes from infected mice identified an IFN-{gamma} signature and enrichment of anti-microbial effectors like IDO, GBP and IRG. Deletion experiments identified a role for Irgm1/m3 in parasite control. Thus, enterocytes promote ILC production of IFN-{gamma} that acts on enterocytes to restrict the growth of C. tyzzeri.

sentences: SC-MEB: spatial clustering with hidden Markov random field using empirical Bayes Spatial transcriptomics has been emerging as a powerful technique for resolving gene expression profiles while retaining tissue spatial information. These spatially resolved transcriptomics make it feasible to examine the complex multicellular systems of different microenvironments. To answer scientific questions with spatial transcriptomics and expand our understanding of how cell types and states are regulated by microenvironment, the first step is to identify cell clusters by integrating the available spatial information. Here, we introduce SC-MEB, an empirical Bayes approach for spatial clustering analysis using a hidden Markov random field. We have also derived an efficient expectation-maximization algorithm based on an iterative conditional mode for SC-MEB. In contrast to BayesSpace, a recently developed method, SC-MEB is not only computationally efficient and scalable to large sample sizes but is also capable of choosing the smoothness parameter and the number of clusters. We performed comprehensive simulation studies to demonstrate the superiority of SC-MEB over some existing methods. We applied SC-MEB to analyze the spatial transcriptome of human dorsolateral prefrontal cortex tissues and mouse hypothalamic preoptic region. Our analysis results showed that SC-MEB can achieve a similar or better clustering performance to BayesSpace, which uses the true number of clusters and a fixed smoothness parameter. Moreover, SC-MEB is scalable to large  sample sizes. We then employed SC-MEB to analyze a colon dataset from a patient with colorectal cancer (CRC) and COVID-19, and further performed differential expression analysis to identify signature genes related to the clustering results. The heatmap of identified signature genes showed that the clusters identified using SC-MEB were more separable than those obtained with BayesSpace. Using pathway analysis, we identified three immune-related clusters, and in a further comparison, found the mean expression of COVID-19 signature genes was greater in immune than non-immune regions of colon tissue. SC-MEB provides a valuable computational tool for investigating the structural organizations of tissues from spatial transcriptomic data.

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Single cell trajectory analysis of human pluripotent stem cells differentiating towards lung and hepatocyte progenitors Understanding the development of human respiratory tissues is crucial for modeling and treating lung disorders. The molecular details for the specification of lung progenitors from human pluripotent stem cells (hPSCs) are unclear. Here, we use single cell RNA-sequencing with high temporal resolution along an optimized differentiation protocol to determine the transcriptional hierarchy of lung specification from human hPSCs and map out the underlying single cell trajectories. We show that Sonic hedgehog, TGF-{beta} and Notch activation are required in an ISL1/NKX2-1 trajectory that gives rise to lung progenitors during the foregut endoderm stage. Induction of HHEX marks an alternative trajectory at the early definitive endoderm stage, which precedes the lung trajectory and generates a major hepatoblast population. Moreover, neither KDR+ nor mesendoderm progenitors are apparent intermediate states of lung and hepatic lineages. Our hierarchical multistep model predicts mechanisms leading to lung organogenesis, and creates a basis for studying early human lung development, as well as hPSC based disease and drug research.

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sentences: Epitranscriptome changes triggered by ammonium nutrition regulate the proteome response of maritime pine roots Epitranscriptomic modifications constitute a gene expression checkpoint in all living organisms. As nitrogen is an essential element for plant growth and development, a reasonable hypothesis is that changes in the epitranscriptome may regulate nitrogen acquisition and metabolism. In this study, epitranscriptomic modifications caused by ammonium nutrition were monitored in maritime pine roots through direct RNA sequencing using Oxford Nanopore Technology. Transcriptomic responses mainly affected transcripts involved in nitrogen and carbon metabolism, defense, hormone synthesis/signaling, and translation. Global detection of epitranscriptomic marks was performed to evaluate this posttranscriptional mechanism in untreated and ammonium-treated seedlings. Increased m6A deposition in the 3-UTR was observed in response to ammonium, which seems to be correlated with poly(A) lengths and changes in the relative abundance of the corresponding proteins. The results showed that m6A deposition and its dynamics seem to be important regulators of translation under ammonium nutrition. These findings suggest that protein translation is finely regulated through epitranscriptomic marks likely by changes in mRNA poly(A) length, transcript abundance and ribosome protein composition. An integration of multiomics data suggests that the epitranscriptome modulates responses to developmental and environmental changes, including ammonium nutrition, through buffering, filtering, and focusing the final products of gene expression.

sentences: Benchmark of Differential Gene Expression Analysis Methods for Inter-species RNA-Seq Data using a Phylogenetic Simulation Framework Inter-species RNA-Seq datasets are increasingly common, and have the potential to answer new questions on gene expression patterns across the evolution. Single species differential expression analysis is a now well studied problem, that benefits from sound statistical methods. Extensive reviews on biological or synthetic datasets have provided the community with a clear picture on the relative performances of the available tools in various settings. Such benchmarks are still missing in the inter-species gene expression context. In this work, we take a first step in this direction by developing and implementing a new simulation framework. This tool builds on both the RNA-Seq and the Phylogenetic Comparative Methods literatures to generate realistic count datasets, while taking into account the phylogenetic relationships between the samples. We illustrate the features of this new framework through a targeted simulation study, that reveals some of the strengths and weaknesses of both the classical and phylogenetic approaches for inter-species differential expression analysis. The tool has been integrated in the R package compcodeR freely available on Bioconductor.

sentences: Amyloid β oligomer selective antibodies for Alzheimers therapeutics and diagnostics Improvements have been made in the diagnosis of Alzheimers disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by MRI and PET scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques - species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid {beta} oligomers (A{beta}Os), rather, are now widely accepted as the A{beta} species most germane to AD onset and progression. Here we report evidence further supporting the role of A{beta}Os as pathological instigators of AD and introduce promising anti-A{beta}O diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, A{beta} oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of A{beta}Os is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these A{beta}Os on memory is in harmony with findings that intraventricular injection of synthetic A{beta}Os into wild type mice induced hippocampal dependent memory dysfunction within 24 hours. Compelling support for the conclusion that endogenous A{beta}Os cause memory loss was found in experiments showing that intranasal inoculation of A{beta}O-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of A{beta}Os in instigating memory loss and salient AD neuropathology, and they demonstrate that A{beta}O selective antibodies have potential both for therapeutics and for diagnostics.

sentences: Mapping structure to function and behavior with individual-level connectome embedding The connectome, a comprehensive map of the brains anatomical connections, is often summarized as a matrix comprising all dyadic connections among pairs of brain regions. This representation cannot capture higher-order relations within the brain graph. Connectome embedding (CE) addresses this limitation by creating compact vectorized representations of brain nodes capturing their context in the global network topology. Here, nodes "context" is defined as random walks on the brain graph and as such, represents a generative model of diffusive communication around nodes. Applied to group-averaged structural connectivity, CE was previously shown to capture relations between inter-hemispheric homologous brain regions and uncover putative missing edges from the network reconstruction. Here we extend this framework to explore individual differences with a novel embedding alignment approach. We test this approach in two lifespan datasets (NKI: n=542; Cam-CAN: n=601) that include diffusion-weighted imaging, resting-state fMRI, demographics and behavioral measures. We demonstrate that modeling functional connectivity with CE substantially improves structural to functional connectivity mapping both at the group and subject level. Furthermore, age-related differences in this structure-function mapping are preserved and enhanced. Importantly, CE captures individual differences by out-of-sample prediction of age and intelligence. The resulting predictive accuracy was higher compared to using structural connectivity and functional connectivity. We attribute these findings to the capacity of the CE to incorporate aspects of both anatomy (the structural graph) and function (diffusive communication). Our novel approach allows mapping individual differences in the connectome through structure to function and behavior.

sentences: Cophylogeny and specificity between cryptic coral species (Pocillopora spp.) at Moorea and their symbionts (Symbiodiniaceae) The congruence between phylogenies of tightly associated groups of organisms (cophylogeny) reflects evolutionary links between ecologically important interactions. However, despite being a classic example of an obligate symbiosis, tests of cophylogeny between scleractinian corals and their photosynthetic algal symbionts have been hampered in the past because both corals and algae contain genetically unresolved and morphologically cryptic species. Here, we studied co-occurring, cryptic Pocillopora species from Moorea, French Polynesia, that differ in their relative abundance across depth. We constructed new phylogenies of the host Pocillopora (using genomic loci, complete mitochondrial genomes, and thousands of single nucleotide polymorphisms) and their Symbiodiniaceae symbionts (using ITS2 and psbAncr markers) and tested for cophylogeny. The analysis supported the presence of five Pocillopora species on the fore-reef at Moorea that mostly hosted either Cladocopium latusorum or C. pacificum. Only Pocillopora species hosting C. latusorum, and that have similar relative abundances across depths, also hosted taxa from Symbiodinium and Durusdinium. In general, the Cladocopium phylogeny mirrored the Pocillopora phylogeny. Within Cladocopium species, lineages also differed in their associations with Pocillopora haplotypes, except those showing evidence of nuclear introgression, and with depth in the two most common Pocillopora species. We also found evidence for a new Pocillopora species (haplotype 10), that has so far only been sampled from French Polynesia, that warrants formal identification. The linked phylogenies of these Pocillopora and Cladocopium species and lineages suggest that symbiont speciation is driven by niche diversification in the host, but there is still evidence for symbiont flexibility in rare cases.

sentences: A Novel Local Ablation Approach for Explaining Multimodal Classifiers With the growing use of multimodal data for deep learning classification in healthcare research, more studies have begun to present explainability methods for insight into multimodal classifiers. Among these studies, few have utilized local explainability methods, which could provide (1) insight into the classification of each sample and (2) an opportunity to better understand the effects of demographic and clinical variables within datasets (e.g., medication of subjects in electrophysiology data). To the best of our knowledge, this opportunity has not yet been explored within multimodal classification. We present a novel local ablation approach that shows the importance of each modality to the correct classification of each class and explore the effects of demographic and clinical variables upon the classifier. As a use-case, we train a convolutional neural network for automated sleep staging with electroencephalogram (EEG), electrooculogram (EOG), and electromyogram (EMG) data. We find that EEG is the most important modality across most stages, though EOG is particular important for non-rapid eye movement stage 1. Further, we identify significant relationships between the local explanations and subject age, sex, and state of medication which suggest that the classifier learned specific features associated with these variables across multiple modalities and correctly classified samples. Our novel explainability approach has implications for many fields involving multimodal classification. Moreover, our examination of the degree to which demographic and clinical variables may affect classifiers could provide direction for future studies in automated biomarker discovery.

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Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, intra-population genomic variation represents the outcome of both stochastic and selective forces, making it difficult to identify whether variants are maintained by adaptive, neutral, or purifying processes. This is partly due to the reliance on gene sequences to interpret variants, which disregards the physical properties of three-dimensional gene products that define the functional landscape on which selection acts. Here we describe an approach to analyze genetic variation in the context of predicted protein structures, and apply it to study a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between the patterns of nonsynonymous polymorphism, selective pressures, and structural properties of proteins such as per-site relative solvent accessibility and distance to ligands, which explain up to 59% of genetic variance in some genes. In glutamine synthetase, a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand binding sites as a function of nitrate concentrations in the environment, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our data also reveals that rare codons are purified from ligand binding sites when genes are under high selection, demonstrating the utility of structure-aware analyses to study the variants that likely impact translational processes. Overall, our work yields insights into the governing principles of evolution that shape the genetic diversity landscape within a globally abundant population, and makes available a software framework for structure-aware investigations of microbial population genetics.

SignificanceIncreasing availability of metagenomes offers new opportunities to study evolution, but the equal treatment of all variants limits insights into drivers of sequence diversity. By capitalizing on recent advances in protein structure prediction capabilities, our study examines subtle evolutionary dynamics of a microbial population that dominates surface oceans through the lens of structural biology. We demonstrate the utility of structure-informed metrics to understand the distribution of nonsynonymous polymorphism, establish insights into the impact of changing nutrient availability on protein evolution, and show that even synonymous variants are scrutinized strictly to maximize translational efficiency when selection is high. Overall, our work illustrates new opportunities for discovery at the intersection between metagenomics and structural bioinformatics, and offers an interactive and scalable software platform to visualize and analyze genetic variants in the context of predicted protein structures and ligand-binding sites.

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Functional Role of Second Heart Field-derived Cells in Aortic Integrity in Mice BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.

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- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.

What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies.

sentences: HIV-induced membraneless organelles orchestrate post-nuclear entry steps. HIV integration occurs in chromatin sites that favor the release of high levels of viral progeny, alternatively the virus is also able to coexist discreetly with the host. The viral infection perturbs the cellular environment inducing the remodeling of the nuclear landscape. Indeed, HIV-1 triggers the nuclear clustering of the host factor CPSF6, but the underlying mechanism is poorly understood. Our data indicate that HIV usurps a recently discovered biological phenomenon, called liquid-liquid phase separation (LLPS), to hijack the host cell. We observed CPSF6 clusters as part of HIV-induced membraneless organelles (HIV-1 MLOs) in macrophages, which are one of the main HIV target cells. We describe that HIV-1 MLOs follow phase separation rules and represent functional biomolecular condensates. We highlight HIV-1 MLOs as hubs of nuclear reverse transcription, while the double stranded viral DNA, once formed, rapidly migrates outside these structures. Transcription-competent proviruses localize outside but near HIV-1 MLOs, in LEDGF-abundant regions, known to be active chromatin sites. Therefore, HIV-1 MLOs orchestrate viral events prior to the integration step and create a favorable environment for the viral replication. This study uncovers single functional host-proviral complexes in their nuclear landscape, which is markedly restructured by HIV-1 to favor viral replication.

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

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

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

sentences: A Genetic History of Continuity and Mobility in the Iron Age Central Mediterranean The Iron Age saw the expansion of Phoenician and Greek colonies across the Mediterranean and the rise of Carthage as the major maritime power of the region. These events were facilitated by the ease of long-distance travel following major advances in seafaring. We know from the archaeological record that trade goods and materials were moving across great distances in unprecedented quantities, but it is unclear how these patterns correlate with human mobility. To investigate population mobility and interactions directly, we sequenced the genomes of 30 ancient individuals from Carthaginian and Etruscan port cities around the central Mediterranean, in Tunisia, Sardinia, and central Italy. At all three locations, there is a meaningful contribution of autochthonous populations (from Bronze Age North Africa, Sardinia, and Italy, respectively), as well as highly heterogeneous ancestry including many individuals with ancestry from other parts of the Mediterranean region. These results highlight both the role of autochthonous populations and the extreme interconnectedness of populations in the Iron Age Mediterranean. By studying these trans-Mediterranean neighbors together, we explore the complex interplay between local continuity and mobility that shaped the Iron Age societies of the central Mediterranean.

sentences: MPDB: a unified multi-domain protein structure database integrating structural analogue detection With the development of protein structure prediction methods and biological experimental determination techniques, the structure of single-domain proteins can be relatively easier to be modeled or experimentally solved. However, more than 80% of eukaryotic proteins and 67% of prokaryotic proteins contain multiple domains. Constructing a unified multi-domain protein structure database will promote the research of multi-domain proteins, especially in the modeling of multi-domain protein structures. In this work, we develop a unified multi-domain protein structure database (MPDB). Based on MPDB, we also develop a server with two functional modules: (1) the culling module, which filters the whole MPDB according to input criteria; (2) the detection module, which identifies structural analogues of the full-chain according to the structural similarity between input domain models and the protein in MPDB. The module can discover the potential analogue structures, which will contribute to high-quality multi-domain protein structure modeling.

sentences: Gestational Early-Time Restricted Feeding Results in Sex-Specific Glucose Intolerance in Adult Male Mice The timing of food intake is a novel dietary component that can impact health. Time-restricted feeding (TRF), a form of intermittent fasting, manipulates food timing. During pregnancy, one may experience disruptions to food intake for diverse reasons (e.g. nausea and vomiting of pregnancy, food insecurity, desire to manage gestational weight gain, disordered eating behaviors, changes in taste and food preferences, etc) and therefore may experience periods of intentional or unintentional fasting similar to TRF protocols. Because interest in TRF is gaining popularity and feeding may be interrupted in those who are pregnant, it is important to understand the long-term effects of TRF during pregnancy on the resultant offspring. Using a mouse model, we tested the effects of gestational exposure to early TRF (eTRF) over the life course of both male and female offspring. Offspring body composition was similar between experimental groups in both males and females from weaning (day 21) to adulthood (day 70), with minor increases in food intake in eTRF females and improved glucose tolerance in males. After 10 weeks of high fat, high sucrose diet feeding, male eTRF offspring were more sensitive to insulin but developed glucose intolerance with impaired insulin secretion. As such, gestational eTRF causes sex-specific deleterious effects on glucose homeostasis after chronic high fat, high sucrose diet feeding in male offspring. Further studies are needed to determine the effect gestational eTRF has on the male pancreas as well as to elucidate the mechanisms that protect females from this metabolic dysfunction.

sentences: Reduction of layer 5 mediated long-range cortical communication by antipsychotic drugs Psychosis is characterized by a diminished ability of the brain to distinguish externally driven activity patterns from self-generated activity patterns. Antipsychotic drugs are a class of small molecules with relatively broad binding affinity for a variety of neuromodulator receptors that, in humans, can prevent or ameliorate psychosis. How these drugs influence the function of cortical circuits and in particular their ability to identify self-generated activity patterns is still largely unclear. Here we used widefield calcium imaging to determine the cell type specific functional effects of antipsychotic drugs in mouse dorsal cortex during visuomotor integration. By comparing cell type specific activation patterns between locomotion onsets that were experimentally coupled to self-generated visual feedback and locomotion onsets that were not coupled, we show that deep cortical layers were differentially activated in these two conditions. We then show that the antipsychotic drug clozapine disrupted visuomotor integration at locomotion onsets also primarily in deep cortical layers. Given that one of the key components of visuomotor integration in cortex are long-range cortico-cortical connections, we tested whether the effect of clozapine was detectable in the correlation structure of activity patterns across dorsal cortex. We found that clozapine, as well as two other antipsychotic drugs, aripiprazole and haloperidol, resulted in a strong reduction in correlations of layer 5 activity between cortical areas and impaired the spread of visuomotor prediction errors generated in visual cortex. Our results are consistent with the interpretation that a major functional effect of antipsychotic drugs is an alteration of the activation of internal representations as a consequence of reduced long-range layer 5 mediated communication.

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Functional Redundancy in Ocean Microbiomes Controls Trait Stability Theory predicts that functional redundancy in microbial communities increases trait stability, meaning that traits or functions are less likely to be lost from the community when species go extinct. However, few experiments have empirically tested this prediction, especially in the context of microbial communities and at the landscape scale. In part, the lack of metrics for functional redundancy in microbial ecosystems has prevented addressing this question. In a companion manuscript we proposed a quantitative metric for functional redundancy called Contribution Evenness (CE) that is optimized to reflect trait stability. Here, we use CE to predict the stability of marine microbial functions to species and transcription loss. Using transcriptomes deposited in the Ocean Microbial Reference Gene Catalog (OM-RGC.v2), a catalog of genes and transcripts sequenced by the TARA Ocean expedition, we quantified the functional redundancy for 4,314 KEGG Orthologs (KOs) across 124 marine sites. Functional redundancy was highly correlated with a latent variable consisting of four ocean physiochemical parameters: oxygen and chlorophyll a concentrations, depth, and salinity. Functional redundancy was higher at the poles than in non-polar regions. Simultaneously, regional {beta}-diversity for individual functions was higher for functions with higher functional redundancy. These observations provide evidence that higher functional redundancy indicates increased stability of microbial ecosystem functions on spatiotemporal scales consistent with surface ocean mixing. We suggest that future changes in ocean physiochemistry could likely influence this stability for functions with lower functional redundancy.

ImportanceFunctional redundancy describes the state of multiple species performing the same function. Theory suggests functional redundancy stabilizes microbial community functions from disturbances leading to species loss or other changes to the microbiome. Previous work suggests that functional redundancy is common in ocean microbiomes which implies traits should be more stable among metacommunities. Some laboratory experiments demonstrate this idea, but it is difficult to test in the natural world. In a companion manuscript, we proposed a functional redundancy metric sensitive to trait stability. Here, we used this metric to show that functional redundancy varied substantially among ocean microbiomes and that regions with higher functional redundancy had higher regional trait stability. Last, we noted that variations in functional redundancy strongly correlated to ocean physiochemistry. Thus, changes in ocean physiochemistry via climate change may alter community traits to become more or less resistant to disturbance relative to contemporary conditions.

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Impaired photoprotection in Phaeodactylum tricornutum KEA3 mutants reveals the proton regulatory circuit of diatoms light acclimation. Diatoms are amongst the most successful clades of oceanic phytoplankton, significantly contributing to photosynthesis on Earth. Their ecological success likely stems from their ability to acclimate to changing environmental conditions, including e.g. variable light intensity. Diatoms are outstanding at dissipating light energy exceeding the maximum photosynthetic electron transfer (PET) capacity of via Non Photochemical Quenching (NPQ). While the molecular effectors of this process, as well as the role of the Proton Motive Force (PMF) in its regulation are known, the putative regulators of the PET/PMF relationship in diatoms remain unidentified. Here, we demonstrate that the H+/K+ antiporter KEA3 is the main regulator of the coupling between PMF and PET in the model diatom Phaeodactylum tricornutum. By controlling the PMF, it modulates NPQ responses at the onset of illumination, during transients and in steady state conditions. Under intermittent light KEA3 absence results in reduced fitness. Using a parsimonious model including only two components, KEA3 and the diadinoxanthin de-epoxidase, we can describe most of the feedback loops observed between PET and NPQ. This two-components regulatory system allows for efficient responses to fast (minutes) or slow (e.g. diel) changes in light environment, thanks to the presence of a regulatory Ca2+-binding domain in KEA3 that controls its activity. This circuit is likely finely tuned by the NPQ effector proteins LHCX, providing diatoms with the required flexibility to thrive in different ocean provinces.

One sentence summaryThe author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Giovanni Finazzi.

sentences: Trading Mental Effort for Confidence in the Metacognitive Control of Value-Based Decision-Making Why do we sometimes opt for actions or items that we do not value the most? Under current neurocomputational theories, such preference reversals are typically interpreted in terms of errors that arise from the unreliable signaling of value to brain decision systems. But, an alternative explanation is that people may change their mind because they are reassessing the value of alternative options while pondering the decision. So, why do we carefully ponder some decisions, but not others? In this work, we derive a computational model of the metacognitive control of decisions or MCD. In brief, we assume that fast and automatic processes first provide initial (and largely uncertain) representations of options values, yielding prior estimates of decision difficulty. These uncertain value representations are then refined by deploying cognitive (e.g., attentional, mnesic) resources, the allocation of which is controlled by an effort-confidence tradeoff. Importantly, the anticipated benefit of allocating resources varies in a decision-by-decision manner according to the prior estimate of decision difficulty. The ensuing MCD model predicts response time, subjective feeling of effort, choice confidence, changes of mind, and choice-induced preference change and certainty gain. We test these predictions in a systematic manner, using a dedicated behavioral paradigm. Our results provide a quantitative link between mental effort, choice confidence, and preference reversals, which could inform interpretations of related neuroimaging findings.

sentences: Horizontal Transfers Lead to the Birth of Momilactone Biosynthetic Gene Clusters in Grass Momilactone A, an important plant labdane-related diterpenoid, functions as a phytoalexin against pathogens and an allelochemical against neighboring plants. The genes involved in biosynthesis of momilactone A are found in clusters, i.e., MABGCs (Momilactone A biosynthetic gene clusters), in the rice and barnyardgrass genomes. How MABGCs originate and evolve is still not clear. Here, we integrated results from comprehensive phylogeny and comparative genomic analyses of the core genes of MABGC-like clusters and MABGCs in 40 monocot plant genomes, providing convincing evidence for the birth and evolution of MABGCs in grass species. The MABGCs found in the PACMAD clade of the core grass lineage (including Panicoideae and Chloridoideae) originated from a MABGC-like cluster in Triticeae (BOP clade) via horizontal gene transfer (HGT) and followed by recruitment of MAS and CYP76L1 genes. The MABGCs in Oryzoideae originated from PACMAD through another HGT event and lost CYP76L1 afterwards. The Oryza MABGC and another Oryza diterpenoid cluster c2BGC are two distinct clusters, with the latter being originated from gene duplication and relocation within Oryzoideae. Further comparison of the expression patterns of the MABGC genes between rice and barnyardgrass in response to pathogen infection and allelopathy provides novel insights into the functional innovation of MABGCs in plants. Our results demonstrate HGT-mediated origination of MABGCs in grass and shed lights into the evolutionary innovation and optimization of plant biosynthetic pathways.

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Ganglioside enriched phospholipid vesicles induce cooperative Aβ oligomerization and membrane disruption A major hallmark of Alzheimer disease (AD) is the accumulation of extracellular aggregates of amyloid-{beta} (A{beta}). Structural polymorphism observed among A{beta} fibrils in AD brains seem to correlate with the clinical sub-types suggesting a link between fibril polymorphism and pathology. Since fibrils emerge from a templated growth of low-molecular weight oligomers, understanding the factors affecting oligomer generation is important. The membrane lipids are key factors that influence early stages of A{beta} aggregation and oligomer generation, and cause membrane disruption. We have previously demonstrated that conformationally discrete A{beta} oligomers can be generated by modulating the charge, composition, chain length of lipids and surfactants. Here, we extend our studies into liposomal models by investigating A{beta} oligomerization on large unilamellar vesicles (LUVs) of total brain extracts (TBE), reconstituted lipid rafts (LRs) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Specifically, we varied the vesicle composition by varying the amount of GM1 gangliosides added as a constituent. We found that liposomes enriched in GM1 induce the formation of toxic, low-molecular weight oligomers that are isolable in a lipid-complexed form. Importantly, the data indicate that oligomer formation and membrane disruption are highly cooperative processes. Numerical simulations on the experimental data confirm cooperativity and reveal that GM1-enriched liposomes form twice as many numbers of pores as those without GM1. Overall, this study uncovers mechanisms of cooperativity between oligomerization and membrane disruption under controlled lipid compositional bias, and refocuses the significance of the early stages of A{beta} aggregation in polymorphism, propagation, and toxicity in AD.

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Non-Coding Genetic Analysis Implicates Interleukin 18 Receptor Accessory Protein 3'UTR in Amyotrophic Lateral Sclerosis The non-coding genome is substantially larger than the protein-coding genome but is largely unexplored by genetic association studies. Here, we performed region-based burden analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole-genomes and 70,403 non-ALS controls. We identified Interleukin-18 Receptor Accessory Protein (IL18RAP) 3'UTR variants significantly enriched in non-ALS genomes, replicated in an independent cohort, and associated with a five-fold reduced risk of developing ALS. Variant IL18RAP 3'UTR reduces mRNA stability and the binding of RNA-binding proteins. Variant IL18RAP 3'UTR further dampens neurotoxicity of human iPSC-derived C9orf72-ALS microglia that depends on NF-{kappa}B signaling. Therefore, the variant IL18RAP 3'UTR provides survival advantage for motor neurons co-cultured with C9-ALS microglia. The study reveals direct genetic evidence and therapeutic targets for neuro-inflammation, and emphasizes the importance of non-coding genetic association studies.

One Sentence SummaryNon-coding genetic variants in IL-18 receptor 3UTR decrease ALS risk by modifying IL-18-NF-{kappa}B signaling in microglia.

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Genomics-informed insights into microbial degradation of N,N-dimethylformamide Effective degradation of N,N-Dimethylformamide (DMF), an important industrial waste product, is challenging as only few bacterial isolates are known to be capable of degrading DMF. Aerobic remediation of DMF has typically been used, whereas anoxic remediation attempts are recently made, using nitrate as one electron acceptor, and ideally include methane as a byproduct. Here, we analyzed 20,762 complete genomes and 28 constructed draft genomes for the genes associated with DMF degradation. We identified 952 genomes that harbor genes involved in DMF degradation, expanding the known diversity of prokaryotes with these metabolic capabilities. Our findings suggest acquisition of DMF-degrading gene via plasmids are important in the order Rhizobiales and genus Paracoccus, but not in most other lineages. Degradation pathway analysis reveals that most putative DMF degraders using aerobic Pathway I will accumulate methylamine intermediate, while members of Paracoccus, Rhodococcus, Achromobacter, and Pseudomonas could potentially mineralize DMF completely under aerobic conditions. The aerobic DMF degradation via Pathway II is more common than thought and is primarily present in -and {beta}-Proteobacteria and Actinobacteria. Most putative DMF degraders could grow with nitrate anaerobically (Pathway III), however, genes for the use of methyl-CoM to produce methane were not found. These analyses suggest that microbial consortia could be more advantageous in DMF degradation than pure culture, particularly for methane production under the anaerobic condition. The identified genomes and plasmids form an important foundation for optimizing bioremediation of DMF-containing wastewaters.

ImportanceDMF is extensively used as a solvent in industries, and is classified as a probable carcinogen. DMF is a refractory compound resistant to degradation, and until now, only few bacterial isolates have been reported to degrade DMF. To achieve effective microbial degradation of DMF from wastewater, it is necessary to identify genomic diversity with the potential to degrade DMF and characterize the genes involved in two aerobic degradation pathways and potential anaerobic degradation for methane production. A wide diversity of organisms has the potential to degrade DMF. Plasmid-mediated degradation of DMF is important for Rhizobiales and Paracoccus. Most DMF degraders could grow anaerobically with nitrate as electron acceptor, while co-cultures are required to complete intermediate methanogenesis for methane production. This is the first genomics-based global investigation into DMF degradation pathways. The genomic database generated by this study provides an important foundation for the bioremediation of DMF in industrial waste waters.

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sentences: Wnt/β-catenin inhibition disrupts drug-tolerance in isogenic carboplatin-resistant models of Triple-Negative Breast Cancer. Triple-Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype, characterized by both limited treatment options and higher relapse rates than hormone-receptor-positive breast cancers. Chemotherapy remains the mainstay treatment for TNBC, and platinum salts have been explored as a therapeutic alternative in neo-adjuvant and metastatic settings. However, primary and acquired resistance to chemotherapy in general and platinum-based regimens specifically strongly hampers TNBC management. In this study, we used carboplatin-resistant in vivo patient-derived xenograft and isogenic TNBC cell-line models and detected enhanced Wnt/{beta}-catenin activity correlating with an induced expression of stem cell markers in both resistant models. In accordance, the activation of canonical Wnt signaling in parental TNBC cell lines increases stem cell markers expression, formation of tumorspheres, and promotes carboplatin resistance. Finally, we prove that Wnt signaling inhibition resensitizes resistant models to carboplatin both in vitro and in vivo, suggesting the synergistic use of Wnt inhibitors and carboplatin as a therapeutic option in TNBC. Here we provide evidence for a prominent role of Wnt signaling in mediating resistance to carboplatin, and we establish that combinatorial targeting of Wnt signaling overcomes carboplatin resistance enhancing chemotherapeutic drug efficacy.

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AutoSmarTrace: Automated Chain Tracing and Flexibility Analysis of Biological Filaments Single-molecule imaging is widely used to determine statistical distributions of molecular properties. One such characteristic is the bending flexibility of biological filaments, which can be parameterized via the persistence length. Quantitative extraction of persistence length from images of individual filaments requires both the ability to trace the backbone of the chains in the images and sufficient chain statistics to accurately assess the persistence length. Chain tracing can be a tedious task, performed manually or using algorithms that require user input and/or supervision. Such interventions have the potential to introduce user-dependent bias into the chain selection and tracing. Here, we introduce a fully automated algorithm for chain tracing and determination of persistence lengths. Dubbed "AutoSmarTrace", the algorithm is built off a neural network, trained via machine learning to identify filaments within images recorded using atomic force microscopy (AFM). We validate the performance of AutoSmarTrace on simulated images with widely varying levels of noise, demonstrating its ability to return persistence lengths in agreement with the ground truth. Persistence lengths returned from analysis of experimental images of collagen and DNA agree with previous values obtained from these images with different chain-tracing approaches. While trained on AFM-like images, the algorithm also shows promise to identify chains in other single-molecule imaging approaches, such as rotary shadowing electron microscopy and fluorescence imaging.

Statement of SignificanceMachine learning presents powerful capabilities to the analysis of large data sets. Here, we apply this approach to the determination of bending flexibility - described through persistence length - from single-molecule images of biological filaments. We present AutoSmarTrace, a tool for automated tracing and analysis of chain flexibility. Built on a neural network trained via machine learning, we show that AutoSmarTrace can determine persistence lengths from AFM images of a variety of biological macromolecules including collagen and DNA. While trained on AFM-like images, the algorithm works well to identify filaments in other types of images. This technique can free researchers from tedious tracing of chains in images, removing user bias and standardizing determination of chain mechanical parameters from single-molecule conformational images.

sentences: A multiplexed cell-free assay to screen for antimicrobial peptides in double emulsion droplets The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analysed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

sentences: Ecological correlates of reproductive status in a guild of Afrotropical understory trees The relative abundance patterns of tropical trees have been of interest since the expeditions of Alfred Russel Wallace, but little is known about how differences in relative abundance relate to reproductive patterns. Flowering is resource-dependent and fitness differences as well as differences in the quality of the abiotic and biotic neighborhood may contribute to the variation in reproductive status responsible for population-level flowering patterns. This variation determines the density and distance between flowering conspecifics and may alter relative abundance extremes among species during reproduction, factors known to influence pollination success. We collected flowering status data for a guild of twenty-three co-occurring tree species that flower in the understory of the Korup Forest Dynamics Plot in Cameroon. We examined how the occurrence and location of reproductive events were related to spatial patterns of adult abundance, focal tree size, neighborhood crowding, and habitat, while accounting for the influence of shared ancestry. Across species, the probability of flowering was higher for individuals of rarer species and for larger individuals but was unrelated to neighborhood crowding or habitat differences. Relative abundance extremes were reduced when only flowering individuals were considered, leading to a negative relationship between plot abundance and flowering probability at the species level that was not structured by shared ancestry. Spatially, flowering conspecifics tended to be overdispersed relative to all adult conspecifics. Rare species are predicted to suffer Allee effects or reduced fitness due to the difficulty of finding mates at low densities and frequencies. Here, however, rare species appear to maximize the size of their mate pool, compared to abundant species. If this partial  leveling of the playing field during reproduction is typical, it has consequences for our understanding of biodiversity maintenance and species coexistence in tropical forests.

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Comprehensive evaluation of human brain gene expression deconvolution methods Gene expression measurements, similar to DNA methylation and proteomic measurements, are influenced by the cellular composition of the sample analysed. Deconvolution of bulk transcriptome data aims to estimate the cellular composition of a sample from its gene expression data, which in turn can be used to correct for composition differences across samples. Although a multitude of deconvolution methods have been developed, it is unclear whether their performance is consistent across tissues with different complexities of cellular composition. The human brain is unique in its transcriptomic diversity, expressing the highest diversity of alternative splicing isoforms and non-coding RNAs. It comprises a complex mixture of cell-types including transcriptionally similar sub-types of neurons, which undergo gene expression changes in response to neuronal activity. However, a comprehensive assessment of the accuracy of transcriptome deconvolution methods on human brain data is currently lacking.

Here we carry out the first comprehensive comparative evaluation of the accuracy of deconvolution methods for human brain transcriptome data, and assess the tissue-specificity of our key observations by comparison with transcriptome data from human pancreas and heart.

We evaluate 8 transcriptome deconvolution approaches, covering all main classes: 4 partial deconvolution methods, each applied with 9 different cell-type signatures, 2 enrichment methods, and 2 complete deconvolution methods. We test the accuracy of cell-type estimates using in silico mixtures of single-cell RNA-seq data, mixtures of neuronal and glial RNA, as well as nearly 2,000 human brain samples.

Our results bring several important insights into the performance of transcriptome deconvolution: (a) We find that cell-type signature data has a stronger impact on brain deconvolution accuracy than the choice of method. (b) We demonstrate that biological factors influencing brain cell-type signature data (e.g. brain region, in vitro cell culturing), have stronger effects on the deconvolution outcome than technical factors (e.g. RNA sequencing platform). (c) We find that partial deconvolution methods outperform complete deconvolution methods on human brain data. To facilitate wider implementation of correction for cellular composition, we develop a webtool that implements the best performing methods, and is available at https://voineagulab.shinyapps.io/BrainDeconvShiny/ .

sentences: Combining Multi-Dimensional Molecular Fingerprints to Predict hERG Cardiotoxicity of Compounds At present, drug toxicity has become a critical problem with heavy medical and economic burdens. acLQTS (acquired Long QT Syndrome) is acquired cardiac ion channel disease caused by drugs blocking the hERG channel. Therefore, it is necessary to avoid cardiotoxicity in the drug design and computer models have been widely used to fix this plight. In this study, we present a molecular fingerprint based on the molecular dynamic simulation and uses it combined with other molecular fingerprints (multi-dimensional molecular fingerprints) to predict hERG cardiotoxicity of compounds. 203 compounds with hERG inhibitory activity (pIC50) were retrieved from a previous study and predicting models were established using four machine learning algorithms based on the single and multi-dimensional molecular fingerprints. Results showed that MDFP has the potential to be an alternative to traditional molecular fingerprints and the combination of MDFP and traditional molecular fingerprints can achieve higher prediction accuracy. Meanwhile, the accuracy of the best model, which was generated by consensus of four algorithms with multi-dimensional molecular fingerprints, was 0.694 (RMSE) in the test dataset. Besides, the number of hydrogen bonds from MDFP has been determined as a critical factor in the predicting models, followed by rgyr and sasa. Our findings provide a new sight of MDFP and multi-dimensional molecular fingerprints in building models of hERG cardiotoxicity prediction.

sentences: If Research is Not the Evidence, What is it? Explanations of the Lack of Research Citations in their Health Vlogs Doctor-influencers have the knowledge needed to understand scientific research, and they have the social media popularity to share it with large audiences. In this article, I explore the treatment of evidence in vlogs by Egyptian doctor-influencers: how they present it and what they use instead of it. I answer the following questions: 1) How do doctor-influencers present evidence on social media? 2) If evidence is not the research, then what is it? The data was collected through in-depth interviews with 12 Egyptian doctor-influencers from different specializations; two focus groups; and a critical discourse analysis of 48 of the influencers most popular and most engaging videos. I found that the doctor-influencers cited academic research only in the videos about medical controversies, new treatments, misinformation, or common medical mistakes. In most of their videos, the Egyptian doctor-influencers scarcely referred to medical research. This is because they believe that their audiences would not understand evidence due to their low educational attainment, weak research skills, and the language barrier (as most medical research is published in English rather than Arabic). These findings highlight the challenges of communicating science in societies with a low level of education. In these settings, communicators may need to move beyond mediating research studies on social media and instead focus on simplifying, translating, and connecting evidence-based information to the cultural context.

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AI-driven high-throughput automation of behavioral analysis and dual-channel wireless optogenetics for multiplexing brain dynamics Advances in behavioral optogenetics are limited by the absence of high-throughput pipelines for the automated analysis of behavior in freely behaving animals. Although a variety of AI algorithms has been proposed that enable automation of behavioral analysis, existing methods are generally low-throughput. In addition, optogenetic manipulation of neural circuits typically requires physical tethers to light sources, which limits the number of brain areas that can be targeted and thus constrains behavioral testing. Here, we develop a new wireless platform that combines a battery-free dual-channel optogenetic implant with an AI algorithm for high-throughput behavioral analysis. In our platform, a customized AI algorithm detected and quantified freezing behavior of rats that had undergone fear conditioning. Notably, our platform allows up to four enclosures to be monitored simultaneously. Wireless dual-channel optogenetic devices were implanted in the basolateral amygdala (BLA) to permit independent modulation of BLA principal neurons (red light, AAV-CaMKII-JAWS) or BLA interneurons (blue light, AAV-mDlx-ChR2) in freely behaving animals. In vivo validation with behaving rats demonstrates the utility of the telemetry system for large-scale optogenetic studies.

SignificanceAI algorithms can enable automation of behavioral analysis and thereby facilitate the progress on behavioral optogenetics. Successful integration of advanced wireless dual-channel optoelectronic devices with biological systems can also yield new tools and techniques for neuroscience research, particularly in the context of techniques for optogenetics. Here, we propose a new approach that combines an advanced AI algorithm with a low power wireless telemetry system, yielding powerful capabilities in the understanding of brain functions and the evaluation of the behavioral consequences of neural circuit manipulations. In vivo studies using optimized systems demonstrate high-throughput automation of behavioral manipulation and analysis via AI-powered wireless telemetry.

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Histone neutralization in a rat model of acute lung injury induced by double-hit lipopolysaccharide BackgroundAcute respiratory distress syndrome (ARDS) remains a challenge because of its high morbidity and mortality. Circulation histones levels in ARDS patients were correlated to disease severity and mortality. This study examined the impact of histone neutralization in a rat model of acute lung injury (ALI) induced by a lipopolysaccharide (LPS) double-hit.

MethodsSixty-eight male Sprague-Dawley rats were randomized to sham (N=8, received saline only) or LPS (N=60). The LPS double-hit consisted of a 0.8 mg/kg intraperitoneal injection followed after 16 hours by 5 mg/kg intra-tracheal nebulized LPS. The LPS group was then randomized into five groups: LPS only (N=12); LPS + 5, 25, or 100 mg/kg intravenous STC3141 every 8 hours (LPS+L, LPS+M, LPS+H, respectively, each N=12); or LPS + intraperitoneal dexamethasone 2.5 mg/kg every 24 hours for 56 hours (LPS+D, N=12) The animals were observed for 72 hours.

ResultsLPS animals developed ALI as suggested by lower oxygenation, lung edema formation, and histological changes compared to the sham animals. Compared to the LPS group, LPS+H and +D animals had significantly lower circulating histone levels; only the LPS+D group had significantly lower bronchoalveolar lavage fluid (BALF) histone concentrations. The LPS+L, +M, +H and +D groups had improved oxygenation compared to the LPS group and the LPS+H and +D groups had a lower lung wet-to-dry ratio. All animals survived.

ConclusionNeutralization of histone using STC3141, especially at high dose, had similar therapeutic effects to dexamethasone in this LPS double-hit rat ALI model, with significantly decreased circulating histone concentration, improved oxygenation, and decreased lung edema formation.

sentences: Benchmarking functional connectivity by the structure and geometry of the human brain The brains structural connectivity supports the propagation of electrical impulses, manifesting as patterns of co-activation, termed functional connectivity. Functional connectivity emerges from the underlying sparse structural connections, particularly through poly-synaptic communication. As a result, functional connections between brain regions without direct structural links are numerous, but their organization is not completely understood. Here we investigate the organization of functional connections without direct structural links. We develop a simple, data-driven method to benchmark functional connections with respect to their underlying structural and geometric embedding. We then use this method to re-weigh and re-express functional connectivity. We find evidence of unexpectedly strong functional connectivity within the canonical intrinsic networks of the brain. We also find unexpectedly strong functional connectivity at the apex of the unimodal-transmodal hierarchy. Our results suggest that both phenomena - functional modules and functional hierarchies - emerge from functional interactions that transcend the underlying structure and geometry. These findings also potentially explain recent reports that structural and functional connectivity gradually diverge in transmodal cortex. Collectively, we show how structural connectivity and geometry can be used as a natural frame of reference with which to study functional connectivity patterns in the brain.

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Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

Author summaryDengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

sentences: Single-cell analysis reveals cellular heterogeneity and molecular determinants of hypothalamic leptin-receptor cells Hypothalamic nuclei which regulate homeostatic functions express leptin receptor (LepR), the primary target of the satiety hormone leptin. Single-cell RNA sequencing (scRNA-seq) has facilitated the discovery of a variety of hypothalamic cell types. However, low abundance of LepR transcripts prevented further characterization of LepR cells. Therefore, we perform scRNA-seq on isolated LepR cells and identify eight neuronal clusters, including three uncharacterized Trh-expressing populations as well as 17 non-neuronal populations including tanycytes, oligodendrocytes and endothelial cells. Food restriction had a major impact on Agrp neurons and changed the expression of obesity-associated genes. Multiple cell clusters were enriched for GWAS signals of obesity. We further explored changes in the gene regulatory landscape of LepR cell types. We thus reveal the molecular signature of distinct populations with diverse neurochemical profiles, which will aid efforts to illuminate the multi-functional nature of leptins action in the hypothalamus.

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Youthful and age-related matreotypes predict drugs promoting longevity The identification and validation of drugs that promote health during aging ( geroprotectors) is key to the retardation or prevention of chronic age-related diseases. Here we found that most of the established pro-longevity compounds shown to extend lifespan in model organisms also alter extracellular matrix gene expression (i.e., matrisome) in human cell lines. To harness this novel observation, we used age-stratified human transcriptomes to define the age-related matreotype, which represents the matrisome gene expression pattern associated with age. Using a  youthful matreotype, we screened in silico for geroprotective drug candidates. To validate drug candidates, we developed a novel tool using prolonged collagen expression as a non-invasive and in-vivo surrogate marker for C. elegans longevity. With this reporter, we were able to eliminate false positive drug candidates and determine the appropriate dose for extending the lifespan of C. elegans. We improved drug uptake for one of our predicted compounds, genistein, and reconciled previous contradictory reports of its effects on longevity. We identified and validated new compounds, tretinoin, chondroitin sulfate, and hyaluronic acid, for their ability to restore age-related decline of collagen homeostasis and increase lifespan. Thus, our innovative drug screening approach - employing extracellular matrix homeostasis - facilitates the discovery of pharmacological interventions promoting healthy aging.

HighlightsO_LIMany geroprotective drugs alter extracellular matrix gene expression
C_LIO_LIDefined young and old human matreotype signatures can identify novel potential geroprotective compounds
C_LIO_LIProlonged collagen homeostasis as a surrogate marker for longevity
C_LI

sentences: Complementing machine learning-based structure predictions with native mass spectrometry The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) has moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may therefore require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations, that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on the small and the large scale.

sentences: A CTP-dependent gating mechanism enables ParB spreading on DNA in Caulobacter crescentus Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA 1. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

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

sentences: RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control It is becoming apparent that translational regulation involves the coordinated actions of RNA binding proteins (RBPs) and non-coding RNAs. For efficient translation, mRNA needs to be circularized, which is catalyzed by RNA binding proteins and translation factors. However, the role of lncRNAs in the process is not yet defined. We first performed RNA-seq and RNA- immunoprecipitation coupled-Seq and identified LSAMP-3 and Flii-1. Moreover, modeling studies suggest enhanced kissing loop interactions including of transcripts that encode angiogenesis and epithelial mesenchymal transition. While intestinal epithelial cell specific RBM3 transgenic mice showed increased LSAMP-3 and Flii-1, this was reduced in knockout mice. Also, RBM3 overexpression increased tumor xenograft growth, this was suppressed by knockdown of the lncRNAs. Also, knockdown of endogenous RBM3 reduced lncRNA levels and tumor xenograft growth. In addition, it reduced colitis-associated cancers. We propose that RBPs such as RBM3 mediate their function through regulatory lncRNAs that enable circularization to control translation.

sentences: KOMB: Graph-Based Characterization of Genome Dynamics in Microbial Communities Characterizing metagenomic samples via kmer-based, database-dependent taxonomic classification methods has provided crucial insight into underlying host-associated microbiome dynamics. However, novel approaches are needed that are able to track microbial community dynamics within metagenomes to elucidate genome flux in response to perturbations and disease states. Here we describe KOMB, a novel approach for tracking homologous regions within microbiomes. KOMB utilizes K-core graph decomposition on metagenome assembly graphs to identify repetitive and homologous regions to varying degrees of resolution. K-core performs a hierarchical decomposition which partitions the graph into shells containing nodes having degree at least K, called K-shells, yielding O(V + E) complexity compared to exact betweenness centrality complexity of O(V E) found in prior related approaches. We show through rigorous validation on simulated, synthetic, and real metagenomic datasets that KOMB accurately recovers and profiles repetitive and homologous genomic regions across organisms in the sample. KOMB can also identify functionally-rich regions in Human Microbiome Project (HMP) datasets, and can be used to analyze longitudinal data and identify pivotal taxa in fecal microbiota transplantation (FMT) samples. In summary, KOMB represents a novel approach to microbiome characterization that can efficiently identify sequences of interest in metagenomes.

sentences: Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Plasticity of Clytia Medusa Cell Types We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica, and determine how its component cell types respond to starvation. Utilizing multiplexed scRNA-seq, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for future studies of development, function, and plasticity in a genetically tractable jellyfish species. Moreover, we introduce a powerful workflow for high-resolution, whole animal, multiplexed single-cell genomics (WHAM-seq) that is readily adaptable to other traditional or non-traditional model organisms.

sentences: Evaluation of heat inactivation of human norovirus in freshwater clams using human intestinal enteroids Foodborne disease attributed to consumption of shellfish contaminated with human norovirus (HuNoV) is one of many global health concerns. Our study aimed to determine conditions of heat-inactivation of HuNoV in freshwater clams (Corbicula japonica) using a recently developed HuNoV cultivation system employing stem-cell derived human intestinal enteroids (HIEs). We first measured the internal temperature of clam tissue in a water bath during boiling at 90 {degrees}C and found that approximately 2 minutes are required for the tissue to reach 90 {degrees}C. Next, GII.4 HuNoV was spiked into the center of the clam tissue followed by boiling at 90 {degrees}C for 1, 2, 3, or 4 minutes. The infectivity of the HuNoV in clam tissue homogenates was evaluated using HIEs. We demonstrated that HuNoV in unboiled clam tissue homogenates replicated in HIEs, whereas infectivity was lost in all boiled samples, indicating that heat treatment at 90 {degrees}C for 1 minute is sufficient to inactivate HuNoV in freshwater clams. To our knowledge, this is the first study to determine the infectivity of HuNoV tolerability in shellfish using HIEs and our results will be informative to develop strategies to inactivate HuNoV in foods.

sentences: Emergent vulnerability to intensive coastal anthropogenic disturbances in mangrove forests Mangrove forests, as one of the most productive coastal ecosystems in tropical and subtropical areas, provide multiple valuable ecosystem services for human well-being. Mangrove coverage has been declining dramatically across much of developing regions due to extensive coastal anthropogenic disturbances such as reclamation, aquaculture, and seawall construction. As coastal human activities increase, there is urgent need to understand not only the direct loss, but also the vulnerability of mangroves to anthropogenic disturbances. In this study, we evaluated spatial pattern of mangrove vulnerability based on the conceptual framework of "Exposure-Sensitivity-Resilience" using geospatial datasets in mainland China. We find that within all 25,829 ha mangroves in five coastal provinces of mainland China in 2015, nearly 76% of mangroves was exposed or threatened by anthropogenic disturbances. Coastal reclamation and aquaculture were the key threats causing mangrove vulnerability. The overall distribution of high, medium and low vulnerability was following similar trend of aquaculture distribution, which suggests aquaculture was the greatest anthropogenic disturbance agent to mangroves. Hotspot regions for mangrove vulnerability are located at the developing provinces such as Guangxi and Hainan. This study provides the first spatially explicit evidence of the vulnerability of mangrove forests to intensive coastal anthropogenic disturbances at national scale, cloud serve as a benchmark for navigating coastal ecological redline management and coastal ecosystem restoration.

sentences: Phosphorylation by casein kinase 2 ensures ER-phagy receptor TEX264 binding to ATG8 proteins Selective autophagy cargos are recruited to autophagosomes primarily by interacting with autophagosomal ATG8 family proteins via the LC3-interacting region (LIR). The upstream sequence of most LIRs contains negatively charged residues such as Asp, Glu, and phosphorylated Ser and Thr. However, the significance of LIR phosphorylation (compared with having acidic amino acids) and the structural basis of phosphorylated LIR-ATG8 binding are not entirely understood. Here, we show that the serine residues upstream of the core LIR of the endoplasmic reticulum (ER)-phagy receptor TEX264 are phosphorylated by casein kinase 2, which is critical for its interaction with ATG8s, autophagosomal localization, and ER-phagy. Structural analysis showed that phosphorylation of these serine residues increased binding affinity by producing multiple hydrogen bonds with ATG8s that cannot be mimicked by acidic residues. This binding mode is different from those of other ER-phagy receptors that utilize a downstream helix, which is absent from TEX264, to increase affinity. These results suggest that phosphorylation of the LIR is critically important for strong LIR-ATG8 interactions, even in the absence of auxiliary interactions.

sentences: A neural circuit basis for binasal input-enhanced chemosensory avoidance Our understanding of how bilaterian animals utilize parallel input channels from paired sensory organs to optimize chemosensory behavior and the underlying neural circuit mechanisms are limited. Here we developed microfluidics-based behavioral and brainwide imaging platforms to study the neural integration of binasal inputs and chemosensory avoidance in larval zebrafish. We show that larval zebrafish efficiently escape from cadaverine-carrying streams by making more frequent swim bouts and larger undirected turns. Binasal inputs are strictly required for the nasal input-dependent component of klinokinesis, while each nasal input additively enhances angular orthokinesis. Throughout brain regions, including those along the olfactory processing pathways, a distributed neural representation with a wide spectrum of ipsilateral-contralateral nasal stimulus selectivity is maintained. Nonlinear sensory information gain with bilateral signal convergence is especially prominent in neurons weakly encoding unilateral cadaverine stimulus, and associated with stronger activation of sensorimotor neurons in the downstream brain regions. Collectively, these results provide insights into how the vertebrate model sums parallel input signals to guide chemosensory avoidance behavior.

sentences: Marine biofilms on different fouling control coating types reveal differences in microbial community composition and abundance Marine biofouling imposes serious environmental and economic impacts on marine applications, especially in the shipping industry. To combat biofouling, protective coatings are applied on vessel hulls which are divided into two major groups: biocidal and non-toxic fouling-release. The aim of the current study was to explore the effect of coating type on microbial biofilm community profiles to better understand the differences between the communities developed on fouling control biocidal antifouling and biocidal-free coatings. Biocidal (Intersmooth(R) 7460HS SPC), fouling-release (Intersleek(R) 900), and inert surfaces were deployed in the marine environment for 4 months and the biofilms that developed on these surfaces were investigated using Illumina NGS sequencing, targeting the prokaryotic 16S rRNA gene. The results confirmed differences in the community profiles between coating types. The biocidal coating supported communities dominated by Alphaproteobacteria (Loktanella, Sphingorhabdus, Erythrobacter) and Bacteroidetes (Gilvibacter), whilst other taxa such as Portibacter and Sva0996 marine group, proliferated on the fouling-release surface. Knowledge of these marine biofilm components on fouling control coatings will serve as a guide for future investigations of marine microfouling as well as informing the coatings industry of potential microbial targets for robust coating formulations.

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Bacillus sp. RZ2MS9, a tropical PGPR, colonizes maize endophytically and alters the plant's production of volatile organic compounds both independently and when co-inoculated with Azospirillum brasilense Ab-V5 Bacillus spp. are among the most efficient known plant growth-promoting rhizobacteria (PGPR). The PGPR Bacillus sp. strain RZ2MS9 is a multi-trait maize growth promoter previously isolated from guarana plants cultivated in the Amazon rainforest. However, there are several aspects of its interaction with the host that need further investigation. To achieve effective performance of microbial inoculants in crop production, it is necessary to monitor the plants colonization by a PGPR and to assess the potential synergy among beneficial strains. Here, we obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We verified that the insertion of the plasmid did not affect either bacterial growth nor its ability to promote maize growth in vitro. Using fluorescent microscopy and qPCR, we demonstrated that RZ2MS9-GFP successfully colonizes maizes roots and leaves endophytically. Subsequently, we evaluated whether RZ2MS9 has a synergistic effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5, a commercial inoculant for maize. The two strains combined enhanced maizes roots and shoots dry weight by 50.8% and 79.6%, respectively, when compared to the non-inoculated control. In addition, we used co-inoculation experiments in glass chambers to analyze the plants Volatile Organic Compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maizes VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the tropical PGPR Bacillus sp. RZ2MS9 and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

ImportanceBacillus sp. RZ2MS9 is a PGPR, previously isolated from guarana plants cultivated in the Brazilian Amazon, which successfully promotes the growth of maize and soybean plants. To improve our knowledge about the interaction between this very promising PGPR and maize, we labelled RZ2MS9 with gfp and monitored its maize colonization. The transformation did not affect either RZ2MS9 growth nor its ability to promote maize growth in vitro. We demonstrated that RZ2MS9 colonizes endophytically maizes roots and leaves. We also verified that the co-inoculation of RZ2MS9 and Azospirillum brasilense Ab-V5, a known commercial maize inoculant enhanced maizes roots and shoots growth. Moreover, the co-inoculation altered the maizes volatile organic compounds, increasing the production of indoles, that is related with decreased upon the reduction of fertilization. Certainly, our research contributed with better Bacillus sp. RZ2MS9 - maize interaction understanding and also provided new information concerning RZ2MS9 activity when applied with A. brasilense Ab-V5.

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Molecular-enriched functional connectivity in the human brain using multiband multi-echo simultaneous ASL/BOLD fMRI Receptor-Enriched Analysis of functional Connectivity by Targets (REACT) is a novel analytical strategy that enriches functional connectivity (FC) information from functional MRI (fMRI) with molecular information on the neurotransmitter distribution density in the human brain, providing a biological basis to the FC analysis. So far, this integrative approach has been used in blood oxygen level-dependent (BOLD) fMRI studies only, providing new insights into the brain mechanisms underlying specific disorders and its response to pharmacological challenges. In this study, we demonstrate that the application of REACT can be further extended to arterial spin labelling (ASL) fMRI. Some of the advantages of this extension include the combination of neurotransmitter specific information provided by molecular imaging with a quantitative marker of neuronal activity, the suitability of ASL for pharmacological MRI (phMRI) studies assessing drug effects on baseline brain function, and the possibility to acquire images that are not affected by susceptibility artifacts in the regions linked to major neurotransmitter systems.

In this work, we tested the feasibility of applying REACT to resting state ASL fMRI and compared the molecular-enriched FC maps derived from ASL data with those derived from BOLD data. We applied REACT to high-resolution, whole-brain simultaneous ASL/BOLD resting-state fMRI data of 29 healthy subjects and estimated the ASL- and BOLD-based FC maps related to six molecular systems, including the transporters of dopamine, noradrenaline, serotonin and vesicular acetylcholine, and the GABA-A and mGlu5 receptors. We then compared the ASL and BOLD FC maps in terms of spatial similarity, using the Dice Similarity Index and the voxel-wise spatial correlation. On a data subsample (N=19) we also evaluated the test-retest reproducibility of each modality using the regional intraclass correlation coefficient, and compared the two modalities.

Our results showed robust spatial patterns of molecular-enriched functional connectivity for both modalities, moderate to high similarity between BOLD- and ASL-derived FC maps and mixed results in terms of reproducibility (i.e., none of the modalities outperformed the other). Overall, our findings show that the ASL signal is as informative as BOLD in detecting functional circuits associated with specific molecular pathways, and that the two modalities may provide complementary information related to these circuits.

Considering the more direct link of ASL imaging with neuronal acrivity compared to BOLD and its suitability for phMRI studies, this new integrative approach could become a valuable asset in clinical studies investigating functional alterations in patients with brain disorders, or in pharmacological studies investigating the effects of new or existing compounds on the brain.

sentences: In Silico Evaluation of Plant Nitrification Suppression Effects on Agroecosystem Nitrogen Loss AO_SCPLOWBSTRACTC_SCPLOWNitrification regulates potential for nitrogen (N) loss from ecosystems because it converts ammonium to nitrate, which is susceptible to leaching and gaseous emissions. Crops can suppress the microbes that perform nitrification by exuding nitrification-inhibiting compounds from their roots and taking up available ammonium, the substrate for nitrification. However, the effect of nitrification suppression on agroecosystem N losses remains poorly characterized, and a lack of temporal synchrony between nitrification, N losses, and nitrification suppression by plants could limit the effect of nitrification suppression. We used the DayCent-CABBI model to evaluate the effectiveness of the suppression of nitrification by sorghum to reduce N2O emissions and nitrate leaching in an energy sorghum/soybean rotation at the Energy Farm in Urbana-Champaign, IL. We simulated nitrification suppression at the measured levels (measNS) and at the maximum measured level applied to the entire growing season (maxNS), and we also explored ways to better utilize nitrification suppression by altering the timing of UAN fertilizer applications. Model experiments showed that most nitrification occurred immediately after fertilizer was applied, whereas nitrification suppression begins to ramp up more than a month after planting. On an annual basis, measNS experiments showed a 1-2% reduction in annual N2O emissions relative to no nitrification suppression (noNS), and maxNS experiments showed a 4-9% reduction in annual N2O emissions relative to noNS. Both nitrification suppression levels showed < 1% reduction in nitrate leaching. Altering the timing of fertilizer applications to better synchronize nitrification suppression with high soil ammonium levels had mixed effects on annual N2O emissions and nitrate leaching and sometimes resulted in increased N losses. The timing of simulated N2O emissions shifted with the timing of fertilization, and N2O emissions from denitrification increased when N2O emissions from nitrification decreased. Increasing N retention during the non-growing season may be more effective and growing-season nitrification suppression for reducing annual N losses in the rainfed Midwest, particularly for NO3- leaching in the early spring. Optimizing the timing of nitrification suppression alongside off-season N retention strategies would best improve the N sustainability of agroecosystems.

sentences: Viral Phrenology We introduce Viral Phrenology, a new scheme for understanding the genomic composition of spherical viruses base on the locations of their structural protrusions. We used icosahedral point arrays to classify 135 distinct viral capsids collected from over 600 capsids available in the VIPERdb. Using gauge points of point arrays, we found 149 unique structural protrusions. We then show how to use the locations of these protrusions to determine the genetic composition of the virus. We then show that ssDNA, dsDNA, dsRNA and ssRNA viruses use different arrangements for distributing their protrusions. We also found that Triangulation number is also partially dependent on the structural protrusions. This analysis begins to tie together Baltimore classification and Triangulation number using point arrays.

sentences: Extracellular matrix stiffness and TGFβ2 regulate YAP/TAZ activity in human trabecular meshwork cells Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGF{beta}2) levels in aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM (GTM) cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGF{beta}2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ transcriptional activity potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGF{beta}2 increased YAP/TAZ nuclear localization in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGF{beta}2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGF{beta}2-induced HTM/GTM hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

sentences: MCIBox: A Toolkit for Single-molecule Multi-way Chromatin Interaction Visualization and Micro-Domains Identification The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for Multi-way Chromatin Interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell cycle associated genes. MCIBox could potentially distinguish the specificity of single-molecule chromatin interaction patterns in various phases of a cell cycle or cell types.

sentences: Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. We find that no single method is best for all cases. Instead, the optimal storage and extraction methods vary by taxa, by tissue, and by down-stream application. Therefore, we provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for Phase 1 of the Vertebrate Genomes Project (VGP), whose ultimate mission is to generate chromosome-level reference genome assemblies of all [~]70,000 extant vertebrate species.

sentences: Pixelwise H-score: a novel digital image analysis based-metric to quantify membrane biomarker expression from immunohistochemistry images Immunohistochemistry (IHC) assays play a central role in evaluating biomarker expression in tissue sections for diagnostic and research applications. Manual scoring of IHC images, which is the current standard of practice, is known to have several shortcomings in terms of reproducibility and scalability to large scale studies. Here, by using a digital image analysis-based approach, we introduce a new metric called the pixelwise H-score (pix H-score) that quantifies biomarker expression from whole-slide scanned IHC images. The pix H-score is an unsupervised algorithm that only requires the specification of intensity thresholds for the biomarker and the nuclear-counterstain channels. We present the detailed implementation of the pix H-score in two different whole-slide image analysis software packages Visiopharm and HALO. We consider three biomarkers P-cadherin, PD-L1, and 5T4, and show how the pix H-score exhibits tight concordance to multiple orthogonal measurements of biomarker abundance such as the biomarker mRNA transcript and the pathologist H-score. We also compare the pix H-score to existing automated image analysis algorithms and demonstrate that the pix H-score provides either comparable or significantly better performance over these methodologies. We also present results of an empirical resampling approach to assess the performance of the pix H-score in estimating biomarker abundance from select regions within the tumor tissue relative to the whole tumor resection. We anticipate that the new metric will be broadly applicable to quantify biomarker expression from a wide variety of IHC images. Moreover, these results underscore the benefit of digital image analysis-based approaches which offer an objective, reproducible, and highly scalable strategy to quantitatively analyze IHC images.

sentences: The ataxia protein sacsin is required for integrin trafficking and synaptic organization Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS, which manifest as a childhood-onset cerebellar ataxia. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament (IF) disorganization, and loss of Purkinje neurons. It is unclear how the loss of SACS causes these deficits, or why they manifest as cerebellar ataxia. We employed a multi-omics approach to characterize molecular and cellular deficiencies in SACS knockout (KO) cells. We identified alterations in microtubule structure and dynamics, protein trafficking, and mislocalization of synaptic and focal adhesion proteins. Targeting PTEN, a negative regulator of focal adhesions, rescued several cellular phenotypes in SACS KO cells. We found sacsin interacts with proteins implicated in vesicle transport, including HSP proteins, and interactions between structural and cell adhesion proteins were diminished in SACS KO cells. In all, this study suggests that trafficking and localization of synaptic adhesion proteins is a causal molecular deficiency in ARSACS.

sentences: Mitogenomic analyses provide further evidence for multiple miniaturization events during the evolution of Seychelles caecilian amphibians Whole mitochondrial genomes have been helpful in estimating phylogenetic relationships in many organismal groups, including caecilian amphibians. Despite the increasing ease of obtaining mitochondrial genome sequences from high-throughput sequencing, several species of caecilian lack this important molecular resource. As part of a targeted-sequence capture project of nuclear ultraconserved elements for a small but substantially diverse radiation of caecilian amphibians found on the granitic Seychelles, we examined off-target sequences to determine if we captured enough mitochondrial fragments to reconstruct mitogenomes. We reconstructed (near-)complete mitogenomes for six of the eight species of Seychelles caecilians and completed 14 independent phylogenetic analyses (Bayesian inference and maximum likelihood) on different mitochondrial datasets assembled using different alignment techniques. As with other studies, we were unable to fully resolve internal phylogenetic relationships of the group. However, we found strong support in most analyses that a recently described miniaturized species, Hypogeophis pti, and another similarly-sized miniaturized species, H. brevis are not sister taxa. Our study suggests that miniature species of caecilians likely evolved at least twice on the Seychelles and highlights the need to revise genus-level taxonomy of Seychelles caecilians while providing further evidence that off-target sequences often contain enough mitochondrial fragments to reconstruct mitogenomes.

sentences: Spatial spread of white-nose syndrome in North America, 2006-2018 White-nose syndrome has caused massive mortality in multiple bat species and spread across much of North America, making it one of the most destructive wildlife diseases on record. This has also resulted in it being one of the most well-documented wildlife disease outbreaks, making it possible to look for changes in the pattern of spatial spread over time. We fit a series of spatial interaction models to the United States county-level observations of the pathogenic fungus, Pseudogymnoascus destructans, that causes white-nose syndrome. Models included the distance between caves, cave abundance, measures of winter length and winter onset, and species richness of all bats and hibernating bats only. We found that the best supported models included all of these factors, but that the particular structure and most informative covariates changed over the course of the outbreak, with winter length displacing winter onset as the most informative measure of winter conditions, and evidence for the effects total species richness and hibernation varying from year to year. We also found that weather had detectable effects on spread. While the effect sizes for cave abundance and species richness were relatively stable over the length of the outbreak, distance became less important as time went on. These findings indicate that although models produced early in the outbreak captured important and consistent aspects of the spatial spread of white-nose syndrome, there were also changes over time in the factors associated with spread, suggesting that forecasts may be improved by iterative model refinement.

sentences: Cellular and genetic drivers of RNA editing variation in the human brain Posttranscriptional adenosine-to-inosine modifications amplify the functionality of RNA molecules in the brain, yet the cellular and genetic regulation of RNA editing is poorly described. We quantified base-specific RNA editing across three major cell populations from the human prefrontal cortex: glutamatergic neurons, medial ganglionic eminence GABAergic neurons, and oligodendrocytes. We found more selective editing and RNA hyper-editing in neurons relative to oligodendrocytes. The pattern of RNA editing was highly cell type-specific, with 189,229 cell type-associated sites. The cellular specificity for thousands of sites was confirmed by single nucleus RNA-sequencing. Importantly, cell type-associated sites were enriched in GTEx RNA-sequencing data, edited [~]twentyfold higher than all other sites, and variation in RNA editing was predominantly explained by neuronal proportions in bulk brain tissue. Finally, we discovered 661,791 cis-editing quantitative trait loci across thirteen brain regions, including hundreds with cell type-associated features. These data reveal an expansive repertoire of highly regulated RNA editing sites across human brain cell types and provide a resolved atlas linking cell types to editing variation and genetic regulatory effects.

sentences: Discovery of Flaviviridae-derived endogenous viral elements in shrew genomes provide novel insights into Pestivirus ancient history As viral genomic imprints in host genomes, endogenous viral elements (EVEs) shed light on the deep evolutionary history of viruses, ancestral host ranges, and ancient viral-host interactions. In addition, they may provide crucial information for calibrating viral evolutionary timescales. In this study, we conducted a comprehensive in silico screening of a large dataset of available mammalian genomes for EVEs deriving from members of the viral family Flaviviridae, an important group of viruses including well-known human pathogens. We identified two novel pestivirus-like EVEs in the reference genome of the Indochinese shrew (Crocidura indochinensis). Homologs of these novel EVEs were subsequently detected in vivo by molecular detection and sequencing in 27 shrew species, including 26 species representing a wide distribution within the Crocidurinae subfamily and one in the Soricinae subfamily. Based on this wide distribution, we estimate that the integration event occurred before the last common ancestor of the subfamily, about 10.8 million years ago, attesting to an ancient origin of pestiviruses and Flaviviridae in general. Moreover, we provide the first description of Flaviviridae-derived EVEs in mammals even though the family encompasses numerous mammal-infecting members, including major human pathogens such as Zika, dengue, or hepatitis C viruses. This also suggests that shrews were past and perhaps also current natural reservoirs of pestiviruses. Taken together, our results expand the current known Pestivirus host range and provide novel insight into the ancient evolutionary history of pestiviruses and the Flaviviridae family in general.

sentences: Speeded detection of spectrotemporal modulations of sound We tested whether joint spectrotemporal sensitivity follows from spectrotemporal separability for normal-hearing conditions and for impaired-hearing simulations. In a manual reaction-time task, normal-hearing listeners had to detect the onset of a ripple (with density between 0-8 cycles/octave and a fixed modulation depth of 50%), that moved up or down the log-frequency axis at constant velocity (between 0-64 Hz), in an otherwise-unmodulated broadband white-noise. Spectral and temporal modulations elicited band-pass filtered sensitivity characteristics, with fastest detection rates around 1 cycle/oct and 32 Hz for normal-hearing conditions. These results closely resemble data from other studies that typically used the modulation-depth threshold as a sensitivity measure for spectral-temporal modulations. To simulate hearing-impairment, stimuli were processed with a 6-channel cochlear-implant vocoder, and a hearing-aid simulation that introduced spectral smearing and low-pass filtering. Reaction times were always much slower compared to normal hearing, especially for the highest spectral densities. Binaural performance was predicted well by the benchmark race model of statistical facilitation of independent monaural channels. For the impaired-hearing simulations this implied a "best-of-both-worlds" principle in which the listeners relied on the hearing-aid ear to detect spectral modulations, and on the cochlear-implant ear for temporal-modulation detection. Although singular-value decomposition indicated that the joint spectrotemporal sensitivity matrix could be largely reconstructed from independent temporal and spectral sensitivity functions, in line with time-spectrum separability, a significant inseparable spectral-temporal interaction was present in all hearing conditions. These results imply that the reaction-time task yields a solid and effective objective measure of acoustic spectrotemporal modulation sensitivity, which may also be applicable to hearing-impaired individuals.

sentences: Distinct ground state and activated state modes of spiking in forebrain neurons Neuronal firing patterns have significant spatiotemporal variability with no agreed upon theoretical framework. Using a combined experimental and modeling approach, we found that spike interval statistics can be described by discrete modes of activity. Of these, a "ground state" (GS) mode of low-rate spiking is universal among forebrain excitatory neurons and characterized by irregular spiking at neuron-specific rates. In contrast, "activated state" (AS) modes consist of spiking at characteristic timescales and regularity that are specific to neuron populations in a given region and brain state. The majority of spiking is contributed by GS mode, while neurons can transiently switch to AS spiking in response to stimuli or in coordination with population activity patterns. We hypothesize that GS spiking serves to maintain a persistent backbone of neuronal activity while AS modes support communication functions.

sentences: The genetic interaction of  REVOLUTA  and  WRKY53  links plant development, senescence, and immune responses In annual plants, tight coordination of successive developmental events is of primary importance to optimize performance under fluctuating environmental conditions. The recent finding of the genetic interaction of WRKY53, a key senescence-related gene with REVOLUTA, a master regulator of early leaf patterning, raises the question of how early and late developmental events are connected. Here, we investigated the developmental and metabolic consequences of an alteration of the REVOLUTA and WRKY53 gene expression, from seedling to fruiting. Our results show that REVOLUTA critically controls late developmental phases and reproduction while inversely WRKY53 determines vegetative growth at early developmental stages. We further show that these regulators of distinct developmental phases frequently, but not continuously, interact throughout ontogeny and demonstrated that their genetic interaction is mediated by the salicylic acid (SA). Moreover, we showed that REVOLUTA and WRKY53 are keys regulatory nodes of development and plant immunity thought their role in SA metabolic pathways, which also highlights the role of REV in pathogen defence. Together, our findings demonstrate how late and early developmental events are tightly intertwined by molecular hubs. These hubs interact with each other throughout ontogeny, and participate to the interplay between plant development and immunity.

sentences: Global distribution of the rooting zone water storage capacity reflects plant adaptation to the environment The rooting zone water storage capacity (S0) extends from the soil surface to the weathered bedrock (the Critical Zone) and determines land-atmosphere exchange during dry periods. Despite its importance to land-surface modeling, variations of S0 across space are largely unknown as they cannot be observed directly. We developed a method to diagnose global variations of S0 from the relationship between vegetation activity (measured by sun-induced fluorescence and by the evaporative fraction) and the cumulative water deficit (CWD). We then show that spatial variations in S0 can be predicted from the assumption that plants are adapted to sustain CWD extremes occurring with a return period that is related to the life form of dominant plants and the large-scale topographical setting. Predicted biome-level S0 distributions, translated to an apparent rooting depth (zr) by accounting for soil texture, are consistent with observations from a comprehensive zr dataset. Large spatial variations in S0 across the globe reflect adaptation of zr to the hydroclimate and topography and implies large heterogeneity in the sensitivity of vegetation activity to drought. The magnitude of S0 inferred for most of the Earths vegetated regions and particularly for those with a large seasonality in their hydroclimate indicates an important role for plant access to water stored at depth - beyond the soil layers commonly considered in land-surface models.

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Genome-wide association mapping of ethanol sensitivity in the Diversity Outbred mouse population BackgroundA strong predictor for the development of alcohol use disorders (AUDs) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled in part by genetic factors. Mice offer a powerful tool for elucidating the genetic basis of behavioral and physiological traits relevant to AUDs; but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations allow for the opportunity to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification.

MethodsWe have taken advantage of the Diversity Outbred (DO) mouse population to identify and precisely map quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 798 male J:DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high density MEGAMuga and GIGAMuga arrays to obtain genotypes ranging from 77,808 - 143,259 SNPs. In addition, we performed RNA sequencing in striatum to map expression QTLs and to identify gene expression-trait correlations.

ResultsWe then applied a systems genetic strategy to identify narrow QTLs and construct the network of correlations that exist between DNA sequence, gene expression values and ethanol-related phenotypes to prioritize our list of positional candidate genes.

ConclusionsOur results can be used to identify alleles that contribute to AUDs in humans, elucidate causative biological mechanisms, or assist in the development of novel therapeutic interventions.

sentences: Why are children so distractible? Development of attentional capacities and motor control from childhood to adulthood. Distractibility is the propensity to behaviorally react to irrelevant information. Though children are more distractible the younger they are, the precise contribution of attentional and motor components to distractibility and their developmental trajectories have not been characterized yet. We used a new behavioral paradigm to identify the developmental dynamics of components contributing to distractibility in a large cohort of French participants balanced, between age groups, in gender and socio-economic status (N=352; age: 6-25). Results reveal that each measure of these components, namely voluntary attention, distraction, impulsivity and motor control, present a distinct maturational timeline. In young children, increased distractibility is mostly the result of reduced sustained attention capacities and enhanced distraction, while in teenagers, it is the result of decreased motor control and increased impulsivity.

sentences: HaloTag8: an engineered protein tag to improve fluorophore performance HaloTag9 is an engineered variant of HaloTag7 with up to 40% higher brightness and increased fluorescence lifetime when labeled with fluorogenic rhodamines. Moreover, combining HaloTag9 with HaloTag7 and other fluorescent probes enabled live-cell multiplexing using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. The increased brightness of HaloTag9 and its use in fluorescence lifetime multiplexing makes it a powerful tool for live-cell imaging.

sentences: Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus Human metapneumovirus (hMPV) is a major cause of acute respiratory tract infections in infants and the elderly for which there are no approved vaccines or antibody therapies. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies, however, little is known about the humoral immune response generated by humans as a result of natural infection. Here, we use stabilized hMPV F proteins to interrogate memory B cells from two elderly donors. We obtained over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal antibody response to hMPV F in these individuals. Characterization of 136 of these monoclonal antibodies revealed broad recognition of the hMPV F surface, with potent neutralizing antibodies targeting each antigenic site. Cryo-EM structures of two neutralizing antibodies reveal the molecular basis for recognition of two prefusion-specific epitopes at the membrane-distal apex of hMPV F. Collectively these results provide new insights into the humoral response to hMPV infection in the elderly and will guide development of novel vaccine antigens.

sentences: Interference with lipoprotein maturation sensitizes methicillin-resistant Staphylococcus aureus to human group IIA secreted phospholipase A2 and daptomycin. Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with faster and increased cell wall penetration of hGIIA. Moreover, lspA deletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced a lspA mutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

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scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

MotivationscWGBS library preparation in a one-cell-per-library format presents practical and economical constraints to the number of cells that can be analyzed in a research project. In addition, most of the current scWGBS methods suffer from low read alignment rates. We present a scWGBS protocol which mitigates these issues, empowering single-cell DNA methylation analysis at an increased scale.

sentences: An automated microfluidic platform integrating functional vascularized organoids-on-chip The development of vascular networks on-chip is crucial for the long-term culture of three-dimensional cell aggregates such as organoids, spheroids, tumoroids, and tissue explants. Despite the rapid advancement of microvascular network systems and organoid technology, vascularizing organoids-on-chips remains a challenge in tissue engineering. Moreover, most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical settings to operate. Considering these constraints, we developed an innovative platform to establish and monitor the formation of endothelial networks around model spheroids of mesenchymal and endothelial cells as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 15 days on-chip. Importantly, these networks were functional, demonstrating intravascular perfusion within the spheroids or vascular organoids connected to neighbouring endothelial beds. This microphysiological system thus represents a viable organ-on-chip model to vascularize biological tissues and should allow to establish perfusion into organoids using advanced microfluidics.

sentences: 3D matrix adhesion composition facilitates nuclear force coupling to drive invasive cell migration Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

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AGO104 is an RdDM effector of paramutation at the maize b1 locus Paramutation is an exception among eukaryotes, in which epigenetic information is conserved through mitosis and meiosis. It has been studied for over 70 years in maize, but the mechanisms involved are largely unknown. Previously described actors of paramutation encode components of the RNA-dependent DNA-methylation (RdDM) pathway all involved in the biogenesis of 24-nt small RNAs. However, no actor of paramutation have been identified in the effector complex of RdDM. Here, through a combination of reverse genetics, immunolocalization and immunoprecipitation (siRNA-IP) we found that ARGONAUTE104 (AGO104), AGO105 and AGO119 are members of the RdDM effector complex in maize and bind siRNAs produced from the tandem repeats required for paramutation at the b1 locus. We also showed that AGO104 is an effector of the b1 paramutation in maize.

Author summaryReprogramming of epigenetic information has been described in both plants and mammals. Here, we show that maize ARGONAUTE (AGO) AGO104 and AGO105/AGO119, respectively the close homologs of A. thaliana AGO9 and AGO4, are required to enable paramutation at the b1 locus in maize. Paramutation is an epigenetic phenomenon that is stable over many generations (both mitotically and meiotically). A classic example is the booster1 (b1) gene in maize, where the weakly expressed Booster (B) allele stably decreases the expression of the Booster-Intense (B-I) allele, and changes it into a new B allele. This new B allele will in turn change B-I into new B in subsequent crosses. Previous research demonstrated that paramutation requires several proteins involved in the biosynthesis of small interfering RNAs (siRNAs) all related to the RNA-dependent DNA-methylation (RdDM) pathway. Yet, few members of the RdDM were functionally identified in maize. Here, we identify two new members of the maize RdDM pathway, and provide evidence that they are also involved in paramutation at the b1 locus.

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Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics.

One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

sentences: Delimitation of the Tick-Borne Flaviviruses. Resolving the Tick-Borne Encephalitis and Louping-Ill Virus Paraphyletic Taxa The tick-borne flavivirus (TBFV) group contains at least 12 members where five of them are important pathogens of humans inducing diseases with varying severity (from mild fever forms to acute encephalitis). The taxonomy structure of TBFV is not fully clarified at present. In particular, there is a number of paraphyletic issues of tick-borne encephalitis virus (TBEV) and louping-ill virus (LIV). In this study, we aimed to apply different bioinformatic approaches to analyze all available complete genome amino acid sequences to delineate TBFV members at the species level. Results showed that the European subtype of TBEV (TBEV-E) is a distinct species unit. LIV, in turn, should be separated into two species. Additional analysis of the diversity of TBEV and LIV antigenic determinants also demonstrate that TBEV-E and LIV are significantly different from other TBEV subtypes. The analysis of available literature provided data on other virus phenotypic particularities that supported our hypothesis. So, within the TBEV+LIV paraphyletic group, we offer to assign four species to get a more accurate understanding of the TBFV interspecies structure according to the modern monophyletic conception.

sentences: Conflict over fertilization underlies the transient evolution of reinforcement When two species meet in secondary contact, the production of low fitness hybrids may be prevented by the adaptive evolution of increased prezygotic isolation, a process known as reinforcement. Theoretical challenges to the evolution of reinforcement are generally cast as a coordination problem--e.g., recombination may break the association between trait and preference loci. However, the evolution of reinforcement also poses a potential conflict between mates. For example, the opportunity costs to hybridization may differ between the sexes or species. This is particularly likely for reinforcement based on postmating prezygotic (PMPZ) incompatibilities, as the ability to fertilize both conspecific and heterospecific eggs is beneficial to male gametes, but heterospecific mating may incur a cost for female gametes. We develop a population genetic model of interspecific conflict over reinforcement inspired by "gametophytic factors", which act as PMPZ barriers among Zea mays subspecies. We demonstrate that this conflict results in the transient evolution of reinforcement--after females adaptively evolve to reject gametes lacking a signal common in conspecific gametes, this gamete signal adaptively introgresses into the other population. Ultimately the male gamete signal fixes in both species, and prezygotic isolation returns to pre-reinforcement levels. We interpret geographic patterns of isolation among Z. mays subspecies in light of these findings, and suggest when and how this conflict can be resolved. Our results suggest that sexual conflict over fertilization may pose an understudied obstacle to the evolution of reinforcement via PMPZ incompatibilities.

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PPML-Omics: a Privacy-Preserving federated Machine Learning system protects patients' privacy from omic data Modern machine learning models towards various tasks with omic data analysis give rise to threats of privacy leakage of patients involved in those datasets. Despite the advances in different privacy technologies, existing methods tend to introduce too much noise, which hampers model accuracy and usefulness. Here, we built a secure and privacy-preserving machine learning (PPML) system by combining federated learning (FL), differential privacy (DP) and shuffling mechanism.

We applied this system to analyze data from three sequencing technologies, and addressed the privacy concern in three major tasks of omic data, namely cancer classification with bulk RNA-seq, clustering with single-cell RNA-seq, and the integration of spatial gene expression and tumour morphology with spatial transcriptomics, under three representative deep learning models. We also examined privacy breaches in depth through privacy attack experiments and demonstrated that our PPML-Omics system could protect patients privacy. In each of these applications, PPML-Omics was able to outperform state-of-the-art systems under the same level of privacy guarantee, demonstrating the versatility of the system in simultaneously balancing the privacy-preserving capability and utility in omic data analysis. Furthermore, we gave the theoretical proof of the privacy-preserving capability of PPML-Omics, suggesting the first mathematically guaranteed model with robust and generalizable empirical performance.

sentences: Validation of the first PGT-whole genome sequencing approach including mitochondrial variants Whole genome screening is currently not available in preimplantation genetic testing (PGT) due to poor performance of whole genome amplification methods. Here, we present the first validation study using our latest whole genome amplification (WGA) protocol, which yields amplified DNA with comparable quality to genomic DNA when perfoming whole genome sequencing assays. We started by validating PGT for aneuploidy (PGT-A) using our WGA protocol on cell lines and donated human embryos, where amplification success rates were >99.9% and 96.2% respectively. Accuracy on both was >99.9% on aneuploidy status. We next validated whole genome sequencing using amplified and genomic DNA from the Genome in the Bottle reference sample NA12878 to demonstrate accuracy, specificity, sensitivity, and precision of our WGA methods. Amplified DNA and genomic DNA are comparable in this case, with 99.99% accuracy, 99.99% specificity, 98.0% sensitivity and 98.1% precision with our WGA protocol. We additionally examined variant calls between biopsies and the remaining embryos from which they were derived. Again, 99.99% accuracy, 99.99% specificity, 98.1% sensitivity and 98.0% precision were observed. Mitochondrial heteroplasmy between biopsies and embryos was validated, and 99.9% accuracy, 100% specificity, 99.1% sensitivity and 100% precision were observed. Finally, we demonstrated the ability to improve the accuracy of certain types of mendelian variants in our data to close to 100% by leveraging parental and a born childs genome to perform linkage analysis.

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A novel hidden Markov approach to studying dynamic functional connectivity states in human neuroimaging IntroductionHidden Markov models are a popular choice to extract and examine recurring patterns of activity or functional connectivity in neuroimaging data, both in terms of spatial patterns and their temporal progression. Although many diverse hidden Markov models have been applied to neuroimaging data, most have defined states based on activity levels (intensity-based states) rather than patterns of functional connectivity between brain areas (connectivity-based states), which is problematic if we want to understand connectivity dynamics: intensity-based states are unlikely to provide useful information about dynamic connectivity patterns.

MethodsWe addressed this problem by introducing a new hidden Markov model that defines states based on full functional connectivity profiles among brain regions. We empirically explored the behavior of this new model in comparison to existing approaches based on intensity-based or summed functional connectivity states using the HCP unrelated 100 functional magnetic resonance imaging "resting state" dataset.

ResultsOur  full functional connectivity model discovered connectivity states with more distinguishable patterns than previous approaches, and recovered simulated connectivity-based states more faithfully.

DiscussionThus, if our goal is to extract and interpret connectivity states in neuroimaging data, our new model outperforms previous methods which miss crucial information about the evolution of functional connectivity in the brain.

Impact statementHidden Markov models can be used to investigate brain states noninvasively. Previous models "recover" connectivity from intensity-based hidden states, or from connectivity  summed across nodes. Here we introduce a novel connectivity-based hidden Markov model and show how it can reveal true connectivity hidden states under minimal assumptions.

sentences: Evidence for Normalization as a Fundamental Operation Across the Human Visual Cortex Divisive normalization of the neural responses by the activity of the neighboring neurons has been proposed as a fundamental operation in the nervous system based on its success in predicting neural responses recorded in primate electrophysiology studies. Nevertheless, experimental evidence for the existence of this operation in the human brain is still scant. Here, using functional MRI, we explored the role of normalization across the visual hierarchy in the human visual cortex. Using stimuli form the two categories of human bodies and houses, we presented objects in isolation or in clutter and asked participants to attend or ignore the stimuli.Focusing on the primary visual area V1, the object-selective regions LO and pFs, and the category-selective regions EBA and PPA, we first modeled single-voxel responses using a weighted sum, a weighted average, and a normalization model and demonstrated that the response to multiple stimuli could best be described by a model that takes normalization into account. We then explored the observed effects of attention on cortical responses and demonstrated that these effects were predicted by the normalization model, but not by the weighted sum or the weighted average models. Our results thus provide compelling evidence for normalization as a canonical computation operating in the human brain.

sentences: Logical and experimental modeling of cytokine and eicosanoid signaling in psoriatic keratinocytes Psoriasis is characterized by chronic inflammation, perpetuated by a Th17-dependent signaling loop between the immune system and keratinocytes that could involve phospholipase A2 (PLA2)-dependent eicosanoid release. A prior knowledge network supported by experimental observations was used to encode the regulatory network of psoriatic keratinocytes in a computational model for studying the mode of action of a cytosolic (c) PLA2 inhibitor. A combination of evidence derived from the computational model and experimental data suggests that Th17 cytokines stimulate pro-inflammatory cytokine expression in psoriatic keratinocytes via activation of cPLA2-PGE2-EP4 signaling, which could be suppressed using the anti-psoriatic calcipotriol. cPLA2 inhibition and calcipotriol showed overlapping and distinct modes of action. Model analyses revealed the immunomodulatory role of Th1 cytokines, the modulation of the physiological states of keratinocytes by Th17 cytokines, and how Th1 and Th17 cells together promote the development of psoriasis. Model simulations additionally suggest novel drug targets, including EP4 and PRKACA, for treatment that may restore a normal phenotype. Our work illustrates how the study of complex diseases can benefit from an integrated systems approach.

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Lifelong single-cell profiling of cranial neural crest diversification The cranial neural crest generates a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head. How neural crest cells acquire such extraordinary lineage potential remains unresolved. By integrating single-cell transcriptome and chromatin accessibility profiles of cranial neural crest-derived cells across the zebrafish lifetime, we observe region-specific establishment of enhancer accessibility for distinct fates. Neural crest-derived cells rapidly diversify into specialized progenitors, including multipotent skeletal progenitors, stromal cells with a regenerative signature, fibroblasts with a unique metabolic signature linked to skeletal integrity, and gill-specific progenitors generating cell types for respiration. By retrogradely mapping the emergence of lineage-specific chromatin accessibility, we identify a wealth of candidate lineage-priming factors, including a Gata3 regulatory circuit for respiratory cell fates. Rather than multilineage potential being an intrinsic property of cranial neural crest, our findings support progressive and region-specific chromatin remodeling underlying acquisition of diverse neural crest lineage potential.

HighlightsO_LISingle-cell transcriptome and chromatin atlas of cranial neural crest
C_LIO_LIProgressive emergence of region-specific cell fate competency
C_LIO_LIChromatin accessibility mapping identifies candidate lineage regulators
C_LIO_LIGata3 function linked to gill-specific respiratory program
C_LI

Graphical Abstract

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sentences: Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines, in primary human adipocytes, fibroblasts and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery and different LNP vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By super-resolution microscopy we could resolve single LNP-mRNA within sub-endosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.

sentences: Characterization of an operon required for growth on cellobiose in Clostridioides difficile Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pull-down experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model- substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

sentences: Chronic Intermittent Hypobaric Hypoxia Prevents Contrast-Induced Acute Kidney Injury By Modulating The HIF-1α Signaling Pathway The aim of this study was to explore the role of CIHH in preventing contrast-induced acute kidney injury (CI-AKI) in rats and its mechanism. Rats mean arterial pressure, heart rate, serum creatinine and blood urea nitrogen levels were measured. The kidney tissue pathological changes, superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, hypoxia inducible factor-1, Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) , cysteiny aspartate specific protease3(caspase3) and poly(ADP-ribose) polymerase (PARP) expression levels were testing. The results showed that CIHH prevented CI-AKI group mean arterial pressure, heart rate, serum creatinine and blood urea nitrogen levels were reduced, kidney tissue SOD activity was increased, MDA levels was reduced, HIF-1,BNIP3,caspase3 and PARP levels were increased than the CI-AKI group. This study indicates that CIHH pretreatment may have a protective effect on contrast-induced early kidney injury by activating the HIF-1/BNIP3 signaling pathway to regulate mitochondrial autophagy and enhance cellular anti-apoptotic and renal antioxidant capacity, for the first time.

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A multiwell-plate Caenorhabditis elegans assay for assessing the therapeutic potential of Bacteriophages against Clinical Pathogens In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffers from batch-to-batch variations and requires manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans which feeds on bacteria, a fast growing and easy to handle organism which can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae and EHEC/ EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages.

ImportanceValidating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism, C. elegans as a quick, reliable and simple alternative for testing phages. The data shows that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

sentences: Set4 coordinates the activity of histone deacetylases and regulates stress-responsive gene expression within subtelomeric regions in yeast The yeast chromatin protein Set4 is a member of the Set3-subfamily of SET domain proteins which play critical roles in the regulation of gene expression in diverse developmental and environmental contexts, although they appear to lack methyltransferase activity. The molecular functions of Set4 are relatively unexplored, likely due to its low abundance in standard growth conditions. We previously reported that Set4 promotes survival during oxidative stress and regulates expression of stress response genes via stress-dependent chromatin localization. In this study, global gene expression analysis and investigation of histone modification status has revealed a role for Set4 in maintaining gene repressive mechanisms within yeast subtelomeres under both normal and stress conditions. We show that Set4 works in a partially overlapping pathway to the SIR complex and the histone deacetylase Rpd3 to maintain proper levels of histone acetylation and expression of stress response genes encoded in subtelomeres. This role for Set4 is particularly critical for cells under hypoxic conditions, and the loss of Set4 decreases cell fitness and cell wall integrity in hypoxia. These findings uncover a new regulator of subtelomeric chromatin that is key to stress defense pathways and demonstrate a function for yeast Set4 in regulating repressive, heterochromatin-like environments.

sentences: EGCG-modified bone graft to modulate the recruitment of M1 macrophage and alleviate the forming of fibrous capsule In response to current trends in the modification of guided bone regeneration (GBR) materials, we aimed to build upon our previous studies on epigallocatechin-3-gallate (EGCG) by immersing a commonly used bone graft primarily composed of hydroxyapatite (HA) in EGCG solution, expecting to obtain superior bone-material integration after implantation. Bone grafts are commonly used for bone repair, in which the bone extracellular matrix is stimulated to promote osteogenesis. However, due to its pro-fibrosis effect, this osteoconductive material commonly exhibits implant failure. In addition to providing a basic release profile of EGCG-modified bone graft (E-HA) to clarify the relationship between this material and the environment, we have examined the integration effect via subcutaneous implantation experiments. In this manner, we have assessed the aggregation of pro-inflammatory macrophages, initial angiogenesis, the formation of fibrous capsules, and an enhanced cell viability observed in cultured RAW 264.7 cells. Among these results, we focus on pro-inflammatory macrophages due to their close relationship with fibrosis, which is the most important process in the immune response. Immunofluorescent staining results showed that E-HA substantially compromised the formation of fibrous capsules in hematoxylin-eosin-stained sections, which exhibited less pro-inflammatory macrophage recruitment; meanwhile, the cell viability and primary angiogenesis were improved. This work lays the foundation for future studies on GBR.

sentences: Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient Even though most axonal cargos are synthesized in the soma, the concentration of many of these cargos is larger at the presynaptic terminal than in the soma. This requires transport of these cargos from the soma to the presynaptic terminal or other active sites in the axon. Axons utilize both bidirectional (for example, slow axonal transport) and unidirectional (for example, fast anterograde axonal transport) modes of cargo transport. Bidirectional transport seems to be less efficient because it requires more time and takes more energy to deliver cargos. In this paper, we studied a family of models which differ by the modes of axonal cargo transport (such as anterograde and retrograde motor-driven transport and passive diffusion) as well as by the presence or absence of pausing states. The models are studied to investigate their ability to describe axonal transport against the cargo concentration gradient. We argue that bidirectional axonal transport is described by a higher-order mathematical model, which allows imposing cargo concentration not only at the axon hillock but also at the axon terminal. The unidirectional transport model allows only for the imposition of cargo concentration at the axon hillock. Due to the great lengths of the axons, anterograde transport mostly relies on molecular motors, such as kinesins, to deliver cargos synthesized in the soma to the terminal and other active sites in the axon. Retrograde transport can be also motor-driven, in which case cargos are transported by dynein motors. If cargo concentration at the axon tip is higher than at the axon hillock, retrograde transport can also occur by cargo diffusion. However, because many axonal cargos are large or they assemble in multiprotein complexes for axonal transport, the diffusivity of such cargos is very small. We investigated the case of a small cargo diffusivity using a perturbation technique and found that for this case the effect of diffusion is limited to a very thin diffusion boundary layer near the axon tip. If cargo diffusivity is decreased in the model, we show that without motor-driven retrograde transport the model is unable to describe a high cargo concentration at the axon tip. To the best of our knowledge, our paper presents the first explanation for the utilization of seemingly inefficient bidirectional transport in neurons.

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Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage The connexin family is a diverse group of highly regulated non-{beta}-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3,5-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 s simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 s of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner.

Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-{beta}-barrel wide-pore channels.

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Terminal heterocyst differentiation in the Anabaena patA mutant as a result of post-transcriptional modifications and molecular leakage The Anabaena genus is a model organism of filamentous cyanobacteria whose vegetative cells can differentiate under nitrogen-limited conditions into a type of cell called heterocyst. These heterocysts lose the possibility to divide and are necessary for the colony because they can fix and share environmental nitrogen. In order to distribute the nitrogen efficiently, heterocysts are arranged to form a quasi-regular pattern whose features are maintained as the filament grows. Recent efforts have allowed advances in the understanding of the interactions and genetic mechanisms underlying this dynamic pattern. However, the main role of the patA and hetF genes are yet to be clarified; in particular, the patA mutant forms heterocysts almost exclusively in the terminal cells of the filament. In this work, we investigate the function of these genes and provide a theoretical model that explains how they interact within the broader genetic network, reproducing their knock-out phenotypes in several genetic backgrounds, including a nearly uniform concentration of HetR along the filament for the patA mutant. Our results suggest a role of hetF and patA in a post-transcriptional modification of HetR which is essential for its regulatory function. In addition, the existence of molecular leakage out of the filament in its boundary cells is enough to explain the preferential appearance of terminal heterocysts, without any need for a distinct regulatory pathway.

Author summaryUnderstanding multicellular pattern formation is key for the study of both natural and synthetic developmental processes. Arguably one of the simplest model systems for this is the filamentous cyanobacterium Anabaena, that in conditions of nitrogen deprivation undergoes a dynamical differentiation process that differentiates roughly one in every ten cells into nitrogen-fixing heterocysts, in a quasi-regular pattern that is maintained as the filament keeps growing. One of the most characteristic mutations affecting this process forms heterocysts mostly constrained to the terminal cells of the filament. We have used experimental observations to propose a mathematical model of heterocyst differentiation able to reproduce this striking phenotype. The model extends our understanding of the regulations in this pattern-forming system and makes several predictions on molecular interactions. Importantly, a key aspect is the boundary condition at the filaments ends: inhibitors of differentiation should be able to leak out of the filament, or otherwise the terminal cells would not differentiate. This highlights, in a very clear example, the importance of considering physical constraints in developmental processes.

sentences: Using dual-calibrated functional MRI to map brain oxygen supply and consumption in multiple sclerosis Evidence suggests that cerebrovascular function and oxygen consumption are altered in multiple sclerosis (MS). Here, we quantified the vascular and oxygen metabolic MRI burden in patients with MS (PwMS) and assessed the relationship between these MRI measures of and metrics of damage and disability. In PwMS and in matched healthy volunteers, we applied a newly developed dual-calibrated fMRI method of acquisition and analysis to map grey matter (GM) cerebral blood flow (CBF), oxygen extraction fraction (OEF), cerebral metabolic rate of oxygen consumption (CMRO2) and effective oxygen diffusivity of the capillary network (DC). We also quantified physical and cognitive function in PwMS and controls. There was no significant difference in GM volume between 22 PwMS and 20 healthy controls (p=0.302). Significant differences in CBF (PwMS vs. controls: 44.91 {+/-} 6.10 vs. 48.90 {+/-} 5.87 ml/100g/min, p=0.010), CMRO2 (117.69 {+/-} 17.31 vs. 136.49 {+/-} 14.48 mol/100g/min p<0.001) and DC (2.70 {+/-} 0.51 vs. 3.18 {+/-} 0.41 mol/100g/mmHg/min, p=0.002) were observed in the PwMS. No significant between-group differences were observed for OEF (PwMS vs. controls: 0.38 {+/-} 0.09 vs. 0.39 {+/-} 0.02, p=0.358). Regional analysis showed widespread reductions in CMRO2 and DC for PwMS compared to healthy volunteers. There was a significant correlation between physiological measures and T2 lesion volume, but no association with current clinical disability. Our findings demonstrate concurrent reductions in oxygen supply and consumption in the absence of an alteration in oxygen extraction that may be indicative of a reduced demand for oxygen (O2), an impaired transfer of O2 from capillaries to mitochondria, and/or a reduced ability to utilise O2 that is available at the mitochondria. With no between-group differences in GM volume, our results suggest that changes in brain physiology may precede MRI-detectable GM loss and thus may be one of the pathological drivers of neurodegeneration and disease progression.

sentences: Investigating trait variability of gene co-expression network architecture in brain by manipulating genomic signatures of schizophrenia risk While the role of genomic risk for schizophrenia on brain gene co-expression networks has been described, the patterns of its manifestations are varied and complex. To acquire a deeper understanding of this issue, we implemented a novel approach to network construction by manipulating the RNA-Seq expression input to "integrate" or "remove" the "modulatory" effects of genomic risk for schizophrenia. We created co-expression networks in DLPFC from the adjusted expression input and compared them in terms of gene overlap and connectivity. We used linear regression models to remove variance explained by RNA quality, cell type proportion, age, sex and genetic ancestry. We also created co-expression networks based on the genomic profile of a normative trait, height, as a "negative control"; we also applied the same analytical approach in two independent samples: LIBD Human Brain Repository (HBR) (N=78 brains, European ancestry) and Common Mind Consortium (CMC) (N=116 brains, European ancestry). In addition to direct comparisons, we explored the biological plausibility of the differential gene clusters between co-expression networks by testing them for enrichment in relevant gene ontologies and gene sets of interest (PGC2-CLOZUK GWAS significant loci genes, height GWAS significant loci genes, genes in synaptic ontologies-SynGO and genes of the "druggable genome"). We identify several key aspects of the role of genomic risk for schizophrenia in brain co-expression networks: 1) Variability of co-expression modules with "integration" or "removal" of genomic profiles of complex traits (normal or pathological); 2) Biological plausibility of gene sets represented in the differential co-expression contrasts and potential relevance for illness etiopathogenesis; 3) Non-preferential mapping of schizophrenia GWAS loci genes to network areas apparently influenced by the genomic risk score. Overall, our study supports the notion that genomic risk for schizophrenia has an extensive and non-linear effect on brain gene co-expression networks that possibly manifests as a molecular background for gene-gene, gene-environment interactions that affect various biological pathways.

sentences: Phase-amplitude coupling and phase synchronization between medial temporal, frontal and posterior brain regions support episodic autobiographical memory recall Episodic autobiographical memory (EAM) is a complex cognitive function that emerges from the coordination of specific and distant brain regions. Specific brain rhythms, namely theta and gamma oscillations and their synchronization, are thought of as putative mechanisms enabling EAM. Yet, the mechanisms of inter-regional interaction in the EAM network remain unclear in humans at the whole brain level. To investigate this, we analyzed EEG recordings of participants instructed to retrieve autobiographical episodes. EEG recordings were projected in the source space, and time-courses of atlas-based brain regions-of-interest (ROIs) were derived. Directed phase synchrony in high theta (7-10 Hz) and gamma (30-80 Hz) bands and high theta-gamma phase-amplitude coupling were computed between each pair of ROIs. Using network-based statistics, a graph-theory method, we found statistically significant networks for each investigated mechanism. In the gamma band, two sub-networks were found, one between the posterior cingulate cortex (PCC) and the medial temporal lobe (MTL) and another within the medial frontal areas. In the high theta band, we found a PCC to ventromedial prefrontal cortex (vmPFC) network. In phase-amplitude coupling, we found the high theta phase of the left MTL biasing the gamma amplitude of posterior regions and the vmPFC. Other regions of the temporal lobe and the insula were also phase biasing the vmPFC. These findings suggest that EAM, rather than emerging from a single mechanism at a single frequency, involves precise spatio-temporal signatures mapping on distinct memory processes. We propose that the MTL orchestrates activity in vmPFC and PCC via precise phase-amplitude coupling, with vmPFC and PCC interaction via high theta phase synchrony and gamma synchronization contributing to bind information within the PCC-MTL sub-network or valuate the candidate memory within the medial frontal sub-network.

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Dual Effects of NV-CoV-2 Biomimetic Polymer: An Antiviral regimen against COVID-19 Remdesivir (RDV) is the only antiviral drug so far approved for COVID-19 therapy by the FDA. However its efficacy is limited in vivo due to its low stability in presence of plasma. This paper compared the stability of RDV encapsulated with our platform technology based polymer NV-387 (NV-CoV-2), in presence of plasma in vitro and in vivo. Furthermore, a non- clinical pharmacology studies of NV-CoV-2 (Polymer) and NV-CoV-2-R (Polymer encapsulated Remdesivir) in both NL-63 infected and uninfected rats were done. In an in vitro cell culture model experiment, antiviral activity of NV-CoV-2 and NV-CoV-2-R are also compared with RDV.

The results are (i) NV-CoV-2 polymer encapsulation protects RDV from plasma- mediated catabolism in vitro and in vivo, too. (ii) Body weight measurements of the normal (uninfected) rats after administration of the test materials (NV-CoV-2, and NV-CoV-2-R) show no toxic effects on them. (iii) NL-63 infected rats body weights and their survival length were like uninfected rats after treatment with NV-CoV-2 and NV-CoV-2-R, and the efficacy as an antiviral regimen were found in the order as below: NV-CoV-2-R > NV-CoV-2 > RDV.

In brief, our platform technology based NV-387-encapsulated-RDV (NV-CoV-2-R) drug has a dual effect on coronaviruses. First, NV-CoV-2 itself as an antiviral regimen. Secondly, RDV is protected from plasma-mediated degradation in transit, rendering altogether the safest and an efficient regimen against COVID-19.

sentences: Computational mechanisms for neural representation of words A critical way for humans to acquire, represent and communicate information is through language, yet the underlying computation mechanisms through which language contributes to our word meaning representations are poorly understood. We compared three major types of word computation mechanisms from large language corpus (simple co-occurrence, graph-space relations and neural-network-vector-embedding relations) in terms of the association of words brain activity patterns, measured by two functional magnetic resonance imaging (fMRI) experiments. Word relations derived from a graph-space representation, and not neural-network-vector-embedding, had unique explanatory power for the neural activity patterns in brain regions that have been shown to be particularly sensitive to language processes, including the anterior temporal lobe (capturing graph-common-neighbors), inferior frontal gyrus, and posterior middle/inferior temporal gyrus (capturing graph-shortest-path). These results were robust across different window sizes and graph sizes and were relatively specific to language inputs. These findings highlight the role of cumulative language inputs in organizing word meaning neural representations and provide a mathematical model to explain how different brain regions capture different types of language-derived information.

sentences: Digenic traits under population admixture and inbreeding The population genetics of digenic genotypes in diploid populations, genotypes based on alleles at two loci, have been studied theoretically for decades with relevant digenic traits of medical interest being known for over 25 years. Given the effects of linkage and linkage disequilibrium on two locus genotypes, it should be expected that these factors can change the expected frequencies of digenic genotypes in many, sometimes unexpected, ways. In particular, the combination of linkage disequilibrium and inbreeding can combine to increase the frequencies of double homozygotes and double heterozygotes significantly over outbred comparisons. Given the prevalence of linkage disequilibrium in recently admixed populations, this can lead to large shifts in trait prevalence such that it can sometimes exceed that of either original pre-admixed population with the combined effects of linkage disequilibrium and inbreeding. Here we investigate the frequencies of digenic genotypes under the combined effects of linkage, linkage disequilibrium, and inbreeding to analyze how these interact to increase or decrease the frequency of the genotypes across two loci.

sentences: Antagonism between killer yeast strains as an experimental model for biological nucleation dynamics Antagonistic interactions are widespread in the microbial world and affect microbial evolutionary dynamics. Natural microbial communities often display spatial structure, which affects biological interactions, but much of what we know about microbial warfare comes from laboratory studies of well-mixed communities. To overcome this limitation, we manipulated two killer strains of the budding yeast Saccharomyces cerevisiae, expressing different toxins, to independently control the rate at which they released their toxins. We developed mathematical models that predict the experimental dynamics of competition between toxin-producing strains in both well-mixed and spatially structured populations. In both situations, we experimentally verified theorys prediction that a stronger antagonist can invade a weaker one only if the initial invading population exceeds a critical frequency or size. Finally, we found that toxin-resistant cells and weaker killers arose in spatially structured competitions between toxin-producing strains, suggesting that adaptive evolution can affect the outcome of microbial antagonism in spatial settings.

sentences: Face dissimilarity judgements are predicted by representational distance in morphable and image-computable models Human vision is attuned to the subtle differences between individual faces. Yet we lack a quantitative way of predicting how similar two face images look, or whether they appear to show the same person. Principal-components-based 3D morphable models are widely used to generate stimuli in face perception research. These models capture the distribution of real human faces in terms of dimensions of physical shape and texture. How well does a "face space" defined to model the distribution of faces as an isotropic Gaussian explain human face perception? We designed a behavioural task to collect dissimilarity and same/different identity judgements for 232 pairs of realistic faces. The stimuli densely sampled geometric relationships in a face space derived from principal components of 3D shape and texture (Basel Face Model, BFM). We then compared a wide range of models in their ability to predict the data, including the BFM from which faces were generated, a 2D morphable model derived from face photographs, and image-computable models of visual perception. Euclidean distance in the BFM explained both similarity and identity judgements surprisingly well. In a comparison against 14 alternative models, we found that BFM distance was competitive with representational distances in state-of-the-art image-computable deep neural networks (DNNs), including a novel DNN trained on BFM identities. Models describing the distribution of facial features across individuals are not only useful tools for stimulus generation. They also capture important information about how faces are perceived, suggesting that human face representations are tuned to the statistical distribution of faces.

sentences: Does Vergence Affect Perceived Size? Since Kepler (1604) and Descartes (1637), its been suggested that  vergence (the angular rotation of the eyes) plays a key role in size constancy. However, this has never been tested divorced from confounding cues such as changes in the retinal image. In our experiment participants viewed a target which grew or shrank over 5 seconds. At the same time the fixation distance specified by vergence was reduced from 50cm to 25cm. The question was whether the reduction in the viewing distance specified by vergence biased the participants judgements of whether the target grew or shrank? We found no evidence of any bias, and therefore no evidence that eye movements affect perceived size. If this is correct, then this finding has three implications: First, perceived size is much more reliant on cognitive influences than previously thought. This is consistent with the argument that visual scale is purely cognitive in nature (Linton, 2017; 2018). Second, it leads us to question whether the vergence modulation of V1 contributes to size constancy. Third, given the interaction between vergence, proprioception, and the retinal image in the Taylor illusion, it leads us to ask whether this cognitive approach could also be applied to multisensory integration.

sentences: Cellular and Molecular Organization of the Rhesus Macaque Dorsal Horn with a Comparison to Mouse The spinal dorsal horn transforms incoming somatosensory information and transmits it supraspinally to generate modality-specific sensory percepts. The lack of an established framework for the molecular and cellular organization of the dorsal horn across species has greatly hampered delineating precisely how this region processes somatosensory information, including pain. Furthermore, the translation potential of the rodent work is unclear without data from higher order species. To fill these gaps, we performed single nucleus RNA-sequencing of Rhesus macaque dorsal horn and compared the results to a recently reported meta-analysis of mouse. Because dorsal horn laminae serve as a key organizing principle for function, we also determined the laminar location of each identified cell type. The work provides a comprehensive cross-species cellular and molecular database that will be critical for decoding the logic of dorsal horn somatosensory circuits and validating preclinical targets.

sentences: Single-cell analysis of the human pancreas in type 2 diabetes using multi-spectral imaging mass cytometry Type 2 diabetes mellitus (T2D) is a chronic age-related disorder characterized by hyperglycemia due to the failure of pancreatic beta cells to compensate for increased insulin demand, typically associated with peripheral insulin resistance. However, despite decades of research, the pathogenic mechanisms underlying T2D remain poorly defined. Imaging mass cytometry (IMC) enables multiplexed assessment of the abundance and subcellular localization of multiple proteins on the same tissue section. Herein, we utilized IMC with a panel of 34 antibodies to quantify key markers of pancreatic exocrine, islet, and immune cells as well as stromal components. We employed this panel to analyze over 2.1 million cells from 16 pancreata obtained from donors with T2D and 13 pancreata from age similar non-diabetic controls. In the T2D pancreata, we observed significant alterations in islet architecture, endocrine cell composition, and surprisingly immune cell constituents. Thus, both HLA-DR positive CD8 T cells and macrophages were enriched intra-islet in the T2D pancreas. These efforts demonstrate the utility of IMC to investigate complex events at the cellular level in order to provide new insights into the pathophysiology of T2D.

sentences: The origins and molecular evolution of SARS-CoV-2 lineage B.1.1.7 in the UK The first SARS-CoV-2 variant of concern (VOC) to be designated was lineage B.1.1.7, later labelled by the World Health Organisation (WHO) as Alpha. Originating in early Autumn but discovered in December 2020, it spread rapidly and caused large waves of infections worldwide. The Alpha variant is notable for being defined by a long ancestral phylogenetic branch with an increased evolutionary rate, along which only two sequences have been sampled. Alpha genomes comprise a well-supported monophyletic clade within which the evolutionary rate is more typical of SARS-CoV-2. The Alpha epidemic continued to grow despite the continued restrictions on social mixing across the UK, and the imposition of new restrictions, in particular the English national lockdown in November 2020. While these interventions succeeded in reducing the absolute number of cases, the impact of these non-pharmaceutical interventions was predominantly to drive the decline of the SARS-CoV-2 lineages which preceded Alpha. We investigate the only two sampled sequences that fall on the branch ancestral to Alpha. We find that one is likely to be a true intermediate sequence, providing information about the order of mutational events that led to Alpha. We explore alternate hypotheses that can explain how Alpha acquired a large number of mutations yet remained largely unobserved in a region of high genomic surveillance: an under-sampled geographical location, a non-human animal population, or a chronically-infected individual. We conclude that the last hypothesis provides the best explanation of the observed behaviour and dynamics of the variant, although we find that the individual need not be immunocompromised, as persistently-infected immunocompetent hosts also display a higher within-host rate of evolution. Finally, we compare the ancestral branches and mutation profiles of other VOCs to each other, and identify that Delta appears to be an outlier both in terms of the genomic locations of its defining mutations, and its lack of rapid evolutionary rate on the ancestral branch. As new variants, such as Omicron, continue to evolve (potentially through similar mechanisms) it remains important to investigate the origins of other variants to identify ways to potentially disrupt their evolution and emergence.

sentences: A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus. Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases.

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Synthetic deconstruction of hunchback regulation by Bicoid For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as the model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation.

By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoids partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

sentences: DNA dynamics dictates p53 functional outcome The tumor suppressor protein p53 is activated in response to cellular stress. An unresolved question in the field is how p53 is able to parse its myriad functions in a timely manner in accordance with the severity of the stress signal. Here we show across the genome that p53 target genes belonging to pathways acting early in the stress response have REs that are significantly more flexible than REs of genes involved in pathways that need to be more strictly regulated, or whose functional outcome occurs later in the response to stress. We validated these findings by several complementary experimental approaches, for six p53 REs belonging to pathways that operate at different times post p53 induction. Our results demonstrate that the flexibility of p53 REs contributes significantly to the temporal expression of p53 target genes and thereby to life versus death decisions in the p53 system.

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Evolutionarily stable gene clusters shed light on the common grounds of pathogenicity in the Acinetobacter calcoaceticus-baumanniicomplex Nosocomial pathogens of the Acinetobacter calcoaceticus-baumannii (ACB) complex are a cautionary example for the world-wide spread of multi- and pan-drug resistant bacteria. Aiding the urgent demand for novel therapeutic targets, comparative genomics studies between pathogens and their apathogenic relatives shed light on the genetic basis of human-pathogen interaction. Yet, existing studies are limited in taxonomic scope, sensing of the phylogenetic signal, and resolution by largely analyzing genes isolated from their functional contexts. Here, we explored more than 3,000 Acinetobacter genomes in a phylogenomic framework integrating orthology-based phylogenetic profiling and microsynteny conservation analyses. This allowed to delineate gene clusters in the type strain A. baumannii ATCC 19606 whose evolutionary conservation indicates a functional integration of the subsumed genes. These evolutionarily stable gene clusters (ESGCs) reveal metabolic pathways, transcriptional regulators residing next to their targets but also tie together sub-clusters with distinct functions to form higher-order functional modules. We shortlisted 150 ESGCs that either co-emerged with, or are found preferentially in, the pathogenic ACB clade. They unveil, at an unprecedented resolution, the genetic makeup that coincides with the manifestation of the pathogenic phenotype in the last common ancestor of the ACB clade. Key innovations are the remodeling of the regulatory-effector cascade connecting LuxR/LuxI quorum sensing via an intermediate messenger to biofilm formation, the extension of micronutrient scavenging systems, and the increase of metabolic flexibility by exploiting carbon sources that are provided by the human host. Specifically, we could show that only members of the ACB clade use kynurenine as a sole carbon and energy source, a substance produced by humans to fine-tune the antimicrobial innate immune response. In summary, this study provides a rich and unbiased set of novel testable hypotheses on how pathogenic Acinetobacter interact with and ultimately infect their human host. They disclose promising routes for future therapeutic strategies.

Author SummaryThe spread of multi- and pan-drug resistant bacterial pathogens is a world-wide threat to human health. Understanding the genetics of host colonization and infection is essential for devising novel ways of treatment. Acinetobacter baumannii, a nosocomial pathogen ranked top by the World Health Organization in the list of bacteria for which novel therapeutic approaches are needed, is a prime example. Here, we have carved out the genetic make-up that distinguishes A. baumannii and its pathogenic next relatives comprising the A. baumannii-calcoaceticus complex from other and mostly apathogenic Acinetobacter species. We found a rich spectrum of pathways and regulatory modules that reveals how the pathogens have modified biofilm formation, iron scavenging, and their carbohydrate metabolism to adapt to their human host. Among these, the capability to metabolize kynurenine is particularly intriguing. Humans produce this substance to contain bacterial invaders and to fine-tune the innate immune response. But A. baumannii and its allies have turned the table. They feed on kynurenine, and thus most likely also dysregulate human immune response. In summary, our study substantially deepens the understanding of how a highly critical pathogen interacts with its host, which substantially eases the identification of novel targets for innovative therapeutic strategies.

sentences: Distrust Before First Sight: Knowledge- and Appearance-Based Effects of Trustworthiness on the Visual Consciousness of Faces Not all visual stimuli processed by the brain reach the level of conscious perception. Previous research has shown that the emotional value of a stimulus is one of the factors that can affect whether it is consciously perceived. Here, we investigated whether social-affective knowledge influences a faces chance to reach visual consciousness. Furthermore, we took into account the impact of facial appearance. Faces differing in facial trustworthiness (i.e., being perceived as more or less trustworthy based on appearance) were associated with neutral or negative socially relevant information. Subsequently, an attentional blink task was administered to examine whether the manipulated factors affect the faces chance to reach visual consciousness under conditions of reduced attentional resources. Participants showed enhanced detection of faces associated with negative as compared to neutral social information. In event-related potentials (ERPs), this was accompanied by effects in the time range of the early posterior negativity (EPN) component. These findings indicate that social-affective person knowledge is processed already before or during attentional selection and can affect which faces are prioritized for access to visual consciousness. In contrast, no clear evidence for an impact of facial trustworthiness during the attentional blink was found. This study was pre-registered using the Open Science Framework (OSF).

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The exchange rates of amide and arginine guanidinium CEST in the mouse brain PurposeTo develop a pipeline for measuring the exchange rates and concentrations of in vivo excgangeable protons, and to demonstrate this for the amide and arginine (Arg) guanidinium (Guan) protons in mobile proteins in the mouse brain.

MethodsAn ultra-short echo (UTE) CEST sequence with a continuous wave presaturation (preRadCEST) was applied to acquire Z-spectra with robustness to motion and physiological fluctuations. AmideCEST and Arginine guanCEST (ArgCEST) were extracted and their proton concentrations and exchange rates obtained using a two-step multi-B1 Bloch fitting approach that included the semisolid macromolecular background. To minimize contamination from the amine protons from creatine and phosphocreatine, ArgCEST measurements were performed on the Guanidinoacetate N-methyltransferase deficiency (GAMT-/-) mouse characterized by low creatine and phosphocreatine concentrations in the brain.

ResultsFor the amideCEST proton pool, the exchange rate and concentrations were found to be 59.6 {+/-} 9.0 s-1 and 41.7 {+/-} 7.0 mM, respectively, with the maximum signal observed at B1 = 0.8 T. For the ArgCEST proton, the guanidinium exchange these were 70.1 {+/-} 5.5 s-1 and 10.1 {+/-} 1.3 mM, respectively, with the maximum effect observed at B1 = 0.9 T. The current study suggests that the inverse pH dependence in GuanCEST of brain is led by the CrCEST component, not ArgCEST.

ConclusionThe current pipeline is expected to have general use for in vivo CEST quantitation and optimization of visible CEST resonances.

sentences: Understanding patterns of HIV multi-drug resistance through models of temporal and spatial drug heterogeneity 1Triple-drug therapies have transformed HIV from a fatal condition to a chronic one. These therapies should prevent HIV drug resistance evolution, because one or more drugs suppress any partially-resistant viruses. In practice, such therapies drastically reduced, but did not eliminate, resistance evolution. In this article, we reanalyze published data from an evolutionary perspective and demonstrate several intriguing patterns about HIV resistance evolution - resistance evolves (1) even after years on successful therapy, (2) sequentially, often via one mutation at a time and (3) in a partially predictable order. We describe how these observations might emerge under two models of HIV drugs varying in space or time. Despite decades of work in this area, much opportunity remains to create models with realistic parameters for three drugs, and to match model outcomes to resistance rates and genetic patterns from patients on triple-drug therapy. Further, lessons from HIV may inform other systems.

sentences: Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

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Raloxifene reduces sex- and age-related intervertebral disc degeneration in mice by estrogen signaling Estrogen agonist raloxifene is an FDA-approved treatment for osteoporosis in postmenopausal women that may also be a promising prophylactic for painful intervertebral disc (IVD) degeneration. Here, we hypothesized that raloxifene would augment IVD structure and reduce neurokinin-1 (substance P) in young and old mice by stimulating estrogen signaling. 2.5 month (male and female) and 22.5 month (female) C57Bl/6J mice were subcutaneously injected with raloxifene hydrochloride (5x/week, 6week, n=7-9/grp). Next, to determine the impact of estrogen-deficiency to IVD structure and substance P, female mice were ovariectomized (OVX) at 4mo and tissues from OVX and sham-operated mice were harvested at 6mo (n=5-6/grp). First, compared to male IVD, female IVD expressed less col2 and osterix transcription, early markers of IVD degeneration. Irrespective of sex, raloxifene increased the transcriptional expression for extracellular matrix anabolism, proliferation, notochordal cells (vs chondrocyte-like cells) and estrogen signaling in young IVD. Next, we determined that biological sex and aging each induced structural features of lumbar IVD degeneration. Therapeutically, injection of raloxifene countered these features by increasing IVD height in young mice, preventing mild sex-related IVD degeneration in young female mice and partially reversing age-related IVD degeneration in old female mice. Further, estrogen agonist raloxifene upregulated er- protein and downregulated substance P protein in young and old IVD. By contrast, estrogen-deficiency by OVX increased IVD degeneration and substance P protein in IVD cells. Similarly, substance P protein in vertebral osteocytes was upregulated in females relative to males and by estrogen-deficiency and downregulated by raloxifene. Overall, raloxifene augmented IVD structure and reduced substance P expression in young and old female murine IVD, whereas estrogen-deficiency increased substance P in the spine. These data suggest that raloxifene may potentially relieve painful IVD degeneration in postmenopausal women induced by biological sex, estrogen-deficiency and advanced age.

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Injection of raloxifene promotes IVD health by engaging estrogen and Wnt signaling to promote cell proliferation and IVD structure. Differential estrogen signaling by raloxifene and ovariectomy regulated nerve signaling protein substance P in the spine. Raloxifene may also bind water to collagen to promote hydration. Acan: aggrecan, AF: annulus fibrosus, NC: notochordal cell, NP: nucleus pulposus

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sentences: Deep learning classification of temporal data in ecology Temporal data is ubiquitous in ecology and ecologists often face the challenge of accurately differentiating these data into predefined classes, such as biological entities or ecological states. The usual approach consists of transforming the time series into user-defined features and then using these features as predictors in conventional statistical or machine learning models. Here we suggest the use of deep learning models as an alternative to this approach. Recent deep learning techniques can perform the classification directly from the time series, eliminating subjective and resource-consuming data transformation steps, and potentially improving classification results. We describe some of the deep learning architectures relevant for time series classification and show how these architectures and their hyper-parameters can be tested and used for the classification problems at hand. We illustrate the approach using three case studies from distinct ecological subdisciplines: i) insect species identification from wingbeat spectrograms; ii) species distribution modelling from climate time series and iii) the classification of phenological phases from continuous meteorological data. The deep learning approach delivered ecologically sensible and accurate classifications demonstrating its potential for wide applicability across subfields of ecology.

sentences: Comparative analysis of high- and low-level deep learning approaches in microsatellite instability prediction Deep learning-based approaches in histopathology can be largely divided into two categories: a high-level approach using an end-to-end model and a low-level approach using feature extractors. Although the advantages and disadvantages of both approaches are well known, direct comparative analysis has rarely been conducted. Using the Cancer Genomic Atlas (TCGA) based dataset, we compared these two different approaches in microsatellite instability (MSI) prediction and analyzed morphological image features associated with MSI. Our high-level approach was based solely on EfficientNet while our low-level approach relied on LightGBM and multiple deep learning models trained on publicly available multi-class tissue, nuclei, and gland datasets. We compared their performance and important image features. Our high-level approach showed a superior performance compared to our low-level approach. In both approaches, debris, lymphocytes, and necrotic cells were revealed as important features of MSI, which is consistent with clinical knowledge. Then, during qualitative analysis, we discovered the weaknesses of our low-level approach and demonstrated that its performance can be improved by using different image features in a complementary way. We performed our study using open-access data, and we believe this study can serve as a useful basis for discovering image biomarkers for clinical application.

sentences: Is virulence phenotype evolution driven exclusively by Lr gene deployment in French Puccinia triticina populations? Puccinia triticina is a highly damaging wheat pathogen. The efficacy of leaf rust control by genetic resistance is mitigated by the adaptive capacity of the pathogen, expressed as changes in its virulence combinations (pathotypes). An extensive P. triticina population survey has been carried out in France over the last 30 years, describing the evolution dynamics of this pathogen in response to cultivar deployment. We analyzed the dataset for the 2006-2016 period to determine the relationship between the Lr genes in the cultivars and virulence in the pathotypes. Rust populations were dominated by a small number of pathotypes, with variations in most of the virulence frequencies related to the corresponding Lr gene frequencies in the cultivated landscape. Furthermore, the emergence and spread of a new virulence matched the introduction and use of the corresponding Lr gene (Lr28), confirming that the deployment of qualitative resistance genes is an essential driver of evolution in P. triticina populations. However, PCA revealed that certain pathotype-cultivar associations cannot be explained solely by the distribution of Lr genes in the landscape. This conclusion is supported by the predominance of a few pathotypes on some cultivars, with the persistence of several other compatible pathotypes at low frequencies. Specific interactions are not, therefore, sufficient to explain the distribution of virulence in rust populations. Our findings suggest that aggressiveness is a driver of changes in pathotype frequencies. Accordingly, the hypothesis of "dual selection", based on both qualitative and quantitative interactions between P. triticina populations and bread wheat cultivars, is favored.

sentences: DeepSpot: a deep neural network for RNA spot enhancement in smFISH microscopy images Detection of RNA spots in single molecule FISH microscopy images remains a difficult task especially when applied to large volumes of data. The small size of RNA spots combined with high noise level of images often requires a manual adaptation of the spot detection thresholds for each image. In this work we introduce DeepSpot, a Deep Learning based tool specifically designed to enhance RNA spots which enables spot detection without need to resort to image per image parameter tuning. We show how our method can enable the downstream accurate detection of spots. The architecture of DeepSpot is inspired by small object detection approaches. It incorporates dilated convolutions into a module specifically designed for the Context Aggregation for Small Object (CASO) and uses Residual Convolutions to propagate this information along the network. This enables DeepSpot to enhance all RNA spots to the same intensity and thus circumvents the need for parameter tuning. We evaluated how easily spots can be detected in images enhanced by our method, by training DeepSpot on 20 simulated and 1 experimental datasets, and have shown that more than 97% accuracy is achieved. Moreover, comparison with alternative deep learning approaches for mRNA spot detection (deepBlink) indicated that DeepSpot allows more precise mRNA detection. In addition, we generated smFISH images from mouse fibroblasts in a wound healing assay to evaluate whether DeepSpot enhancement can enable seamless mRNA spot detection and thus streamline studies of localized mRNA expression in cells.

sentences: A Chalcone Synthase-Like Bacterial Protein Catalyzes Heterocyclic C-Ring Cleavage of Naringenin to Alter Bioactivity Against Nuclear Receptors in Colonic Epithelial Cells The human gut microbiota contributes enzymatic functions that are unavailable to host cells and play crucial roles in host metabolism, nutrient processing and regulating immune functions. As dietary compounds that are only partially absorbed, flavonoids are available for metabolism by gut microbiota, leading to diverse bioactive products. Combining prediction of enzyme promiscuity, metabolomics, and in vitro model systems, we identified a bacterial enzyme that can catalyze heterocyclic C-ring cleavage of naringenin. Culture experiments using a wild-type and mutant strain of Bacillus subtilis confirmed that the enzyme is a chalcone synthase-like polyketide synthase. The prediction-validation methodology developed in this work could be used to systematically characterize the products of gut bacterial flavonoid metabolism and identify the responsible enzymes and species. Further, we demonstrated that naringenin and its ring cleavage metabolites differentially engage the AhR and NR4A in intestinal epithelial cells. Our results suggest that the abundance of selected gut bacterial species impacts the profile of bioactive flavonoids and flavonoid-derived metabolites and thereby influences inflammatory responses in the intestine. These results are significant for understanding the mechanisms of gut microbiota-dependent effects of dietary flavonoids.

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Post-stroke administration of the p75 neurotrophin receptor modulator, LM11A-31, attenuates chronic changes in brain metabolism, increases neurotransmitter levels, and improves recovery The aim of this study was to test whether post-stroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks following stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31 treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31 treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75 neurotrophin receptor for preserving neuronal health and function during stroke recovery.

SIGNIFICANCE STATEMENTThe findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimers disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.

sentences: Single-cell transcriptome reveals the redifferentiation trajectories of the early stage of de novo shoot regeneration in Arabidopsis thaliana De novo shoot regeneration from a callus plays a crucial role in both plant biotechnology and the fundamental research of plant cell totipotency. Recent studies have revealed many regulatory factors involved in this developmental process. However. our knowledge of the cell heterogeneity and cell fate transition during de novo shoot regeneration is still limited. Here, we performed time-series single-cell transcriptome experiments to reveal the cell heterogeneity and redifferentiation trajectories during the early stage of de novo shoot regeneration. Based on the single-cell transcriptome data of 35,669 cells at five-time points, we successfully determined seven major cell populations in this developmental process and reconstructed the redifferentiation trajectories. We found that all cell populations resembled root identities and undergone gradual cell-fate transitions. In detail, the totipotent callus cells differentiated into pluripotent QC-like cells and then gradually developed into less differentiated cells that have multiple root-like cell identities, such as pericycle-like cells. According to the reconstructed redifferentiation trajectories, we discovered that the canonical regeneration-related genes were dynamically expressed at certain stages of the redifferentiation process. Moreover, we also explored potential transcription factors and regulatory networks that might be involved in this process. The transcription factors detected at the initial stage, QC-like cells, and the end stage provided a valuable resource for future functional verifications. Overall, this dataset offers a unique glimpse into the early stages of de novo shoot regeneration, providing a foundation for a comprehensive analysis of the mechanism of de novo shoot regeneration.

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From mass spectral features to molecules in molecular networks: a novel workflow for untargeted metabolomics. In the context of untargeted metabolomics, molecular networking is a popular and efficient tool which organizes and simplifies mass spectrometry fragmentation data (LC-MS/MS), by clustering ions based on a cosine similarity score. However, the nature of the ion species is rarely taken into account, causing redundancy as a single compound may be present in different forms throughout the network. Taking advantage of the presence of such redundant ions, we developed a new method named MolNotator. Using the different ion species produced by a molecule during ionization (adducts, dimers, trimers, in-source fragments), a predicted molecule node (or neutral node) is created by triangulation, and ultimately computing the associated molecules calculated mass. These neutral nodes provide researchers with several advantages. Firstly, each molecule is then represented in its ionization context, connected to all produced ions and indirectly to some coeluted compounds, thereby also highlighting unexpected widely present adduct species. Secondly, the predicted neutrals serve as anchors to merge the complementary positive and negative ionization modes into a single network. Lastly, the dereplication is improved by the use of all available ions connected to the neutral nodes, and the computed molecular masses can be used for exact mass dereplication. MolNotator is available as a Python library and was validated using the lichen database spectra acquired on an Orbitrap, computing neutral molecules for >90% of the 156 molecules in the dataset. By focusing on actual molecules instead of ions, MolNotator greatly facilitates the selection of molecules of interest.

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sentences: Single Cell Biological Microlasers Powered by Deep Learning Cellular lasers are cutting-edge technologies for biomedical applications. Due to the enhanced interactions between light and cells in microcavities, cellular properties and subtle changes of cells can be significantly reflected by the laser emission characteristics. In particular, transverse laser modes from single-cell lasers which utilize Fabry-Perot cavities are highly correlated to the spatial biophysical properties of cells. However, the high chaotic and complex variation of laser modes limits their practical applications for cell detections. Deep learning technique has demonstrated its powerful capability in solving complex imaging problems, which is expected to be applied for cell detections based on laser mode imaging. In this study, deep learning technique was applied to analyze laser modes generated from single-cell lasers, in which a correlation between laser modes and physical properties of cells was built. As a proof-of-concept, we demonstrated the predictions of cell sizes using deep learning based on laser mode imaging. In the first part, bioinspired cell models were fabricated to systematically study how cell sizes affect the characteristics of laser modes. By training a convolutional neuron network (CNN) model with laser mode images, predictions of cell model diameters with a sub-wavelength accuracy were achieved. In the second part, deep learning was employed to study laser modes generated from biological cells. By training a CNN model with laser mode images acquired from astrocyte cells, predictions of cell sizes with a sub-wavelength accuracy were also achieved. The results show the great potential of laser mode imaging integrated with deep learning for cell analysis and biophysical studies.

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Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.

AUTHOR SUMMARYThis work identifies a novel function of lung-resident MSCs during virus-induced acute lung injury. These findings contribute to the understanding of host response and lung repair mechanisms during a highly prevalent clinical situation and may have therapeutic implications.

sentences: Heterogeneous interactions and polymer entropy decide organization and dynamics of chromatin domains Chromatin is known to be organized into multiple domains of varying sizes and compaction. While these domains are often imagined as static structures, they are highly dynamic and show cell-to-cell variability. Since processes such as gene regulation and DNA replication occur in the context of these domains, it is important to understand their organization, fluctuation and dynamics. To simulate chromatin domains, one requires knowledge of interaction strengths among chromatin segments. Here, we derive interaction strength parameters from experimentally known contact maps and use them to predict chromatin organization and dynamics. Taking two domains on the human chromosome as examples, we investigate its 3D organization, size/shape fluctuations, and dynamics of different segments within a domain, accounting for hydrodynamic effects. Considering different cell types, we quantify changes in interaction strengths and chromatin shape fluctuations in different epigenetic states. Perturbing the interaction strengths systematically, we further investigate how epigenetic-like changes can alter the spatio-temporal nature of the domains. Our results show that heterogeneous weak interactions are crucial in determining the organization of the domains. Computing effective stiffness and relaxation times, we investigate how perturbations in interactions affect the solid-like and liquid-like nature of chromatin domains. Quantifying dynamics of chromatin segments within a domain, we show how the competition between polymer entropy and interaction energy influence the timescales of loop formation and maintenance of stable loops.

sentences: Targeted RNA-seq improves efficiency, resolution, and accuracy of allele specific expression for human term placentas Genomic imprinting is an epigenetic mechanism that results in allele specific expression (ASE) based on parent of origin. It is known to play a role in the prenatal and postnatal allocation of maternal resources in mammals. ASE detected by whole transcriptome RNA-seq (wht-RNAseq) has been widely used to analyze imprinted genes using reciprocal crosses in mice to generate large numbers of informative SNPs. Studies in humans are more challenging due to the paucity of SNPs and the poor preservation of RNA in term placentas and other tissues. Targeted RNA-seq (tar-RNAseq) can potentially mitigate these challenges by focusing sequencing resources on the regions of interest in the transcriptome. Here we compared tar-RNAseq and wht-RNAseq in a study of ASE in known imprinted genes in placental tissue collected from a healthy human cohort in Mali, West Africa. As expected, tar-RNAseq substantially improved the coverage of SNPs. Compared to wht-RNAseq, tar-RNAseq produced on average four times more SNPs in twice as many genes per sample and read depth at the SNPs increased 4-fold. In previous research on humans, discordant ASE values for SNPs of the same gene have limited the ability to accurately quantify ASE. We show that tar-RNAseq reduces this limitation as it unexpectedly increased the concordance of ASE between SNPs of the same gene, even in cases of degraded RNA. Studies aimed at discovering associations between individual variation in ASE and phenotypes in mammals and flowering plants will benefit from the improved power and accuracy of tar-RNAseq.

sentences: Adaptation to chronic ER stress enforces pancreatic β-cell plasticity Pancreatic {beta}-cells undergo high levels of endoplasmic reticulum (ER) stress due to their role in insulin secretion. Hence, they require sustainable and efficient adaptive stress responses to cope with the stress. Whether duration and episodes of chronic ER stress directly compromises {beta}-cell identity is largely unknown. We show that under reversible, chronic ER stress, {beta}-cells undergo a distinct transcriptional and translational reprogramming. During reprogramming, expression of master regulators of {beta}-cell function and identity and proinsulin processing is impaired. Upon recovery from stress, {beta}-cells regain their identity, highlighting a high-degree of adaptive {beta}-cell plasticity. Remarkably, when stress episodes exceed a certain threshold, {beta}-cell identity is gradually lost. Single cell RNA-seq analysis of islets from type 1 diabetes (T1D) patients, identifies the severe deregulation of the chronic stress-adaptation program, and reveals novel biomarkers for progression of T1D. Our results suggest {beta}-cell adaptive exhaustion ({beta}EAR) is a significant component of the pathogenesis of T1D.

sentences: Predictive evolution of metabolic phenotypes using model-designed selection niches Traits lacking fitness benefit cannot be directly selected for under Darwinian evolution. Thus, features such as metabolite secretion are currently inaccessible to adaptive laboratory evolution. Here, we utilize environment-dependency of trait correlations to enable Darwinian selection of fitness-neutral or costly traits. We use metabolic models to design selection niches and to identify surrogate traits that are genetically correlated with cell fitness in the selection niche but coupled to the desired trait in the target niche. Adaptive evolution in the selection niche and subsequent return to the target niche is thereby predicted to enhance the desired trait. We experimentally validate the theory by evolving Saccharomyces cerevisiae for increased secretion of aroma compounds in wine fermentation. Genomic, transcriptomic, and proteomic changes in the evolved strains confirmed the predicted flux re-routing to aroma biosynthesis. The use of model-designed selection niches facilitates the predictive evolution of fitness-costly traits for ecological and biotechnological applications.

sentences: The influence of adaptation to life at high-altitude on condition dependent sexual shape and size dimorphism in Drosophila melanogaster Sexual dimorphism is common despite factors such as inter-sex genetic correlations and sex-specific patterns of selection that might limit its evolution. Sexual dimorphism can be phenotypically plastic and condition dependent, that themselves may be targets of selection. However, it remains unclear how sexual dimorphism, its plasticity and condition dependence evolves, in particular during rapid adaptation to a new environment. Furthermore, the interplay between SSD and other forms of dimorphism, such as shape dimorphism co-evolves. Using Sub-Saharan populations of Drosophila melanogaster that vary for size and shape as a result of adaptation to high altitude environments, we examined sex specific patterns of developmental plasticity. We raised strains of Drosophila from low (Zambia) and high (Ethiopia) altitude populations varying for food quality or rearing temperature. We observed expected differences in wing size and shape due to population, sex and plasticity. While larval mass showed substantial evolved changes for sex specific condition dependence, effects on wing size and shape were modest. We examined shape-size allometric effects between groups. Allometric effects were generally similar across sexes, but differed substantially due to population of origin and plasticity. We discuss findings within the context of the evolution of plasticity for SSD, condition dependence and allometric relationships.

sentences: Pyramidal cell types drive functionally distinct cortical activity patterns during decision-making Understanding how cortical circuits generate complex behavior requires investigating the cell types that comprise them. Functional differences across pyramidal neuron (PyN) types have been observed within cortical areas, but it is not known whether these local differences extend throughout the cortex, nor whether additional differences emerge when larger-scale dynamics are considered. We used genetic and retrograde labeling to target pyramidal tract (PT), intratelencephalic (IT) and corticostriatal projection neurons and measured their cortex-wide activity. Each PyN type drove unique neural dynamics, both at the local and cortex-wide scale. Cortical activity and optogenetic inactivation during an auditory decision task also revealed distinct functional roles: all PyNs in parietal cortex were recruited during perception of the auditory stimulus, but, surprisingly, PT neurons had the largest causal role. In frontal cortex, all PyNs were required for accurate choices but showed distinct choice-tuning. Our results reveal that rich, cell-type-specific cortical dynamics shape perceptual decisions.

sentences: An automated approach to quantify chemotaxis index in Caenorhabditis nematodes Chemotaxis assays are used extensively to study behavioral responses of Caenorhabditis nematodes to environmental cues. These assays result in a chemotaxis index (CI) that denotes the behavioral response of a population of nematodes to a particular compound and can range from 1 (maximum attraction) to -1 (maximum avoidance). Traditional chemotaxis assays have low throughput because researchers must manually setup experimental populations and score CIs. Here, we describe an automated methodology that increases throughput by using liquid-handling robots to setup experimental populations and a custom image analysis package, ct, to automate the scoring of CIs from plate images.

sentences: Biological, Molecular And Phiysiological Characterization Of Four Soybean mosaic virus Isolates Present In Argentine Soybean Crops Soybean mosaic virus (SMV) causes systemic infections in soybean plants, leading to chlorotic mosaic and producing significant yield losses. The virus is widely distributed in all soybean production areas in the world. In Argentina, three geographical isolates were identified: Marcos Juarez (MJ), Manfredi (M), and North Western Argentina (NOA), and another isolate named "Planta Vinosa" (PV), which causes severe necrosis symptoms in some cultivars. Here, the biological, molecular and physiological characterization of these isolates was performed for the first time. Three of the four isolates showed a low genetic divergence in the evaluated genes (P1, CI and CP). Although SMV-NOA and SMV-PV had high homology at the sequence level, they showed wide differences in pathogenicity, seed mottling and the ability of transmission by seeds or aphids, as well as in physiological effects. SMV-NOA caused early alterations (before symptom appearance, BS) in {Phi}PSII and MDA content in leaves with respect to the other isolates. After the appearance of macroscopic symptoms (late symptoms, LS), SMV-M caused a significant increase in the content of MDA, total soluble sugars, and starch with respect to the other isolates. Thus, early alterations of {Phi}PSII and soluble sugars might have an impact on late viral symptoms. Likewise, SMV-MJ developed more severe symptoms in the susceptible Davis cultivar than in DM 4800. Therefore, our results show differences in genome, biological properties and physiological effects among SMV isolates as well as different interactions of SMV-MJ with two soybean cultivars.

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Selective Laser Sintering of a Photosensitive Drug: Impact of Processing and Formulation Parameters on Degradation, Solid-State, and Quality of 3D Printed Dosage Forms This research study utilized a light-sensitive drug, nifedipine (NFD), to understand the impact of processing parameters, and formulation composition on drug degradation, crystallinity, and quality attributes (dimensions, hardness, disintegration time) of selective laser sintering (SLS) based 3D printed dosage forms. Selective laser sintering (SLS), in most cases, uses an ultraviolet laser source, and drugs tend to absorb radiation at varying intensities around this wavelength (455 nm). This phenomenon may lead to chemical degradation, and solid-state transformation, which was assessed for nifedipine in formulations with varying amounts of vinyl pyrrolidone-vinyl acetate copolymer (Kollidon(R) VA 64) and potassium aluminum silicate-based pearlescent pigment (Candurin(R)), processed under different SLS conditions in the presented work. After preliminary screening Candurin(R), surface temperature (ST), and laser speed (LS) were identified as the significant independent variables. Further, using the identified independent variables a 17-run, randomized, Box-Behnken design was developed to understand the correlation trends and quantify the impact on degradation (%), crystallinity, quality attributes (dimensions, hardness, disintegration time) employing qualitative and quantitative analytical tools. The design of experiments (DoE) and statistical analysis observed that LS and Candurin(R) (%wt) had a strong negative correlation on drug degradation, hardness, and weight, whereas ST had a strong positive correlation with, drug degradation, amorphous conversion, and hardness of the 3D printed dosage form. From this study, it can be concluded that formulation and processing parameters have a critical impact on stability and performance; hence these parameters should be evaluated and optimized before exposing light-sensitive drugs to the SLS processes.

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Use of object detection in camera trap image identification: assessing a method to rapidly and accurately classify human and animal detections for research and application in recreation ecology Camera traps are increasingly used to answer complex ecological questions. However, the rapidly growing number of images collected presents technical challenges. Each image must be classified to extract data, requiring significant labour, and potentially creating an information bottleneck. We applied an object-detection model (MegaDetector) to camera trap data from a study of recreation ecology in British Columbia, Canada. We tested its performance in detecting humans and animals relative to manual image classifications, and assessed efficiency by comparing the time required for manual classification versus a modified workflow integrating object-detection with manual classification. We also evaluated the reliability of using MegaDetector to create an index of human activity for application to the study of recreation impacts to wildlife. In our application, MegaDetector detected human and animal images with 99% and 82% precision, and 95% and 92% recall respectively, at a confidence threshold of 90%. The overall time required to process the dataset was reduced by over 500%, and the manual processing component was reduced by 840%. The index of human detection events from MegaDetector matched the output from manual classification, with a mean 0.45% difference in estimated human detections across site-weeks. Our test of an open-source object-detection model showed it performed well in partially classifying a camera trap dataset, significantly increasing processing efficiency. We suggest that this tool could be integrated into existing camera trap workflows to accelerate research and application by alleviating data bottlenecks, particularly for surveys processing large volumes of human images. We also show how the model and workflow can be used to anonymize human images prior to classification, protecting individual privacy.

Impact StatementWe developed and tested a workflow for classifying camera trap images that integrated an existing object-detection model with manual image classification. Our workflow demonstrates an increase in efficiency of 500% over manual labelling, and additionally includes a method to anonymize human images prior to archiving and classification. We provide an example of the application of these tools to ease data processing, particularly for studies focused on recreation ecology which record high volumes of human images. Data lags due to processing delays have the potential to result in sub-optimal conservation decisions, which may be alleviated by accelerated processing. To our knowledge, this is the first in-depth assessment of the practical application of such technology to real world workflows focused on human detections.

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Analysis of SARS-CoV-2 synonymous codon usage evolution throughout the COVID-19 pandemic. SARS-CoV-2, the seventh coronavirus known to infect humans, can cause severe life-threatening respiratory pathologies. To better understand SARS-CoV-2 evolution, genome-wide analyses have been made, including the general characterization of its codons usage profile. Here we present a bioinformatic analysis of the evo-lution of SARS-CoV-2 codon usage over time using complete genomes collected since December 2019. Our results show that SARS-CoV-2 codon usage pattern is antagonistic to, and it is getting farther away from that of the human host. Further, a selection of deoptimized codons over time, which was accompanied by a decrease in both the codon adaptation index and the effective number of codons, was observed. All together, these findings suggest that SARS-CoV-2 could be evolving, at least from the perspective of the synonymous codon usage, to become less pathogenic.

Graphical Abstract

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Evidence for a Potential Pre-Pandemic SARS-like Coronavirus Among Animals in North America In late 2019, a novel coronavirus began circulating within humans in central China. It was designated SARS-CoV-2 because of its genetic similarities to the 2003 SARS coronavirus (SARS-CoV). Now that SARS-CoV-2 has spread worldwide, there is a risk of it establishing new animal reservoirs and recombination with native circulating coronaviruses. To screen local animal populations in the United States for exposure to SARS-like coronaviruses, we developed a serological assay using the receptor binding domain (RBD) from SARS-CoV-2. SARS-CoV-2s RBD is antigenically distinct from common human and animal coronaviruses allowing us to identify animals previously infected with SARS-CoV or SARS-CoV-2. Using an indirect ELISA for SARS-CoV-2s RBD, we screened serum from wild and domestic animals for the presence of antibodies against SARS-CoV-2s RBD. Surprisingly pre-pandemic feline serum samples submitted to the University of Tennessee Veterinary Hospital were [~]50% positive for anti-SARS RBD antibodies. Some of these samples were serologically negative for feline coronavirus (FCoV), raising the question of the etiological agent generating anti-SARS-CoV-2 RBD cross-reactivity. We also identified several white-tailed deer from South Carolina with anti-SARS-CoV-2 antibodies. These results are intriguing as cross-reactive antibodies towards SARS-CoV-2 RBD have not been reported to date. The etiological agent responsible for seropositivity was not readily apparent, but finding seropositive cats prior to the current SARS-CoV-2 pandemic highlights our lack of information about circulating coronaviruses in other species.

ImportanceWe report cross-reactive antibodies from pre-pandemic cats and post-pandemic South Carolina white-tailed deer that are specific for that SARS-CoV RBD. There are several potential explanations for this cross-reactivity, each with important implications to coronavirus disease surveillance. Perhaps the most intriguing possibility is the existence and transmission of an etiological agent (such as another coronavirus) with similarity to SARS-CoV-2s RBD region. However, we lack conclusive evidence of pre-pandemic transmission of a SARS-like virus. Our findings provide impetus for the adoption of a One Health Initiative focusing on infectious disease surveillance of multiple animal species to predict the next zoonotic transmission to humans and future pandemics.

sentences: Balancing quality and quantity of social relationships in volatile social environments Cooperation does not occur in a vacuum: interactions develop over time in social groups that undergo demographic changes. Intuition suggests that stable social environments favour developing few but strong reciprocal relationships (a  focused strategy), while volatile social environments favour the opposite: more but weaker social relationships (a  diversifying strategy). We model reciprocal investments under a quality-quantity tradeoff for social relationships. We find that volatility, counterintuitively, can favour a focused strategy. This result becomes explicable through applying the theory of antagonistic pleiotropy, originally developed for senescence, to social life. Diversifying strategies show superior performance later in life, but with costs paid at young ages while the social network is slowly being built. Under volatile environments, many individuals die before reaching sufficiently old ages to reap the benefits. Social strategies that do well early in life are then favoured: a focused strategy leads individuals to form their first few social bonds quickly and to make strong use of existing bonds. Our model highlights the importance of pleiotropy and population age structure for the evolution of cooperative strategies and other social traits, and shows that it is not sufficient to reflect on the fate of survivors only, when evaluating the benefits of social strategies.

sentences: Origami: Single-cell oriented 3D shape dynamics of folding epithelia from fluorescence microscopy images A common feature of morphogenesis is the formation of three-dimensional structures from the folding of two-dimensional epithelial sheets aided by spatio-temporal cell shape changes at the cellular-level. Studying cell shape dynamics and polarised processes that underpin them, requires orienting cells within the epithelial sheet. In epithelia with highly curved surfaces, assigning cell orientation can be difficult to automate in silico. We present  Origami, a MATLAB-based image analysis pipeline to compute oriented cell shape-features. Our automated method accurately computed cell orientation in regions with opposing curvature in synthetic epithelia and fluorescence images of zebrafish embryos. As proof of concept, we identified different cell shape signatures in the developing zebrafish inner ear, where the epithelium deforms in opposite orientations to form different structures. Origami is designed to be user-friendly and is generally applicable to fluorescence images of curved epithelia.

sentences: Clemastine fumarate enhances myelination and promotes functional recovery in a syndromic ASD mouse model of Pitt-Hopkins Syndrome Pitt-Hopkins syndrome (PTHS) is an autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 gene (TCF4). One pathobiological process caused by Tcf4 mutation is a cell autonomous reduction in oligodendrocytes (OLs) and myelination. In this study, we show that clemastine is effective at restoring myelination defects in a PTHS mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells (OPCs) and normalized OL density. In vivo, two-week intraperitoneal administration of clemastine also normalized OPC and OL density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in uncompacted myelin. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behavior. Together, these results provide preclinical evidence that remyelination therapies may be beneficial in PTHS and potentially other neurodevelopmental disorders characterized by demyelination.

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An inactivated multivalent influenza A virus vaccine is broadly protective in mice and ferrets Influenza A viruses (IAVs) present major public health threats from annual seasonal epidemics, from pandemics caused by novel virus subtypes, and from viruses adapted to a variety of animals including poultry, pigs and horses. Vaccines that broadly protect against all such IAVs, so-called "universal" influenza vaccines, do not currently exist, but are urgently needed. This study demonstrates that an inactivated, multivalent whole virus vaccine, delivered intramuscularly or intranasally, is broadly protective against challenges with multiple IAV HA/NA subtypes in both mice and ferrets, including challenges with IAV subtypes not contained in the vaccine. This vaccine approach indicates the feasibility of eliciting broad "universal" IAV protection, and identifies a promising candidate for influenza vaccine clinical development.

One-Sentence SummaryAn inactivated, whole avian influenza virus vaccine delivered intramuscularly or intranasally provides extremely broad protection against antigenically divergent viral challenge and is a promising candidate for a "universal" influenza virus vaccine.

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

sentences: Non-invasive Fluorescence Imaging of Gut Commensal Bacteria in Live Mice In mammals, gut commensal microbiota interact extensively with the host and the same interactions can be dysregulated in diseased states. The development of methods to monitor gut microbiota in vivo can lead to improved foundational understanding of the biological events underpinning these interactions. The current standard for non-invasive monitoring of gut bacteria entails classification by 16S rRNA sequencing from fecal samples. This method has many advantages but also has serious limitations, especially for monitoring dynamic changes in the gut of live animals. In recent years, several imaging techniques have been widely adopted that afford non-invasive assessment of animal subjects - most notably in cancer biology; however, these technical gains have not translated to the imaging of gut bacterial communities. Herein, we describe a method to non-invasively image commensal bacteria based on the specific metabolic labeling of bacterial cell walls to illuminate the gut bacteria of live mice. This tagging strategy may additionally provide unprecedented insight into cell wall turnover of gut commensals, which has implications for bacterial cellular growth and division, in a live animal.

sentences: Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to induce autophagy The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and works with the SEA complex to regulate autophagy. Furthermore, we found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during autophagy. Additionally, we found the SEA complex becomes destabilized in vps501{Delta}sea1{Delta} cells, which resulted in aberrant TORC1 hyperactivity and misregulation of autophagy induction.

sentences: Effect of ambient fluid rheology on oscillatory instabilities in filament-motor systems Filaments and filament bundles such as microtubules or actin interacting with molecular motors such as dynein or myosin constitute a common motif in biology. Synthetic mimics, examples being artificial muscles and reconstituted active networks, also feature active filaments. A common feature of these filament-motor systems is the emergence of stable oscillations as a collective dynamic response. Here, using a combination of classical linear stability analysis and non-linear numerical solutions, we study the dynamics of a minimal filament-motor system immersed in model viscoelastic fluids. We identify steady states, test the linear stability of these states, derive analytical stability boundaries, and investigate emergent oscillatory solutions and their properties. We show that the interplay between motor activity, aggregate elasticity and fluid viscoelasticity allows for stable oscillations or limit cycles to bifurcate from steady states. For highly viscous Newtonian media, frequencies at onset decay with viscosity  as [Formula]. In viscoelastic fluids that have the same viscosity as the Newtonian fluid but additionally allow for temporary energy storage, emergent limit cycles are associated with higher frequencies. The magnitude of the increase in the frequency depends on motor mechanochemistry and the interplay between fluid relaxation time-scales and time-scales associated with motor binding and unbinding. Our results suggest that stability and dynamical response in filamentous active systems may be controlled by tailoring the rheology of the ambient environment.

sentences: The novel conserved NAD+ binding micropeptide SGHRT regulates mitochondrial function and metabolism in human cardiomyocytes AbstractsNicotinamide adenine dinucleotide (NAD) is a critical metabolite and coenzyme for multiple metabolic pathways and cellular processes (1-4). In this study, we identified Singheart, SGHRT as a nuclear genome-encoded NAD+-binding mitochondrial micropeptide. SGHRT, present in both monomeric and dimeric forms, binds directly to NAD, but not NADH or flavin adenine dinucleotide (FAD). Localized to the inner mitochondrial membrane and mitochondrial matrix, SGHRT interacts with the mitochondrial enzymes Succinate-CoA Ligase and Succinate Dehydrogenase. SGHRT deletion in human embryonic stem cell derived cardiomyocytes disrupted mitochondria morphology, decreased total NAD and ATP abundance, and resulted in defective TCA cycle metabolism, the electron transport chain and in Ox-Phos processes. These results comprise the first report of an NAD+-binding micropeptide, SGHRT, required for mitochondrial function and metabolism.

sentences: Plakoglobin and HMGB1 mediate intestinal epithelial cell apoptosis induced by Clostridioides difficile TcdB Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNAi screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or {gamma}-catenin, a member of the catenin family, as a novel host factor, and a previously known cell death-related chromatin factor, high mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondria-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with Bcl-XL after stimulation by TcdB, suggesting a role of JUP in cell death signaling through mitochondria. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis.

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Antibody response patterns to Helicobacter pylori infection in a rural Ugandan population cohort. BackgroundHelicobacter pylori (H. pylori) establishes life-long infection in humans in the absence of treatment and has been associated with a variety of gastrointestinal conditions including peptic ulcer and gastric cancer. Antibody responses to H. pylori antigens are found to be associated with disease risk, however, data from Africa are scarce.

MethodsTo assess the seroprevalence of H. pylori and characterise antibody response patterns, we measured serum IgG antibody levels to 14 antigens among 7,211 individuals in a rural Ugandan population cohort. Multivariate-adjusted linear regression models were fitted to investigate the influence of age, sex, and co-infection on antibody seroreactivity levels.

ResultsH. pylori seroprevalence was 95% in our study population, with 94% of individuals seropositive in childhood (<15 years). In H. pylori positive individuals, we found a markedly high seroprevalence (~99%) and antibody levels to the high-risk antigens CagA and VacA, in addition to Cag{delta}. HSV-2 co-infection was significantly associated with higher IgG levels of CagA and VacA (OR=1.10, 95% C. I=1.05-1.16). HIV infection was associated with lowered IgG levels to CagA (OR=0.86, 95% C.I.=0.80-0.93), and HPV infection was associated with increased IgG levels to VacA (OR=1.16, 95% C.I.=1.11-1.21).

ConclusionsH. pylori in this population is ubiquitous from childhood, with a high prevalence and high seroreactivity levels of high-risk antigens, suggesting chronic active inflammatory responses in individuals that are indicative of risk of disease. Further investigation is warranted to fully understand the relationship between host, immunogenicity, and clinical outcomes to better stratify by risk and improve treatment.

Key MessagesO_LIAntibody responses to H. pylori antigens are found to be associated with risk of gastric cancer, however, despite the high seroprevalence in African populations, data from Africa are scarce. This is the first study of antibody response patterns and their determinants from an African population.
C_LIO_LIOur study shows a population where H. pylori is ubiquitous from childhood, and seroprevalence of virulent antigens is distinctively high suggesting an increased of disease compared to other populations.
C_LIO_LIWe observe inter-individual variation in virulent antibody responses partly influenced by co-infection.
C_LIO_LIWe highlight crucial insights into antibody-based biomarkers of disease risk and reinforce the need for population-based H. pylori screening and treatment programmes for gastric cancer control.
C_LI

sentences: Flotillin-2 regulates EGFR activation, degradation, and cancer growth Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

sentences: Magnesium Force Fields for OPC Water with Accurate Solvation, Ion-Binding, and Water-Exchange Properties: Successful Transfer from SPC/E Magnesium plays a vital role in a large variety of biological processes. To model such processes by molecular dynamics simulations, researchers rely on accurate force field parameters for Mg2+ and water. OPC is one of the most promising water models yielding an improved description of biomolecules in water. The aim of this work is to provide force field parameters for Mg2+ that lead to accurate simulation results in combination with OPC water. Using twelve different Mg2+ parameter sets, that were previously optimized with different water models, we systematically assess the transferability to OPC based on a large variety of experimental properties. The results show that the Mg2+ parameters for SPC/E are transferable to OPC and closely reproduce the experimental solvation free energy, radius of the first hydration shell, coordination number, activity derivative, and binding affinity toward the phosphate oxygens on RNA. Two optimal parameter sets are presented: MicroMg yields water exchange in OPC on the microsecond timescale in agreement with experiments. NanoMg yields accelerated exchange on the nanosecond timescale and facilitates the direct observation of ion binding events for enhanced sampling purposes.

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Microclimate is a strong predictor of the native and invasive plant-associated soil microbiota on San Cristobal Island, Galapagos archipelago Understanding the major drivers that influence soil bacterial and fungal communities is essential to mitigate the impacts of human activity on vulnerable ecosystems, like those found on the Galapagos Islands. Located ~1000 km off the coast of Ecuador, the volcanically formed islands are situated within distinct oceanic currents, which provide seasonal weather patterns and unique microclimates within small spatial scales across the islands. Although much is known about the impacts of human activity, such as climate change and invasive plant species, on above ground biodiversity of the Galapagos Islands, little is known about the resident soil microbial communities and the drivers that shape these communities. Here, our goal was to investigate the bacterial and fungal communities found in soil located in three distinct microclimates: Mirador (arid), Cerro Alto (semi-arid), and El Junco (humid), and associated with native and invasive plant types. At each site, we collected soil at three depths (rhizosphere, 5 cm, and 15 cm) associated with the invasive plant, Psidium guajava (guava), and native plant types. We determined that the sampling location (microclimate) was the strongest driver of both bacterial and fungal communities (74 and 38%, respectively), with additional minor but significant impacts from plant type and soil depth. This study highlights the continued need to explore microbial communities across diverse environments and demonstrates the weight of different abiotic and biotic factors impacting soil microbial communities across San Cristobal Island in the Galapagos archipelago.

IMPORTANCE/SIGNIFICANCEHuman activity such as climate change, pollution, introduction of invasive species, and deforestation, poses a huge threat to biodiverse environments. Soil microbiota are an essential component to maintaining healthy ecosystems. However, a greater understanding of factors that alter these microbial communities is needed in order to find ways to mitigate and reverse the impacts imposed by human activity. The Galapagos Islands are a unique real-world laboratory, in that the islands biogeography and physical locations in the Pacific Ocean provide distinct microclimates within small geographic distances. Harnessing these distinct environments allowed us to investigate the influence of microclimates, soil depth, and vegetation cover on bacterial and fungal community composition.

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Information flows from hippocampus to cortex during replay of verbal working memory items. The maintenance of items in working memory relies on a widespread network of cortical areas and hippocampus where synchronization between electrophysiological recordings reflects functional coupling.

We investigated the direction of information flow between auditory cortex and hippocampus while participants heard and then mentally replayed strings of letters in working memory by activating their phonological loop. We recorded LFP from the hippocampus, reconstructed beamforming sources of scalp EEG, and - additionally in 3 participants - recorded from subdural cortical electrodes. When analyzing Granger causality, the information flow was from auditory cortex to hippocampus with a peak in the 4-8 Hz range while participants heard the letters. This flow was subsequently reversed during maintenance while participants maintained the letters in memory. The functional interaction between hippocampus and the cortex and the reversal of information flow provide a physiological basis for the encoding of memory items and their active replay during maintenance.

sentences: Thermally tolerant symbionts may explain Caribbean octocoral resilience to heat stress Coral reef ecosystems are under threat from the frequent and severe impacts of anthropogenic climate change, particularly rising sea surface temperatures. The effects of thermal stress may be ameliorated by adaptation and/or acclimation of the host, symbiont, or holobiont (host + symbiont) to increased temperatures. We examined the role of the symbiont in promoting thermal tolerance of the holobiont, using Antillogorgia bipinnata (octocoral host) and Breviolum antillogorgium (symbiont) as a model system. We identified five distinct genotypes of B. antillogorgium from symbiont populations isolated from Antillogorgia colonies in the Florida Keys. Three symbiont genotypes were cultured and maintained at 26{degrees}C (ambient historical temperature) and two were cultured and maintained at 30{degrees}C (elevated historical temperature) for two years. Following culturing, we analyzed the growth rate and carrying capacity of each symbiont genotype at both ambient and elevated temperatures in culture (in vitro). All genotypes grew well at both temperatures, indicating thermal tolerance among these B. antillogorgium cultures. Prior culturing at the elevated temperature, however, did not result in increased thermal tolerance. We then inoculated juvenile A. bipinnata polyps with each of the five symbiont genotypes, and reared these polyps at both ambient and elevated temperatures (in hospite experiment). All genotypes were able to establish symbioses with polyps in both temperature treatments. Survivorship of polyps at 30{degrees}C was significantly lower than survivorship at 26{degrees}C, but all treatments had surviving polyps at 56 days post-infection, suggestive of broad thermal tolerance in B. antillogorgium, which may play a part in the increased resilience of Caribbean octocorals during heat stress events.

sentences: GABAergic neuronal IL-4R mediates T cell effect on memory Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying effects of T cells on synaptic function and episodic memory. Depletion of CD4 T cells led to memory deficits and impaired long term potentiation. Severe combined immune-deficient (SCID) mice exhibited amnesia, which was reversible by repopulation with T cells from wild-type but not from IL-4-knockout mice. This rescue was mediated via IL-4 receptors (IL-4R) expressed on neurons. Exploration of snRNAseq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways and genes regulated by immune cells and molecules. IL-4R knockout in inhibitory neurons impaired contextual fear memory, suggesting participation of an IL-4-associated switch in regulating synaptic function and promoting contextual fear memory. These findings provide insights into neuroimmune interactions at the transcriptional and functional levels in neurons.

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OmicsView: omics data analysis through interactive visual analytics With advances in NGS technologies, transcriptional profiling of human tissue across many diseases is becoming more routine, leading to the generation of petabytes of data deposited in public repositories. There is a need for bench scientists with little computational expertise to be able to access and mine this data to understand disease pathology, identify robust biomarkers of disease and the effect of interventions (in vivo or in vitro). To this end we release an open source analytics and visualization platform for expression data called OmicsView, http://omicsview.org.

This platform comes preloaded with 1000s of samples across many disease areas and normal tissue, including the GTEx database, all processed with a harmonized pipeline. We demonstrate the power and ease-of-use of the platform by means of a Crohns disease data mining exercise where we can quickly uncover disease pathology and identify strong biomarkers of disease and response to treatment.

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

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Secretory MPP3 reinforce myeloid differentiation trajectory and amplify myeloid cell production Recent lineage tracing analyses revealed multipotent progenitors (MPP) to be major functional contributors to steady-state hematopoiesis (1-6). However, we are still lacking a precise resolution of myeloid differentiation trajectories and cellular heterogeneity in MPPs. Here, we found that myeloid-biased MPP3 (2, 3) are functionally and molecularly heterogeneous, with a distinct subset of myeloid-primed secretory cells with high endoplasmic reticulum (ER) volume and Fc{gamma}R expression. We show that Fc{gamma}R+/ERhigh MPP3 are a transitional population for rapid production of granulocyte/macrophage progenitors (GMP), which directly amplify myelopoiesis through inflammation-triggered secretion of cytokines in the local bone marrow (BM) microenvironment. Our results identify a novel regulatory function for a subset of secretory MPP3 that controls myeloid differentiation through lineage-priming and cytokine production, and act as a self-reinforcing amplification compartment in stress and disease conditions.

One-Sentence SummaryA secretory subset of multipotent hematopoietic progenitors augment myelopoiesis in stress and diseases conditions.

sentences: Aerosphere occupancy ({psi}) model of lesser short nosed fruit bat (Cynopterus brachyotis Muller, 1838) related to tree species in Asia mountainous paddy fields Bat is animal that occupies aerosphere, especially fruit bats that forage on the space around the trees. The fruit bats use whether narrow space below tree canopy or in edge space on the edge of canopy. Whereas the aerosphere occupancy of fruits bats related to the specific tree species is poorly understood. Here, this paper aims to assess and model the association of fruit bat Cynopterus brachyotis aerosphere occupancy ({Psi}) with tree species planted in mountainous paddy fields in West Java. The studied tree species including Alianthus altissima, Acacia sp., Cocos nucifera, Mangifera indica, Pinus sp., and Swietenia macrophylla. The result shows that the tree species diversity has significantly (x2= 27.67, P < 0.05) affected the C. brachyotis aerosphere occupancy. According to values of {Psi} and occupancy percentage, high occupancy of narrow space by C. brachyotis was observed in Swietenia macrophylla ({Psi} = 0.934, 78%), followed by Alianthus altissima ({Psi} = 0.803, 57%), and Mangifera indica ({Psi} = 913, 55%). While high occupancy of edge space was observed in Mangifera indica ({Psi} = 0.685, 41%), followed by Pinus sp. ({Psi} = 0.674, 38%), and Alianthus altissima sp. ({Psi} = 0.627, 36%). The best model for explaining C. brachyotis occupation in narrow space is the tree height with preferences on high tree ({Psi}~tree height, AIC = 1.574, R2= 0.5535, Adj. R = 0.4047). While for edge space occupant, the best model is also the tree height ({Psi}~tree height, AIC = -26.1510, R2= 0.7944, Adj. R = 0.7258).

sentences: Regional and cell type-specific afferent and efferent projections of the mouse claustrum The claustrum (CLA) is a conspicuous subcortical structure interconnected with cortical and subcortical regions. However, its regional anatomy and cell-type-specific connections in the mouse remain largely undetermined. Here, we accurately delineated the boundary of the mouse CLA and quantitatively investigated its inputs and outputs brain-wide using anterograde and retrograde viral tracing and fully reconstructed single claustral principal neurons. At a population level, the CLA reciprocally connects with all isocortical modules. It also receives inputs from at least 35 subcortical structures but sends projections back to only a few of them. We found that cell types projecting to the CLA are differentiated by cortical areas and layers. We classified single CLA principal neurons into at least 9 cell types that innervate the diverse sets of functionally linked cortical targets. Axons of interneurons within the CLA arborize along almost its entire anteroposterior extent. Together, this detailed wiring diagram of the cell-type-specific connections of the mouse CLA lays a foundation for studying its functions.

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Using BERT to identify drug-target interactions from whole PubMed BackgroundDrug-target interactions (DTIs) are critical for drug repurposing and elucidation of drug mechanisms, and they are collected in large databases, such as ChEMBL, BindingDB, DrugBank and DrugTargetCommons. However, the number of studies providing this data (~0.1 million) likely constitutes only a fraction of all studies on PubMed that contain experimental DTI data. Finding such studies and extracting the experimental information is a challenging task, and there is a pressing need for machine learning for the extraction and curation of DTIs. To this end, we developed new text mining document classifiers based on the Bidirectional Encoder Representations from Transformers (BERT) algorithm. Because DTI data intimately depends on the type of assays used to generate it, we also aimed to incorporate functions to predict the assay format.

ResultsOur novel method identified and extracted DTIs from 2.1 million studies not previously included in public DTI databases. Using 10-fold cross-validation, we obtained ~99% accuracy for identifying studies containing drug-target pairs. The accuracy for the prediction of assay format is ~90%, which leaves room for improvement in future studies.

ConclusionThe BERT model in this study is robust and the proposed pipeline can be used to identify new and previously overlooked studies containing DTIs and automatically extract the DTI data points. The tabular output facilitates validation of the extracted data and assay format information. Overall, our method provides a significant advancement in machine-assisted DTI extraction and curation. We expect it to be a useful addition to drug mechanism discovery and repurposing.

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Restoration of motor-evoked cortical activity is a distinguishing feature of the most effective rehabilitation therapy after stroke BackgroundAn ischemic stroke is followed by the remapping of motor representation and extensive changes in cortical excitability involving both hemispheres. Although stimulation of the ipsilesional motor cortex, especially when paired with motor training, facilitates plasticity and functional restoration, the mechanisms underneath the reshaping of cortical functionality are widely unknown.

ObjectiveWe investigated the spatio-temporal features of motor-evoked cortical activity associated with generalized recovery after stroke, and its dependence on the type of rehabilitative treatment.

MethodsWe designed a novel rehabilitative treatment that combines neuro-plasticizing intervention with motor training. Specifically, optogenetic stimulation of peri-infarct excitatory neurons expressing Channelrhodopsin-2 was associated with daily motor training on a robotic device. The effectiveness of the combined therapy was compared with spontaneous recovery and with the single treatments (i.e. individually administered optogenetic stimulation or motor training).

ResultsWe found that only the combined therapy promotes generalized recovery of forelimb function and the rescue of spatio-temporal features of motor-evoked activity. Generalized recovery results from a new excitatory/inhibitory balance between hemispheres as revealed by the augmented motor response flanked by the increased expression of parvalbumin positive neurons in the peri-infarct area.

ConclusionsOur findings demonstrate that though behavioral recovery is not necessarily associated with the restoration of pre-stroke motor-evoked activity, the reestablishment of pre-stroke activation transients was a distinguishing feature of the most efficient therapeutic approach, the combined therapy.

sentences: ECM dimensionality tunes actin tension to modulate the endoplasmic reticulum and spheroid phenotype Primary tissue organoids and cell spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a three dimensional laminin-rich extracellular matrix supports the polarized, stress resilient spheroid phenotype of mammary epithelial cells. Cells within a three dimensional laminin-rich extracellular matrix decreased and redistributed the actin crosslinker filamin to reduce their cortical actin tension. Cells with low cortical actin tension had increased plasma membrane protrusions that promoted negative plasma membrane curvature and fostered protein associations with the plasma membrane, consistent with efficient protein secretion. By contrast, cells engaging a laminin-rich extracellular matrix in two dimensions had high filamin-dependent cortical actin tension, exhibited compromised endoplasmic reticulum function including increased expression of PKR-like Endoplasmic Reticulum Kinase signaling effectors, and had compromised protein secretion. Cells with low filamin-mediated cortical actin tension and reduced endoplasmic reticulum stress response signaling secreted, and assembled, a polarized endogenous basement membrane and survived better, and their spheroids were more resistant to exogenous stress. The findings implicate filamin-dependent cortical actin tension in endoplasmic reticulum function and highlight a role for mechanics in organoid homeostasis.

sentences: Benchmarking brain organoid recapitulation of fetal corticogenesis Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols, including different degrees of heterochronicity in differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as an accessible resource to query for modelling human corticogenesis and the neuropsychiatric outcomes of its alterations.

sentences: NMR solution structure and analysis of isolated S3b-S4a motif of repeat IV of the human cardiac sodium channel Voltage-gated sodium channels are membrane proteins that play an important role in the propagation of electrical signals by mediating the rising phase of an action potential. Numerous diseases, including epilepsy, extreme pain, and certain cardiac arrhythmias have been linked to defects in these channels. The S3b-S4a helix-turn-helix motif (paddle motif) is a region of the channel that is involved in voltage sensing and undergoes significant structural changes during gating. It is also the binding site for many gating-modifier toxins. We determined the solution structure of the paddle motif from the fourth repeat of NaV1.5 in dodecylphosphocholine micelles by NMR spectroscopy and investigated its dynamics and micelle interactions. The structure displays a helix hairpin with a short connecting loop, and likely represents the activated conformation with three of the first four gating charges facing away from S3. Furthermore, paramagnetic relaxation measurements show that the paddle motif is mainly interacting with the interface region of the micelle. NMR relaxation studies reveal that the paddle motif is mostly rigid, with some residues around the loop region and the last 4 residues on the C-terminus displaying heightened mobility. The structural findings reported here allowed the interpretation of three disease-causing mutations in this region of the human cardiac sodium channel, S1609W, F1617del and T1620M. The establishment of this model system for NMR studies of the paddle region offers a promising platform for future toxin interaction studies in the cardiac sodium channels, and similar approaches may be applied to other sodium channel isoforms.

sentences: Potassium effects on NCC are attenuated during inhibition of Cullin E3-ubiquitin ligases The thiazide sensitive sodium-chloride co-transporter (NCC) plays a vital role in maintaining sodium (Na+) and potassium (K+) homeostasis. NCC activity is modulated by the with-no-lysine kinases 1 and 4 (WNK1 and WNK4), the abundance of which are controlled by the RING-type E3 ligase Cullin 3 (Cul3) and its substrate adapter Kelch-like protein 3. Dietary K+ intake has an inverse correlation with NCC activity, but the mechanism underlying this phenomenon remains to be fully elucidated. Here, we investigated the involvement of other members of the Cullin family in mediating K+ effects on NCC phosphorylation (active form) and abundance. In kidneys from mice fed diets varying in K+ content, there were negative correlations between NCC (phosphorylated and total) and active (neddylated) forms of Cullins (Cul1, 3, 4 and 5). High dietary K+ effects on phosphorylated NCC were attenuated in Cul3 mutant mice (CUL3-Het/{Delta}9). Short-term (30 min) and long-term (24 h) alterations in the extracellular K+ concentration did not affect Cullin neddylation levels in ex vivo renal tubules. Short-term, the ability of high extracellular K+ to decrease NCC phosphorylation was preserved in the presence of MLN4924 (pan Cullin inhibitor), but the response to low extracellular K+ was absent. Long-term, MLN4924 attenuated the effects of high extracellular K+ on NCC phosphorylation and responses to low extracellular K+ were absent. Our data suggest that in addition to Cul3, other Cullins are involved in mediating the effects of K+ on NCC phosphorylation and abundance.

sentences: The human SKI complex prevents DNA-RNA hybrid-associated telomere instability Super killer (SKI) complex is a well-known cytoplasmic 3' to 5' mRNA decay complex that functions with the exosome to degrade excessive and aberrant mRNAs. Recently, SKIV2L, the 3' to 5' RNA helicase of the human SKI (hSKI) complex has been implicated in the degradation of nuclear non-coding RNAs escaping to the cytoplasm. Here, we show that hSKI is present in the nucleus, on chromatin and in particular at telomeres during the G2 cell cycle phase. In cells, SKIV2L prevents telomeric loss, and DNA damage response activation, and its absence leads to DNA-RNA hybrid-mediated telomere fragility. Moreover, we demonstrate that purified hSKI complex preferentially unwinds telomeric DNA-RNA hybrids in vitro. Taken together, our results provide a nuclear function of the hSKI complex in overcoming replication stress caused by aberrant processing of telomeric DNA-RNA hybrids and thus maintaining telomere stability.

sentences: Estimating the rates of crossover and gene conversion from individual genomes Recombination can occur either as a result of crossover or gene conversion events. Population genetic methods for inferring the rate of recombination from patterns of linkage disequilibrium generally assume a simple model of recombination that only involves crossover events and ignore gene conversion. However, distinguishing the two processes is not only necessary for a complete description of recombination, but also essential for understanding the evolutionary consequences of inversions and other genomic partitions in which crossover (but not gene conversion) is reduced. We present heRho, a simple composite likelihood scheme for co-estimating the rate of crossover and gene conversion from individual diploid genomes. The method is based on analytic results for the distance-dependent probability of heterozygous and homozygous states at two loci. We apply heRho to simulations and data from the house mouse Mus musculus castaneus, a well studied model. Our analyses show i) that the rates of crossover and gene conversion can be accurately co-estimated at the level of individual chromosomes and ii) that previous estimates of the population scaled rate of recombination [Formula] under a pure crossover model are likely biased.

sentences: The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid- host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.

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Apis mellifera cognition as a tool for scientific engagement In addition to the crowdsourcing of experimental data, citizen science, and scientific engagement more broadly, serve as a bridge between researchers and the wider community. This serves to foster a greater understanding of the scientific method and science-based solutions generally. Apis mellifera (honeybees) are a well-established model for the study of learning and cognition and can serve as an engaging outreach system for this wider community. Here, we developed and implemented a protocol using well established honeybee conditioning protocols to safely study the effects of caffeine and dopamine on learning performance. Using this protocol, a group of high-school aged students as part of the Ryukyu Girls program demonstrated that caffeine, but not dopamine, significantly reduced the number of trials required for a successful conditioning response. This allowed these students to explore the scientific method in a relatable and engaging way.

Simple SummaryGlobal scientific literacy can be improved through widespread and effective community engagement by researchers. We propose Apis mellifera (honeybee) as an public engagement tool due to widespread awareness of colony collapse and the bees importance in food production. Moreover, their cognitive abilities make for engaging experiments. Their relative ease of cultivation means that studies can be performed cost-effectively, especially when partnering with local aperists. Using a proxy for honeybee learning, a group of non-specialist high-school-aged participants obtained data suggesting that caffeine, but not dopamine, improved learning. This hands-on experience facilitated student understanding of the scientific method, factors that shape learning and the importance of learning for hive health.

sentences: Neurons undergo pathogenic metabolic reprograming in models of familial ALS Normal cellular function requires a rate of ATP production sufficient to meet demand. In most neurodegenerative diseases (including Amyotrophic Lateral Sclerosis, ALS), mitochondrial dysfunction is postulated raising the possibility of impaired ATP production and a need for compensatory maneuvers to sustain the ATP production/demand balance. We find in our rodent models of familial ALS (fALS), impairment in neuronal glycolytic flux with maintained or enhanced activity of the citric acid cycle. This rewiring of metabolism is associated with normal ATP levels and redox status, supporting the notion that mitochondrial function is not compromised in neurons expressing fALS genes. Genetic loss-of-function manipulation of individual steps in the glycolysis and the pentose phosphate pathway blunt the negative phenotypes seen in various fALS models. We propose that neurons adjust fuel utilization in the setting of neurodegenerative disease-associated mitochondrial dysfunction in a baleful manner and targeting this process can be healthful.

sentences: Palmitoylation targets the Calcineurin phosphatase to the Phosphatidylinositol 4-kinase complex at the plasma membrane Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. To discover novel calcineurin-regulated processes we examined an understudied isoform, CNA{beta}1. We show that unlike canonical cytosolic calcineurin, CNA{beta}1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNA{beta}1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a new calcineurin-regulated signaling pathway highlighting the PI4KA complex as a regulatory target and revealing that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNA{beta}1.

sentences: Population dynamics and ranging behaviors of provisioned silvered langur (Trachypithecus cristatus) in Peninsular Malaysia Tourists are attracted to the Bukit Melawati Kuala Selangor (BMKS) of Peninsular Malaysia, a small hill park, both for its status as a historical site and the free-ranging silvered langurs (Trachypithecus cristatus) that come for provisioning. We assessed the population trends and group sizes of T. cristatus over 10 years in the BMKS and examined their ranging patterns. Comparisons of observed populations between 2005 (190 individuals) and 2017 (193 individuals) revealed the stable demography and group sizes of the six T. cristatus groups in the BMKS. Based on a total of 185 location points of the six groups in 2017, their mean ranging area was 3.6 ha with a range of 0.86 to 6.93 ha with extensive spatial overlap. We also found a significant positive relationship between the six groups ranges and group sizes in 2017. Additionally, qualitative ecological comparisons with a previous study on T. cristatus in 1965 (before provisioning) suggest that the artificial food supply in the study area could modify the population dynamics and socioecology of T. cristatus. The modifications might alter their range size and territoriality in the BMKS. Overall, we found that provisioning had negative effects on the ecology of T. cristatus in the BMKS. Therefore, modifying management policies, such as banning feeding and implementing educational programs, may contribute to their proper conservation.

sentences: Sensitizing Staphylococcus aureus to antibacterial host defense by decoding and blocking the lipid flippase MprF The pandemic of antibiotic resistance represents a major human health threat demanding new antimicrobial strategies. MprF is the synthase and flippase of the phospholipid lysyl-phosphatidylglycerol that increases virulence and resistance of methicillin-resistant Staphylococcus aureus (MRSA) and other pathogens to cationic host defense peptides and antibiotics. With the aim to design MprF inhibitors that could sensitize MRSA to both, human antimicrobials and antibiotics and support the clearance of staphylococcal infections with minimal selection pressure, we developed MprF-targeting monoclonal antibodies, which bound and blocked the MprF flippase subunit. Antibody M-C7.1 targeted a specific loop in the flippase domain that proved to be exposed at both sides of the bacterial membrane, thereby enhancing the mechanistic understanding into bacterial lipid translocation. M-C7.1 rendered MRSA susceptible to host antimicrobial peptides and antibiotics such as daptomycin. Moreover, it impaired MRSA survival in human phagocytes, which recommends MprF inhibitors for new anti-MRSA approaches. MprF-directed monoclonal antibodies provide a proof of concept for development of precisely targeted anti-virulence approaches, which block bacterial antimicrobial resistance mechanisms.

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Global non-random abundance of short tandem repeats in rodents and primates. BackgroundWhile of predominant abundance across vertebrate genomes and significant biological implications, the relevance of short tandem repeat (STR) abundance to speciation remains largely elusive and attributed to random coincidence for the most part. In a model study, here we collected whole-genome abundance of mono-, di-, and trinucleotide STRs in nine species, encompassing rodents and primates, including rat, mouse, olive baboon, gelada, macaque, gorilla, chimpanzee, bonobo, and human. The obtained unnormalized and normalized data were used to analyze hierarchical clustering of the STR abundances in the selected species.

ResultsWe found massive differential abundances between the rodent and primate orders. In addition, while numerous STRs had random abundance across the nine selected species, the global abundance conformed to three consistent <clusters>, as follows: <rat, mouse>, <gelada, macaque, olive baboon>, <gorilla, chimpanzee, bonobo, human>, which coincided with the phylogenetic distances of the selected species (p< 4E-05). Exceptionally, in the trinucleotide STR compartment, human was significantly distant from all other species.

ConclusionWe propose that the global abundance of STRs is non-random in rodents and primates, and probably had a determining impact on the speciation of the two orders. We also propose the STRs and STR lengths which specifically coincided with the phylogeny of the selected species.

sentences: 3D Reconstruction of Bird Flight Using a Single Video Camera Video cameras are finding increasing use in the study and analysis of bird flight over short ranges. However, reconstruction of flight trajectories in three dimensions typically requires the use of multiple cameras and elaborate calibration procedures. We present an alternative approach that uses a single video camera and a simple calibration procedure for the reconstruction of such trajectories. The technique combines prior knowledge of the wingspan of the bird with a camera calibration procedure that needs to be used only once in the lifetime of the system. The system delivers the exact 3D coordinates of the position of the bird at the time of every full wing extension and uses interpolated height estimates to compute the 3D positions of the bird in the video frames between successive wing extensions. The system is inexpensive, compact and portable, and can be easily deployed in the laboratory as well as the field.

sentences: iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation Since multiple ribosomes can engage a single mRNA, nonuniform ribosome progression can result in collisions. Ribosome collisions during translation elongation elicit a multifaceted ribosome-associated quality control (RQC) response. Despite advanced mechanistic understanding of translation initiation, a parallel RQC pathway that acts on collided preinitiation complexes has not been described. Here, we show that blocking progression of scanning or elongating ribosomes past the start codon triggers uS3 and uS5 ribosomal ubiquitylation. We demonstrate that conditions that activate the integrated stress response can also induce preinitiation complex collisions. The ubiquitin ligase, RNF10, and the deubiquitylating enzyme, USP10, are the key regulators of uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in an autophagy-independent manner. This study identifies a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on pervasive ribosome collisions during translation initiation to modulate translation activity and capacity.

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AlphaTims: Indexing trapped ion mobility spectrometry - time of flight data for fast and easy accession and visualization High resolution mass spectrometry-based proteomics generates large amounts of data, even in the standard liquid chromatography (LC) - tandem mass spectrometry configuration. Adding an ion mobility dimension vastly increases the acquired data volume, challenging both analytical processing pipelines and especially data exploration by scientists. This has necessitated data aggregation, effectively discarding much of the information present in these rich data sets. Taking trapped ion mobility spectrometry (TIMS) on a quadrupole time-of-flight platform (Q-TOF) as an example, we developed an efficient indexing scheme that represents all data points as detector arrival times on scales of minutes (LC), milliseconds (TIMS) and microseconds (TOF). In our open source AlphaTims package, data are indexed, accessed and visualized by a combination of tools of the scientific Python ecosystem. We interpret unprocessed data as a sparse 4D matrix and use just-in-time compilation to machine code with Numba, accelerating our computational procedures by several orders of magnitude while keeping to familiar indexing and slicing notations. For samples with more than six billion detector events, a modern laptop can load and index raw data in about a minute. Loading is even faster when AlphaTims has already saved indexed data in a HDF5 file, a portable scientific standard used in extremely large-scale data acquisition. Subsequently, data accession along any dimension and interactive visualization happen in milliseconds. We have found AlphaTims to be a key enabling tool to explore high dimensional LC-TIMS-QTOF data and have made it freely available as an open-source Python package with a stand-alone graphical user interface at https://github.com/MannLabs/alphatims or as part of the AlphaPept  ecosystem.

HighlightsO_LIEasy visualization and fast accession of LC-TIMS-QTOF data
C_LIO_LIFreely available graphical user interface, command-line interface and Python module on Windows, Linux and macOS.
C_LI

sentences: Multivariate mining of an alpaca immune repertoire identifies potent cross-neutralising SARS-CoV-2 nanobodies Conventional approaches to isolate and characterize nanobodies are laborious and cumbersome. Here we combine phage display, multivariate enrichment, and novel sequence analysis techniques to annotate an entire nanobody repertoire from an immunized alpaca. We combine this approach with a streamlined screening strategy to identify numerous anti-SARS-CoV-2 nanobodies, and use neutralization assays and Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX-MS) epitope mapping to characterize their potency and specificity. Epitope mapping revealed that the binding site is a key determinant of neutralization potency, rather than affinity alone. The most potent nanobodies bind to the receptor binding motif of the RBD, directly preventing interaction with the host cell receptor ACE2, and we identify two exceptionally potent members of this category (with monomeric IC50s around 13 and 16 ng/ml). Other nanobodies bind to a more conserved epitope on the side of the RBD, and are able to potently neutralize the SARS-CoV-2 founder virus (42 ng/ml), the beta variant (B.1.351/501Y.V2) (35 ng/ml), and also cross-neutralize the more distantly related SARS-CoV-1 (0.46 g/ml). The approach presented here is well suited for the screening of phage libraries to identify functional nanobodies for various biomedical and biochemical applications.

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Substrate ligand density modulates gap junction intercellular communication during mesenchymal cell condensation To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis.

Materials & MethodsPreviously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid (RGD) were used as cell culture substrates to control cell adhesion at the nanoscale. We conducted in vitro chondrogenesis of mesenchymal stem cells on the nanopatterns. We evaluated cohesion and GJIC in cell condensates.

ResultsMechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling.

ConclusionCell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

sentences: Contextual expectations shape cortical reinstatement of sensory representations When making a turn at a familiar intersection, we know what items and landmarks will come into view. These perceptual expectations, or predictions, come from our knowledge of the context, however its unclear how memory and perceptual systems interact to support the prediction and reactivation of sensory details in cortex. To address this, human participants learned the spatial layout of animals positioned in a cross maze. During fMRI, participants navigated between animals to reach a target, and in the process saw a predictable sequence of five animal images. Critically, to isolate activity patterns related to item predictions, rather than bottom-up inputs, one quarter of trials ended early, with a blank screen presented instead. Using multivariate pattern similarity analysis, we reveal that activity patterns in early visual cortex, posterior medial regions, and the posterior hippocampus showed greater similarity when seeing the same item compared to different items. Further, item effects in posterior hippocampus were specific to the sequence context. Critically, activity patterns associated with seeing an item in visual cortex and posterior medial cortex, were also related to activity patterns when an item was expected, but omitted, suggesting sequence predictions were reinstated in these regions. Finally, multivariate connectivity showed that patterns in the posterior hippocampus at one position in the sequence were related to patterns in early visual cortex and posterior medial cortex at a later position. Together, our results support the idea that hippocampal representations facilitate sensory processing by modulating visual cortical activity in anticipation of expected items.

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Comparative small RNA and degradome sequencing provide new insights into antagonistic interactions in the biocontrol fungus Clonostachys rosea Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small-RNAs (sRNAs) are vital components of post-transcriptional gene regulation, although their role in gene expression regulation during mycoparasitism remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tags sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were down-regulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing de-suppression (up-regulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated gene targets for 282 differentially expressed sRNAs. Analysis of sRNA gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. While the regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum highlighting common and mycohosts-specific gene regulation of C. rosea. By combining these results with transcriptome data collected in similar experimental conditions during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism.

ImportanceSmall-RNAs (sRNAs) are emerging as key players in pathogenic and symbiotic fungus-plant interactions, however, their role in fungal-fungal interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and plant pathogenic mycohots Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel miRNAs and their potential gene targets at endogenous level, and at a cross-species level in B. cinerea and F. graminearum indicating a role of cross-species RNAi in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

sentences: Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N-(NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we showed that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that was consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

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Annealing synchronizes the 70S ribosome into a minimum-energy conformation Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70S ribosome from Escherichia coli as a model, wherein the 30S subunit undergoes a thermally driven inter-subunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70S ribosome homogeneity and improves local resolution on the 30S subunit. After annealing, the 70S ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes, and exhibits a minimum energy in the free energy landscape. One can readily crystallize these minimum-energy ribosomes, which have great potential for synchronizing proteins on a single-molecule level. Our experimental results are consistent with theoretical analysis on the temperature-dependent Boltzmann distribution, and offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing can be extended to study protein folding and explore conformational and energy landscape.

Significance statementIn metallurgy, annealing heats a metal or alloy to a predetermined temperature, holding for a certain time, and then cooling to room temperature to change the physical and sometimes also the chemical properties of the material. Researchers introduce the similar concept as simulated annealing to predict minimum-energy conformations of biological macromolecules. In this work, we experimentally verify that annealing at a fast cooling rate can synchronize the 70S ribosome into a nonrotated state with a minimum energy in the free energy landscape. Our results not only offer a facile yet robust approach to stabilize proteins for high-resolution structural analysis, but also contribute to the understanding of protein folding and temperature adaptation.

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Combining SLA 3D printing and soft lithography for fast, versatile, and accessible high-resolution fabrication of customised multiscale cell culture devices with complex designs Commercially available cell culture devices are designed to increase the complexity of simple cell culture models to provide better experimental platforms for biological systems. From microtopography, microwells, plating devices and microfluidic systems to larger constructs for specific applications like live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology labs. However, the techniques used for their fabrication can be out of reach for most wet labs due to cost and availability of specialised equipment or the need for engineering expertise. Moreover, these techniques also have technical limitations to the volumes, shapes and dimensions they can generate. For these reasons, creating customisable devices tailored to lab-specific biological questions remains difficult to apply.

Taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft-lithography we have developed an optimised microfabrication pipeline capable of generating a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This technique enables the manufacture of complex devices across scales bridging the gap between microfabrication and fused deposition moulding (FDM) printing. The method we describe allows for the efficient treatment of resin-based 3D printed constructs for PDMS curing, using a combination of curing steps, washes and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we provide several proof-of-principle applications ranging from simple 2D culture devices to large tissue engineering constructs and organoid formation systems.

We believe this methodology will be applicable in any wet lab, irrespective of prior expertise or resource availability and will therefore enable a wide adoption of tailored microfabricated devices across many fields of biology.

sentences: HOW 1 CONCERNING IS A SARS-COV-2 VARIANT OF2 CONCERN? COMPUTATIONAL PREDICTIONS AND3 THE VARIANTS LABELING SYSTEM. We herein report a study to evaluate the use of computational prediction of SARS-CoV-2 genetic variations in improving the current variants labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the adaptations made to it by the United States Center of Diseases Control (CDC). We observed that the labeling system is based upon the virus major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two health management agencies. Consequently, discrepancies exist between the labels given by WHO and CDC to same variants. Our observations suggest that giving the VOC label to a new variant is premature and might not be appropriate. Therefore, we carried out a comparative computational study to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO and the CDC for variant labeling, we ascertained that comparative computational predictions of the impact of genetic variations are a better ground for rapid and more accurate labelling of SARS-CoV-2 variants. We propose to label all emergent variants VUM or VBM and to carry out computational predictive studies and thorough variants comparison, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variants labeling system would be globally beneficial to communicate about and fight COVID-19 pandemic.

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Full-length de novo protein structure determination from cryo-EM maps using deep learning Advances in microscopy instruments and image processing algorithms have led to an increasing number of cryo-EM maps. However, building accurate models for the EM maps at 3-5 [A] resolution remains a challenging and time-consuming process. With the rapid growth of deposited EM maps, there is an increasing gap between the maps and reconstructed/modeled 3-dimensional (3D) structures. Therefore, automatic reconstruction of atomic-accuracy full-atom structures from EM maps is pressingly needed. Here, we present a semi-automatic de novo structure determination method using a deep learning-based framework, named as DeepMM, which builds atomic-accuracy all-atom models from cryo-EM maps at near-atomic resolution. In our method, the main-chain and C positions as well as their amino acid and secondary structure types are predicted in the EM map using Densely Connected Convolutional Networks. DeepMM was extensively validated on 40 simulated maps at 5 [A] resolution and 30 experimental maps at 2.6-4.8 [A] resolution as well as an EMDB-wide data set of 2931 experimental maps at 2.6-4.9 [A] resolution, and compared with state-of-the-art algorithms including RosettaES, MAINMAST, and Phenix. Overall, our DeepMM algorithm obtained a significant improvement over existing methods in terms of both accuracy and coverage in building full-length protein structures on all test sets, demonstrating the efficacy and general applicability of DeepMM.

Availabilityhttps://github.com/JiahuaHe/DeepMM

Supplementary informationSupplementary data are available.

sentences: CAN CARBON NANOFIBERS AFFECT ANUROFAUNA? STUDY INVOLVING NEOTROPICAL Physalaemus cuvieri (Fitzinger, 1826) TADPOLES Although carbon nanotubes (CNTs) toxicity in different experimental systems (in vivo and in vitro) is known, little is known about the toxic effects of carbon nanofibers (CNFs) on aquatic vertebrates. We herein investigated the potential impact of CNFs (1 and 10 mg/L) by using Physalaemus cuvieri tadpoles as experimental model. CNFs were able to induce nutritional deficit in animals after 48-h exposure to them, and this finding was inferred by reductions observed in body concentrations of total soluble carbohydrates, total proteins, and triglycerides. The increased production of hydrogen peroxide, reactive oxygen species and thiobarbituric acid reactive substances in tadpoles exposed to CNFs has suggested REDOX homeostasis change into oxidative stress. This process was correlated to the largest number of apoptotic and necrotic cells in the blood of these animals. On the other hand, the increased superoxide dismutase and catalase activity has suggested that the antioxidant system of animals exposed to CNFs was not enough to maintain REDOX balance. In addition, CNFs induced increase in acetylcholinesterase and butyrylcholinesterase activity, as well as changes in the number of neuromats evaluated on body surface (which is indicative of the neurotoxic effect of nanomaterials on the assessed model system). To the best of our knowledge, this is the first report on the impact of CNFs on amphibians; therefore, it broadened our understanding about ecotoxicological risks associated with their dispersion in freshwater ecosystems and possible contribution to the decline in the populations of anurofauna species.

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Single molecule tracking of bacterial cell surface cytochromes reveals dynamics that impact long-distance electron transport Using a series of multiheme cytochromes, the metal-reducing bacterium Shewanella oneidensis MR-1 can perform extracellular electron transfer (EET) to respire redox-active surfaces, including minerals and electrodes outside the cell. While the role of multiheme cytochromes in transporting electrons across the cell wall is well established, these cytochromes were also recently found to facilitate long-distance (micrometer-scale) redox conduction along outer membranes and across multiple cells bridging electrodes. Recent studies proposed that long-distance conduction arises from the interplay of electron hopping and cytochrome diffusion, which allows collisions and electron exchange between cytochromes along membranes. However, the diffusive dynamics of the multiheme cytochromes have never been observed or quantified in vivo, making it difficult to assess their hypothesized contribution to the collision-exchange mechanism. Here we use quantum dot labeling, total internal reflection fluorescence microscopy, and single-particle tracking to quantify the lateral diffusive dynamics of the outer membrane-associated decaheme cytochromes MtrC and OmcA, two key components of EET in S. oneidensis. We observe confined diffusion behavior for both quantum dot-labeled MtrC and OmcA along cell surfaces (diffusion coefficients DMtrC = 0.0192 {+/-} 0.0018 m2/s, DOmcA = 0.0125 {+/-} 0.0024 m2/s) and the membrane extensions thought to function as bacterial nanowires. We find that these dynamics can trace a path for electron transport via overlap of cytochrome trajectories, consistent with the long-distance conduction mechanism. The measured dynamics inform kinetic Monte Carlo simulations that combine direct electron hopping and redox molecule diffusion, revealing significant electron transport rates along cells and membrane nanowires.

SignificanceMultiheme cytochromes in Shewanella oneidensis MR-1 transport electrons across the cell wall, in a process called extracellular electron transfer. These electron conduits can also enable electron transport along and between cells. While the underlying mechanism is thought to involve a combination of electron hopping and lateral diffusion of cytochromes along membranes, these diffusive dynamics have never been observed in vivo. Here, we observe the mobility of quantum dot-labeled cytochromes on living cell surfaces and membrane nanowires, quantify their diffusion with single-particle tracking techniques, and simulate the contribution of these dynamics to electron transport. This work reveals the impact of redox molecule dynamics on bacterial electron transport, with implications for understanding and harnessing this process in the environment and bioelectronics.

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Soft tissue deformations explain most of the mechanical work variations of human walking Humans perform mechanical work during walking, some by leg joints actuated by muscles, and some by passive, dissipative soft tissues. Dissipative losses must be restored by active muscle work, potentially in amounts sufficient to cost substantial metabolic energy. The most dissipative, and therefore costly, walking conditions might be predictable from the pendulum-like dynamics of the legs. If pendulum behavior is systematic, it may also predict the work distribution between active joints and passive soft tissues. We therefore tested whether the overall negative work of walking, and the fraction due to soft tissue dissipation, are both predictable by a pendulum model across a wide range of conditions. The model predicts whole-body negative work from the leading legs impact with ground (termed the Collision), to increase with the squared product of walking speed and step length. We experimentally tested this in humans (N = 9) walking in 26 different combinations of speed (0.7 - 2.0 m{middle dot}s-1) and step length (0.5 - 1.1 m), with recorded motions and ground reaction forces. Whole-body negative Collision work increased as predicted (R2 = 0.73), with a consistent fraction of about 63% (R2 = 0.88) due to soft tissues. Soft tissue dissipation consistently accounted for about 56% of the variation in total whole-body negative work. During typical walking, active work to restore dissipative losses could account for 31% of the net metabolic cost. Soft tissue dissipation, not included in most biomechanical studies, explains most of the variation in negative work of walking, and could account for a substantial fraction of the metabolic cost.

Summary statementSoft tissue deformations dissipate substantial energy during human walking, as predicted by a simple walking model.

sentences: Low generalizability of polygenic scores in African populations due to genetic and environmental diversity African populations are vastly underrepresented in genetic studies but have the most genetic variation and face wide-ranging environmental exposures globally. Because systematic evaluations of genetic prediction had not yet been conducted in ancestries that span African diversity, we calculated polygenic risk scores (PRS) in simulations across Africa and in empirical data from South Africa, Uganda, and the UK to better understand the generalizability of genetic studies. PRS accuracy improves with ancestry-matched discovery cohorts more than from ancestry-mismatched studies. Within ancestrally and ethnically diverse South Africans, we find that PRS accuracy is low for all traits but varies across groups. Differences in African ancestries contribute more to variability in PRS accuracy than other large cohort differences considered between individuals in the UK versus Uganda. We computed PRS in African ancestry populations using existing European-only versus ancestrally diverse genetic studies; the increased diversity produced the largest accuracy gains for hemoglobin concentration and white blood cell count, reflecting large-effect ancestry-enriched variants in genes known to influence sickle cell anemia and the allergic response, respectively. Differences in PRS accuracy across African ancestries originating from diverse regions are as large as across out-of-Africa continental ancestries, requiring commensurate nuance.

sentences: SARS-CoV-2 mutations among minks show reduced lethality and infectivity to humans SARS-CoV-2 infection in minks has become a serious problem, as the virus may mutate and reinfect humans; some countries have decided to cull minks. Here, the virus sequencing data in minks were analysed and compared to those of human-virus. Although the mink-virus maintained the characteristics of human-virus, some variants rapidly mutated, adapting to minks. Some mink-derived variants infected humans, which accounted for 40% of the total SARS-CoV-2 cases in the Netherlands. These variants appear to be less lethal and infective compared to those in humans. Variants that have mutated further among minks were not found in humans. Such mink-viruses might be suitable for vaccination for humans, such as in the case of the smallpox virus, which is less infective and toxic to humans.

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The Streptococcus pyogenes hyaluronic acid capsule promotes experimental nasal and skin infection by preventing neutrophil-mediated clearance Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S. pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S. pyogenes infections, mice infected with a {Delta}hasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post{-}nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S. pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo, highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice, but not macrophage depletion, recovered the reduced burden by the {Delta}hasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S. pyogenes and demonstrates that its predominant function is to protect S. pyogenes against neutrophil-mediated killing.

AUTHOR SUMMARYStreptococcus pyogenes is a globally disseminated and human-adapted bacterial pathogen that has evolved an arsenal of evasion strategies to overcome and escape host immune clearing mechanisms. Many strains of S. pyogenes are covered by a polysaccharide capsule composed of hyaluronic acid (HA) that is widely recognized to promote severe infections. In this study, we demonstrate using the encapsulated S. pyogenes MGAS8232 strain that the HA capsule is a key virulence factor that facilitates non-invasive infections of the nasopharynx and skin. Although bacterial adhesion and entry into host cells was impeded by HA capsule expression, we show that the key function for both nasal and skin infections is to protect S. pyogenes from neutrophil-mediated killing. Depletion of neutrophils, but not macrophages, recovered the low bacterial burden by unencapsulated S. pyogenes at both sites of infection. Our findings outline an important interaction between the HA capsule and neutrophils in the establishment of acute upper respiratory and skin infections by S. pyogenes.

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Quantitative monitoring of nucleotide sequence data from genetic resources in context of their citation in the scientific literature BackgroundLinking nucleotide sequence data (NSD) to scientific publication citations can enhance understanding of NSDs provenance, scientific use, and re-use in the community. By connecting publications with NSD records, NSD geographical provenance information, and author geographical information, it becomes possible to assess the contribution of NSD to infer trends in scientific knowledge gain at the global level.

FindingsFor this data note, we extracted and linked records from the European Nucleotide Archive to citations in open-access publications aggregated at Europe PubMed Central. A total of 8,464,292 ENA accessions with geographical provenance information were associated with publications. We conducted a data quality review to uncover potential issues in publication citation information extraction and author affiliation tagging and developed and implemented best-practice recommendations for citation extraction. Flat data tables and an data warehouse with an interactive web application were constructed to enable ad hoc exploration of NSD use and summary statistics.

ConclusionsThe extraction and linking of NSD with associated publication citations enables transparency. The quality review contributes to enhanced text mining methods for identifier extraction and use. Furthermore, the global provision and use of NSD enables scientists around the world to join literature and sequence databases in a multidimensional fashion. As a concrete use case, statistics of country clusters were visualized with respect to NSD access in the context of discussions around digital sequence information under the United Nations Convention on Biological Diversity.

sentences: Divergent assembly trajectories: a comparison of the plant and soil microbiome with plant communities in a glacier forefield Community assembly is a result of dispersal, abiotic and biotic characteristics of the habitat as well as stochasticity. A direct comparison between the assembly of microbial and  macrobial organisms is hampered by the sampling of these communities in different studies, at different sites, or on different scales. In a glacier forefield in the Austrian Alps, we recorded the soil and plant microbiome (bacteria and fungi) and plants that occurred in the same landscape and in close proximity in the same plots. We tested five predictions deduced from assembly processes and revealed deviating patterns of assembly in these community types. In short, microbes appeared to be less dispersal limited than plants, soil microbes and plants strongly responded to abiotic factors whereas the leaf microbiome was plant species-specific and well buffered from environmental conditions. The observed differences in community assembly processes may be attributed to the organisms dispersal abilities, the exposure of the habitats to airborne propagules, and habitat characteristics. The finding that assembly is conditional to the characteristics of the organisms, the habitat, and the spatial scale under consideration is thus central for our understanding about the establishment and the maintenance of biodiversity.

sentences: Maintaining and escaping feedback control in hierarchically organised tissue: a case study of the intestinal epithelium The intestinal epithelium is one of the fastest renewing tissues in mammals. It shows a hierarchical organisation, where intestinal stem cells at the base of crypts give rise to rapidly dividing transit amplifying cells that in turn renew the pool of short-lived differentiated cells. Upon injury and stem-cell loss, cells can also dedifferentiate. Tissue homeostasis require a tightly regulated balance of differentiation and stem cell proliferation, and failure can lead to tissue extinction or to unbounded growth and cancerous lesions. Here, we present a two-compartment mathematical model of intestinal epithelium population dynamics that includes a known feedback inhibition of stem cell differentiation by differentiated cells. The model shows that feedback regulation stabilises the number of differentiated cells as these become invariant to changes in their apoptosis rate. Stability of the system is largely independent of feedback strength and shape, but specific thresholds exist after unbounded growth occurs. When dedifferentiation is added to the model, we find that the system can recover more gracefully after certain external perturbations. However, dedifferentiation makes the system more prone to loosing homoeostasis. Taken together, our mathematical model shows how a feedback-controlled hierarchical tissue can maintain homeostasis and can be robust to many external perturbations.

sentences: A single cell atlas of the cycling murine ovary The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum (CL) formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle, and the paracrine factors that help coordinate this process. Herein we used single-cell RNA sequencing to evaluate the transcriptome of > 34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

sentences: MLF2 modulates phase separated nuclear envelope condensates that provoke dual proteotoxicity DYT1 dystonia is a highly debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is presently unknown. We report that FG-nucleoporins in the bleb lumen undergo phase separation and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitinated proteins that are normally rapidly degraded in healthy cells partition into the bleb lumen and become stabilized. Additionally, blebs selectively sequester a chaperone network composed of HSP70s and HSP40s. The composition of this chaperone network is altered by the bleb component MLF2. We further demonstrate that MLF2 is a catalyst of phase separation that suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our studies identify unprecedented, dual mechanisms of proteotoxicity in the context of liquid-liquid phase separation with direct implications for our understanding of disease etiology and treatment.

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A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology.

In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

sentences: Detecting natural selection in trait-trait coevolution No phenotypic trait evolves independently of all other traits, but the cause of trait-trait coevolution is poorly understood. While the coevolution could arise simply from pleiotropic mutations that simultaneously affect the traits concerned, it could also result from multivariate natural selection favoring certain trait relationships. To gain a general mechanistic understanding of trait-trait coevolution, we examine the evolution of 220 cell morphology traits across 16 natural strains of the yeast Saccharomyces cerevisiae and the evolution of 24 wing morphology traits across 110 fly species of the family Drosophilidae, along with the variations of these traits among gene deletion or mutation accumulation lines (a.k.a. mutants). For numerous trait pairs, the phenotypic correlation among evolutionary lineages differs significantly from that among mutants. Specifically, we find hundreds of cases where the evolutionary correlation between traits is strengthened or reversed relative to the mutational correlation, which, according to our population genetic simulation, is likely caused by multivariate selection. Furthermore, we detect selection for enhanced modularity of the yeast traits analyzed. Together, these results demonstrate that trait-trait coevolution is shaped by natural selection and suggest that the pleiotropic structure of mutation is not optimal. Because the morphological traits analyzed here are chosen largely because of their measurability, our conclusion is likely general.

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Disrupted development and altered hormone signaling in male Padi2/Padi4 double knockout mice BackgroundPeptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo.

MethodsPreliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice for a range of reproductive defects.

ResultsControlled breeding trials found that DKO breeding pairs, particularly males, appeared to take longer to have their first litter than wild-type FVB controls (WT), and that pups and DKO male weanlings weighed significantly less than their WT counterparts. Additionally, DKO males took significantly longer than WT males to reach puberty and had lower serum testosterone levels. Furthermore, DKO males had smaller testes than WT males with increased rates of germ cell apoptosis.

ConclusionsThe Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

sentences: Deciphering PD1 activation mechanism from molecular docking and molecular dynamic simulations The activation of T cells is normally accompanied by inhibitory mechanisms within which the PD1 receptor stands out. Upon binding the ligands PDL1 and PDL2, PD1 drives T cells to an unresponsive state called exhaustion characterized by a markedly decreased capacity to exert effector functions. For this reason, PD1 has become one of the most important targets in cancer immunotherapy. Despite the numerous studies about PD1 signaling modulation, how the PD1 signaling is activated upon the ligands binding remains an open question. Several experimental facts suggest that the activation of the PD1-PLD1 pathway depends on the interaction with an unknown partner at the cellular membrane. In this work, we investigate the possibility that the target of PD1-PDL1 is the same PD1-PDL1 complex. We combined molecular docking to explore different binding modes with molecular dynamics and umbrella sampling simulations to assess the complexes stability. We found a high molecular weight complex that explains the activation of PD1 upon PDL1 binding. This complex has an affinity comparable to the PD1-PDL1 interaction and resembles the form of a linear lattice.

sentences: NetAct: a computational platform to construct core transcription factor regulatory networks using gene activity A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform for constructing core transcription-factor regulatory networks, named NetAct, using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers the activities of regulators using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. We show that NetAct outperforms existing algorithms in inferring transcriptional activity from our benchmark tests. We illustrate the application of NetAct to model networks driving TGF-beta induced Epithelial-Mesenchymal transition and macrophage polarization.

sentences: A missense KCNQ1 Mutation Impairs Insulin Secretion in Neonatal Diabetes KCNQ1/Kv7 is a voltage-gated K+ channel that regulates heart rhythm, glucose and salt homeostasis. Mutations of KCNQ1 are primarily associated with long-QT syndrome and type 2 diabetes; however, thus far KCNQ1 mutations have not been associated with monogenetic diabetes. Here, we identified a homozygous KCNQ1 missense mutation (R397W) in an individual with permanent neonatal diabetes (PND). To identify the mechanisms that link the mutation to the disease, we introduced the mutation into human embryonic stem cells (hESCs), and used them to derived pancreatic {beta}-like cells (hESC-{beta} cell). In early {beta}-like cells, we observed atypical membrane electrical activity, increased levels of cytoplasmic Ca2+, and a hypersecretion of insulin. Upon extended culture, their insulin secretion decreased and the number of apoptotic cells increased, resulting in a reduction in the numbers of {beta}-like cells. Late-stage {beta}-like cells exhibited a decrease in the expression of metabolic genes, e.g. HNF4, PDX1 and GLUT1, providing a possible mechanism for {beta}-cell dysfunction. Our study identifies KCNQ1 as a novel candidate gene of monogenetic diabetes and shows that KCNQ1 regulates {beta}-cell function and survival.

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Weak Task Synchronization of Default Mode Network in Task Based Paradigms Default mode network (DMN) are the areas of the brain that get deactivated upon the presentation of externally focused, attention demanding tasks. Interestingly, these areas are consistently deactivated irrespective of the nature of the task. Recent evidence shows that the behavior of the DMN may be more nuanced than what was earlier believed. We wanted to investigate the dynamics of DMN further and utilized novel statistical approaches to study the same. We analysed four publicly available datasets (total 223 subjects) across seven tasks related to different cognitive modalities and found out that there is a large amount of variability in the presumed task-negative network across subjects, trials and regions. We designed a method called Inter Trial Temporal Synchronization Analysis (IT-TSA) and Inter Subject TSA (IS-TSA) to analyse variability across trials and subjects respectively. We found out that all task-negative regions and even weak task-positive regions have low synchronization across trials and subjects. We hypothesize that the DMN variability might be more due to neural differences rather than structural or HRF variations. We also investigated the use of non GLM technique like the Inter Subject Correlation (ISC) and found that it may only be suitable to study tightly synchronized task-positive regions. Our study challenges the understanding of DMN as a task-negative region and advances the findings that DMN acts as an active component associated with a unique set of self-referential, autobiographical processes which are deactivated differentially and non linearly across trials and subjects in the presence of extrinsic processes.

HighlightsO_LIDefault Mode Network is weakly synchronized across trials and subjects in task paradigms.
C_LIO_LIInter Subject Correlation is not suited to detect task-based activations in DMN.
C_LIO_LISynchronization Analysis reveals temporal structure in fMRI data.
C_LI

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IDOL deficiency inhibits cholesterol-rich diet-induced atherosclerosis in rabbits BACKGROUNDThe E3 ubiquitin ligase IDOL (Inducible Degrader of the LDL-Receptor) contributes to regulation of cholesterol metabolism through degradation of LDLR, VLDLR and ApoER2. Human genetic studies support the hypothesis that IDOL could serve as a target for the treatment of dyslipidemia. However, species-specific differences in overall lipid metabolism and IDOL regulation require new preclinical models to realize its therapeutic potential. We leveraged the advantages afforded by the rabbit model to address those limitations and generated a novel rabbit IDOL knockout, which we characterized in the context of atherosclerosis.

METHODSIDOL-/- rabbits were generated by CRISPR/Cas9 technology. IDOL-/- and wildtype littermates, on standard (SD) and atherogenic high-cholesterol diets (HCDs) were compared through assessment of lipid and lipoprotein profiles, triglyceride clearance, lipoprotein lipase (LPL) activity, liver pathology, atherosclerosis development, and fecal cholesterol, with bile acid contents assessed by mass spectrometry.

ResultsHepatic IDOL expression was increased in response to hypercholesterolemia and hypertriglyceridemia induced by HCD. On SD, loss of IDOL increased LDLR stability with reduced total cholesterol in plasma. On HCD, IDOL-/- rabbits showed simultaneous and remarkable reduction in hypercholesterolemia and hypertriglyceridemia associated with enhanced lipid clearance and LPL activity as well as increased bile acid excretion in feces. IDOL-/- rabbits presented markedly reduced HCD-induced atherosclerosis in the aorta and left coronary artery, without enhanced liver steatosis.

CONCLUSIONSLoss of IDOL in rabbits recapitulates human genetic findings, thus setting the stage to accelerate preclinical studies towards development of strategies targeting IDOL for the treatment of atherosclerotic cardiovascular disease.

sentences: Speed fluctuations of bacterial replisomes Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly five-fold between 17{degrees}C and 37{degrees}C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate. We interpret this correlation as a speed-error trade-off in DNA replication. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

sentences: Salicylaldehyde ester-mediated protein semi-synthesis enables studies on the tetra-acetylation of HMGB1 To answer how protein post-translational modifications (PTMs) affect protein function, conformation, stability, localization and interaction with binders remains important in the biological study. However, the related study has been dramatically hindered by the difficulty in obtaining homogenous proteins with site-specific PTMs of interest. Herein, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation). This methodology has enabled us to generate Lys (2/6/7/11) tetra-acetylated HMGB1 (high-mobility group box 1) protein, a 25 kDa proinflammatory protein, in high purity. Further studies revealed that the tetra-acetylation may represent a regulatory switch to control the HMGB1 signaling pathway by abolishing its interaction with lipopoly-saccharide (LPS) and accelerating its degradation, consequently preventing cells from pyroptosis and lethality upon infectious injury.

sentences: Modeling lung cell development using human pluripotent stem cells Human pluripotent stem cells (hPSC) differentiations can capture developmental phenotypes and processes. They are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show temporal development of lung cell lineages using hPSC that recapitulate developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial spheroids, and the functional utility of the lung models at different differentiation stages for cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and containing basal stem cells that generate renewable airway spheroids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult airways, including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime and RNA velocity analyses of the integrated datasets from fetal and ALI stages reveal both previously identified and new cell lineage relationships. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

sentences: ICoRD: Iterative Correlation-Based ROI Detection Method for the Extraction of Neural Signals in Calcium Imaging In vivo calcium imaging is a standard neuroimaging technique that allows selective observation of target neuronal activities. In calcium imaging, neuron activation signals provide key information for the investigation of neural circuits. For efficient extraction of the calcium signals of neurons, selective detection of the region of interest (ROI) pixels corresponding to the active subcellular region of the target neuron is essential. However, current ROI detection methods for calcium imaging data exhibit a relatively low signal extraction performance from neurons with a low signal-to-noise power ratio (SNR). This is problematic because a low SNR is unavoidable in many biological experiments. Therefore, we propose an iterative correlation-based ROI detection (ICoRD) method that robustly extracts the calcium signal of the target neuron from a calcium imaging series with severe noise. ICoRD extracts calcium signals closer to the ground-truth calcium signal than the conventional method from simulated calcium imaging data in all low SNR ranges. Additionally, this study confirmed that ICoRD robustly extracts activation signals against noise, even within in vivo environments. ICoRD showed reliable detection from neurons with a low SNR and sparse activation, which were not detected by conventional methods. ICoRD will facilitate our understanding of neural circuit activity by providing significantly improved ROI detection in noisy images.

sentences: Caveat in Fisher's theorem reveals fitness can change without mutations The fitness of microbial genotypes is linked to their reproduction rate: Mutants reproducing faster than their wild-type counterparts are favoured by selection, but otherwise the mutation is lost. Here I show that relative differences in fitness between two mutants can change over time without invoking new mutations, contravening textbook population genetics theory. Widespread fitness measurements assume the ratio between Malthusian reproduction rates of competing genotypes is constant. But they are not. Here I competed two constructs of Escherichia coli, one harbouring the non-transmissible plasmid pGW155B. The plasmid protects the bacterium against tetracycline, and yet, it was maintained without using the antibiotic. pGW155B imposes a slower reproduction rate due to carriage costs but it also prompts a known trade-off between reproduction and survival, so the construct harbouring pGW155B attains higher densities at the equilibrium. Consequently, if selection favours survival the bacterium could maintain the plasmid, despite its slower reproduction, without using tetracycline. Exclusive reliance on reproduction rates may therefore yield inaccurate fitness measurements, altering our intuition about how natural selection operates, unless competing genotypes reach their equilibrium. This means that selection for plasmid carriage could be stronger than previously thought.

sentences: Overexpression of miR5505 enhanced drought and salt resistance in rice (Orayza sativa) Rice is one of the most important crops in the world and half of the world population consumes it as their staple food. The abiotic stresses caused by drought, salt and other stresses have severely impacted rice production. MicroRNAs (miRNAs) are a type of small non-coding RNAs which widely reported as gene regulators, suppressing genes expression by degradation mRNA or translation inhibition. Previously, high-throughput sequencing has found a conserved miRNA miR5505 responding to drought stress in Dongxiang wild rice (DXWR). Several other studies also revealed that miR5505 was involved in rice stress responses. We further studied the effect of miRNA in drought and salt tolerance by overexpression it in rice. 2 in 18 successfully transformed transgenic lines with higher miR5505 expression were selected and then drought and salt resistance ability were evaluated. Both transgenic lines showed stronger drought and salt tolerance than wild-type (WT). Putative targets of miR5505 were identified by psRNATarget and several of them were found stress-related. RNA-seq found 1,980 differentially expressed genes (DEGs) in transgenic lines. Among them, 978 genes were down-regulated. Three genes were predicted by psRNATarget and two of them might be stress-related. We also found various environmental stress cis-acting elements in upstream of miR5505 promoter through Software PlantCARE. In all, we improved rice drought and salt tolerance by overexpressing miR5505, and the generated putative targets and cis-acting elements also suggested miR5505 might play important roles in the regulation of drought and salt responses.

sentences: Day-night and seasonal variation of human gene expression across tissues Circadian and circannual cycles trigger physiological changes whose reflection on human transcriptomes remains largely uncharted. We used the time and season of death of 932 individuals from GTEx to jointly investigate transcriptomic changes associated with those cycles across multiple tissues. Overall, most variation across tissues during day-night and among seasons was unique to each cycle. Although all tissues remodeled their transcriptomes, brain and gonadal tissues exhibited the highest seasonality, whereas those in the thoracic cavity showed stronger day-night regulation. Core clock genes displayed marked day-night differences across multiple tissues, which were largely conserved in baboon and mouse, but adapted to their nocturnal or diurnal habits. Seasonal variation of expression affected multiple pathways and it was enriched among genes associated with the immune response, consistent with the seasonality of viral infections. Furthermore, they unveiled cytoarchitectural changes in brain regions. Altogether, our results provide the first combined atlas of how transcriptomes from human tissues adapt to major cycling environmental conditions.

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NLRP3 activation in response to disrupted endocytic traffic Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3.

One-Sentence SummaryNLRP3 senses disruptions in endosome trafficking to trigger the formation of an inflammasome complex and initiate an inflammatory response.

sentences: Effect of reduced graphene oxide (rGO) compaction degree and concentration on rGO-polymer composites printability and cell interactions Graphene derivatives combined with polymers have attracted enormous attention for bone tissue engineering applications. Among others, reduced graphene oxide (rGO) is one of the preferred graphene-based fillers for the preparation of composites via melt compounding, and their further processing into 3D scaffolds, due to its established large-scale production method, thermal stability, and electrical conductivity. In this study, rGO (low bulk density 10g/L) was compacted by densification using a solvent (either acetone or water) prior to melt compounding, to simplify its handling and dosing into a twin-screw extrusion system. The effects of rGO bulk density (medium and high), densification solvent, and rGO concentration (3, 10 and 15% in weight) on rGO dispersion within the composite, electrical conductivity, printability and cell-material interactions were studied. High bulk density rGO (90 g/L) occupied a low volume fraction within polymer composites, offering poor electrical properties but a reproducible printability up to 15 wt% rGO. On the other hand, the volume fraction within the composites of medium bulk density rGO (50 g/L) was higher for a given concentration, enhancing rGO particle interactions and leading to enhanced electrical conductivity, but compromising the printability window. For a given bulk density (50 g/L), rGO densified in water was more compacted and offered poorer dispersability within the polymer than rGO densified in acetone, and resulted in scaffolds with poor layer bonding or even lack of printability at high rGO percentages. A balance in printability and electrical properties was obtained for composites with medium bulk density rGO densified in acetone. Here, increasing rGO concentration led to more hydrophilic composites with a noticeable increase in protein adsorption. Moreover, scaffolds prepared with such composites presented antimicrobial properties even at low rGO contents (3 wt%). In addition, the viability and proliferation of human mesenchymal stromal cells (hMSCs) was maintained on scaffolds with up to 15% rGO and with enhanced osteogenic differentiation on 3% rGO scaffolds.

sentences: Invadopodia enable cooperative invasion and metastasis of breast cancer cells Invasive and non-invasive cancer cells can invade together during the cooperative invasion. However, the events leading to it, role of EMT and the consequences this may have on metastasis are unknown. In this study, we demonstrate that the isogenic 4T1 and 67NR breast cancer cells sort from each other in 3D spheroids, followed by cooperative invasion. By time-lapse microscopy, we show that the invasive 4T1 cells move more persistently compared to non-invasive 67NR, sorting and accumulating at the spheroid-ECM interface, a process dependent on cell-ECM adhesions and independent from E-cadherin cell-cell adhesions. Elimination of invadopodia in 4T1 cells blocks invasion, demonstrating that invadopodia requirement is limited to leader cells. Importantly, we demonstrate that cells with and without invadopodia can also engage in cooperative metastasis in preclinical mouse models. Altogether, our results suggest that a small number of cells with invadopodia can drive the metastasis of heterogeneous cell clusters.

sentences: Live imaging YAP signaling in vivo with fusion reporter mice YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATs kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behavior of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signaling in vivo in many different situations.

sentences: Using the UniFrac metric on Whole Genome Shotgun data The UniFrac metric has proven useful in revealing diversity across metagenomic communities. Due to the phylogeny-based nature of this measurement, UniFrac has historically only been applied to 16S rRNA data. Simultaneously, Whole Genome Shotgun (WGS) metagenomics has been increasingly widely employed and proven to provide more information than 16S data, but a UniFrac-like diversity metric suitable for WGS data has not previously been developed. The main obstacle for UniFrac to be applied directly to WGS data is the absence of phylogenetic distances in the taxonomic relationship derived from WGS data. In this study, we demonstrate a method to overcome this intrinsic difference and compute the UniFrac metric on WGS data by assigning branch lengths to the taxonomic tree obtained from input taxonomic profiles. We conduct a series of experiments to demonstrate that this WGSUniFrac method is comparably robust to traditional 16S UniFrac and is not highly sensitive to branch lengths assignments, be they data-derived or model-prescribed. Code implementing a prototype of WGSUniFrac along with paper reproducible are available at https://github.com/KoslickiLab/WGSUniFrac.

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Booster vaccines protect hamsters with waning immunity from Delta VOC infection, disease, and transmission Waning immunity to COVID-19 vaccination is associated with increased risk of breakthrough infection, especially with highly transmissible variants of concern (VOC). Booster vaccination generates rapid immune recall in humans, which real-world observational studies suggest protects against VOC infection and associated disease, and modeling studies suggest could mitigate community spread. We directly tested the impact of booster vaccination on protection against Delta VOC infection, disease, and transmission to naive cohorts in golden Syrian hamsters. Animals with waning immunity to bnt162b2 generated rapid immune recall and strong protection against upper- and lower-respiratory tract infection when boosted with bnt126b2, mRNA-1273 or AZD1222. Boosting with either mRNA vaccine generated moderate protection against lung inflammation and virus transmission to unvaccinated animals. Our data support booster vaccination as a tool to address emerging VOC in the COVID-19 pandemic.

One-Sentence SummaryA booster vaccine delivered 9 months after primary bnt162b2 vaccination protects hamsters from Delta VOC infection, disease, and transmission.

sentences: Interplay between brassinosteroids and TORC signaling in Arabidopsis revealed by integrated multi-dimensional analysis Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major processes coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we performed transcriptome, proteome, and phosphoproteome profiling of Arabidopsis mutants with altered levels of either BIN2 or RAPTOR1B, two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Additionally, we performed Multiplexed Assay for Kinase Specificity (MAKS), which provided a system-wide view of direct BIN2 substrates. Furthermore, phosphoproteomic data was used to reconstruct a kinase-signaling network and to identify novel proteins dependent on BR and/or TORC signaling pathways. Loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results provide genome-wide evidence for crosstalk between BR and TORC signaling and established a kinase signaling network that defines the molecular mechanisms of BR and TORC interactions in the regulation of plant growth/stress balance.

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Behavior-dependent spatial maps enable efficient theta phase coding Navigation through space involves learning and representing relationships between past, current and future locations. In mammals, this might rely on the hippocampal theta phase code, where in each cycle of the theta oscillation, spatial representations start behind the animals location and then sweep forward. However, the exact relationship between phase and represented and true positions remains unclear. Developing a quantitative framework for the theta phase code, we formalize two previous notions: in spatial sweeps, different phases of theta encode positions at fixed distances behind or ahead of the animal, whereas in temporal sweeps, they encode positions reached at fixed time intervals into the past or future. These two schemes predict very different position representations during theta depending on the animals running speed. Paradoxically, in some studies sweep length has been shown to increase proportionally with running speed, consistent with temporal sweeps, whereas in other studies individual place field parameters such as field size and phase precession slope were shown to remain constant with speed, consistent with spatial sweeps. Here, we introduce a third option: behavior-dependent sweeps, according to which sweep length and place field properties vary across the environment depending on the running speed characteristic of each location. Analyzing single-cell and population variables in parallel in recordings from rat CA1 place cells and comparing them to model simulations, we show that behavior-dependent sweeps uniquely account for all relevant variables. This coding scheme combines features and advantages of both spatial and temporal sweeps, revealing an efficient hippocampal code.

SignificanceTo learn the structure of the world and the consequences of our actions, information about the past must be carried through to the present and linked to what is currently happening. To plan, desired future states and the predicted outcomes of actions must be represented. In mammals, including humans, hippocampal neurons are thought to encode such representations of past, present and future states at different phases of the theta oscillation. However, the precise hippocampal phase code remains unknown. We show that two previous ideas are incompatible with each other and with rat experimental data. So, we propose a new coding scheme that synthesizes features from both ideas and accounts for all relevant observations.

sentences: Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins Proteoform-resolved information, obtained by top-down (TD) "intact protein" proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (SEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5-155 kDa) prior to nRPLC-MS. The coupling of SEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns pre- and post-SEC columns to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) pre-fractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5-30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100x less CSF than previous OFFGEL studies. Furthermore, the modular design of the SEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.

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Genome features of common vetch (Vicia sativa) in natural habitats Wild plants are often tolerant to biotic and abiotic stresses in their natural environments, whereas domesticated plants such as crops frequently lack such resilience. This difference is thought to be due to the high levels of genome heterozygosity in wild plant populations and the low levels of heterozygosity in domesticated crop species. In this study, common vetch (Vicia sativa) was used as a model to examine this hypothesis. The common vetch genome (2n = 14) was estimated as 1.8 Gb in size. Genome sequencing produced a reference assembly that spanned 1.5 Gb, from which 31,146 genes were predicted. Using this sequence as a reference, 24,118 single nucleotide polymorphisms were discovered in 1,243 plants from 12 natural common vetch populations in Japan. Common vetch genomes exhibited high heterozygosity at the population level, with lower levels of heterozygosity observed at specific genome regions. Such patterns of heterozygosity are thought to be essential for adaptation to different environments. These findings suggest that high heterozygosity at the population level would be required for wild plants to survive under natural conditions while allowing important gene loci to be fixed to adapt the conditions. The resources generated in this study will provide insights into de novo domestication of wild plants and agricultural enhancement.

HighlightSequence analysis of the common vetch (Vicia sativa) genome and SNP genotyping across natural populations revealed nucleotide diversity levels associated with native population environments.

sentences: A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.

sentences: A low repeated dose of Δ9-tetrahydrocannabinol affects memory performance through serotonergic signalling in mice Cannabis is the most widely used illicit drug worldwide. Its principal psychoactive component, {Delta}9-tetrahydrocannabinol (THC), acts as a partial agonist of the main cannabinoid receptor in the brain, the cannabinoid type-1 receptor (CB1R), that is responsible for the central effects of THC including memory impairment. CB1Rs may form heterodimers with the serotonin 5-HT2A receptor (5-HT2AR) which were found responsible for the memory impairment produced by acute high dose of THC in mice. In this study we investigated whether a repeated low dose of THC (1 mg/kg), with no acute consequence on memory performance, could eventually have deleterious cognitive effects. We found that this dose of THC impaired novel object-recognition memory and fear conditioning memory 24 h after the last of 7 consecutive daily treatments. At that time, a general enhancement of c-Fos expression was also observed in several brain regions of THC-exposed animals, as well as a decreased dendritic spine density on hippocampal CA1 pyramidal neurons that was accompanied by reduced long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Interestingly, an up-regulation in the expression of CB1R/5-HT2AR heterodimers was observed in the hippocampus after THC exposure and pre-treatment with the 5-HT2AR antagonist MDL 100,907 (0.01 mg/kg) prevented the enhanced heterodimerization and the THC-associated memory impairment. Together, these results reveal the significance of serotonergic signalling through 5-HT2ARs in the memory-impairing effects of repeated low doses of THC.

sentences: Orchestrating and sharing large multimodal data for transparent and reproducible research Reproducibility is essential to open science, as there is limited relevance for findings that can not be reproduced by independent research groups, regardless of its validity. It is therefore crucial for scientists to describe their experiments in sufficient detail so they can be reproduced, scrutinized, challenged, and built upon. However, the intrinsic complexity and continuous growth of biomedical data makes it increasingly difficult to process, analyze, and share with the community in a FAIR (findable, accessible, interoperable, and reusable) manner. To overcome these issues, we created a cloud-based platform called ORCESTRA (orcestra.ca), which provides a flexible framework for the reproducible processing of multimodal biomedical data. It enables processing of clinical, genomic and perturbation profiles of cancer samples through automated processing pipelines that are user-customizable. ORCESTRA creates integrated and fully documented data objects with persistent identifiers (DOI) and manages multiple dataset versions, which can be shared for future studies.

sentences: Machine learning meets classical computer vision for accurate cell identification High-parameter multiplex immunostaining techniques have revolutionized our ability to image healthy and diseased tissues with unprecedented depth; however, accurate cell identification and segmentation remain significant downstream challenges. Identifying individual cells with high precision is a requisite to reliably and reproducibly interpret acquired data. Here we introduce CIRCLE, a cell identification pipeline that combines classical and modern machine learning-based computer vision algorithms to address the shortcomings of current cell segmentation tools for 2D images. CIRCLE is a fully automated hybrid cell detection model, eliminating subjective investigator bias and enabling high-throughput image analysis. CIRCLE accurately distinguishes cells across diverse tissues microenvironments, resolves low-resolution structures, and can be applied to any 2D image that contains nuclei. Importantly, we quantitatively demonstrate that CIRCLE outperforms current state-of-the-art image segmentation tools using multiple accuracy measures. As high-throughput multiplex imaging grows closer toward standard practice for histology, integration of CIRCLE into analysis protocols will deliver unparalleled segmentation quality.

sentences: Conjugative transfer of naturally occurring plasmids in Mycolicibacterium sp. Conjugation is considered the main horizontal gene transfer (HGT) mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard in relation to this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids (sizes of 21 and 274 kb), and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.

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

sentences: Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

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Hotter chili peppers by hybridisation: heterosis and reciprocal effects The selection of pure lines followed by crosses to create superior hybrids is one of the oldest strategies in crop breeding. However, in domesticated species of the Capsicum genus hybrid breeding has lagged, in part due to a lack of detailed information about the phenotypic and metabolic consequences of hybridization. Here, we performed reciprocal crosses between four inbred varieties of two species of cultivated C. chinense (cv. Habanero and Biquinho) and C. annuum var. annuum (cv. Jalapeno and cv. Cascadura Ikeda). These varieties were specifically selected for their highly divergent traits, including plant growth habit, fruit size, shape and pungency: Habanero and Jalapeno peppers are highly pungent forms, particularly popular in Mexico. The Biquinho cultivar of C. chinense and the Cascadura Ikeda bell pepper are traditional sweet cultivars from Brazil. From the parental genotypes and from the progeny of the reciprocal crosses, we measured 28 phenotypic traits, including plant growth, and yield, 32 fruit shape parameters, and 50 fruit pericarp and placenta metabolites, including capsaicinoids. We found large differences for agronomic and metabolic traits between the genotypes, including heterosis for pungency and reciprocal effects for agronomic traits. We also show that the strong association between fruit shape and pungency can be broken in intraspecific hybrids, paving the way for the precision breeding of novel varieties.

Once sentence summaryHybrids of Capsicum peppers display heterosis and reciprocal effects for agronomic traits and fruit pungency

sentences: Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein -actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model which lacks both dystrophin and sarcomere -actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%) and this correlates with a graded force loss over three eccentric contractions for dKO muscles ([~]35% after first contraction, [~]66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction ([~]73% after first contraction, [~]89% after three contractions). In dKO protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

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Hidden pathway for cytokine receptor activation: Structural insights into a marine sponge-derived lectin that activates the thrombopoietin receptor via recognition of the fucose moiety N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism has not been studied because no external agonists existed. We describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. ThC-induced activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. MPL is subject to sugar-mediated activation, where the kinetics differ from those of cytokines. This result suggests the presence of diverse receptor activation pathways in human thrombopoiesis.

One-sentence summaryA marine sponge lectin catalyzes thrombopoietin receptor dimerization and activation, exhibiting strong synergy with thrombopoietin, and modulates internalization of the receptor.

sentences: PROTEOMIC SIGNATURES OF MICROBIAL ADAPTATION TO THE HIGHEST UV -IRRADIATION ON EARTH: LESSONS FROM A SOIL ACTINOBACTERIUM In the Puna region, the total solar irradiation and the UV incidence is the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. In this work, we present the molecular events involved in the adaptive response of the model HAAL extremophilic actinobacterium Nesterenkonia sp. Act20 under artificial UV-B radiation, herein called as UV-resistome. Proteomic profiles of cultures exposed to different UV-experimental conditions showed that the leading systems for adaptation to the UV-challenge in-vitro are DNA repair and antioxidant mechanisms.

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Chromatin landscape dynamics in development of the plant parasitic nematode Meloidogyne incognita In model organisms, epigenome dynamics underlies a plethora of biological processes. The role of epigenetic modifications in development and parasitism in nematode pests remains unknown. The root-knot nematode Meloidogyne incognita adapts rapidly to unfavorable conditions, despite its asexual reproduction. However, the mechanisms underlying this remarkable plasticity and their potential impact on gene expression remain unknown. This study provides the first insight into contribution of epigenetic mechanisms to this plasticity, by studying histone modifications in M. incognita. The distribution of five histone modifications revealed the existence of strong epigenetic signatures, similar to those found in the model nematode Caenorhabditis elegans. We investigated their impact on chromatin structure and their distribution relative to transposable elements (TE) loci. We assessed the influence of the chromatin landscape on gene expression at two developmental stages: eggs, and pre-parasitic juveniles. H3K4me3 histone modification was strongly correlated with high levels of expression for protein-coding genes implicated in stage-specific processes during M. incognita development. We provided new insights in the dynamic regulation of parasitism genes kept under histone modifications silencing. In this pioneering study, we establish a comprehensive framework for the importance of epigenetic mechanisms in the regulation of the genome expression and its stability in plant-parasitic nematodes.

Author summaryThe nematode Meloidogyne incognita is one of the most destructive plant parasites worldwide. Its ability to infect a wide range of hosts and its high adaptability contribute to its parasitic success. We investigated the role of epigenetic mechanisms -- specifically post-translational histone modifications -- in the parasitic life cycle. We showed these modifications are linked to gene expression regulation and likely contribute to nematode development and pathogenicity.

sentences: Mitochondrial ATP production is required for endothelial cell control of vascular tone Arteries and veins are lined by non-proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully-formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation disrupts endothelial control of vascular tone. The role for endothelial cell energy production is independent of species, sex, or vascular bed. Basal, mechanically-activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. These data show that mitochondrial ATP is necessary for calcium-dependent, nitric oxide mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

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Disturbance by soil mixing decreases microbial richness and supports homogenizing community assembly processes The spatial heterogeneity of soils microhabitats warrants the study of ecological patterns and community assembly processes in the context of community coalescence, or the combining and restructuring of communities and their environment. By mixing soil at various frequencies in a 16-week lab incubation, we explored the effects of mixing disturbance on soil bacterial richness, community composition, and community assembly processes. We hypothesized that well-mixed soil would harbor less richness, dominated by homogenizing dispersal and homogeneous selection. Using 16S rRNA gene sequencing, we inferred ecological processes, estimated richness and differential abundance, calculated compositional dissimilarity, and constructed co-occurrence networks. Findings supported our hypotheses, with >20% decrease in soil bacterial richness in well-mixed soil. While soil mixing resulted in increasingly dissimilar communities compared to unmixed soil (Bray-Curtis dissimilarity; 0.75 vs. 0.25), well-mixed soil communities were increasingly self-similar. Our results imply that vast soil diversity may be attributed to the unmixed and spatially heterogeneous nature of soil, and also provide insight into soil communities following coalescence events. By isolating and better understanding the effect of spatial heterogeneity and dysconnectivity on soil microbial communities, we may better extrapolate how anthropogenic disturbances, such as climate change or land use change, may affect broad soil functions.

One sentence summarySoil mixing decreases bacterial richness as several taxa dominate the community, providing evidence for homogenizing community assembly processes.

sentences: Anticipatory regulation of cardiovascular system on emergence of auditory-motor interaction in young infants Humans develop auditory-motor interaction to produce a variety of rhythmic sounds using body movements, which are often produced and amplified with tools, such as drumming. The extended production of sounds allows us to express a wide range of emotions, accompanied by physiological changes. According to previous studies, even young infants enhance movements in response to auditory feedback. However, their exhibition of physiological adaptation on emergence of auditory-motor interaction is unclear. We investigated the movement and cardiac changes associated with auditory feedback to spontaneous limb movements in 3-month-old infants. The results showed that infants increased the frequency of limb movements inducing auditory feedback, while they exhibited the more regular rhythm of the limb movements. Furthermore, heart rate increase associated with the limb movement was first inhibited immediately after the timing of the auditory feedback, which may reflect sustained attention to the auditory stimuli. Then, through auditory-motor experience, the heart rate increase was inhibited even prior to the auditory feedback, leading to suppression on the peak intensity of the heart rate increase. These findings suggest that young infants regulate the cardiovascular system as well as limb movements in anticipation of the auditory feedback. The anticipatory regulation associated with movement and attentional changes may contribute to reduced cardiovascular stress in auditory-motor interaction, and provide a developmental basis for more sophisticated goal-directed behavior of producing rhythmic sounds.

sentences: State Dependent Anionic Pore Currents Conducted by Single Countercharge Mutants in a Voltage-Sensing Phosphatase Mutating gating charge residues in the S4 segment of voltage-sensing domains (VSDs) can cause ionic leak currents through the VSDs. These leak currents, known as gating pore or omega currents, play important pathophysiological role in many diseases. Here, we show that mutations in a key countercharge residue, D129, in the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) facilitate conduction of unique anionic omega currents. Neutralization of D129 causes a dramatic positive shift of activation, facilitates the formation of a continuous water path through the intermediate state VSD, and creates a positive electrostatic potential landscape inside the VSD leading to anion selectivity. Increasing the population or duration of the conducting state by a high external pH or an engineered Cd2+ bridge markedly increases the current magnitude. Our findings uncover a new role of countercharge residues and could inform on the mechanisms of channelopathies linked to countercharge residue mutations.

sentences: Key mutations on spike protein altering ACE2 receptor utilization and potentially expanding host range of emerging SARS-CoV-2 variants Increasing evidence supports inter-species transmission of SARS-CoV-2 variants from human to domestic or wild animals during the ongoing COVID-19 pandemic, which is posing great challenges to epidemic control. Clarifying the host range of emerging SARS-CoV-2 variants will provide instructive information for the containment of viral spillover. The spike protein (S) of SARS-CoV-2 is the key determinant of receptor utilization, and therefore amino acid mutations on S will probably alter viral host range. Here, in order to evaluate the impact of S mutations, we constructed 20 Hela cell lines stably expressing ACE2 orthologs from different animals, and prepared 27 pseudotyped SARS-CoV-2 carrying different spike mutants, among which 20 bear single mutation and the other 7 were cloned from emerging SARS-CoV-2 variants, including D614G, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.135), Lambda (B.1.429) and Mu (B.1.525). Using pseudoviral reporter assay, we identified that the substitutions of T478I and N501Y enabled the pseudovirus to utilize chicken ACE2, indicating potential infectivity to avian species. Furthermore, the S mutants of real SARS-CoV-2 variants comprising N501Y showed significantly acquired abilities to infect cells expressing mouse ACE2, indicating a critical role of N501Y in expanding SARS-CoV-2 host range. In addition, A262S and T478I significantly enhanced the utilization of various mammals ACE2. In summary, our results indicated that T478I and N501Y substitutions were two S mutations important for receptor adaption of SARS-CoV-2, potentially contributing to spillover of the virus to many other animal hosts. Therefore, more attention should be paid to SARS-CoV-2 variants with these two mutations.

sentences: Genome-wide mechanisms of rediploidization in paleopolyploid teleosts revealed by a high-resolution comparative atlas across 74 fishes Teleost fishes are ancient tetraploids stemming from an ancestral whole-genome duplication that may have contributed to the impressive diversification of this clade. Whole-genome duplications can occur via self-doubling (autopolyploidy) or via hybridization between different species (allopolyploidy). The mode of tetraploidization conditions subsequent evolutionary processes by which descendant genomes return to a diploid state through gene losses and functional divergence. How teleosts became tetraploid remains however unresolved, leaving a fundamental gap to interpret their functional evolution. As legacy of the whole genome duplication, identifying orthologous and paralogous genomic regions across teleosts is challenging, hindering genome-wide investigations into their polyploid history. Here, we combine tailored gene phylogeny methodology together with the state-of-the-art ancestral karyotype reconstruction to establish the first high-resolution comparative atlas of paleopolyploid regions across 74 teleost genomes. We then leverage this atlas to investigate the genome-wide mechanisms of rediploidization after tetraploidization in teleosts. We uncover that meiotic recombination persisted between duplicated chromosomes for over 60 million years, with entire chromosomes only functionally diverging after the separation of major teleost families. This evidence suggests that the teleost ancestor was an autopolyploid. Further, ancient duplicated chromosomes have evolved asymmetrically in teleosts, disputing unbalanced evolution as a hallmark of polyploidization through hybridization.

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Hypothalamic Overexpression of Neurosecretory Protein GL Leads to Obesity in Mice IntroductionThe mechanisms underlying obesity are not fully understood, necessitating the creation of novel animal models for investigation of metabolic disorders from the cellular-molecular to behavioral levels of analysis. We have previously found that neurosecretory protein GL (NPGL), a newly-identified hypothalamic neuropeptide, is involved in feeding behavior and fat accumulation in rats. Given the broad availability of genetic tools in mice, the present investigation sought to establish a mouse model of NPGL-induced obesity.

MethodsWe overexpressed the NPGL-precursor gene (Npgl) in the hypothalamus using adeno-associated virus in C57BL/6J mice fed normal chow (NC) or a high-calorie diet (HCD). After 9 weeks of Npgl overexpression, we measured adipose tissues, muscle, and several organ masses in addition to food intake and body mass. To assess the effects of Npgl overexpression on peripheral tissues, we analyzed mRNA expression of lipid metabolism-related genes by quantitative RT-PCR.

ResultsNpgl overexpression increased food intake, body mass, adipose tissues and liver masses, food efficiency, and circulating insulin levels under both NC and HCD, resulting in obesity observable within 8 weeks. Furthermore, we observed fat accumulation in adipose tissues and liver. Additionally, mRNA expression of lipid metabolism-related factors was increased in white adipose tissue and the liver after Npgl overexpression.

ConclusionTaken together, the present study suggests that NPGL is an endogenous obesogenic factor that acts within a short period of time in mice. As a result, this animal model can be widely applied to study the etiology of obesity from genes to behavior.

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Systemic regulation of nitrogen acquisition and use in Oryza longistaminata ramets under nitrogen heterogeneity Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability by using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin, a cytokinin, was up-regulated in response to the nitrogen supply, but trans-zeatin appears not to target the compensatory regulation. Our results also implied that the O. longistaminata ortholog of OsCEP1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory up-regulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

One sentence summaryOryza longistaminata, a rhizomatous wild rice, systemically regulates ammonium acquisition and use in response to spatially heterogeneous nitrogen availability via inter-ramet communication.

sentences: Plant-based biosensors for detecting CRISPR-mediated genome engineering CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

sentences: Epigenetic dynamics of centromeres and neocentromeres in Cryptococcus deuterogattii Deletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres do not and instead span actively expressed genes. To explore the epigenetic organization of neocentromeres, we analyzed the distribution of the heterochromatic histone modification H3K9me2, 5mC DNA methylation and the euchromatin mark H3K4me2. Native centromeres are enriched for both H3K9me2 and 5mC DNA methylation marks and are devoid of H3K4me2, while neocentromeres do not exhibit any of these features. Neocentromeres in cen10{Delta} mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is inactivated and the native centromere of the chromosome fusion partner remains as the sole, active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted the re-activation of the inactive neocentromere. Our results show that the inactive neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To study the impact of transcription on centromere stability, the actively expressed URA5 gene was introduced into the CENP-A bound regions of a native centromere. The introduction of the URA5 gene led to a loss of CENP-A from the native centromere, and a neocentromere formed adjacent to the native centromere location. Remarkably, the inactive, native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. A cumulative analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unreported phenomenon in fungi. The CENP-A-binding shifted within the ORF-free regions and showed a possible association with a truncated transposable element. Taken together, our findings reveal that neocentromeres in C. deuterogattii are highly unstable and are not marked with an epigenetic memory, distinguishing them from native centromeres.

sentences: Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-{kappa}B from DNA is achieved through accelerated dissociation by the inhibitor protein I{kappa}B. Using single-molecule FRET, we observe a continuum of conformations of NF-{kappa}B in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-{kappa}B is partially bound, allowing I{kappa}B invasion to facilitate DNA dissociation. I{kappa}B induces a locked conformation where the DNA-binding domains of NF-{kappa}B are too far apart to bind DNA, whereas the loss-of-function I{kappa}B mutant retains the NF-{kappa}B conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

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Minor deviations from randomness have huge repercussions on the functional structuring of sequence space Approaches based on molecular evolution have organized natural proteins into a hierarchy of families, superfamilies, and folds, which are often pictured as islands in a great sea of unrealized and generally non-functional polypeptides. In contrast, approaches based on information theory have substantiated a mostly random scatter of natural proteins in global sequence space. We evaluate these opposing views by analyzing fragments of a given length derived from either a natural dataset or different random models. For this, we compile distances in sequence space between fragments within each dataset and compare the resulting distance distributions between sets. Even for 100-mers, more than 95% of distances can be accounted for by a random sequence model that incorporates the natural amino acid frequency of proteins. When further accounting for the specific residue composition of the respective fragments, which would include biophysical constraints of protein folding, more than 99% of all distances can be modeled. Thus, while the local space surrounding a protein is almost entirely shaped by common descent, the global distribution of proteins in sequence space is close to random, only constrained by divergent evolution through the requirement that all intermediates connecting two forms in evolution must be functional.

Significance StatementWhen generating new proteins by evolution or design, can the entire sequence space be used, or do viable sequences mainly occur only in some areas of this space? As a result of divergent evolution, natural proteins mostly form families that occupy local areas of sequence space, suggesting the latter. Theoretical work however indicates that these local areas are highly diffuse and do not dramatically affect the statistics of sequence distribution, such that natural proteins can be considered to effectively cover global space randomly, though extremely sparsely. By comparing the distance distribution of natural sequences to that of various random models, we find that they are indeed distributed largely randomly, provided that the amino acid composition of natural proteins is respected.

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Idefix: identifying accidental sample mix-ups in biobanks using polygenic scores Structured abstractIdentifying sample mix-ups in biobanks is essential to allow the repurposing of genetic data for clinical pharmacogenetics. Pharmacogenetic advice based on the genetic information of another individual is potentially harmful. Existing methods for identifying mix-ups are limited to datasets in which additional omics data (e.g. gene expression) is available. Cohorts lacking such data can only use sex, which can reveal only half of the mix-ups. Here, we describe Idefix, a method for the identification of accidental sample mix-ups in biobanks using polygenic scores. In the Lifelines population-based biobank we calculated polygenic scores (PGSs) for 25 traits for 32,786 participants. Idefix then compares the actual phenotypes to PGSs and uses the relative discordance that is expected for mix-ups, compared to correct samples.

In a simulation, using induced mix-ups, Idefix reaches an AUC of 0.90 using 25 polygenic scores and sex. This is a substantial improvement over using only sex, which has an AUC of 0.75. Idefix therefore is not yet able to identify every sample mix-up. However, this will likely improve soon, with highly powered GWAS summary statistics that will likely become available for more commonly measured traits.

Nevertheless, Idefix can already be used to identify a high-quality set of participants for whom it is very unlikely that they reflect sample mix-ups, and therefore could be offered a pharmacogenetic passport. For instance, when selecting the 10% of participants for whom predicted phenotypes adhere best to the actually measured phenotypes, we estimate that the proportion of sample mix-ups is reduced 250-fold.

Availability and implementationIdefix is freely available at https://github.com/molgenis/systemsgenetics/wiki/Idefix

Contactl.h.franke@umcg.nl

sentences: High genetic diversity in the pelagic deep-sea fauna of the Atacama Trench revealed by environmental DNA A current paradigm in marine biodiversity states that faunal richness decreases with depth. However, the deep-ocean ecosystem has been significantly under-sampled, hindering a complete view of its biodiversity. This situation is accentuated in ultra-deep waters, where the remote and extreme conditions unfit the traditional sampling methods. Using environmental DNA, we assessed the pelagic metazoan diversity of the Atacama Trench from the high-productive near-surface level down to ~8000 m depth. Our results show that waters deeper than 4000 m contributed up to 50% of the overall genetic diversity. These findings contrast with similar observations in the less-productive Kermadec Trench, where the diversity in deep waters was lower than in shallower waters. Moreover, both deep pelagic ecosystems exhibited some unknown phylogenetic clades within the dominant taxonomic groups: hydrozoans and copepods. The deep-ocean biota may thus contribute to global biodiversity far more than hitherto suggested, especially in zones influenced by high primary production. Our results underline the need for increased effort to study these remote ecosystems and improve our understanding of their contribution to the ecology and biogeochemistry of the deep-sea pelagic and benthic realms.

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Fast analyzing, exploring and sharing quantitative omics data using omicsViewer SummaryTo facilitate the biological interpretation of omics data, we developed a general omics data visualization platform termed omicsViewer, enabling interactive visualization of statistical results and performing downstream analyses directly in response to features and samples selected. Multiple hypotheses and parameters can be evaluated in a few clicks. In addition, users can share the platform and statistical results with collaborators and the public in different ways.

Availability and ImplementationThe project page is https://github.com/mengchen18/omicsViewer

Contactchen.meng@tum.de

sentences: MTCL2 promotes asymmetric microtubule organizationby crosslinking microtubules on the Golgi membrane The Golgi complex plays an active role in organizing asymmetric microtubule arrays essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled- coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.

sentences: Optimizing microtubule arrangements for rapid cargo capture Cellular functions such as autophagy, cell signaling and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow diffusive movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus-ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus-ends relative to the initial cargo position. We use analytic mean first passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus-ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule ar-rangements that minimize mean cargo capture times. Namely, we show that configurations with a single long microtubule and broad distribution of additional microtubule plus-ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus-ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between microtubule length distribution and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

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The AIRE G228W mutation results in a longer-lasting AIRE-SIRT1 interaction The in silico and in vitro binding of a peptide covering a part of the autoimmune regulator (AIRE) SAND domain with the SIRT1 protein provides a powerful model system for studying the mechanism of the dominant SAND G228W mutation, which is the causative of APS-1 autoimmune syndrome. It is known that the mutant G228W AIRE protein accumulates more within the nucleus of cells than its wild-type counterpart does. This accumulation is not physiological and is associated with loss of AIRE function. However, the precise molecular mechanism that leads to AIRE accumulation is not yet known. AIRE works as a tetramer and interacts with partner proteins to form the "AIRE complex" that pushes RNA Pol II stalling in the chromatin of medullary thymic epithelial cells. Under normal conditions, the SIRT1 protein temporarily interacts with AIRE and deacetylates Lys residues of the SAND domain. Once AIRE is deacetylated, the binding with SIRT1 is undone, allowing the complex to proceed downstream. Here, we integrate molecular modeling, docking, dynamics, and surface plasmon resonance approaches to compare the structure and energetics of binding/release between AIRE G228 (wild-type) or W228 (mutant) peptides to SIRT1. We find that the proximity of G228W mutation to a K aminoacid residue in the SAND domain promotes a longer-lasting AIRE-SIRT1 interaction. The lasting interaction might cause a delay in the AIRE SAND domain to be released from the SIRT1 catalytic site, which might cause accumulation of the defective AIRE mutant protein in the nuclei of cells.

SignificanceThis report reveals the mechanism of the pathogenic and dominant G228W mutation in the AIRE SAND domain. The G228W mutation is found in APS-1 syndrome patients, and it is critical to understand the molecular basis of loss of self-representation, a challenging aspect for immunology. Through modeling, molecular dynamics, and protein binding kinetics, we found that the G228W mutation leads to a stronger physical interaction between the AIRE SAND domain and the SIRT1 protein when compared to the equivalent wild-type segment. The short-term consequence of this stronger interaction is that the release of the AIRE-SIRT1 binding is slower. This might explain the reason that cells carrying the G228W mutation accumulate AIRE protein in their nuclei. This finding reveals with precision the AIRE-SIRT1 binding and the molecular mechanism of the human AIRE G228W mutation.

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An Archaea-Specific c-type Cytochrome Maturation Machinery is Crucial for Methanogenesis in Methanosarcina acetivorans C-type cytochromes (cyt c) are proteins that covalently bind heme and are integral to electron transport chains. A growing body of evidence suggests that cyt c play a vital role in both intra- and extra-cellular electron transfer processes in Archaea, especially in members that metabolize methane and other short chain alkanes. Elaborate mechanisms for the biogenesis of cyt c are known in Bacteria and Eukarya but this process remains largely uncharacterized in Archaea. Here, we have used the model methanogenic archaeon Methanosarcina acetivorans to characterize a distinct form of the system I cyt c maturation machinery (referred to as the Ccm machinery henceforth) that is broadly distributed in members of the Archaea. Phenotypic analyses of M. acetivorans mutants deficient in essential components of the Ccm machinery reveal that cyt c are broadly important for growth and methanogenesis, but the magnitude of their impact can vary substantially depending on the growth substrate. Heterologous expression of a synthetic operon with the Ccm machinery (CcmABCEF) from M. acetivorans is both necessary and sufficient for cyt c biogenesis in a non-native host (M. barkeri Fusaro) that is incapable of cyt c biogenesis. Even though components of the Ccm machinery are universally conserved across the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this pathway through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we have demonstrated the convergent evolution of a novel Archaea-specific Ccm machinery for cyt c biogenesis and its role in methane metabolism.

Significance StatementMicroorganisms belonging to the domain Archaea play an especially important role in regulating atmospheric methane levels. Specifically, methanogens are the primary source of biogenic methane and anaerobic methanotrophic archaea (ANME) consume a substantial proportion of methane released in marine sediments. Genomic studies have implicated a class of electron-transfer proteins called c-type cytochromes as being crucial in mediating archaeal methane metabolism in the environment. However, neither the biogenesis nor the role of c-type cytochromes in methane metabolism has ever been investigated. Here, we have used a model methanogen, Methanosarcina acetivorans, to characterize a distinct pathway for maturation of c-type cytochromes that seems to be uniformly conserved across the Archaea and have also identified substrate-specific functional roles for c-type cytochromes during methanogenesis.

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Multimodal Magnetic Resonance Histology and Light Sheet Imaging for Quantitative Neurogenetics of the Mouse Paul Lauterbur closed his seminal paper on MRI with the statement that "zeugmatographic (imaging) techniques should find many useful applications in studies of the internal structures, states and composition of microscopic objects" [1]. Magnetic resonance microscopy was subsequently demonstrated in 1986 by three groups [2] [3] [4]. The application of MRI to the study of tissue structure, i.e. magnetic resonance histology (MRH) was suggested in 1993 [5]. MRH, while based on the same physical principals as MRI is something fundamentally different than the clinical exams which are typically limited to voxel dimensions of ~ 1 mm3. Preclinical imaging systems can acquire images with voxels ~ 1000 times smaller. The MR histology images presented here have been acquired at yet another factor of 1000 increase in spatial resolution. Figure S1 in the supplement shows a comparison of a state-of-the-art fractional anisotropy images of a C57 mouse brain in vivo @ 150 m resolution (voxel volume of 3.3 x10-3 mm3) with the atlas we have generated for this work at 15 m spatial resolution (voxel volume of 3.3 x 10-6 mm3). In previous work, we have demonstrated the utility of MR histology in neurogenetics at spatial/angular resolution of 45 m /46 angles [6]. At this spatial/angular resolution it is possible to map whole brain connectivity with high correspondence to retroviral tracers [7]. But the MRH derived connectomes can be derived in less than a day where the retroviral tracer studies require months/years [8]. The resolution index (angular samples/voxel volume) for this previous work was >500,000 [9]. Figure S2 shows a comparison between that previous work and the new atlas presented in this paper with a resolution index of 32 million.

Light sheet microscopy (LSM) has undergone similar rapid evolution over the last 20 years. The invention of tissue clearing, advances in immunohistochemistry and development of selective plane illumination microscopy (SPIM) now make it possible to acquire whole mouse brain images at submicron spatial resolution with a vast array of cell specific markers [10] [11] [12] [13]. And these advantages can be realized in scan times of < 6hrs. The major limitation from these studies is the distortion in the tissue from dissection from the cranium, swelling from clearing and staining, and tissue damage from handling.

We report here the merger of these two methods: O_LIMRH with the brain in the skull to provide accurate geometry, cytoarchitectural measures using scalar imaging metrics and whole brain connectivity at 15 m isotropic spatial resolution with super resolution track density images @ 5 m isotropic resolution;
C_LIO_LIwhole brain multichannel LSM @ 1.8x1.8x4.0 m;
C_LIO_LIa big image data infrastructure that enables label mapping from the atlas to the MR image, geometric correction to the light sheet data, label mapping to the light sheet volumes and quantitative extraction of regional cell density. These methods make it possible to generate a comprehensive collection of image derived phenotypes (IDP) of cells and circuits covering the whole mouse brain with throughput that can be scaled for quantitative neurogenetics.
C_LI

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Fatal neuroinvasion of SARS-CoV-2 in K18-hACE2 mice is partially dependent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis.

sentences: SRF-MYOCD axis is the targetable driver of a well differentiated aggressive subtype of leiomyosarcomas Differentiated phenotypes in solid tumors mostly lead to a better outcome than in their more immature counterparts. In leiomyosarcoma (LMS), a very aggressive tumor for which neither chemotherapy nor targeted or immune therapies have demonstrated efficacy, a relatively transcriptionally uniform subgroup of well differentiated tumors has been described and is associated with poor survival. In this disease, differentiation does not systematically hinder tumor progression, so it is a good model for studying the relation between these two mechanisms. By applying ad hoc bioinformatic methods, we conducted a comprehensive analysis in order to detect subgroups of leiomyosarcoma and finely investigate their transcriptomic and genomic profiles. We first used unsupervised hierarchical clustering to identify the most transcriptionally homogeneous LMS subgroup, which we named  hLMS. We derived a transcriptional signature that could consistently stratify patients with similar clinical and genomic characteristics in TCGA and ICGC cohorts. Functional analysis of the signature showed over-expression of SRF and E2Fs targets harboring histone modifications that were specifically identified in smooth muscles in hLMS. By integrating multi-omics data, we identified punctual and structural variations as well as the potential cellular origins of each group of LMS using GTEX normal tissue gene expression. Altogether, our results suggest that hLMS, but not oLMS (other LMS), may originate from vascular smooth muscle cells in which oncogenesis is driven by the MYOCD/SRF axis, which is essential for hLMS survival. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway overactivation could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

sentences: NMDAR-mediated transcriptional control of gene expression during the development of medial ganglionic eminence-derived interneurons Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+ and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical and physiological properties. However, the molecular mechanisms regulating their diversity remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates subtype-specific transcriptional regulation of gene expression in MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific conditional Grin1 loss results in a systemic downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders, particularly schizophrenia. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

sentences: ATR facilitates the degradation of Api5 through the ubiquitin-proteasome pathway via FBXW2 to regulate apoptosis upon DNA damage Apoptosis inhibitor 5 (Api5) is an inhibitor of apoptosis, which is found to be upregulated in several cancers and promotes invasion as well as metastasis. Over-expression of Api5 is positively co-related with poor survival of cancers and inhibition of DNA damage induced apoptosis in cancerous cells. Acetylation at lysine 251 (K251) on Api5 facilitates the stability of the protein and thus functionally provides resistance to cancer cells against chemotherapeutic or anti-cancerous agents. However, the regulation of Api5 upon DNA damage is not yet known. In this study, we demonstrate that Api5 undergoes degradation following DNA damage via the ubiquitin-proteasome system. Upon DNA damage, ATR was observed to phosphorylate Api5 at serine 138 which led to the cytoplasmic localisation of Api5. The E3-ubiquitin ligase, SCF-FBXW2 ubiquitinates Api5 leading to its proteasomal degradation.

sentences: Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of an N-arylbenzdiimidazole ligand's rescue of Parkinson's-associated cell toxicity The development of phenotypic models of Parkinsons disease (PD) has enabled screening and identification of phenotypically active small molecules that restore complex biological pathways affected by PD toxicity. While these phenotypic screening platforms are powerful, they do not inherently enable direct identification of the cellular targets of promising lead compounds. To overcome this, chemoproteomic platforms like Thermal Proteome Profiling (TPP) and Stability of Proteins from Rates of Oxidation (SPROX) can be implemented to reveal protein targets of biologically active small molecules. Here we utilize both of these chemoproteomic strategies to identify targets of an N-arylbenzdiimidazole compound, NAB2, which was previously identified for its ability to restore viability in cellular models of PD-associated -synuclein toxicity. The combined results from our TPP and SPROX analyses of NAB2 and the proteins in a neuroblastoma-derived SHSY5Y cell lysate reveal a previously unrecognized protein target of NAB2. This newly recognized target, Rab1a, is a small GTPase that acts as a molecular switch to regulate ER-to-Golgi trafficking, a process that is disrupted by -synuclein toxicity and restored by NAB2 treatment. Further validation reveals that NAB2 binds to Rab1a with selectivity for its GDP-bound form and that NAB2 treatment phenocopies Rab1a overexpression in alleviation of -synuclein toxicity. Finally, we conduct a preliminary investigation into the relationship between Rab1a and the E3 ubiquitin ligase, Nedd4, a previously identified NAB2 target. Together, these efforts expand our understanding of the mechanism of NAB2 in the alleviation of -synuclein toxicity and reinforce the utility of chemoproteomic identification of the targets of phenotypically active small molecules that regulate complex biological pathways.

sentences: AC2P20 selectively kills M. tuberculosis  at acidic pH by depleting free thiols Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a [~]220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.

sentences: Live Imaging Reveals the Cellular Events Downstream of SARM1 Activation SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here we used live imaging with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.

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

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The effects of protein crowders on small molecule drug diffusion Crowded environments affect the pharmacokinetics of drug molecules. Here, we investigate how three macromolecular protein crowders, bovine serum albumin, hen egg-white lysozyme and myoglobin, influence the translational diffusion rates and interactions of four low molecular-weight drugs, fluorescein, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763 and quinacrine. Using Fluorescence Recovery After Photo-bleaching in Line mode (Line FRAP), Brownian dynamics simulations and molecular docking, we find that the diffusive behavior of the small molecules is highly affected by self-aggregation, interactions with the proteins, and surface adhesion. Fluorescein diffusion is decreased by protein crowders due to their interactions. On the other hand, for doxorubicin, the presence of protein crowders increases diffusion by reducing surface interactions. SB216763 shows a third scenario, where BSA, but not myoglobin or lysozyme, reduces self-aggregation, resulting in faster diffusion. Quinacrine was the only small molecule whose diffusion was not affected by the presence of protein crowders. The mechanistic insights gained here into the effects of interactions of small molecules with proteins and surfaces on the translational diffusion of small molecules can assist in optimizing the design of compounds for higher mobility and lower occlusion in complex macromolecular environments.

Significance statementThe activity of small molecules is directly related to their active concentration. This, in turn, relates to their molar concentration and their activity coefficient. Any deviations from ideal behavior affects the small molecule activity. This is particularly important for drugs, where we seek to optimize their molar activity. Here, we follow the diffusional activity of four small molecule drugs in various solutions, with and without protein crowders. The study, which involves experimental measurements and theoretical simulations, shows that 3 of the 4 drugs do not diffuse normally, either due to aggregation, protein binding or surface adhesion. We show the mechanisms driving the abnormal diffusion, and how it can be reduced. Lessons learned from this study can be implemented into drug design.

sentences: Hematopoietic stem and progenitor cells improve survival from sepsis by boosting immunomodulatory cells New therapeutic strategies to reduce sepsis-related mortality are urgently needed, as sepsis accounts for 1 in 5 deaths worldwide. Since hematopoietic stem and progenitor cells (HSPCs) are responsible for producing blood and immune cells, including in response to immunological stress, we explored their potential for treating sepsis. In a mouse model of Group A Streptococcus (GAS)-induced sepsis, severe immunological stress was associated with significant depletion of bone marrow HSPCs and mortality within approximately 5-7 days. We hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation and depletion that can be rescued by infusion of donor HSPCs. Indeed, infusion of 10,000 naive HSPCs into GAS-infected mice resulted in rapid myelopoiesis and a 50-60% increase in overall survival. Surprisingly, mice receiving donor HSPCs displayed a similar pathogen load compared to untreated mice. Flow cytometric analysis revealed a significantly increased number of myeloid-derived suppressor cells in HSPC-infused mice, which correlated with reduced inflammatory cytokine levels and restored HSPC levels. These findings suggest that HSPCs play an essential immunomodulatory role that may translate into new therapeutic strategies for sepsis.

sentences: Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutations Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate (dNTP) pools by ribonucleotide reductase (RNR) and repair of replication errors by the mismatch repair (MMR) system. We identified genetic interactions between rnr1 alleles that elevate dNTP levels and MMR. We then utilized a targeted deep-sequencing approach to determine mutational signatures associated with MMR pathway defects. By combining rnr1 and msh mutations to increase dNTP levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.

sentences: Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 - 33 copies/{micro}L (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72-16.95 copies/oocyte), Hprt1 (1.05-19.05 copies/oocyte) and ATPase 6, (5.55-32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

sentences: DNA metabarcoding reveals host-specific communities of arthropods residing in fungal fruit bodies Biological communities within living organisms are structured by their hosts traits. How host traits affect biodiversity and community composition is poorly explored for some associations, such as arthropods within fungal fruit bodies. Using DNA metabarcoding, we revealed the arthropod communities in living fruit bodies of eleven wood-decay fungi from boreal forests and investigated how they were affected by different fungal traits. Arthropod diversity was higher in fruit bodies with a larger surface area-to-volume ratio, suggesting that colonisation is crucial to maintain arthropod populations. Diversity was not higher in long-lived fruit bodies, most likely because these fungi invest in physical or chemical defences against arthropods. Arthropod community composition was structured by all measured host traits, namely fruit body size, thickness, surface area, morphology and toughness. Notably, we identified a community gradient where soft and short-lived fruit bodies harboured more true flies, while tougher and long-lived fruit bodies had more oribatid mites and beetles, which might reflect different development times of the arthropods. Ultimately, close to 75% of the arthropods were specific to one or two fungal hosts. Besides revealing surprisingly diverse and host-specific arthropod communities within fungal fruit bodies, our study provided insight on how host traits structure communities.

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Tiara: Deep learning-based classification system for eukaryotic sequences MotivationWith a large number of metagenomic datasets becoming available, the eukaryotic metagenomics emerged as a new challenge. The proper classification of eukaryotic nuclear and organellar genomes is an essential step towards the better understanding of eukaryotic diversity.

ResultsWe developed Tiara, a deep-learning-based approach for identification of eukaryotic sequences in the metagenomic data sets. Its two-step classification process enables the classification of nuclear and organellar eukaryotic fractions and subsequently divides organellar sequences to plastidial and mitochondrial. Using test dataset, we have shown that Tiara performs similarly to EukRep for prokaryotes classification and outperformed it for eukaryotes classification with lower calculation time. Tiara is also the only available tool correctly classifying organellar sequences.

Availability and implementationTiara is implemented in python 3.8, available at https://github.com/ibe-uw/tiara and tested on Unix-based systems. It is released under an open-source MIT license and documentation is available at https://ibe-uw.github.io/tiara. Version 1.0.1 of Tiara has been used for all benchmarks.

sentences: Resource-diversity relationships in bacterial communities reflect the network structure of microbial metabolism The relationship between the number of available nutrients and community diversity is a central question for ecological research that remains unanswered. Here, we studied the assembly of hundreds of soil-derived microbial communities on a wide range of well-defined resource environments, from single carbon sources to combinations of up to 16. We found that, while single resources supported multispecies communities varying from 8 to 40 taxa, mean community richness increased only one-by-one with additional resources. Cross-feeding could reconcile these seemingly contrasting observations, with the metabolic network seeded by the supplied resources explaining the changes in richness due to both the identity and the number of resources, as well as the distribution of taxa across different communities. By using a consumer-resource model incorporating the inferred cross-feeding network, we provide further theoretical support to our observations and a framework to link the type and number of environmental resources to microbial community diversity.

sentences: The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2 Sotrovimab (VIR-7831) and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sotrovimab and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an "LS" mutation in the Fc region to prolong serum half-life. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. Sotrovimab and VIR-7832 neutralize wild-type and variant pseudotyped viruses and authentic virus in vitro. In addition, they retain activity against monoclonal antibody resistance mutations conferring reduced susceptibility to previously authorized mAbs. The sotrovimab/VIR-7832 epitope continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with sotrovimab had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that sotrovimab and VIR-7832 are key agents in the fight against COVID-19.

sentences: Cortical Depth-Dependent Modeling of Visual Hemodynamic Responses A physiologically based three-dimensional (3D) hemodynamic model is used to predict the experimentally observed blood oxygen level dependent (BOLD) responses versus the cortical depth induced by visual stimuli. Prior 2D approximations are relaxed in order to analyze 3D blood flow dynamics as a function of cortical depth. Comparison of the predictions with experimental data for typical stimuli demonstrates that the full 3D model matches at least as well as previous approaches while requiring significantly fewer assumptions and model parameters (e.g., there is no more need to define depth-specific parameter values for spatial spreading, peak amplitude, and hemodynamic velocity).

sentences: The histone methyltransferase NSD3 contributes to cohesin loading during mitotic exit During the cell cycle, dynamic post-translational modifications modulate the association of the cohesin complex with chromatin. Phosphorylation / dephosphorylation and acetylation / deacetylation of histones and of cohesin components ensure correct establishment of cohesion during S phase and its proper dissolution during mitosis. In contrast, little is known about the contribution of methylation to the regulation of sister chromatid cohesion. We performed a RNA interference-mediated inactivation screen against 14 histone methyltransferases of the SET domain family that highlighted NSD3 as a factor essential for sister chromatid cohesion in mitosis. We established that NSD3 ensures proper level of the cohesin loader MAU2 and of cohesin itself onto chromatin at mitotic exit. Consistent with its implication in the loading of kollerin and cohesin complexes onto chromatin, we showed that NSD3 associates with chromatin in early anaphase prior to that of MAU2 and RAD21 and dissociates from chromatin upon cells entry into prophase. Finally, we demonstrated that of the two NSD3 variant that exist in somatic cells, the long form that carries the methyltransferase activity is the one that acts in cohesion regulation. Taken together, these results describe a novel factor associated with histone methylation in cohesin loading.

sentences: Normalizing and denoising protein expression data from droplet-based single cell profiling Multimodal single-cell protein and transcriptomic profiling (e.g. CITE-seq) holds promise for comprehensive dissection of cellular heterogeneity, yet protein counts measured by oligo-conjugated-antibody can have substantial noise that masks biological variations. Here we integrated experiments and computational analysis to reveal two major noise sources: protein-specific noise from unbound antibodies and cell-specific noise captured by the shared variance of isotype controls and background protein counts. We provide an open source R package (dsb) to denoise and normalize CITE-seq data based on these findings. (https://cran.r-project.org/web/packages/dsb/index.html).

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'rbioacc': an R-package to analyse toxicokinetic data O_LI rbioacc is an  R-package dedicated to the analysis of experimental data collected from bioaccumulation tests during which organisms are exposed to a chemical (exposure phase) and then put into a clean media (depuration phase). Internal concentrations are regularly measured over time all along the experiment.
C_LIO_LI rbioacc provides ready-to-use functions to visualize and fully analyze such data. Under a Bayesian framework, this package fits a generic one-compartment toxicokinetic (TK) model automatically built from the data. It provides TK parameter estimates (appropriate uptake and elimination rates) and bioaccumulation metrics (e.g., BCF, BSAF, BMF). All parameter estimates, bioaccumulation metrics as well as predictions of internal concentrations into organisms are delivered with their uncertainty.
C_LIO_LIBioaccumulation metrics are primarily provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve marketing applications of chemical substances.
C_LIO_LIThis paper gives brief worked examples of the use of  rbioacc from data collected through standard bioaccumulation tests, and publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyze any new data set, uploaded in the right format.
C_LI

sentences: Widening the landscape of transcriptional regulation of algal photoprotection Availability of light and CO2, substrates of microalgae photosynthesis, is frequently far from optimal. Microalgae activate photoprotection under strong light, to prevent oxidative damage, and the CO2 Concentrating Mechanism (CCM) under low CO2, to raise intracellular CO2 levels. The two processes are interconnected; yet, the underlying transcriptional regulators remain largely unknown. By employing a large transcriptomics data compendium of Chlamydomonas reinhardtiis responses to different light and carbon supply we reconstructed a consensus genome-scale gene regulatory network from complementary inference approaches and used it to elucidate the transcriptional regulation of photoprotection. We showed that the CCM regulator LCR1 also controls photoprotection, and that QER7, a Squamosa Binding Protein, suppresses photoprotection- and CCM-gene expression under the control of the blue light photoreceptor Phototropin. Along with demonstrating the existence of regulatory hubs that channel light- and CO2-mediated signals into a common response, our study provides a unique resource to dissect gene expression regulation in this microalga.

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NIfTI-MRS: A standard format for magnetic resonance spectroscopic data PurposeThe use of multiple data formats in the MRS community currently hinders data sharing and integration. NIfTI-MRS is proposed as a standard MR spectroscopy data format, which is implemented as an extension to the neuroimaging informatics technology initiative (NIfTI) format.

Using this standardised format will facilitate data sharing, ease algorithm development, and encourage the integration of MRS analysis with other imaging modalities.

MethodsA file format based on the NIfTI header extension framework was designed to incorporate essential spectroscopic metadata and additional encoding dimensions. A detailed description of the specification is provided. An open-source command-line conversion program is implemented to enable conversion of single-voxel and spectroscopic imaging data to NIfTI-MRS. To provide visualisation of data in NIfTI-MRS, a dedicated plugin is implemented for FSLeyes, the FSL image viewer.

ResultsAlongside online documentation, ten example datasets are provided in the proposed format. In addition, minimal examples of NIfTI-MRS readers have been implemented. The conversion software, spec2nii, currently converts fourteen formats to NIfTI-MRS, including DICOM and vendor proprietary formats.

ConclusionThe proposed format aims to solve the issue of multiple data formats being used in the MRS community. By providing a single conversion point, it aims to simplify the processing and analysis of MRS data, thereby lowering the barrier to use of MRS. Furthermore, it can serve as the basis for open data sharing, collaboration, and interoperability of analysis programs. It also opens possibility of greater standardisation and harmonisation. By aligning with the dominant format in neuroimaging, NIfTI-MRS enables the use of mature tools present in the imaging community, demonstrated in this work by using a dedicated imaging tool, FSLeyes, as a viewer.

sentences: Blood flow modeling under LVAD physiology. From global circulation to local hemodynamics This document presents the modeling strategy to address the in-silico study of different LVAD patho-physiological scenarios. The proposed approach employs a closed-loop lumped-parameter compartmental representation of the global circulation in the cardiovascular system (CVS). The CVS is coupled to a HeartMate 3 LVAD, and different cardiovascular conditions are proposed by modification of model parameters. Once the simulation for these conditions are performed, the cardiac function is analyzed in detail, and the global circulation model delivers flow rate waveforms which are employed as boundary conditions in a 3D hemodynamic simulation. This local circulation model is built using a patient-specific geometry of the aortic arch, containing 7 inlet/outlet boundaries, namely: LVAD cannula, aortic root, left and right subclavian arteries, left and right common carotid arteries and thoracic aorta. This model is exploited to investigate the impact of global cardiovascular conditions in the local hemodynamic features, particularly the wall shear stress (WSS) in different spatial regions.

sentences: Opposing mechanisms of competition determine species invasions and functional diversity Understanding how species phenotypic differences affect competition is key to explaining community assembly and predicting biodiversity responses. Many studies overlook the variable effects that species trait differences can have on the direction of competitive exclusion, which reverses depending on the specific mechanism at play. We performed a comprehensive trait-based study of an ant invasion integrating morphological, dietary, physiological and behavioral analyses. We found that trait differences between invasive and resident species were not only associated with niche differences which promoted the coexistence of dissimilar species, but also competitive ability differences which acted in the opposite fashion. Furthermore, competition along separate trait axes led to complex and contrasting patterns in the invaded assemblages, where species were at once similar (clustered) in some traits but also dissimilar (overdispersed) in others. Our results reveal that different aspects of phenotype may distinctly modulate the effect of competition in structuring ecological communities and functional diversity.

sentences: Comprehensive analysis of mutational signatures in pediatric cancers Analysis of mutational signatures can reveal the underlying molecular mechanisms of the processes that have imprinted the somatic mutations found in a cancer genome. Here, we present a pan-cancer mutational signatures analysis of single base substitutions (SBS) and small insertion and deletions (ID) in pediatric cancers encompassing 537 whole genome sequenced tumors from 20 molecularly defined cancer subtypes. We identified only a small number of mutational signatures active in pediatric cancers when compared to the previously analyzed adult cancers. Further, we report a significant difference in the proportion of pediatric tumors which show homologous recombination repair defect signature SBS3 compared to prior analyses. Correlating genomic alterations with signature activities, we identified an association of TP53 mutation status with substitution signatures SBS2, SBS8, SBS13 and indel signatures ID2 and ID9, as well as chromothripsis associated with SBS8, SBS40 and ID9. This analysis provides a systematic overview of COSMIC v.3 SBS and ID mutational signatures active across pediatric cancers, which is highly relevant for understanding tumor biology as well as enabling future research in defining biomarkers of treatment response.

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Genomic Copy Number Signatures Based Classifiers for Subtype Identification in Cancer Copy number aberrations (CNA) are one of the most important classes of genomic mutations related to oncogenetic effects. In the past three decades, a vast amount of CNA data has been generated by molecular-cytogenetic and genome sequencing based methods. While this data has been instrumental in the identification of cancer-related genes and promoted research into the relation between CNA and histo-pathologically defined cancer types, the heterogeneity of source data and derived CNV profiles pose great challenges for data integration and comparative analysis. Furthermore, a majority of existing studies have been focused on the association of CNA to pre-selected "driver" genes with limited application to rare drivers and other genomic elements.

In this study, we developed a bioinformatics pipeline to integrate a collection of 44,988 high-quality CNA profiles of high diversity. Using a hybrid model of neural networks and attention algorithm, we generated the CNA signatures of 31 cancer subtypes, depicting the uniqueness of their respective CNA landscapes. Finally, we constructed a multi-label classifier to identify the cancer type and the organ of origin from copy number profiling data. The investigation of the signatures suggested common patterns, not only of physiologically related cancer types but also of clinico-pathologically distant cancer types such as different cancers originating from the neural crest. Further experiments of classification models confirmed the effectiveness of the signatures in distinguishing different cancer types and demonstrated their potential in tumor classification.

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Impact of South African 501.V2 Variant on SARS-CoV-2 Spike Infectivity and Neutralization: A Structure-based Computational Assessment MotivationThe SARS-CoV-2 variants emerging from South Africa (501.V2) and the UK (B.1.1.7) necessitate rapid assessment of the effects of the corresponding amino acid substitutions in the spike (S) receptor-binding domain (RBD) of the variants on the interactions with the human ACE2 receptor and monoclonal antibodies (mAbs) reported earlier to neutralize the spike.

ResultsMolecular modeling and simulations reveal that N501Y, shared by both variants, increases ACE2 binding affinity, and may impact the collective dynamics of the ACE2-RBD complex, occupying a central hinge site that modulates the overall dynamics of the complex. In contrast, the substitutions K417N and E484K in the South African variant 501.V2 would reduce the ACE2-binding affinity by abolishing two interfacial salt bridges that facilitate RBD binding to ACE2, K417(S)-D30(ACE2) and E484 (S)-K31(ACE2). These two mutations may thus be more than compensating the attractive effect induced by N501Y, overall resulting in an ACE2-binding affinity comparable to that of the wildtype RBD. Further analysis of the impact of these mutations on the interactions with mAbs targeting the spike indicate that the substitutions K417N and E484K may also abolish the salt bridges between the spike and selected mAbs, such as REGN10933, BD23, H11_H4, and C105, thus reducing the binding affinity and effectiveness of these mAbs.

Contactbahar@pitt.edu

Supplementary informationSupplementary data are available at Bioinformatics online.

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Model-based inference of neutralizing antibody avidities against influenza virus To assess the response to vaccination, quantity (concentration) and quality (avidity) of neutralizing antibodies are the most important parameters. Specifically, an increase in avidity indicates germinal center formation, which is required for establishing long-term protection. For influenza, the classical hemagglutination inhibition (HI) assay, however, quantifies a combination of both, and to separately determine avidity requires high experimental effort. We developed from first principles a biophysical model of hemagglutination inhibition to infer IgG antibody avidities from measured HI titers and IgG concentrations. The model accurately describes the relationship between neutralizing antibody concentration/avidity and HI titer, and explains quantitative aspects of the HI assay, such as robustness to pipetting errors and detection limit. We applied our model to infer avidities against the pandemic 2009 H1N1 influenza virus in vaccinated patients (n=45) after hematopoietic stem cell transplantation (HSCT) and validated our results with independent avidity measurements using an enzyme-linked immunosorbent assay with urea elution. Avidities inferred by the model correlated with experimentally determined avidities ({rho}=0.54, 95% CI=[0.31, 0.70], P<10-4). The model predicted that increases in IgG concentration mainly contribute to the observed HI titer increases in HSCT patients and that immunosuppressive treatment is associated with lower baseline avidities. Since our approach requires only easy-to-establish measurements as input, we anticipate that it will help to disentangle causes for poor vaccination outcomes also in larger patient populations. This study demonstrates that biophysical modelling can provide quantitative insights into agglutination assays and complement experimental measurements to refine antibody response analyses.

Author SummaryInfluenza vaccines are assessed based on the induced antibody response, where antibody quantity (concentration) and antibody binding strength (avidity) determine the potency to neutralize the virus. In addition, an increase in avidity indicates a successful germinal center reaction, which is required for establishing long-term protection. However, the hemagglutination inhibition (HI) assay - traditionally used to assess influenza vaccines - measures a combination of both antibody concentration and avidity, and to separately determine avidity requires high experimental effort. We developed a biophysical model of the HI assay, which enables the inference of antibody avidities from measured HI titers and antibody concentrations. We applied our approach to a vaccinated population of immunocompromised patients after blood stem cell transplantation and validated our results experimentally. The model predicted that vaccination induced an increase in avidity in only a few patients and that patients under immunosuppressive treatment show lower baseline avidities. Since our approach requires only easily measurable data as input, it can facilitate the investigation of vaccine responses in larger populations. This study demonstrates that biophysical modelling can complement experimental data and provide additional details on agglutination experiments and antibody responses.

sentences: Parental care allows genetic variation to accumulate by relaxing selection Benign social environments are thought to relax selection against deleterious mutations, enabling them to accumulate in the genome. However, it has not proved straightforward to demonstrate causality in this relationship. We allowed burying beetle populations to evolve with and without the benign effects of parental care for over 20 generations, before inbreeding lineages that were seeded from these experimental populations to expose any differences in their mutation load. We found that inbred lineages with a history of parental care went extinct most quickly, indicating that they harboured more recessive deleterious alleles.

sentences: No evidence for long-range male sex pheromones in two malaria mosquitoes AO_SCPLOWBSTRACTC_SCPLOWCues involved in mate seeking and recognition prevent hybridization and can be involved in speciation processes. In malaria mosquitoes, females of the two sibling species Anopheles gambiae s.s. and An. coluzzii mate in monospecific male swarms and hybrids are rare. Long-range sex pheromones driving this behavior have been debated in literature but to date, no study has proven their existence or their absence. Here, we attempted to bring to light their existence. To put all the odds in our favor, we used different chemical ecology methods such as behavioral and electrophysiological assays as well chemical analyses, and we worked with mosquitoes at their optimal physiological mating state i.e. with swarming males during their natural swarming windows. Despite all our efforts, our results support the absence of long-range sex pheromones involved in swarm detection and recognition by females. We briefly discuss the implications of this finding in ecology, evolution and for control strategies.

sentences: Specialized neurons in the right habenula mediate response to aversive olfactory cues Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the right dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The unilateral lratd2a-expressing neurons are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure of adult zebrafish to the aversive odorant cadaverine that provokes avoidance behavior. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show protracted avoidance to cadaverine. Mutants with left-isomerized dHb that lack these neurons are less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in processing of aversive olfactory cues and modulating resultant behavioral responses.

sentences: Emotion Recognition in a Multi-Componential Framework: The Role of Physiology Emotions are rich and complex experiences involving various behavioral and physiological responses. While many empirical studies have focused on discrete and dimensional representations of emotions, these representations do not fully reconcile with recent neuroscience studies that increasingly suggest a multi-process mechanism underlying emotional experience. Moreover, the latter view accords with psychological theories that consider emotions as multicomponent phenomena, such as appraisal theories. Although there is no complete consensus on the specific components of emotions and fundamental principles defining their organization, the Component Process Model (CPM) is well established framework describing an emotion as a dynamic process with five major highly interrelated components: cognitive appraisal, expression, motivation, physiology and feeling. Yet, few studies have systematically investigated a range of discrete emotions through this full multi-componential view. In the present study, we therefore elicited various emotions during movie watching and measured their manifestation across these components. Our primary goal was to investigate the relationship between physiological measures and the theoretically defined components of emotions. In addition, we also investigated whether discrete emotions could be predicted from information provided by the multicomponent response patterns, as well as the specific contributions of each component in such predictions. Results suggest that physiological features are interrelated to all other components of emotion, but the least significant predictors for emotion classification. Overall, emotion prediction was significantly higher when classifiers were trained with all five components. The findings therefore support a description of emotion as a dynamic multicomponent process, in which the emergence of a conscious feeling state requires the integration of all the components.

sentences: Live imaging of delamination in Drosophila shows that epithelial cell motility and invasiveness are independently regulated Delamination requires cells to undergo changes in cell-cell adhesion and in cell polarity, motility, and protrusions. This complex process must be precisely regulated during development as well as in pathogenic conditions. To determine the requirements for epithelial delamination, we analyzed the delamination of Drosophila ovary border cells, in which cells delaminate from the epithelial layer and begin to migrate collectively as is also seen in cancer metastasis. We used live imaging to examine cellular dynamics in delamination-defective mutants during the period in which delamination occurs in the wild-type ovary. We found that border cells in slow border cells (slbo), a delaminationdefective mutant, lacked the properties of invasive cellular extensions but acquired motility while JAK/STAT-inhibited border cells lost both cellular properties, suggesting that the invasiveness and motility required for delamination are regulated independently. Our reconstruction experiments showed that motility is not a prerequisite for acquiring invasiveness.

sentences: A trans-acting long non-coding RNA represses flowering in Arabidopsis Eukaryotic genomes give rise to thousands of long non-coding RNAs (lncRNAs), yet the purpose of lncRNAs remains largely enigmatic. Functional characterization of lncRNAs is challenging due to multiple orthogonal hypothesis for molecular activities of lncRNA loci. Here, we identified a flowering associated intergenic lncRNA (FLAIL) that represses flowering in Arabidopsis. An allelic series of flail loss-of-function mutants generated by CRISPR/Cas9 and T-DNA mutagenesis showed an early flowering phenotype. Gene expression analyses in flail mutants revealed differentially expressed genes linked to the regulation of flowering. A genomic rescue fragment of FLAIL introduced in flail mutants complemented gene expression defects and early flowering, consistent with trans-acting effects of the FLAIL RNA. Knock-down of FLAIL RNA levels using the artificial microRNA approach revealed an early flowering phenotype shared with genomic mutations, indicating a trans-acting role of FLAIL RNA in the repression of flowering time. Genome-wide detection of FLAIL-DNA interactions by ChIRP-seq suggested that FLAIL may directly bind genomic regions. FLAIL bound to genes involved in regulation of flowering that were differentially expressed in flail, consistent with the interpretation of FLAIL as a trans-acting lncRNA directly shaping gene expression. Our findings highlight FLAIL as a trans-acting lncRNA that affects flowering in Arabidopsis, likely through mediating transcriptional regulation of genes directly bound by FLAIL.

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TENET: Gene network reconstruction using transfer entropy reveals key regulatory factors from single cell transcriptomic data Accurate prediction of gene regulatory rules is important towards understanding of cellular processes. Existing computational algorithms devised for bulk transcriptomics typically require a large number of time points to infer gene regulatory networks (GRNs), are applicable for a small number of genes, and fail to detect potential causal relationships effectively. Here, we propose a novel approach  TENET to reconstruct GRNs from single cell RNA sequencing (scRNAseq) datasets. Employing transfer entropy (TE) to measure the amount of causal relationships between genes, TENET predicts large-scale gene regulatory cascades/relationships from scRNAseq data. TENET showed better performance than other GRN reconstructors, in identifying key regulators from public datasets. Specifically from scRNAseq, TENET identified key transcriptional factors in embryonic stem cells (ESCs) and during direct cardiomyocytes reprogramming, where other predictors failed. We further demonstrate that known target genes have significantly higher TE values, and TENET predicted higher TE genes were more influenced by the perturbation of their regulator. Using TENET, we identified and validated that Nme2 is a culture condition specific stem cell factor. These results indicate that TENET is uniquely capable of identifying key regulators from scRNAseq data.

Key PointsO_LITENET measures putative causal relationships between genes using transfer entropy.
C_LIO_LITENET shows outstanding performance in identifying key regulators compared to existing methods.
C_LIO_LITENET can reveal previously uncharacterized regulators.
C_LI

sentences: Avoiding tensional equilibrium in cells migrating on a matrix with cell-scale stiffness-heterogeneity Intracellular stresses affect various cell functions, including proliferation, differentiation and movement, which are dynamically modulated in migrating cells through continuous cell-shaping and remodeling of the cytoskeletal architecture induced by spatiotemporal interactions with extracellular matrix stiffness. When cells migrate on a matrix with cell-scale stiffness-heterogeneity, which is a common situation in living tissues, what intracellular stress dynamics (ISD) emerge? In this study, to explore this issue, finite element method-based traction force microscopy was applied to cells migrating on microelastically patterned gels. Two model systems of microelastically patterned gels (stiff/soft stripe and stiff triangular patterns) were designed to characterize the effects of a spatial constraint on cell-shaping and of the presence of different types of cues to induce competing cellular taxis (usual and reverse durotaxis) on the ISD, respectively. As the main result, the prolonged fluctuation of traction stress on a whole-cell scale was markedly enhanced on single cell-size triangular stiff patterns compared with homogeneous gels. Such ISD enhancement was found to be derived from the interplay between the nomadic migration of cells to regions with different degrees of stiffness and domain shape-dependent traction force dynamics, which should be an essential factor for keeping cells far from tensional equilibrium.

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The yeast claudin Dcv1 is important for demarcating the front domain of pheromone-responding yeast cells Cell polarization in response to chemical gradients is important in development and homeostasis across eukaryota. Chemosensing cells orient toward or away from gradient sources by polarizing along a front-rear axis. Using the chemotropic mating response of the budding yeast S. cerevisiae as a model of environmentally-induced cell polarization, we found that Dcv1, a claudin homolog, is a determinant of front-rear polarity. Although Dcv1 localized uniformly on the plasma membrane (PM){dagger} of vegetative cells, it was confined to the rear of cells responding to pheromone, away from the pheromone receptor and mating projection. Deletion of DCV1 conferred mislocalization of sensory, polarity, and trafficking proteins, as well as the PM lipids ergosterol, phosphatidylinositol-4,5-bisphosphate (PIP2), and phosphatidylserine (PS). These phenotypes correlated with defects in pheromone-gradient tracking and cell fusion. We propose that the novel claudin-like and rear-domain protein, Dcv1, demarcates the mating-specific front domain primarily by restricting PM lipid distribution. Consistent with this hypothesis, a mutation that blocks ergosterol biosynthesis partially phenocopies dcv1{Delta}.

Summary statementThe yeast claudin Dcv1 facilitates the proper localization of plasma membrane lipids and proteins that are required for front-rear polarity, efficient chemotropism, and cell fusion.

sentences: Ecdysone coordinates plastic growth with robust pattern in the developing wing Animals develop in unpredictable, variable environments. In response to environmental change some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesise ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally-regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels, and confers robustness by initiating patterning only once ecdysone peaks exceeds a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.

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Eliminating Aedes aegypti from its southern margin in Australia: insights from genomic data and simulation modeling A rare example of a successful long-term elimination of the mosquito Aedes aegypti is in Brisbane, Queensland, where the legislatively-enforced removal of rainwater tanks drove its disappearance by the mid-1950s. However, a decade-long drought led to the mass installation of rainwater tanks throughout the region, re-introducing critical breeding sites for the mosquitos persistence in this subtropical region. With Ae. aegypti re-invading towns just 150 km north of Brisbane, we examined the potential for their sustained elimination. Through genomic analyses, we estimated historical expansion and current isolation between neighboring populations as close as 15 kilometers. The estimated recent migration rate, entomological and meteorological data were used to calibrate the simulations of elimination campaigns in the two southernmost populations. Our simulations indicate that Ae. aegypti could be eliminated with moderate release numbers of incompatible Wolbachia-infected (IIT) males (sorted with an error rate [≤]10-6) if non-compliant rainwater tanks are removed first. With this combined campaign, highly effective suppression (>99%) was predicted in both towns, and complete elimination was predicted in 35% of simulations in one town. Without tank removal, however, IIT led to a moderate suppression (61-93%) even with a 40:1 ratio of released IIT males to local males. Moreover, with a ratio of >20:1, Wolbachia establishment was predicted when the sorting error was >10-7. Our conservative estimates of intervention outcomes inform the planning of Ae. aegypti elimination in the region, and offer insight into the effective combinations of conventional and novel control tools, particularly for vulnerable mosquito populations at range margins.

SignificanceAfter decades of range stagnation in Australia, the Aedes aegypti mosquito is expanding southward, approaching the most-densely-populated areas of Queensland. Using population genomics and simulation modeling of elimination campaigns, we show that Australias southernmost populations of this disease vector are genetically isolated and could be eliminated with moderate releases of incompatible Wolbachia-infected males if major larval breeding sites (non-compliant rainwater tanks) are removed first. The risk of Wolbachia establishment for this approach is low, and so is the risk of quick mosquito re-invasion. Our conservative estimates of intervention outcomes inform the planning of Ae. aegypti elimination in the region, and offer new insight into the benefits of combining conventional and novel control tools, particularly for mosquito populations at range margins.

sentences: On the prediction of human intelligence from neuroimaging: A systematic review of methods and reporting Human intelligence is one of the main objects of study in cognitive neuroscience. Reviews and meta-analyses have proved to be fundamental to establish and cement neuroscientific theories on intelligence. The prediction of intelligence using in vivo neuroimaging data and machine learning has become a widely accepted and replicated result. Here, we present a systematic review of this growing area of research, based on studies that employ structural, functional, and/or diffusion MRI to predict human intelligence in cognitively normal subjects using machine-learning. We performed a systematic assessment of methodological and reporting quality, using the PROBAST and TRIPOD assessment forms and 30 studies identified through a systematic search. We observed that fMRI is the most employed modality, resting-state functional connectivity (RSFC) is the most studied predictor, and the Human Connectome Project is the most employed dataset. A meta-analysis revealed a significant difference between the performance obtained in the prediction of general and fluid intelligence from fMRI data, confirming that the quality of measurement moderates this association. The expected performance of studies predicting general intelligence from fMRI was estimated to be r = 0.42 (CI95% = [0.35, 0.50]) while for studies predicting fluid intelligence obtained from a single test, expected performance was estimated as r = 0.15 (CI95% = [0.13, 0.17]). We further enumerate some virtues and pitfalls we identified in the methods for the assessment of intelligence and machine learning. The lack of treatment of confounder variables, including kinship, and small sample sizes were two common occurrences in the literature which increased risk of bias. Reporting quality was fair across studies, although reporting of results and discussion could be vastly improved. We conclude that the current literature on the prediction of intelligence from neuroimaging data is reaching maturity. Performance has been reliably demonstrated, although extending findings to new populations is imperative. Current results could be used by future works to foment new theories on the biological basis of intelligence differences.

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Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSC). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Further, pharmacological inhibition of the FOS/JUN AP1 complex binding to its targets, decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its reexpression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 reexpression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; further, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.

Significance statementProliferation and maintenance of NSC are essential during normal brain development, and, postnatally, for the maintenance of hippocampal function and memory until advanced age. Little is known about the molecular mechanisms that maintain the critical aspects of NSC biology (quiescence and proliferation) in postnatal age. Our work provides a methodology, transduction of genes deregulated following Sox2 deletion, that allows to test many candidate genes for their ability to sustain NSC proliferation. In principle, this may have interesting implications for identifying targets for pharmacological manipulations.

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Modeling drug combination effects via latent tensor reconstruction AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSMotivationC_ST_ABSCombination therapies have emerged as a powerful treatment modality to overcome drug resistance and improve treatment efficacy. However, the number of possible drug combinations increases very rapidly with the number of individual drugs in consideration which makes the comprehensive experimental screening infeasible in practice. Machine learning models offer time- and cost-efficient means to aid this process by prioritising the most effective drug combinations for further pre-clinical and clinical validation. However, the complexity of the underlying interaction patterns across multiple drug doses and in different cellular contexts poses challenges to the predictive modelling of drug combination effects.

ResultsWe introduce comboLTR, highly time-efficient method for learning complex, nonlinear target functions for describing the responses of therapeutic agent combinations in various doses and cancer cell-contexts. The method is based on a polynomial regression via powerful latent tensor reconstruction. It uses a combination of recommender system-style features indexing the data tensor of response values in different contexts, and chemical and multi-omics features as inputs. We demonstrate that comboLTR outperforms state-of-the-art methods in terms of predictive performance and running time, and produces highly accurate results even in the challenging and practical inference scenario where full dose-response matrices are predicted for completely new drug combinations with no available combination and monotherapy response measurements in any training cell line.

Availability and implementationcomboLTR code is available at https://github.com/aalto-ics-kepaco/ComboLTR

Contacttianduanyi.wang@aalto.fi; juho.rousu@aalto.fi

sentences: Activation of the adhesion GPCR GPR133 (ADGRD1) by antibodies targeting the N-terminus We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein-coupled receptor (aGPCR) whose canonical signaling raises cytosolic cAMP, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We showed that dissociation of autoproteolytically generated N-terminal and C-terminal fragments (NTF and CTF) of GPR133 at the plasma membrane promotes receptor activation and increases signaling. Toward developing biologics modulating GPR133 function, we tested antibodies against the N-terminus of GPR133 for effects on receptor signaling. Treatment of HEK293T cells overexpressing GPR133 with such antibodies increased cAMP levels in a concentration-dependent manner. Analysis of supernatants following antibody treatment revealed complexes of the antibodies with the autoproteolytically cleaved NTF of GPR133. Cells expressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. The antibody-mediated stimulation of wild-type GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that NTF-CTF dissociation promotes receptor activation and signaling.

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A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469. Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

Summary statementA mouse model of Brittle Cornea Syndrome was created to elucidate molecular mechanisms underlying pathology of this rare connective tissue disorder in which extremely thin corneas rupture, causing irreversible blindness.

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Workflows for rapid functional annotation of diverse arthropod genomes Genome sequencing of a diverse array of arthropod genomes is already underway and these genomes will be used to study human health, agriculture, biodiversity and ecology. These new genomes are intended to serve as community resources and provide the foundational information that is required to apply  omics technologies to a more diverse set of species. However, biologists require genome annotation to use these genomes and derive a better understanding of complex biological systems. Genome annotation incorporates two related but distinct processes: demarcating genes and other elements present in genome sequences (structural annotation); and associating function with genetic elements (functional annotation). While there are well established and freely available workflows for structural annotation of gene identification in newly assembled genomes, workflows for providing the functional annotation required to support functional genomics studies are less well understood. Genome-scale functional annotation is required for functional modeling (enrichment, networks, etc.) and a first-pass genome-wide functional annotation effort can rapidly identify under-represented gene sets for focused community annotation efforts. We present an open source, open access and containerized pipeline for genome-scale functional annotation of insect proteomes and apply it to a diverse range of arthropod species. We show that the performance of the predictions is consistent across a set of arthropod genomes with varying assembly and annotation quality.Complete instructions for running each component of the functional annotation pipeline on the command line, a high performance computing cluster and the CyVerse Discovery Environment can be found at the readthedocs site (https://agbase-docs.readthedocs.io/en/latest/agbase/workflow.html).

Simple summaryGenomic technologies are accumulating information about genes at a faster rate than ever before, and sequencing initiatives like the Earth Biogenome Project, i5k and Ag100Pest are expected to increase this rate of acquisition. However, if genomic sequencing is to be used for improvement of human health, agriculture and our understanding of biological systems, it is necessary to identify genes and understand how they contribute to biological outcomes. While there are several well-established workflows for assembling genomic sequences and identifying genes, understanding gene function is essential to create actionable knowledge. Moreover this functional annotation process must be easily accessible and provide information at a genomic scale to keep up with new sequence data. We report a well defined workflow for rapid functional annotation of whole proteomes to produce Gene Ontology and pathways information. We test this workflow on a diverse set of arthropod genomes and compare it to common arthropod reference genomes. The workflow we described is freely and publicly available via a web interface on CyVerse or as biocontainers that can be deployed scalably on local computing systems.

sentences: Interferon-γ Increases Sensitivity to Chemotherapy and Provides Immunotherapy Targets in Models of Metastatic Castration-Resistant Prostate Cancer Interferon-{gamma} (IFN{gamma}) is a cytokine with limited evidence of benefit in cancer clinical trials to date. However, it could potentially play a role in potentiating anti-tumor immunity in the immunologically "cold" metastatic castration-resistant prostate cancer (mCRPC) by inducing antigen presentation pathways and concurrently providing targets for immune checkpoint blockade therapy. Moreover, it could additionally increase sensitivity to chemotherapy based on its pleiotropic effects on cell phenotype. Here, we show that IFN{gamma} treatment induced expression of major histocompatibility class-I (MHC-I) genes and PD-L1 in prostate cancer cells in vitro. Furthermore, IFN{gamma} treatment led to a decrease in E-cadherin expression with a consequent increase in sensitivity to chemotherapy in vitro. In an in vivo murine tumor model of spontaneous metastatic prostate cancer, IFN{gamma} systemic pretreatment upregulated the expression of HLA-A and decreased E-cadherin expression in the primary tumor, and more importantly in the metastatic site led to increased apoptosis and limited micrometastases in combination with paclitaxel treatment compared to diffuse metastatic disease in control and monotherapy treatment groups. These findings suggest that IFN{gamma} may be useful in combinatorial regimens to induce sensitivity to immunotherapy and chemotherapy in hepatic metastases of mCRPC.

sentences: KLF5-regulated extracellular matrix remodeling secures biliary epithelial tissue integrity against cholestatic liver injury Abstract / Introductory paragraphTubular epithelial tissues in the body play fundamental roles as infrastructure constituting conduits to transport various types of biological fluids, for which contiguous and integrated epithelial tissue structures should be maintained continuously and even under stressed conditions. Compared to tissue morphological processes that take place during ontogeny, the mechanisms whereby tubular epithelial tissues maintain their structural integrity in adulthood remains largely unclear. Here, we show that the transcription factor Klf5 is crucial for maintaining the biliary epithelial integrity in tissue remodeling processes induced under cholestatic injury conditions in the adult liver. Loss of Klf5 in the biliary epithelia led to tissue collapse in vivo in injured mouse livers, as well as in vitro in bile ductular organoids in a tissue-autonomous manner and independent of cell proliferation. Klf5 regulated cell junction organization and cell adhesion, along with extracellular matrix remodeling around the expanding biliary epithelia through deposition of Lamb3-containing laminin complexes. Targeting the Lamb3 expression in biliary epithelia in mice recapitulated the tissue collapse phenotype. Together, our results highlight a novel mechanism whereby the epithelial tissue maintains its integrity while undergoing unstable structural transformation.

sentences: A comparative baseline of coral disease across the central Red Sea The Red Sea is a unique environment for corals with a strong environmental gradient characterized by temperature extremes and high salinities, but minimal terrestrial runoff or riverine input and their associated pollution. Disease surveys were conducted along 22 reefs in the central Red Sea along the Saudi Arabian coast in October 2015, which coincided with a bleaching event. Our objectives were to 1) document types, prevalence, and distribution of coral diseases in a region with minimal terrestrial input, 2) compare regional differences in diseases and bleaching along a latitudinal gradient of environmental conditions, and 3) use histopathology to characterize disease lesions at the cellular level. Coral reefs of the central Red Sea had a widespread but a surprisingly low prevalence of disease (<0.5%), based on the examination of >75,750 colonies. Twenty diseases were recorded affecting 16 coral taxa and included black band disease, white syndromes, endolithic hypermycosis, skeletal eroding band, growth anomalies and focal bleached patches. The three most common diseases were Acropora white syndrome (59.1% of the survey sites), Porites growth anomalies (40.9%), and Porites white syndrome (31.8%). Over half of the coral genera within transects had lesions and corals from the genera Acropora, Millepora and Lobophyllia were the most commonly affected. Cell-associated microbial aggregates were found in four coral genera resembling patterns found in the Indo-Pacific. Differences in disease prevalence, coral cover, amount of heat stress as measured by degree heating weeks (DHW) and extent of bleaching was evident among sites. Disease prevalence was not explained by coral cover or DHW, and a negative relationship between coral bleaching and disease prevalence was found. The northern-most sites off the coast of Yanbu had the highest average DHW values but absence of bleaching and the highest average disease prevalence was recorded. Our study provides a foundation and baseline data for coral disease prevalence in the Red Sea, which is projected to increase as a consequence of increased frequency and severity of ocean warming.

sentences: Population structure analysis and laboratory monitoring of Shigella with a standardised core-genome multilocus sequence typing scheme: a validation study The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. We studied over 4,000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes were grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognised the natural groupings for Shigella. By contrast, the serotyping scheme was affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will enhance the laboratory surveillance of Shigella infections.

sentences: Comparative reproductive biology of deep-sea ophiuroids inhabiting polymetallic-nodule fields in the Clarion-Clipperton Fracture Zone Deep-sea mining in the Pacific Clarion-Clipperton Fracture Zone (CCZ), a low-energy sedimentary habitat with polymetallic nodules, is expected to have considerable and long-lasting environmental impact. The CCZ hosts extraordinarily high species diversity across representatives from all Domains of Life. Data on species biology and ecology remain scarce, however. The current study describes the reproductive biology of Ophiosphalma glabrum (Lutken & Mortensen, 1899) (Ophiosphalmidae) and Ophiacantha cosmica (Lyman, 1878) (Ophiacanthidae), two ophiuroids frequently found in the CCZ. Specimens collected in Spring 2015 and 2019 in four contract areas were examined morphologically and histologically. Size-class frequencies (disc diameter and oocytes feret diameters), sex ratios, gametogenic status, putative reproductive mode and a simple proxy for fecundity are presented. Habitat use differs in each. While Ophiosphalma glabrum is epibenthic, occurring as single individuals, Ophiacantha cosmica often form size-stratified groups living on stalked sponges, suggesting gregarious settlement or retention of offspring (though no brooding individuals were found). Further molecular analyses are needed to establish whether O. cosmica groups are familial. In Ophiosphalma glabrum, for which sample sizes were larger, sex ratios approximated a 1:1 ratio with no size-structuring. In both species, individuals were at various stages of gametogenic maturity but no ripe females were identified. Based on this, O. glabrum is most probably gonochoric. Reproductive mode remains inconclusive for Ophiacantha cosmica. Both species are presumptively lecithotrophic, with vitellogenic-oocyte feret diameters exceeding 250 {micro}m. Oocyte feret diameters at times exceeded 400 {micro}m in Ophiosphalma glabrum, indicating substantial yolk reserves. Estimates of instantaneous fecundity (vitellogenic specimens of O. glabrum only) were confounded by interindividual variability in gonad characteristics. The well-furnished lecithotrophic larvae of O. glabrum would be capable of dispersing even under food-impoverished conditions. The current study examines ophiuroid reproductive biology over multiple localities in the CCZ concurrently for the first time, at sites characterised by differing productivity regimes. The reproductive biology of each species is thus discussed with reference to past evolutionary (habitat stability), contemporary (food supply) and future environmental drivers (potential impacts of deep-sea mining).

sentences: Is male dimorphism under sexual selection in humans? A meta-analysis Humans are sexually dimorphic: men and women differ in body build and composition, craniofacial structure, and voice pitch, likely mediated in part by developmental testosterone. Sexual selection hypotheses posit that, ancestrally, more  masculine men may have acquired more mates and/or sired more viable offspring. Thus far, however, evidence for either association is unclear. Here, we meta-analyze the relationships between six masculine traits and mating/reproductive outcomes (96 studies, 474 effects, N = 177,044). Voice pitch, height, and testosterone all predicted mating; however, strength/muscularity was the strongest and only consistent predictor of both mating and reproduction. Facial masculinity and digit ratios did not significantly predict either. There was no clear evidence for any effects of masculinity on offspring viability. Our findings support arguments that strength/muscularity can be considered sexually selected in humans, but raise concerns over other forms of masculinity and highlight the need to increase tests of evolutionary hypotheses outside of industrialized populations.

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Processing Stage Flexibility of the SNARC effect: Task Relevance or Magnitude Relevance? Previous studies have shown that the processing stage of the spatial-numerical association of response codes (SNARC) effect was flexible. Two recent studies by Nan et al. (2021) and Yan et al. (2021) used the same experimental paradigm to check whether the SNARC effect occurred in the semantic-representation stage but reached contradictory conclusions, showing that the SNARC effect was influenced by a magnitude Stroop effect in a magnitude comparison task but not by a parity Stroop effect in a parity judgment task. The two studies had two distinct operational factors: the task type (magnitude comparison task or parity judgment task, with the numerical magnitude information task-relevant or task-irrelevant) and the semantic representation stage-related interference information (magnitude or parity Stroop effect, with the interference information magnitude-relevant or magnitude-irrelevant). To determine which factor influenced the SNARC effect, in the present study, the Stroop effect was switched in the two tasks based on the previous studies. The findings of four experiments consistently showed that the SNARC effect was not influenced by the parity Stroop effect in the magnitude comparison task but was influenced by the magnitude Stroop effect in the parity judgment task. Combined with the results of Nan et al. (2021) and Yan et al. (2021), the findings indicated that regardless of the task type or the task-relevance of numerical magnitude information, magnitude-relevant interference information was the primary factor to affect the SNARC effect. Furthermore, a two-stage processing model that explained the observed flexibility of the SNARC effect was proposed and discussed.

Public Significance StatementPrevious studies have shown that the spatial-numerical association of response codes (SNARC) effect is flexible in the direction and processing stage. The task type and interference information might be two influential factors for the flexibility of the SNARC effect. The present study reported that magnitude-relevant interference information, regardless of task type, was a crucial role to affect the SNARC effect. Moreover, a two-stage processing model was proposed to reveal the processing pathway of the SNARC effect and provided a possible explanation for the longstanding debate about the processing stage of the SNARC effect.

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Deep Learning Achieves Neuroradiologist-Level Performance in Detecting Hydrocephalus Requiring Treatment PurposeIn large clinical centers a small subset of patients present with hydrocephalus that requires surgical treatment. We aimed to develop a screening tool to detect such cases from the head MRI with performance comparable to neuroradiologists.

MethodsWe leveraged 496 clinical MRI exams collected retrospectively at a single clinical site from patients referred for any reason. This diagnostic dataset was enriched to have 259 hydrocephalus cases. A 3D convolutional neural network was trained on 16 manually segmented exams (ten hydrocephalus) and subsequently used to automatically segment the remaining 480 exams and extract volumetric anatomical features. A linear classifier of these features was trained on 240 exams to detect cases of hydrocephalus that required treatment with surgical intervention. Performance was compared to four neuroradiologists on the remaining 240 exams. Performance was also evaluated on a separate screening dataset of 451 exams collected from a routine clinical population to predict the consensus reading from four neuroradiologists using images alone. The pipeline was also tested on an external dataset of 31 exams from a 2nd clinical site.

ResultsThe most discriminant features were the Magnetic Resonance Hydrocephalic Index (MRHI), ventricle volume, and the ratio between ventricle and brain volume. At matching sensitivity, the specificity of the machine and the neuroradiologists did not show significant differences for detection of hydrocephalus on either dataset (proportions test, p > 0.05). ROC performance compared favorably with the state-of-the-art (AUC 0.90-0.96), and replicated in the external validation.

ConclusionHydrocephalus cases requiring treatment can be detected automatically from MRI in a heterogeneous patient population based on quantitative characterization of brain anatomy with performance comparable to that of neuroradiologists.

sentences: FOD-Net: A Deep Learning Method for Fiber Orientation Distribution Angular Super Resolution Mapping the human connectome using fiber-tracking permits the study of brain connectivity and yields new insights into neuroscience. However, reliable connectome reconstruction using diffusion magnetic resonance imaging (dMRI) data acquired by widely available clinical protocols remains challenging, thus limiting the connectome / tractography clinical applications. Here we develop fiber orientation distribution (FOD) network (FOD-Net), a deep-learning-based framework for FOD angular super-resolution. Our method enhances the angular resolution of FOD images computed from common clinical-quality dMRI data, to obtain FODs with quality comparable to those produced from advanced research scanners. Super-resolved FOD images enable superior tractography and structural connectome reconstruction from clinical protocols. The method was trained and tested with high-quality data from the Human Connectome Project (HCP) and further validated with a local clinical 3.0T scanner. Using this method, we improve the angular resolution of FOD images acquired with typical single-shell low-angular-resolution dMRI data (e.g., 32 directions, b = 1000 s/mm2) to approximate the quality of FODs derived from time-consuming, multi-shell high-angular-resolution dMRI research protocols. We also demonstrate tractography improvement, removing spurious connections and bridging missing connections. We further demonstrate that connectomes reconstructed by super-resolved FODs achieve comparable results to those obtained with more advanced dMRI acquisition protocols, on both HCP and clinical 3.0T data. Advances in deep-learning approaches used in FOD-Net facilitate the generation of high quality tractography / connectome analysis from existing clinical MRI environments. Our code is freely available at https://github.com/ruizengalways/FOD-Net.

sentences: Divergent functional and conformational outcomes in an ion channel-peptide interaction Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

sentences: Global organization of neuronal activity only requires unstructured local connectivity Modern electrophysiological recordings simultaneously capture single-unit spiking activities of hundreds of neurons spread across large cortical distances. Yet this massively parallel activity is often confined to relatively low-dimensional manifolds. This implies strong coordination also among neurons that are most likely not even connected. Here, we combine in vivo recordings with network models and theory to characterize the nature of mesoscopic coordination patterns in macaque motor cortex and to expose their origin: We find that heterogeneity in local connectivity supports network states with complex long-range cooperation between neurons that arises from multi-synaptic, short-range connections. Our theory explains the experimentally observed spatial organization of covariances in resting state recordings as well as the behaviorally related modulation of covariance patterns during a reach-to-grasp task. The ubiquity of heterogeneity in local cortical circuits suggests that the brain uses the described mechanism to flexibly adapt neuronal coordination to momentary demands.

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Plasmacytoid dendritic cells produce type I interferon and reduce viral replication in airway epithelial cells after SARS-CoV-2 infection Infection with SARS-CoV-2 has caused a pandemic of unprecedented dimensions. SARS-CoV-2 infects airway and lung cells causing viral pneumonia. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with inborn errors of type I IFN response or auto-antibodies against IFN-. Plasmacytoid dendritic cells (pDCs) are a unique immune cell population specialized in recognizing and controlling viral infections through the production of high concentrations of type I IFN. In this study, we isolated pDCs from healthy donors and showed that pDCs are able to recognize SARS-CoV-2 and rapidly produce large amounts of type I IFN. Sensing of SARS-CoV-2 by pDCs was independent of viral replication since pDCs were also able to recognize UV-inactivated SARS-CoV-2 and produce type I IFN. Transcriptional profiling of SARS-CoV-2 and UV-SARS-CoV-2 stimulated pDCs also showed a rapid type I and III IFN response as well as induction of several chemokines, and the induction of apoptosis in pDCs. Moreover, we modeled SARS-CoV-2 infection in the lung using primary human airway epithelial cells (pHAEs) and showed that co-culture of pDCs with SARS-CoV-2 infected pHAEs induces an antiviral response and upregulation of antigen presentation in pHAE cells. Importantly, the presence of pDCs in the co-culture results in control of SARS-CoV-2 replication in pHAEs. Our study identifies pDCs as one of the key cells that can recognize SARS-CoV-2 infection, produce type I and III IFN and control viral replication in infected cells.

ImportanceType I interferons (IFNs) are a major part of the innate immune defense against viral infections. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with defects in the type I IFN response. Interestingly, many cells are not able to produce type I IFN after being infected with SARS-CoV-2 and cannot control viral infection. In this study we show that plasmacytoid dendritic cells are able to recognize SARS-CoV-2 and produce type I IFN, and that pDCs are able to help control viral infection in SARS-CoV-2 infected airway epithelial cells.

sentences: AlphaFold-aware prediction of protein-DNA binding sites using graph transformer Protein-DNA interactions play crucial roles in the biological systems, and identifying protein-DNA binding sites is the first step for mechanistic understanding of various biological activities (such as transcription and repair) and designing novel drugs. How to accurately identify DNA-binding residues from only protein sequence remains a challenging task. Currently, most existing sequence-based methods only consider contextual features of the sequential neighbors, which are limited to capture spatial information. Based on the recent breakthrough in protein structure prediction by AlphaFold2, we propose an accurate predictor, GraphSite, for identifying DNA-binding residues based on the structural models predicted by AlphaFold2. Here, we convert the binding site prediction problem into a graph node classification task and employ a transformer-based variant model to take the protein structural information into account. By leveraging predicted protein structures and graph transformer, GraphSite substantially improves over the latest sequence-based and structure-based methods. The algorithm is further confirmed on the independent test set of 181 proteins, where GraphSite surpasses the state-of-the-art structure-based method by 16.4% in AUPR and 11.2% in MCC, respectively. We provide the datasets, the predicted structures, and the source codes along with the pre-trained models of GraphSite at https://github.com/biomed-AI/GraphSite. The GraphSite web server is freely available at https://biomed.nscc-gz.cn/apps/GraphSite.

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CISH targeting in NK cells activates natural cytotoxicity receptor signaling and reduce cell exhaustion to unsilence primary anti-tumor response Cytokine inducible SH2-containing protein (CISH) is a natural killer (NK) cell negative regulator of cytokine signaling pathway. To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). We detected no developmental or homeostatic difference in NK cells.

However, global gene expression of Cishfl/flNcr1Ki/+ NK cells compared to Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways. Indeed, CISH protein expression level increases upon NCR triggering. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis. Remarkably, we found that Cishfl/flNcr1Ki/+ mice were also more resistant to primary breast cancer growth in addition to superior control of spontaneous tumor metastasis. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, we argue that specifically enhancing NK cell function is sufficient to boost anti-tumor response to both primary and secondary tumor models. Using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. According to the results in our mouse model, CISH deletion favors NCR signaling and anti-tumor functions in human NK cells. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

sentences: Are microbes growing on flowers evil? Effects of old flower microbes on fruit set in a wild ginger with one-day flowers, Alpinia japonica (Zingiberaceae) Flowers are colonized and inhabited by diverse microbes. Plants rapidly replace flowers with short lifespan, and old flowers senesce. This may contribute to avoiding adverse effects of the microbes. In this study, we investigate if the flower microbial community on old flowers impedes fruit and seed production in a wild ginger with one-day flowers. We inoculated newly opened flowers with old flower microbes, and monitored the effects on fruit and seed set. We also assessed prokaryotic communities on the flowers using amplicon sequencing. We found six bacterial amplicon sequence variants (ASVs) whose proportions were increased on the inoculated flowers. These ASVs were also found on flower buds and flowers that were bagged by net or paper during anthesis, suggesting that they had been present in small numbers prior to flowering. Fruit set was negatively associated with the proportions of these ASVs, while seed set was not. The results suggest that old flowers harbor microbial communities different from those at anthesis, and that the microbes abundant on old flowers negatively affect plant reproduction. Though the short lifespan of flowers has gotten little attention, it might be an essential defense mechanism to cope with antagonistic microbes that rapidly proliferate on the flowers.

sentences: Whokaryote: distinguishing eukaryotic and prokaryotic contigs in metagenomes based on gene structure Metagenomics has become a prominent technology to study the functional potential of all organisms in a microbial community. Most studies focus on the bacterial content of these communities, while ignoring eukaryotic microbes. Indeed, many metagenomics analysis pipelines silently assume that all contigs in a metagenome are prokaryotic. However, because of marked differences in gene structure, prokaryotic gene prediction tools fail to accurately predict eukaryotic genes. Here, we developed a classifier that distinguishes eukaryotic from prokaryotic contigs based on foundational differences between these taxa in gene structure. We first developed a random forest classifier that uses intergenic distance, gene density and gene length as the most important features. We show that, with an estimated accuracy of 97%, this classifier with principled features grounded in biology can perform almost as well as the classifiers EukRep and Tiara, which use k-mer frequencies as features. By re-training our classifier with Tiara predictions as additional feature, weaknesses of both types of classifiers are compensated; the result is an enhanced classifier that outperforms all individual classifiers, with an F1-score of 1.00 on precision, recall and accuracy for both eukaryotes and prokaryotes, while still being fast. In a reanalysis of metagenome data from a disease-suppressive plant endosphere microbial community, we show how using Whokaryote to select contigs for eukaryotic gene prediction facilitates the discovery of several biosynthetic gene clusters that were missed in the original study. Our enhanced classifier, which we call  Whokaryote, is wrapped in an easily installable package and is freely available from https://git.wageningenur.nl/lotte.pronk/whokaryote.

sentences: An ensemble deep learning framework to refine large deletions in linked-reads The detection of structural variants (SVs) remains challenging due to inconsistencies in detected breakpoints and biological complexity of some rearrangements. Linked-reads have demonstrated their superiority in diploid genome assembly and SV detection. Recently developed tools Aquila and Aquila_stLFR use a reference sequence and linked-reads to generate a high quality diploid genome assembly, using which they then detect and phase personal genetic variations. However, they both produce a substantial proportion of false positive deletion SV calls. To take full advantage of linked-reads, an effective downstream filtering and refinement framework is needed pressingly. In this work, we propose AquilaDeepFilter to filter large deletion SVs from Aquila and Aquila_stLFR. AquilaDeepFilter relies on a deep learning ensemble approach by integrating six state-of-the-art CNN backbones. The filtering of deletion SVs is formulated as a binary classification task on image data that are generated through the extraction of multiple alignment signals, including read depth, split reads and discordant read pairs. Three linked-reads libraries sequenced from the well-studied sample NA24385 and the gold standard of GiaB benchmark were used to perform thorough experiments on our proposed method. The results demonstrated that AquilaDeepFilter could increase the precision rate of Aquila while the recall rate of Aquila decreased only slightly, and the overall F1 improved by 20%. Furthermore, AquilaDeepFilter outperformed another deep learning based method for SV filtering, DeepSVFilter. Even though we designed AquilaDeepFilter for linked-reads, the framework could also be used to improve SV detection on short reads.

sentences: Intracrine Vascular Endothelial Growth Factor Maintains Hippocampal Neural Stem Cell Quiescence In the dentate gyrus (DG) of the adult mouse hippocampus, neural stem cells (NSCs) balance self-renewal and differentiation to produce neurons that support hippocampal function. Vascular endothelial growth factor (VEGF) is a well-known supporting factor for adult neurogenesis, but conflicting studies have left it uncertain how VEGF signals to NSCs. Here, we identified a VEGF-VEGFR2 intracrine signaling mechanism within adult DG NSCs that prevents their exhaustion. We show both in vitro and in vivo that NSC-VEGF loss caused cell-autonomous exhaustion of adult DG NSCs. In contrast, extracellular VEGF was neither necessary nor sufficient to maintain NSC quiescence or to stimulate VEGFR2 signaling, most likely due to sheddase-mediated cleavage of extracellular VEGFR2 ligand binding domains. Our findings support an exclusively intracellular mechanism for VEGF signaling in adult DG NSCs, thereby providing resolution to previously conflicting studies and suggesting cellular source can dictate the functional impact of soluble ligands in DG NSCs.

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Stress alters the activity of Leydig cells dependently on the diurnal time BackgroundThe increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored.

ObjectiveThe purpose of the study was to analyze the stress-response of the testosterone-producing Leydig cells depending on the stressful events time.

Materials and methodsThe study was designed to follow the effects of 3-hour immobilization (IMO) applied at different periods during the day. The IMO performed once (1xIMO) or repeated in 10 consecutive days (10xIMO). Principal-component-analysis (PCA) followed the expression study of the clock and steroidogenic-related genes in Leydig cells.

ResultsBoth types of IMO in all investigated periods increased corticosterone and decreased testosterone blood level. Transcriptional analysis revealed different sensitivity to IMO events depending on the circadian time. The majority of steroidogenesis-related genes (Lhcgr, Cyp11a1, Cyp17a1, Hsd3b1/2) were down-regulated in the inactive but unchanged or even up-regulated in the active phase of the day. Both types of IMO potentiated the expression of clock elements Bmal1/BMAL1, Per1/PER1 regardless of the days stage and reduced Reverba in the inactive phase. The PCA confirmed a major shift, for both IMO-types, in the transcription of steroidogenesis and clock genes across the inactive/active phase. Further, the diurnal pattern of the glucocorticoid receptor (Nr3c1/GR) expression in Leydig cells was increased in the inactive phase due to 10xIMO. The observed time-dependent IMO-response of the Leydig cells correlated with different GR engagements.

DiscussionStress- and the circadian-system coordinatively shape Leydig cells physiology, assuming diverse GR engagement as a possible factor in mediating the diurnal effect of stress.

ConclusionThe Leydig cells stress-response depends on the time of the stressful situation, emphasizing the importance of circadian activity in supporting androgen homeostasis and male fertility.

sentences: Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

sentences: Complement facilitates developmental microglial pruning of astrocyte and vascular networks Microglia, the resident immune cell of the central nervous system (CNS), play a pivotal role in facilitating neurovascular development through mechanisms that are not fully understood. This current work resolves a previously unknown role for microglia in facilitating the developmental pruning of the astrocytic template resulting in a spatially organized retinal superficial vascular bed. Mechanistically, our study identified that local microglial expression of complement (C)3 and C3aR is necessary for the regulation of astrocyte patterning and vascular growth during retinal development. Ablation of retinal microglia and deficiency of C3 or C3aR reduced developmental pruning and clearance of astrocytic bodies leading to increased astrocyte density, altered vascular patterning, and elevated gene expression levels of extracellular matrix proteins involved in vascular growth during retinal development. These data demonstrate that C3/C3aR signaling is an important checkpoint required for the finetuning of vascular density during neuroretinal development.

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

sentences: Insights into the role of lipoteichoic acids in Bacillus subtilis- a new function for MprF In Bacillus subtilis, the cell is protected from the environment by a cell envelope, which comprises of layers of peptidoglycan that maintain the cell shape and anionic teichoic acids polymers whose biological function remains unclear. In B. subtilis, loss of all Class A Penicillin-Binding Proteins (aPBPs) which function in peptidoglycan synthesis is conditionally lethal. Here we show that this lethality is associated with an alteration of the lipoteichoic acids (LTA) and the accumulation of the major autolysin LytE in the cell wall. We provide the first evidence that the length and abundance of LTA acts to regulate the cellular level of LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF which acts to modulate LTA biosynthesis in B. subtilis and in the pathogen Staphylococcus aureus. This finding has implications for our understanding of antimicrobial peptide resistance (particularly daptomycin) in clinically relevant bacteria and MprF-associated virulence in pathogens, such as methicillin resistant S. aureus.

sentences: Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases Mechanically dependent processes are essential in cancer metastases. However, reliable mechanical characterisation of metastatic cancer remains challenging whilst maintaining the tissue complexity and an intact sample. Using atomic force microscopy, we quantified the micro-mechanical properties of relatively intact metastatic breast tumours and their surrounding bone microenvironment isolated from mice, and compared with other breast cancer models both ex vivo and in vitro. A unique mechanical distribution of extremely low elastic modulus and viscosity was identified on metastatic tumours, which were significantly more compliant than both 2D in vitro cultured cancer cells and subcutaneous tumour explants. The presence of mechanically distinct metastatic tumour did not result in alterations of the mechanical properties of the surrounding microenvironment at meso-scale distances (> 200 {micro}m). These findings demonstrate the utility of atomic force microscopy in studies of complex tissues and provide new insights into the mechanical properties of cancer metastases in bone.

sentences: Functional plasticity coupled with structural predispositions in auditory cortex shape successful music category learning Categorizing sounds into meaningful groups helps listeners more efficiently process the auditory scene and is a foundational skill for speech perception and language development. Yet, how auditory categories develop in the brain through learning, particularly for nonspeech sounds, is not well understood. Here, we asked musically naive listeners to complete a brief ([~]20 min) training session where they learned to identify sounds from a nonspeech continuum (minor-major 3rd musical intervals). We used multichannel EEG to track behaviorally relevant neuroplastic changes in the auditory event-related potentials (ERPs) pre- to post-training. To rule out mere exposure-induced changes, neural effects were evaluated against a control group of 14 nonmusicians who did not undergo training. We also compared individual categorization performance with structural volumetrics of bilateral primary auditory cortex (PAC) from MRI to evaluate neuroanatomical substrates of learning. Behavioral performance revealed steeper (i.e., more categorical) identification functions in the posttest that correlated with better training accuracy. At the neural level, improvement in learners behavioral identification was characterized by smaller P2 amplitudes at posttest, particularly over right hemisphere. Critically, learning-related changes in the ERPs were not observed in control listeners, ruling out mere exposure effects. Learners also showed smaller and thinner PAC bilaterally, indicating superior categorization was associated with structural differences in primary auditory brain regions. Collectively, our data suggest successful auditory categorical learning of nonspeech sounds is characterized by short-term functional changes (i.e., greater post-training efficiency) in sensory coding processes superimposed on preexisting structural differences in bilateral auditory cortex.

sentences: KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.

sentences: Extension of bacterial rDNA sequencing to concurrent epigenetic analysis and its application to 16S meta-epigenetics Although polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA region has been used in a wide range of scientific fields, it does not provide DNA methylation information. We describe a simple add-on method to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical samples or flora. Single-stranded bacterial DNA after bisulfite conversion was preferentially amplified with multiple displacement amplification (MDA) at pH neutral, and the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing. 16S rDNA bisulfite sequencing can provide clinically important bacterial DNA methylation status concurrently with intact 16S rDNA sequence information. We used this approach to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii. Next, we analyzed bacterial flora from clinical specimens of small amount and identified different methylation motifs among Enterococcus faecalis strains. The method developed here, referred to as "add-on" to the conventional 16S rDNA analysis, is the most clinically used bacterial identification genetic test, which provides additional information that could not be obtained with the conventional method. Since the relationship between drug resistance in bacteria and DNA methylation status has been reported, bacterial epigenetic information would be useful in clinical testing as well. Our analysis suggests that M. MmnI has a promotive effect on erythromycin resistance. 16S rDNA bisulfite PCR and sequencing coupled with MDA at pH neutral is a useful add-on tool for analyzing 16S meta-epigenetics.

sentences: Environmental genomics points to non-diazotrophic Trichodesmium species abundant and widespread in the open ocean Filamentous and colony-forming cells within the cyanobacterial genus Trichodesmium might account for nearly half of nitrogen fixation in the sunlit ocean, a critical mechanism that sustains planktons primary productivity at large-scale. Here, we report the genome-resolved metagenomic characterization of two newly identified marine species we tentatively named  Ca. Trichodesmium miru and  Ca. Trichodesmium nobis. Near-complete environmental genomes for those closely related candidate species revealed unexpected functional features including a lack of the entire nitrogen fixation gene apparatus and hydrogen recycling genes concomitant with the enrichment of nitrogen assimilation genes and apparent acquisition of the nirb gene from a non-cyanobacterial lineage. These comparative genomic insights were cross-validated by complementary metagenomic investigations. Our results contrast with the current paradigm that Trichodesmium species are necessarily capable of nitrogen fixation. The black queen hypothesis could explain gene loss linked to nitrogen fixation among Trichodesmium species, possibly triggered by gene acquisitions from the colony epibionts. Critically, the candidate species are not only widespread in the 3-2000 m planktonic size fraction of the surface of the oceans and seas, but might also substantially expand the ecological niche of Trichodesmium, stressing the need to disconnect taxonomic signal for this genus from a microbial communitys ability to fix nitrogen. Especially, differentiating diazotrophic from non-diazotrophic populations when counting Trichodesmium filaments and colonies might help refine our understanding of the marine nitrogen balance. While culture representatives are needed to move beyond metagenomic insights, we are reminded that established links between taxonomic lineages and functional traits might not always hold true.

sentences: Generic Approach For Mathematical Model of Multi-Strain Pandemics Pandemics with multi-strain have become a major concern. We introduce a new model for assessing the connection between multi-strain pandemic and the mortality rate, basic reproduction number, and the maximum of infected individuals. The proposed model provides a general mathematical approach for representing multi-strain pandemics, generalizing for an arbitrary number of strains. We show the proposed model fits well with epidemiological historical data world health over a long period. From a theoretical point of view, we show that the increasing number of strains increases logarithmically the maximum number of infected individuals and the mean mortality rate. Moreover, the mean basic reproduction number is statistically identical to the single, most aggressive strain pandemic for multi-strain pandemics.

sentences: Identification and Validation of Novel Microtubule Suppressors with an Imidazopyridine Scaffold through Structure Based Virtual Screening and Docking. Targeting the colchicine binding site of alpha/beta tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure based pharmacophore approach and docking using three programmes MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-Hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b] pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6 micromolar, inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.

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Identification of the functions of 4-coumarate-CoA ligase/ acyl-CoA synthetase paralogs in potato BackgroundThe 4CL/ ACS protein family is well known for its 4-coumarate-CoA ligase (4CL) enzymes but there are many aspects of this family that are still unclear or generally known. Cytosolic class I and class II 4CL enzymes control the biosynthesis of lignin/ suberin and flavonoids, respectively. Many 4CL homologs have broad substrate permissiveness in vitro and have no clear cut function. However, it has been demonstrated unequivocally that a peroxisomal 4CL-like homolog from Arabidopsis efficiently uses p-coumarate for ubiquinone biosynthesis. Another homolog has been shown to act as a fatty acyl-CoA synthetase and yet another as OPDA-CoA ligase. Hence, despite this knowledge, most homologs remain annotated as "4CL-like" whereas other researches study the ACS protein family.

ResultsWe set out identify the specific functions of 4CL/ ACS homologs, specifically in order to study the 4CL family in Solanum tuberosum. An in depth phylogenetic analysis was done. Using clustering techniques, functional annotation and taxonomic signals, three major clades were depicted. Clade 1 is composed of class I from monocotyledons, class I from dicotyledons and class II canonical 4CL enzymes subclades. Specificity determining positions and 3D structure analysis shows that clade 2 cytosolic 4CL-like enzymes show a rather different binding cleft and presumably use medium- to long-chain fatty acids. Clade 3 is composed of five subclades, four of which have a broad taxonomic contribution and a similar binding cleft as 4CLs whereas a fifth, specific for dicotyledons shows a significantly different binding pocket. The potato 4CL family comprises four class I (St4CL-I(A-D)) and one class II (St4CL-II) members. Transcript levels of St4CLs and of marker genes of the flavonoid (chalcone synthase, CHS) and suberin (feruloyl-CoA transferase, FHT) pathways were determined by qRT-PCR in flesh and skin from Andean varieties. St4CL-IA was barely detected in the skin of some varieties whereas St4CL-IB did not show a clear pattern. St4CL-IC and St4CL-ID could not be detected. St4CL-II expression pattern was similar to CHS. St4CL-IA and St4CL-IB were induced by wounding as did FHT whereas St4CL-II and CHS expression was repressed. Constitutive and wound-induced expression suggests that St4CL-IA and St4CL-IB isoforms are likely involved in soluble and/ or suberin-bound phenolic compounds while St4CL-II appears to be involved in flavonoid biosynthesis.

sentences: Neutral competition within a long-lived population of symmetrically dividing cells shapes the clonal composition of cerebral organoids Cerebral organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progeny arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This exceeds what can be explained by existing stochastic models of corticogenesis, indicating the existence of an additional source of stochasticity. We propose the SAN model in which this additional source of stochasticity is the survival time of a lineage which a long-lived population of symmetrically dividing cells under neutral competition. We demonstrate this model to explain the experimentally observed variability of lineage sizes and derive a formula that captures the quantitative relationship between survival time and lineage size. Finally, we show that our model implies the existence of a regulatory mechanism to keeps the size of the symmetrically dividing cell population constant.

sentences: Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein In previous work, Hsieh and Higgins presented a novel structure of antibodies identified from malaria-exposed individuals, in which the extracellular immunoglobulin (Ig)-like domain of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is presented on the third complementarity determining regions (CDR3) of the Ig heavy chain. Here we develop an Antibody Display technology based on this LAIR1-containing antibody, by grafting proteins of interest (POI) onto the heavy chain CDR3 while retaining the biological properties of the POI. As a proof of principle, we displayed the second extracellular domain of Tetraspanin12 (Tspan12EC2) and the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the heavy chain CDR3. Our data revealed that Antibody Display Tspan12EC2 bound to Norrie Disease Protein (Norrin) and Antibody Display SARS-CoV-2 RBD bound to angiotensin-converting enzyme 2 (ACE2) and neutralizing nanobodies. Collectively, Antibody Display technology offers the general strategy of designing novel antibodies by grafting POI onto the CDR3.

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Revisiting the STRmixlikelihood ratio probability interval coverage considering multiple factors In previously reported work a method for applying a lower bound to the variation induced by the Monte Carlo effect was trialled. This is implemented in the widely used probabilistic genotyping system, STRmix. The approach did not give the desired 99% coverage.

However, the method for assigning the lower bound to the MCMC variability is only one of a number of layers of conservativism applied in a typical application. We tested all but one of these sources of variability collectively and term the result the near global coverage. The near global coverage for all tested samples was greater than 99.5% for inclusionary average LRs of known donors. This suggests that when included in the probability interval method the other layers of conservativism are more than adequate to compensate for the intermittent underperformance of the MCMC variability component. Running for extended MCMC accepts was also shown to result in improved precision.

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Towards end-to-end disease prediction from raw metagenomic data Analysis of the human microbiome using metagenomic sequencing data has demonstrated high ability in discriminating various human diseases. Raw metagenomic sequencing data require multiple complex and computationally heavy bioinformatics steps prior to data analysis. Such data contain millions of short sequences read from the fragmented DNA sequences and are stored as fastq files. Conventional processing pipelines consist in multiple steps including quality control, filtering, alignment of sequences against genomic catalogs (genes, species, taxonomic levels, functional pathways, etc.). These pipelines are complex to use, time consuming and rely on a large number of parameters that often provide variability and impact the estimation of the microbiome elements.

Training Deep Neural Networks directly from raw sequencing data is a promising approach to bypass some of the challenges associated with mainstream bioinformatics pipelines. Most of these methods use the concept of word and sentence embeddings that create a meaningful and numerical representation of DNA sequences, while extracting features and reducing the dimensionality of the data. In this paper we present an end-to-end approach that classifies patients into disease groups directly from raw metagenomic reads: metagenome2vec. This approach is composed of four steps (i) generating a vocabulary of k-mers and learning their numerical embeddings; (ii) learning DNA sequence (read) embeddings; (iii) identifying the genome from which the sequence is most likely to come and (iv) training a multiple instance learning classifier which predicts the phenotype based on the vector representation of the raw data. An attention mechanism is applied in the network so that the model can be interpreted, assigning a weight to the influence of the prediction for each genome. Using two public real-life data-sets as well a simulated one, we demonstrated that this original approach reaches high performance, comparable with the state-of-the-art methods applied directly on processed data though mainstream bioinformatics workflows. These results are encouraging for this proof of concept work. We believe that with further dedication, the DNN models have the potential to surpass mainstream bioinformatics workflows in disease classification tasks.

sentences: A brain-based universal measure of attention: predicting task-general and task-specific attention performance and their underlying neural mechanisms from task and resting state fMRI Attention is central for many aspects of cognitive performance, but there is no singular measure of a persons overall attentional functioning across tasks. To develop a universal measure that integrates multiple components of attention, we collected data from more than 90 participants performing three different attention-demanding tasks during fMRI. We constructed a suite of whole-brain models that can predict a profile of multiple attentional components - sustained attention, divided attention and tracking, and working memory capacity - from a single fMRI scan type within novel individuals. Multiple brain regions across the frontoparietal, salience, and subcortical networks drive accurate predictions, supporting a universal (general) attention factor across tasks, which can be distinguished from task-specific attention factors and their neural mechanisms. Furthermore, connectome-to-connectome transformation modeling enhanced predictions of an individuals attention-task connectomes and behavioral performance from their rest connectomes. These models were integrated to produce a new universal attention measure that generalizes best across multiple, independent datasets, and which should have broad utility for both research and clinical applications.

sentences: vRhyme enables binning of viral genomes from metagenomes Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, no approach exists for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identity nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

sentences: Two parallel pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans Ultrasound has been shown to affect the function of both neurons and non-neuronal cells. However, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act in parallel to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4 mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that these two pathways act in parallel. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.

sentences: Absence of electroencephalographic evidence of implicit action-effect intentional binding with varying effect probability The subjective experience of an attraction in time of an action, and the event caused by the action, is known as the intentional binding phenomenon. Intentional binding is a robust phenomenon and has previously been associated with subjective sense of agency, but recent studies have shown that binding can take place in the absence of action intentions. In this study, we tested possible electrophysiological equivalents to the intentional binding phenomenon under a simple action-effect task, where pressing of a button caused tones to occur at different pitches or delays with different probabilities. Changing the probabilities of the effect of an action has in some previous studies shown to influence the intentional binding phenomenon. We tested whether changes in action-effect probability gave rise to differences in movement related cortical potentials (MRCP) slopes, peak latency and auditory event related potential (aERP) changes of amplitude or latency of the N1, P2, P3 and N4 components of the central aERP, of which some has been related to sense of agency or intentional binding. We also tested differences in MRCP across the whole scalp prior to movements, and to differences in aERP across the whole scalp after the tone is played. We found no electrophysiological indications of intentional binding when action-effect contingencies were changed in accordance with conditions that have given rise to intentional binding in previous experiments. Our results are in line with several recent studies that have questioned whether intentional binding follows all voluntary actions and can be related to sense of agency at all.

sentences: Butterfly eyespots evolved via co-option of the antennal gene-regulatory network Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via co-option of the antennal gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster with antennae relative to multiple other tissues. Furthermore, three genes essential for eyespot development (Distal-less (Dll), spalt (sal), and Antennapedia (Antp)) share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots and antennae, and also legs and wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely re-used the ancient antennal GRN, a network previously implicated also in the development of legs and wings.

sentences: CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 {micro}g or 5 {micro}g of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 g or 5 g of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.

sentences: High-throughput single-cell TCR - pMHC dissociation rate measurements performed by an autonomous microfluidic cellular processing unit We developed an integrated microfluidic cellular processing unit (mCPU) capable of autonomously isolating single cells, perform, measure, and on-the-fly analyze cell-surface dissociation rates, followed by recovery of selected cells. We performed proof-of-concept, high-throughput single-cell experiments characterizing pMHC-TCR interactions on live CD8+ T cells. The mCPU platform analyzed TCR-pMHC dissociation rates with a throughput of 50 cells per hour and hundreds of cells per run, and we demonstrate that cells can be selected, enriched, and easily recovered from the device.

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

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Influence of DNA extraction kits on freshwater fungal DNA metabarcoding BackgroundEnvironmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes.

MethodsDNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples.

ResultsNo PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

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Skin specimen as an alternative to brain tissue for post mortem rabies diagnosis in animals to scale up animal rabies surveillance BackgroundDetection of the virus or some of its specific components using WHO and OIE recommended standard laboratory tests is the only way to get a reliable diagnosis of rabies. Brain tissue is the preferred specimen for post-mortem diagnosis of rabies in both humans and animals. Higher biosecurity requirements, skill and transportation facilities required for collection and transport of brain or whole carcass to the laboratory is one of the reasons for the poor rabies surveillance in animals. Point of care testing with simple, reliable and easy to operate devices would be an ideal approach for providing rapid results.

MethodsThe study evaluated diagnostic performance of two reference tests, DFAT and RTPCR on skin specimen, to assess its suitability as an alternative of brain tissue for post mortem rabies diagnosis in animals. Brain tissue and skin sample belonging to different species of animals (n=90) collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis.

ResultsValidation of RT-PCR on skin and DFAT on skin in comparison with DFAT on brain as gold standard gave a sensitivity of 98% (95% CI:94.1-100) and 80% (95% CI:71.8-88.2) respectively. Specificity was 100% in both tests.

ConclusionThe findings highlight the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

Author SummaryThe present study was undertaken to define a reliable protocol for diagnosing animal rabies using skin specimen, a superficial tissue sample which is collected in a non-invasive manner for post-mortem diagnosis. Our aim was to design a protocol to replace the classical post-mortem diagnostic method that uses brain biopsy with an ultimate target of stepping up rabies surveillance in animals. Brain tissue and skin sample belonging to different species of animals collected at necropsy were compared using Fluorescent Antibody Test and RT PCR, internationally approved methods for rabies diagnosis. The study established that RT PCR on skin specimen is rapid, sensitive and specific, opening its potential as an ideal rabies surveillance tool overcoming the logistical challenges of carcass transportation to reference laboratories and alleviating biosafety concerns associated with brain collection. The study highlights the potential of skin specimen for improving rabies surveillance in animals especially in resource poor countries.

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Experimental evaluation of thermodynamic speed limit in living cells via information geometry Chemical reactions are responsible for information processing in living cells, and thermodynamic trade-off relations can explain their accuracy and speed. Its experimental test in living cells had not existed despite its importance because it is hard to justify sample size sufficiency. This paper reports the first experimental test of the thermodynamic trade-off relation, namely the thermodynamic speed limit, in living systems at the single-cell level where the sample size is relatively small. Due to the information-geometric approach, we can demonstrate the thermodynamic speed limit for the extracellular signal-regulated kinase phosphorylation using time-series fluorescence imaging data. Our approach quantifies the intrinsic speed of cell proliferation and can potentially apply other signal transduction pathways to detect their information processing speed.

One-Sentence SummaryExperimental measurement of information thermodynamic speed by fluorescence imaging in living cells

sentences: Elucidating the neural mechanisms of Learning-to-Learn Learning-to-learn, a progressive acceleration of learning while solving a series of similar problems, represents a core process of knowledge acquisition that draws attention in both neuroscience and artificial intelligence. To investigate its underlying brain mechanism, we trained a recurrent neural network model on arbitrary sensorimotor mappings. The network displayed an exponential speedup in learning. The neural substrate of a schema emerges within a low-dimensional subspace of population activity. Its reuse in new problems facilitates learning by limiting connection weight changes. Since the population trajectory of a recurrent network produces behavior, learning is determined by the vector field changes. We propose a novel analysis of weight-driven vector field changes, which showed that novel stimuli in new problems can distort the schema representation. Weight changes eliminate such distortions and improve the invariance of the reused representations in future learning. The accumulation of such weight changes across problems underlies the learning-to-learn dynamics.

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Mayaro Virus Infection Elicits an Innate Immune Response and Represses Autophagy in Anopheles stephensi Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naive regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint suggested activation of the Toll pathway at 7 dpi and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.

AUTHOR SUMMARYMayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discover novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species.

sentences: The endothelial-specific LINC00607 mediates endothelial angiogenic function Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1.

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Classification of the plant-associated phenotype of Pseudomonas strains using genome properties and machine learning The rhizosphere, the region of soil surrounding roots of plants, is colonized by a unique population of Plant Growth Promoting Rhizobacteria (PGPR). By enhancing nutrient uptake from the soil and through modulation of plant phytohormone status and metabolism, PGPR can increase the stress tolerance, growth and yield of crop plants. Many important PGPR as well as plant pathogens belong to the genus Pseudomonas. There is, however, uncertainty on the divide between phytobeneficial and phytopathogenic strains as previously thought to be signifying genomic features have limited power to separate these strains. Here the Genome properties (GP) common biological pathways annotation system was applied to establish the relationship between the genome wide GP composition and the plant-associated phenotype of 91 Pseudomonas strains representing both phenotypes. GP enrichment analysis, Random Forest model fitting and feature selection revealed 28 discriminating features. A validation dataset of 67 new strains confirmed the importance of the selected features for classification. A number of unexpected discriminating features were found, suggesting involvement of novel molecular mechanisms. The results suggest that GP annotations provide a promising computational tool to better classify the plant-associated phenotype.

Author summaryWith a growing population the need to double the agricultural food production is specified. Simultaneously, there is an urgent need to implement sustainable and climate change resilient agricultural practices that preserve natural ecosystems. Cooperative microbiomes play important positive roles in plant growth development and fitness. Properly tuned, these microbiomes can significantly reduce the need for synthetic fertilizers and can replace chemicals in crop pest control. To select beneficial candidates, their traits need to be described and likewise, potential detrimental traits should be avoided. Here we applied GP-based comparative functional genomics, enrichment analysis and Random Forest model fitting to compare known phytobeneficial and phytopathogenic Pseudomonas strains. A number of unexpected discriminating features were found suggesting the involvement of novel molecular mechanisms.

sentences: Assessing HD-EEG functional connectivity states using ahuman brain computational model Electro/Magnetoencephalography (EEG/MEG) source-space network analysis is increasingly recognized as a powerful tool to track fast electrophysiological brain dynamics. However, an objective and quantitative evaluation of the various steps, from source localization and functional connectivity to clustering algorithms, is challenging, due to the lack of realistic  controlled data. Here, we used a human brain computational model containing both physiologically-based cellular GABAergic and Glutamatergic circuits coupled through Diffusion Tensor Imaging - based structural connectivity, to generate realistic High Density-EEG (256 channels) recordings. We designed a scenario of successive gamma-band oscillations in distinct cortical areas in order to emulate a virtual picture naming task. We identified the fast time-varying network states and quantified the performance of the key steps involved in the pipeline: (1) inverse models to reconstruct cortical-level sources, (2) functional connectivity measures to compute statistical interdependency between regional time series, and (3) dimensionality reduction methods to derive dominant brain network states (BNS). Using a systematic evaluation of the different independent/principal/non-negative decomposition techniques along with a clustering approach, results show significant variability among the tested algorithms in terms of spatial and temporal accuracy. We outlined the spatial precision, the temporal sensitivity, as well as the global accuracy of the extracted BNS relative to each method. Our findings suggest a good performance of wMNE/PLV combination to elucidate the appropriate functional networks and ICA techniques to derive relevant dynamic brain network states. Our aim here is twofold: 1) to provide quantitative assessment on the advantages and the limitations of each of the analyzed techniques and 2) to introduce (and share) a complete framework that can be used to optimize the entire pipeline of EEG/MEG source connectivity. With such framework, other tasks can be generated and used for validation and other methodological points can be also addressed.

sentences: Comparison of Three Individual Identification Algorithms for Sperm Whales (Physeter macrocephalus) after Automated Detection Photo-identification of individual sperm whales (Physeter macrocephalus) is the primary technique for mark-recapture-based population analyses for the species The visual appearance of the fluke -- with its distinct nicks and notches -- often serves as the primary visual differentiator, allowing humans to make recorded sightings of specific individuals. However, the advent of digital photography and the significant increase in volume of images from multiple projects in combination with pre-existing historical catalogs has made applying the method more challenging.with the required human labor for de-duplication (reduction of Type II errors) and reconciliation of sightings between large datasets too cost- and time-prohibitive. To address this, we trained and evaluated the accuracy of PIE v2 (a triplet loss network) along with two existing fluke trailing edge-matching algorithms, CurvRank v2 and Dynamic Time Warping (DTW), as a mean to speed comparison among a high volume of photographs. Analyzed data were collected from a curated catalog of well-known sperm whales sighted across years (2005-2018) off the island of Dominica. The newly-trained PIE model outperformed the older CurvRank and DTW algorithms, and PIE provided the following top-k individual ID matching accuracy on a standard min-3/max-10 sighting training data set: Rank-1: 87.0%, Rank-5: 90.5%, and Rank-12: 92.5%. An essential aspect of PIE is that it can learn new individuals without network retraining, which can be immediately applied in the presence of (and for the resolution of) duplicate individuals in overlapping catalogs. Overall, our results recommend the use of PIE v2 and CurvRank v2 for ID reconciliation in combination due to their complementary performance.

sentences: Community composition of coral associated Symbiodiniaceae is driven by fine scale environmental gradients The survival of reef-building corals is dependent upon a symbiosis between the coral and the community of Symbiodiniaceae. Montipora capitata, one of the main reef building coral species in Hawaii, is known to host a diversity of symbionts, but it remains unclear how they change spatially and whether environmental factors drive those changes. Here, we surveyed the Symbiodiniaceae community in 600 M. capitata colonies from 30 sites across K[a]neohe Bay and tested for host specificity and environmental gradients driving spatial patterns of algal symbiont distribution. We found that the Symbiodiniaceae community differed markedly across sites, with M. capitata in the most open-ocean (northern) site hosting few or none of the genus Durusdinium, whereas individuals at other sites had a mix of Durusdinium and Cladocopium. Our study shows that the algal symbiont community composition responds to fine-scale differences in environmental gradients; depth and temperature variability were the most significant predictor of Symbiodiniaceae community, although environmental factors measured in the study explained only about 20% of observed variation. Identifying and mapping Symbiodiniaceae community distribution at multiple scales is an important step in advancing our understanding of algal symbiont diversity, distribution and evolution, and the potential responses of corals to future environmental change.

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Puf3p facilitates fermentative mitochondrial functions via monosome-enriched nuclear-encoded mitochondrial mRNAs in budding yeast Yeasts generally grow with a highly glycolytic metabolism and restrain mitochondrial biogenesis except for some Fe/S proteins. Respiratory mitochondrial functions and biosynthesis pathways are well studied, however how cells coordinate basal fermentative mitochondrial functions is not fully understood. We show that a part of nuclear-encoded mitochondrial mRNAs, which preferentially translated in monosomes, are regulated by Puf3p upon glucose-rich media. Especially, of those monosome-enriched nuclear-encoded mitochondrial mRNAs, CAT5/COQ7 mRNA has a variant of the canonical Puf3p binding site on its 3-UTR. Western blot analysis showed that Puf3p represses the translation of Cat5p regardless of fermentable or respiratory media. In vitro binding assay revealed that Puf3p directly binds to CAT5 mRNA via the non-canonical Puf3p binding site.

Mutants harboring the substitution of the non-canonical Puf3p binding site in CAT5 mRNA grew normally but impaired Cat5p expressions apparently, indicating CAT5 mRNA is a bona fide Puf3p target. Overall, Puf3p, a general key modulator for nuclear-encoded mitochondrial mRNAs, fine-tunes translation of a subset of nuclear-encoded mitochondrial mRNAs including mRNAs with non-canonical Puf3p binding sites under the fermentation. This may be required to keeping the fundamental functions of yeast mitochondria at proper levels.

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Exploring the natural origins of SARS-CoV-2 The lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2, and the large geographical distance between Wuhan and where the closest evolutionary related coronaviruses circulating in horseshoe bats (Sarbecoviruses) have been identified, is fuelling speculation on the natural origins of SARS-CoV-2. We have comprehensively analysed phylogenetic relations between SARS-CoV-2, and the related bat and pangolin Sarbecoviruses sampled so far. Determining the likely recombination events reveals a highly reticulate evolutionary history within this group of coronaviruses. Clustering of the inferred recombination events is non-random with evidence that Spike, the main target for humoral immunity, is beside a recombination hotspot likely driving antigenic shift in the ancestry of bat Sarbecoviruses. Coupled with the geographic ranges of their hosts and the sampling locations, across southern China, and into Southeast Asia, we confirm horseshoe bats, Rhinolophus, are the likely SARS-CoV-2 progenitor reservoir species. By tracing the recombinant sequence patterns, we conclude that there has been relatively recent geographic movement and co-circulation of these viruses ancestors, extending across their bat host ranges in China and Southeast Asia over the last 100 years or so. We confirm that a direct proximal ancestor to SARS-CoV-2 is yet to be sampled, since the closest relative shared a common ancestor with SARS-CoV-2 approximately 40 years ago. Our analysis highlights the need for more wildlife sampling to (i) pinpoint the exact origins of SARS-CoV-2s animal progenitor, and (ii) survey the extent of the diversity in the related Sarbecoviruses phylogeny that present high risk for future spillover.

HighlightsO_LIThe origin of SARS-CoV-2 can be traced to horseshoe bats, genus Rhinolophus, with ranges in both China and Southeast Asia.
C_LIO_LIThe closest known relatives of SARS-CoV-2 exhibit frequent transmission among their Rhinolophus host species.
C_LIO_LISarbecoviruses have undergone extensive recombination throughout their evolutionary history.
C_LIO_LIAccounting for the mosaic patterns of these recombinants is important when inferring relatedness to SARS-CoV-2.
C_LIO_LIBreakpoint patterns are consistent with recombination hotspots in the coronavirus genome, particularly upstream of the pike open reading frame with a coldspot in S1.
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Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the contacts that silence transcription by RNA polymerase, we combined {middle dot}OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching in which linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contact sites.

HighlightsO_LITethered-nuclease mapping (TEN-map) of H-NS DNA-binding domains detects DNA contacts
C_LIO_LIBridged and linear H-NS filaments use the same DNA contact sites
C_LIO_LIH-NS-DNA contacts are unevenly spaced with ~10 bp average separation
C_LIO_LIAT-steps, minor groove width, and electrostatic potential best predict contact sites
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sentences: Modeling microRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses.

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Harmonized-Multinational qEEG Norms (HarMNqEEG) This paper extends our frequency domain quantitative electroencephalography (qEEG) methods pursuing higher sensitivity to detect Brain Developmental Disorders. Prior qEEG work lacked integration of cross-spectral information omitting important functional connectivity descriptors. Lack of geographical diversity precluded accounting for site-specific variance, increasing qEEG nuisance variance. We ameliorate these weaknesses. i) Create lifespan Hermitian Riemannian multinational qEEG norms for cross-spectral tensors. These norms result from the HarMNqEEG project fostered by the Global Brain Consortium. We calculate the norms with data from 9 countries, 12 devices, and 14 studies, including 1564 subjects. Instead of raw data, only anonymized metadata and EEG cross-spectral tensors were shared. After visual and automatic quality control developmental equations for the mean and standard deviation of qEEG traditional and Hermitian Riemannian descriptive parameters were calculated using additive mixed-effects models. We demonstrate qEEG "batch effects" and provide methods to calculate harmonized z-scores. ii) We also show that the multinational harmonized Hermitian Riemannian norms produce z-scores with increased diagnostic accuracy to predict brain dysfunction at school-age produced by malnutrition only in the first year of life. We provide data and software for constructing norms. iii) We offer open code and data to calculate different individual z-scores from the HarMNqEEG dataset. These results contribute to developing bias-free, low-cost neuroimaging technologies applicable in various health settings.

HighlightsO_LIWe create lifespan Hermitian Riemannian qEEG norms for cross-spectral tensors.
C_LIO_LIThe norms are based on 9 countries, 12 devices, and 14 studies, with 1564 subjects.
C_LIO_LIWe demonstrate qEEG "batch effects", providing harmonization methods to remove them.
C_LIO_LIMultinational harmonized z-scores increase diagnostic accuracy of brain dysfunction.
C_LIO_LIData and software are available for norm and individual z-scores calculation.
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sentences: Apoplastic effector candidates of a foliar forest pathogen trigger cell death in host and non-host plants Forests are under threat from pests, pathogens, and changing climate. One of the major forest pathogens worldwide is Dothistroma septosporum, which causes dothistroma needle blight (DNB) of pines. D. septosporum is a hemibiotrophic fungus related to well-studied Dothideomycete pathogens, such as Cladosporium fulvum. These pathogens use small secreted proteins, termed effectors, to facilitate the infection of their hosts. The same effectors, however, can be recognised by plants carrying corresponding immune receptors, resulting in resistance responses. Hence, effectors are increasingly being exploited to identify and select disease resistance in crop species. In gymnosperms, however, such research is scarce. We predicted and investigated apoplastic D. septosporum candidate effectors (DsCEs) using bioinformatics and plant-based experiments. We discovered secreted proteins that trigger cell death in the angiosperm Nicotiana spp., suggesting their recognition by immune receptors in non-host plants. In a first for foliar forest pathogens, we also developed a novel protein infiltration method to show that tissue-cultured pine shoots can respond with a cell death response to one of our DsCEs, as well as to a reference cell death-inducing protein. These results contribute to our understanding of forest pathogens and may ultimately provide clues to disease immunity in both commercial and natural forests.

sentences: Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

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Dietary folic acid deficiency impacts hippocampal morphology and cortical acetylcholine metabolism in adult male and female mice ObjectiveOne-carbon metabolism is a metabolic network that integrates nutritional signals with biosynthesis, redox homeostasis, and epigenetics. There are sex differences in hepatic one-carbon metabolism. However, it is unclear whether there are sex differences in dietary deficiencies of one-carbon metabolism in the brain. The aim of this study was to investigate if sex modulates the effects of dietary folic acid deficiency in brain tissue using a mouse model.

MethodsMale and female C57Bl/6J mice were placed on a folic acid deficient (FD) or control diet (CD) at six weeks of age. Mice were maintained on these diets for six months, after which animals were euthanized and brain tissue and serum were collected for analysis. Serum folate levels were measured. In brain tissue, hippocampal volume and morphology including Cornu Ammonis 1 and 3 (CA1; CA3), and dentate gyrus thickness were measured. Apoptosis within the hippocampus was assessed using active caspase-3 immunofluorescence staining. Additionally, cortical acetylcholine metabolism was measured in brain tissue using immunofluorescence staining of acetylcholinesterase (AChE), or choline acetyltransferase (ChAT), and neuronal nuclei (NeuN).

ResultsMale and female FD mice had reduced serum levels of folate. Both males and females maintained on a FD showed a decrease in the thickness of the hippocampal CA1-CA3 region. Interestingly, there was a sex difference in the levels of active caspase-3 within the CA3 region of the hippocampus. In cortical tissue, there were increased levels of neuronal ChAT and reduced levels of AChE in FD females and male mice.

ConclusionsThe results indicated that FD impacts hippocampal morphology and cortical neuronal acetylcholine metabolism. The data from our study indicate that there was only one sex difference and that was in hippocampal apoptosis. Our study provides little evidence that sex modulates the effects of dietary folate deficiency on hippocampal morphology and cortical neuronal acetylcholine metabolism.

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The Breast Cancer Epigenomics Track Hub BackgroundPioneering research has shown that high-throughput epigenomics assays such as ChlP-seq and ATAC-seq are applicable to patient-derived breast tumor samples. A host of public data has been accumulated since then, which are potentially of high value for basic research as well as personalized medicine. Such data sets constitute encyclopedias of biological knowledge. However, their impact has so far been limited by access obstacles, especially with regard to extraction and visualization of small portions of data that could potentially answer specific questions arising in a research context.

ResultsWe developed the breast cancer epigenomics track hub (BC hub), a resource intended to make it easy for occasional users to find, access and view data of their interest. The BC hub harbors ChIP-seq, ATAC-seq and copy number data from breast tumors, normal breast cells, patient-derived xenografts and breast cancer cell lines in a genome browsable track format. The tracks can be accessed via hyperlinks that automatically configure customized views for different interest groups. Here, we present a detailed description of the resource and informative use cases illustrating its potential in answering specific biological questions.

ConclusionsWe show that track hubs constitute a powerful way of bringing epigenomics data to the user who could benefit from them. The examples presented highlight the added-value of joint visualization of breast cancer data from different sources. The proof-of-concept provided here exemplifies and underscores the importance of efforts to make biological data FAIR (findable, accessible, interoperable and reusable), and may serve as an encouragement of similar bottom-up initiatives in other research fields. The BC hub is freely accessible at https://bchub.epfl.ch.

sentences: Surface electric fields increase human osteoclast resorption through improved wettability on carbonate-incorporated apatite Osteoclast-mediated bioresorption can be of an efficient means of incorporating the dissolution of biomaterials in the bone remodeling process. Because of compositionally and structurally close resemblance of biomaterials with the natural mineral phases of the bone matrix, synthetic carbonate-substituted hydroxyapatite (CA) is considered as an ideal clinical biomaterial. The present study therefore investigated the effects of electrical polarization on the surface characteristics and interactions with human osteoclasts of hydroxyapatite (HA) and CA. Electrical polarization was found to improve the surface wettability of these materials by increasing the surface free energy, and this effect was maintained for one month. Analyses of human osteoclast cultures established that CA subjected to a polarization treatment accelerated osteoclast resorption but did not affect the early differentiation phase or the adherent morphology of the osteoclasts as evaluated by staining. These data suggest that the surface characteristics of the CA promoted osteoclast resorption. The results of this work are expected to contribute to the design of cell-mediated biomaterials that can be resorbed by osteoclasts after fulfilling their primary function as a scaffold for bone regeneration.

sentences: Nucleosome induced homology recognition in chromatin One of the least understood properties of chromatin is the ability of its similar regions to recognise each other through weak interactions. Theories based on electrostatic interactions between helical macromolecules suggest that the ability to recognize sequence homology is an innate property of the non-ideal helical structure of DNA. However, this theory does not account for nucleosomal packing of DNA. Can homologous DNA sequences recognize each other while wrapped up in the nucleosomes? Can structural homology arise at the level of nucleosome arrays? Here we present a theoretical investigation of the recognition-potential-well between chromatin fibers sliding against each other. This well is different to the one predicted and observed for bare DNA; the minima in energy do not correspond to literal juxtaposition, but are shifted by approximately half the nucleosome repeat length. The presence of this potential-well suggests that nucleosome positioning may induce mutual sequence recognition between chromatin fibers and facilitate formation of chromatin nanodomains. This has implications for nucleosome arrays enclosed between CTCF-cohesin boundaries, which may form stiffer stem-like structures instead of flexible entropically favourable loops. We also consider switches between chromatin states, e.g., through acetylation/deacetylation of histones, and discuss nucleosome-induced recognition as a precursory stage of genetic recombination.

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SimpleForest - a comprehensive tool for 3d reconstruction of trees from forest plot point clouds The here-on presented SimpleForest is written in C++ and published under GPL v3. As input data SimpleForest utilizes forestry scenes recorded as terrestrial laser scan clouds. SimpleForest provides a fully automated pipeline to model the ground as a digital terrain model, then segment the vegetation and finally build quantitative structure models of trees (QSMs) consisting of up to thousands of topologically ordered cylinders. These QSMs allow us to calculate traditional forestry metrics such as diameter at breast height, but also volume and other structural metrics that are hard to measure in the field. Our volume evaluation on three data sets with destructive volumes show high prediction qualities with concordance correlation coefficient CCC [Formula] of 0.91 (0.87), 0.94 (0.92) and 0.97 (0.93) for each data set respectively.

We combine two common assumptions in plant modeling "The sum of cross sectional areas after a branch junction equals the one before the branch junction" (Pipe Model Theory) and "Twigs are self-similar" (West, Brown and Enquist model). As even sized twigs correspond to even sized cross sectional areas for twigs we define the Reverse Pipe Radius Branchorder (RPRB) as the square root of the number of supported twigs. The prediction model radius = B0 * RPRB relies only on correct topological information and can be used to detect and correct overestimated cylinders. In QSM building the necessity to handle overestimated cylinders is well known. The RPRB correction performs better with a CCC [Formula] of 0.97 (0.93) than former published ones 0.80 (0.88) and 0.86 (0.85) in our validation.

We encourage forest ecologists to analyze output parameters such as the GrowthVolume published in earlier works, but also other parameters such as the GrowthLength, VesselVolume and RPRB which we define in this manuscript.

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sentences: Polycomb Repressive Complexes occupancy reveals PRC2-independent PRC1 critical role in the control of limb development The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1), in which PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidence pointed to cPRC1 and vPRC1 sharing a significant subset of targets. How the functional interplay between PRC2, cPRC1 and vPRC1 contributes to gene regulation remains partially understood. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, as the majority of PRC2- bound loci are bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of genes retaining CBX2 occupancy as well as a few genes with apparent gain of CBX2 binding. Importantly, among these genes, 56 of them correspond to developmental genes known for playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings emphasize the primary role of PRC2-independent PCR1 function in regulating developmental genes and questions the role of PRC2/cPRC1 in controlling developmental programs.

sentences: UMINT: Unsupervised Neural Network For Single Cell Multi-Omics Integration AO_SCPLOWBSTRACTC_SCPLOWMulti-omics studies have enabled us to understand the mechanistic drivers behind complex disease states and progressions, thereby providing novel and actionable biological insights into health status. However, integrating data from multiple modalities is challenging due to the high dimensionality of data and noise associated with each platform. Non-overlapping features and technical batch effects in the data make the task of learning more complicated. Conventional machine learning (ML) tools are not quite effective against such data integration hazards. In addition, existing methods for single cell multi-omics integration are computationally expensive. This has encouraged the development of a novel architecture that produces a robust model for integration of high-dimensional multi-omics data, which would be capable of learning meaningful features for further downstream analysis. In this work, we have introduced a novel Unsupervised neural network for single cell Multi-omics INTegration (UMINT). UMINT serves as a promising model for integrating variable number of single cell omics layers with high dimensions, and provides substantial reduction in the number of parameters. It is capable of learning a latent low-dimensional embedding that can capture useful data characteristics. The effectiveness of UMINT has been evaluated on benchmark CITE-seq (paired RNA and surface proteins) datasets. It has outperformed existing state-of-the-art methods for multi-omics integration.

sentences: Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection. Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

sentences: Performance of Core Genome Multilocus Sequence Typing Compared to Capillary-Electrophoresis PCR Ribotyping and SNP Analysis of Clostridioides difficile Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary-electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better, but backward compatibility with CE-PCR ribotyping must be assessed. Using a well-characterized collection of diverse strains (N=630; 100 unique ribotypes [RTs]), we aimed to investigate PCR ribotyping prediction from core genome multilocus sequence typing (cgMLST). Additionally, we compared the discriminatory power of cgMLST (SeqSphere & EnteroBase) and whole genome MLST (wgMLST) (EnteroBase) with single nucleotide polymorphism (SNP) analysis). A unique cgMLST profile (>6 allele differences) was observed in 82/100 ribotypes, indicating sufficient backward compatibility. Intra-RT allele difference varied per ribotype and MLST clade. Application of cg/wgMLST and SNP analysis in two outbreak settings with ribotypes RT078 and RT181 (known with a low intra-ribotype allele difference) showed no distinction between outbreak- and non-outbreak strains, in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize and offers higher discrimination. However, in some ribotype complexes adjusted cut-off thresholds and epidemiological data are necessary to recognize outbreaks. We propose to decrease the current threshold of 6 to 3 alleles to better identify outbreaks.

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Plasticity of Neural Connections Underlying Oxytocin-mediated Parental Behaviors of Male Mice The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naive male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Using viral-genetic approaches, here we establish hypothalamic oxytocin neurons as key regulators of parental caregiving behaviors of male mice. We then used rabies virus-mediated unbiased screen to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region classically assumed to be hard-wired.

Highlight- OT is indispensable for parental caregiving behavior of male mice
- Activation of OT neurons triggers paternal caregiving behavior in otherwise infanticidal sexually-naive male mice partly via OT ligand
- Unbiased rabies virus-mediated screening reveals enhanced connectivity originated from excitatory LHA neurons to OT neurons in fathers.
- This structural plasticity can support behavioral plasticity

sentences: Rheb-mTOR Activation Rescues Amyloid Beta-Induced Cognitive Impairment and Memory Function by Restoring miR-146 Activity in Glial Cells Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimers disease patients brain suggests ineffective target cytokine mRNA suppression by existing microRNPs in diseased brain. Exploring the mechanism of amyloid beta induced cytokine expression, we have identified how the inactivation of the repressive miR-146 microRNPs causes increased production of cytokines in amyloid beta exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer induced sequestration of mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling and retarded Ago2-cytokine mRNA interaction in rat astrocyte cells. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 microRNPs in amyloid beta exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimers disease.

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Use Of Canine Olfactory Detection For COVID-19 Testing Study On U.A.E. Trained Detection Dog Sensitivity This study aimed to evaluate the sensitivity of 21 dogs belonging to different United Arab Emirates (UAE) Ministry of Interior (MOI), trained for COVID-19 olfactory detection.

The study involved 17 explosives detection dogs, two cadaver detection dogs and two dogs with no previous detection training. Training lasted two weeks before starting the validation protocol. Sequential five and seven-cone line-ups were used with axillary sweat samples from symptomatic COVID-19 individuals (SARS-CoV-2 PCR positive) and from asymptomatic COVID-19 negative individuals (SARS-CoV-2 PCR negative). A total of 1368 trials were performed during validation, including 151 positive and 110 negative samples. Each line-up had one positive sample and at least one negative sample. The dog had to mark the positive sample, randomly positioned behind one of the cones. The dog, handler and data recorder were blinded to the positive sample location.

The calculated overall sensitivities were between 71% and 79% for three dogs, between83% and 87% for three other dogs, and equal to or higher than 90% for the remaining 15 dogs (more than two thirds of the 21 dogs).

After calculating the overall sensitivity for each dog using all line-ups, "matched" sensitivities were calculated only including line-ups containing COVID-19 positive and negative samples strictly comparable on confounding factors such as diabetes, anosmia, asthma, fever, body pain, diarrhoea, sex, hospital, method of sweat collection and sampling duration. Most of the time, the sensitivities increased after matching.

Pandemic conditions in the U.A.E., associated with the desire to use dogs as an efficient mass-pretesting tool has already led to the operational deployment of the study dogs.

Future studies will focus on comparatives fields-test results including the impact of the main COVID-19 comorbidities and other respiratory tract infections.

sentences: Genotype and age influence cortical-FKBP5 at multiple regulatory and single-cell-type levels in severe psychiatric disorders Deducing genes capable of classifying biologically-distinct psychiatric subtypes, and their targets for treatment, is a priority approach in psychiatry. FKBP5 is one such gene with strong evidence of utility to delineate a trans-diagnostic psychiatric subtype. Yet how brain-expressed FKBP5 is affected in psychiatric disorders in humans is not fully understood and critical for propelling FKBP5-targeting treatment development. We performed a large-scale postmortem study (n=895) of FKBP5 using dorsolateral prefrontal cortex samples derived from individuals with severe psychiatric disorders with a comprehensive battery of bulk/single-cell omics and histological analyses. We observed consistently heightened FKBP5 mRNA and protein in psychopathology, moderated by genotype and age, and accompanied by DNA methylation changes in key enhancers. These effects were most prominent in superficial-layer pyramidal cells. Heightened FKBP5 was also differentially associated with downstream pathways according to age, being specifically associated with synaptic transmission in early adulthood, and neuroinflammation and neurodegeneration in later life.

sentences: A pore-forming β-trefoil lectin with specificity for the tumor-related glycosphingolipid Gb3 Lectins are efficient multivalent glycan receptors, deciphering the glyco-code on cell surfaces. The {beta}-trefoil fold, characterized by three lobe-shaped repeats, is adopted by several classes of lectins, often associated with other domains having enzymatic or toxic activity. Based on the UniLectin3D database classification, the sequence signature of trefoil lobes was defined and used to predict 44714 lectins from 4497 species. Among them, SaroL-1 from the lower eukaryote Salpingoeca rosetta was predicted to contain both {beta}-trefoil and aerolysin-like pore-forming domain. Recombinant SaroL-1 binds to galactose and derivatives, with a stronger affinity for cancer-related -galactosylated epitopes such as glycosphingolipid Gb3 embedded in giant unilamellar vesicles or cell membranes. Crystal structures in complex with Gb3 trisaccharide and GalNAc show similarity with pore-forming toxins. Recognition of the Gal epitope on glycolipids was necessary for hemolysis of rabbit erythrocytes and toxicity on cancer cells through carbohydrate-dependent pore-formation.

sentences: TPX2 Amplification-Driven Aberrant Mitosis in Long-Term Cultured Human Embryonic Stem Cells Although human embryonic stem cells (hESCs) are equipped with highly effective machinery for the maintenance of genome integrity, the frequency of genetic aberrations during long-term in vitro hESC culture has been a serious issue that raises concerns over their safety in future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in the late-passaged hESCs (LP-hESCs) in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs). Through high-resolution genome-wide approaches and by following transcriptome analysis, we found that LP-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2 (targeting protein for Xklp2), a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stability, misaligned chromosomes, and polyploidy. This data suggests that the amplification and increased transcription of the TPX2 gene at 20q11.21 could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

sentences: Mapping Protein Numbers in Living Cells We introduce a new, robust method to map the numbers of proteins in living cells. The method can be applied to laser scanning, spinning disk, and lattice light-sheet microscopes in a robust, reproducible, and scalable fashion. The method uses calibrated EGFP solutions that are imaged with the appropriate microscope modality to create a calibration curve that is then applied to convert the fluorescence intensities from 3D microscope images into molecule numbers. We applied this method to human induced pluripotent stem cells in which proteins representing key cellular structures were endogenously tagged with mEGFP. We used the ratio of mEGFP-tagged proteins to total proteins to create 3D maps of live cells showing the density of total proteins measured in molecules per {micro}m3. The method opens the door to new quantitative single cell analyses of cellular protein numbers in the context of single cell gene expression, associations with cellular complexes, and changes in cellular behaviors. The method is capable of quantifying protein numbers, over three orders of magnitude, in the cytoplasm or within various cellular structures while offering the unique advantages of each microscopy modality.

sentences: H3K36 methylation and DNA-binding are critical for Ioc4 recruitment and Isw1b remodeller function The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promote the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs.

sentences: Exogenous attention generalizes location transfer of perceptual learning in adults with amblyopia Visual perceptual learning (VPL) is a behavioral manifestation of brain neuroplasticity. However, its practical effectiveness is limited because improvements are often specific to the trained conditions and require significant time and effort. It is critical to understand the conditions that promote learning and transfer. Covert endogenous (voluntary) and exogenous (involuntary) spatial attention help overcome VPL location specificity in neurotypical adults, but whether they also do so for people with atypical visual development is unknown. This is the first study to investigate the role of exogenous attention during VPL in adults with amblyopia, an ideal population given their asymmetrically developed, but highly plastic, visual cortex. Here we show that training on a discrimination task leads to improvements in foveal contrast sensitivity, acuity, and stereoacuity. Remarkably, exogenous attention helps generalize learning beyond trained spatial locations. Our findings reveal that attention can enhance the effectiveness of perceptual learning during rehabilitation of visual disorders.

sentences: Fear of parasitism affects the functional role of ecosystem engineers Fear is an integrated part of predator-prey interactions with cascading effects on the structure and function of ecosystems. Fear of parasitism holds a similar ecological potential but our understanding of the underlying mechanisms in host-parasite interactions is limited by lack of empirical examples. Here, we experimentally test if blue mussels Mytilus edulis respond behaviourally to the mere presence of infective transmission stages of the trematode Himasthla elongata by ceasing filtration activity, thereby avoiding infection. Our results show that blue mussels reduced clearance rates by more than 30% in presences of parasites. The reduced filtration activity resulted in lower infection rates in experimental mussels. The identified parasite-specific avoidance behaviour can be expected to play a significant role in regulating the ecosystem engineering function of blue mussels in coastal habitats.

sentences: Unfolding of RNA via Translocation Through a Nanopore RNA unfolding and refolding are important biological phenomena, which occur during the transfer of genetic information from DNA to RNA to proteins. During these processes, RNA is found in single stranded, secondary and tertiary structures, including secondary conformations like hairpins and pseudoknots. Understanding the diverse conformations of RNA and how these influence the dynamics of unfolding and refolding is crucial to gain insight to fundamental biological processes. In this work, we employ coarse-grained Langevin dynamics simulations of a simple model of different RNA hairpins passing through a geometric nanopore to find the influence of structural changes on the translocation dynamics. The threshold voltage of unfolding depends on the length of the hairpin attached to the tail. The lag time to unfold is longer for smaller applied voltages and for the architectures containing a longer hairpin attached to the tail. Chain translocation dynamics for different architectures are largely collapsed by the threshold. A distinct signature for the base unfolding time was observed for the bases around the unpaired bases in all the RNA hairpin models. These results can motivate future technologies or experiments that use translocation to predict secondary structures of polynucleotides.

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High-frequency hearing is required for generating a topographic map of auditory space in the mouse superior colliculus A topographic map of auditory space is a feature found in the superior colliculus (SC) of many species, including CBA/CaJ mice. In this genetic background, high-frequency monaural spectral cues and interaural level differences are used to generate spatial receptive fields (RFs) that form a topographic map along the azimuth. Unfortunately, C57BL/6 mice, a strain widely used for transgenic manipulation, display age-related hearing loss (AHL) due to an inbred mutation in the Cadherin 23 gene (Cdh23) that affects hair cell mechanotransduction. To overcome this problem, researchers have used young C57BL/6 mice in their studies, as they have been shown to have normal hearing thresholds. However, important details of the auditory response characteristics of the SC such as spectral responses and spatial localization, have not been characterized in young C57BL/6 mice.

Here we show that 2-4-month C57BL/6 mice lack neurons with frontal auditory RFs and therefore lack a topographic representation of auditory space in the SC. Analysis of the spectrotemporal receptive fields (STRFs) of the SC auditory neurons shows that C57BL/6 mouse SC neurons lack the ability to detect the high-frequency (>40kHz) spectral cues that are needed to compute frontal RFs. We also show that crossing C57BL/6 mice with CBA/CaJ mice or introducing one copy of the wild-type Cdh23 to C57BL/6 mice rescues the high-frequency hearing deficit and improves the topographic map of auditory space. Taken together, these results demonstrate the importance of high-frequency hearing in computing a topographic map of auditory space.

Significance StatementDespite the strains age-dependent hearing loss, C57BL/6 mice are widely used in auditory studies because of the development of transgenic reporter and Cre lines in this genetic background. Here we examined the topographic map of auditory space and spectrotemporal properties of neurons in the SC of C57BL/6 mice. We found an early-onset high-frequency hearing deficit that results in the loss of SC neurons with frontal RFs and, consequently, an absence of a topographic map of auditory space. These findings stress the importance of high-frequency hearing in generating spatially restricted receptive fields and serve as a caution to researchers that doing auditory-related research using the C57BL/6 genetic background may not be representative of true wild-type mice.

sentences: Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

sentences: Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing In insects and mammals, chronic exposure to odors at high concentrations in early life alters olfactory function, but the role of odor experience-dependent plasticity in more naturalistic contexts is less clear. We investigated olfactory plasticity in the Drosophila antennal lobe by exposing flies to odors at concentrations that are typically encountered in natural odor sources. These stimuli also strongly and selectively activated only a single class of olfactory receptor neuron (ORN) input, facilitating the investigation of input-specific plasticity. Overall, chronic exposure to three such odors elicited limited plasticity in the odor responses of second-order projection neurons (PNs). Exposure to some odors elicited mild increases in PN responses to weak stimuli, extending the lower bound of the dynamic range of PN signaling. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. Chronic E2-hexenal exposure did not affect PN intrinsic properties, local inhibitory innervation, ORN responses, or ORN-PN synaptic strength, but modestly increased broad lateral excitation evoked by some odors. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input and highlight the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment.

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Structural Genetics of circulating variants affecting the SARS CoV-2 Spike / human ACE2 complex SARS-CoV-2 entry in human cells is mediated by the interaction between the viral Spike protein and the human ACE2 receptor. This mechanism evolved from the ancestor bat coronavirus and is currently one of the main targets for antiviral strategies. However, there currently exist several Spike protein variants in the SARS-CoV-2 population as the result of mutations, and it is unclear if these variants may exert a specific effect on the affinity with ACE2 which, in turn, is also characterized by multiple alleles in the human population. In the current study, the GBPM analysis, originally developed for highlighting host-guest interaction features, has been applied to define the key amino acids responsible for the Spike/ACE2 molecular recognition, using four different crystallographic structures. Then, we intersected these structural results with the current mutational status, based on more than 295,000 sequenced cases, in the SARS-CoV-2 population. We identified several Spike mutations interacting with ACE2 and mutated in at least 20 distinct patients: S477N, N439K, N501Y, Y453F, E484K, K417N, S477I and G476S. Among these, mutation N501Y in particular is one of the events characterizing SARS-CoV-2 lineage B.1.1.7, which has recently risen in frequency in Europe. We also identified five ACE2 rare variants that may affect interaction with Spike and susceptibility to infection: S19P, E37K, M82I, E329G and G352V.

Significance StatementWe developed a method to identify key amino acids responsible for the initial interaction between SARS-CoV-2 (the COVID-19 virus) and human cells, through the analysis of Spike/ACE2 complexes. We further identified which of these amino acids show variants in the viral and human populations. Our results will facilitate scientists and clinicians alike in identifying the possible role of present and future Spike and ACE2 sequence variants in cell entry and general susceptibility to infection.

sentences: Genetic control of neuronal activity enhances axonal growth only on permissive substrates Neural tissue has limited regenerative ability, to cope with that, in the recent years a diverse set of novel tools have been used to tailor neurostimulation therapies and promote functional regeneration after axonal injuries. In this report, we explore cell-specific methods to modulate neuronal activity, including opto- and chemogenetics to assess the effect of specific neuronal stimulation in the promotion of axonal regeneration after injury. We found that opto- or chemogenetic modulations of neuronal activity on both dorsal root ganglia and corticospinal motor neurons increase their axonal growth capacity only on permissive substrates.

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Every individual makes a difference: A trinity derived from linking individual brain morphometry, connectivity and mentalising ability Mentalising ability, indexed as the ability to understand others beliefs, feelings, intentions, thoughts, and traits, is a pivotal and fundamental component of human social cognition. How-ever, considering the multifaceted nature of mentalising ability, little research has focused on characterising individual differences in different mentalising components. And even less research has been devoted to investigating how the variance in the structural and functional patterns of the amygdala and hippocampus, two vital subcortical regions of the  social brain, are related to inter-individual variability in mentalising ability. Here, as a first step toward filling these gaps, we exploited inter-subject representational similarity analysis (IS-RSA) to assess relationships between amygdala and hippocampal morphometry (surface-based multivariate morphometry statistics, MMS), connectivity (resting-state functional connectivity, rs-FC) and mentalising ability (interactive mentalisation questionnaire (IMQ) scores) across the participants (N = 24). In IS-RSA, we proposed a novel pipeline, i.e., computing patching and pooling operations-based surface distance (CPP-SD), to obtain a decent representation for high-dimensional MMS data. On this basis, we found significant correlations (i.e., secondorder isomorphisms) between these three distinct modalities, indicating that a trinity existed in idiosyncratic patterns of brain morphometry, connectivity and mentalising ability. Notably, a region-related mentalising specificity emerged from these associations: self-self and self-other mentalisation are more related to the hippocampus, while other-self mentalisation shows a closer link with the amygdala. Furthermore, by utilising the dyadic regression analysis, we observed significant interactions such that subject pairs with similar morphometry had even greater mentalising similarity if they were also similar in rs-FC. Altogether, we demonstrated the feasibility and illustrated the promise in using IS-RSA to study individual differences, deepening our understanding of how individual brains give rise to their mentalising abilities.

O_QD But this general character is contained in every individual character; without individual character there can be no general character. If all individual character were removed, what general character would remain?

C_QD

(MO_SCPLOWAOC_SCPLOW TO_SCPLOWSEC_SCPLOWO_SCPCAP-C_SCPCAPO_SCPLOWTUNGC_SCPLOW - OO_SCPLOWNC_SCPLOW CO_SCPLOWONTRADICTIONC_SCPLOW1)

sentences: High-throughput variant detection using a color-mixing strategy Many diseases are related to multiple genetic alterations along a single gene. Probing for highly multiple (>10) variants in a single qPCR tube is not possible due to a limited number of fluorescence channels and one variant per channel, so many more tubes are needed. Here, we experimentally validate our novel color-mixing strategy that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously. The color-mixing strategy relies on a simple intra-tube assay that can probe for 15 variants as part of an inter-tube assay that can probe for an exponentially increased number of variants. The color-mixing strategy is achieved using multiplex double-stranded toehold probes modified with fluorophores and quenchers; the probes are designed to be quenched or luminous after binding to wildtype or variant templates. We used the color-mixing strategy to probe for 21 pathogenic mutations in thalassemia and to distinguish between heterozygous and homozygous variants in 6 tubes, with a specificity of 99% and a sensitivity of 94%. To support tuberculosis diagnosis, we used the same strategy to simultaneously probe in Mycobacterium tuberculosis for rifampicin-resistance mutations occurring within one 81-bp region and one 48-bp region in rpoB gene, plus five isoniazid-resistance mutations in inhA and katG genes.

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

sentences: Trunk neural crest migratory position and asymmetric division predict terminal differentiation The generation of complex structures during embryogenesis requires the controlled migration and differentiation of cells from distant origins. How migration and differentiation are coordinated and impact each other to form functional structures is not fully understood. Neural crest cells migrate extensively giving rise to many cell types. In the trunk, neural crest migrate collectively forming chains comprised of cells with distinct migratory identities: one leader cell at the front of the group directs migration, while followers track the leader forming the body of the chain. Herein we analysed the relationship between trunk neural crest migratory identity and terminal differentiation. We found that trunk neural crest migration and fate allocation is coherent. Leader cells that initiate movement give rise to the most distal derivativities. Interestingly, the asymmetric division of leaders separates migratory identity and fate. The distal daughter cell retains the leader identity and clonally forms the Sympathetic Ganglia. The proximal sibling migrates as a follower and gives rise to Schwann cells. The sympathetic neuron transcription factor phox2bb is strongly expressed by leaders from early stages of migration, suggesting that specification and migration occur concomitantly and in coordination. Followers divide symmetrically and their fate correlates with their position in the chain.

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Prediction of antibiotic resistant strains of bacteria from their beta-lactamases protein Number of beta-lactamase variants have ability to deactivate ceftazidime antibiotic, which is the most commonly used antibiotic for treating infection by Gram-negative bacteria. In this study an attempt has been made to develop a method that can predict ceftazidime resistant strains of bacteria from amino acid sequence of beta-lactamases. We obtained beta-lactamases proteins from the {beta}-lactamase database, corresponding to 87 ceftazidime-sensitive and 112 ceftazidime-resistant bacterial strains. All models developed in this study were trained, tested, and evaluated on a dataset of 199 beta-lactamases proteins. We generate 9149 features for beta-lactamases using Pfeature and select relevant features using different algorithms in scikit-learn package. A wide range of machine learning techniques (like KNN, DT, RF, GNB, LR, SVC, XGB) has been used to develop prediction models. Our random forest-based model achieved maximum performance with AUROC of 0.80 on training dataset and 0.79 on the validation dataset. The study also revealed that ceftazidime-resistant beta-lactamases have amino acids with non-polar side chains in abundance. In contrast, ceftazidime-sensitive beta-lactamases have amino acids with polar side chains and charged entities in abundance. Finally, we developed a webserver "ABCRpred", for the scientific community working in the era of antibiotic resistance to predict the antibiotic resistance/susceptibility of beta-lactamase protein sequences. The server is freely available at (http://webs.iiitd.edu.in/raghava/abcrpred/).

Key PointsO_LICeftazidime is commonly used to treat infection caused by Gram-negative bacteria.
C_LIO_LIBeta-lactamase is responsible for lysing ceftazidime, make it resistant to bacteria.
C_LIO_LIComparison of resistant and sensitive variants of beta-lactamase.
C_LIO_LIClassification of sensitive and resistant strain of bacteria based on beta-lactamase.
C_LIO_LIPrediction models have been developed using different machine learning techniques.
C_LI

sentences: Serum CSF1 levels are elevated in patients following partial hepatectomy and acetaminophen induced acute liver failure: Reanalysis of previous data A previous study analysed serum CSF1 concentration in patients who had undergone partial hepatectomy and patients with acute liver failure (ALF) induced by acetaminophen. A correlation between low CSF1 and poor prognosis was reported in ALF. In May 2018, an allegation of research misconduct was made against the second author of the published study, D.J.Antoine, who carried out the CSF1 analysis. The current study describes the independent reanalysis of circulating CSF1 concentration in sera from the same patient cohorts. Patient serum CSF1 levels increased following partial hepatectomy. By contrast to the published report, serum CSF1 concentration was greatest in ALF patients who died compared to survivors. The results confirm the relationship between liver injury and circulating serum CSF1.

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A genetically linked pair of NLR immune receptors show contrasting patterns of evolution Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained trans-species in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to responds to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Significance statementPlants have evolved sophisticated defense mechanisms to fend off pathogens. Plant nucleotide-binding leucine-rich repeat receptor (NLR) proteins play crucial roles in detecting pathogen molecules inside plant cells and mounting defense responses. Here, we identified the Pias gene from rice, which encodes the NLR pair Pias-1 "helper" and Pias-2 "sensor." These proteins function together to detect the pathogen molecule AVR-Pias of Magnaporthe oryzae and defend against rice blast disease. Pias is allelic to the previously reported Pia gene. A comparison of Pias/Pia alleles among Oryza species showed that Pias/Pia helper is evolutionarily and functionally conserved, whereas the Pias/Pia sensor shows highly dynamic evolution, with various host domains integrated into similar positions, allowing it to detect a wide variety of pathogen molecules.

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

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

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

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

sentences: Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization Synthetic genomics provides a distinct approach to systematically explore the process of genome evolution in a dynamic manner. SCRaMbLE is an evolutionary system intrinsic to the synthetic yeast genome that can rapidly drive structural variations. Here, we detected over 260,000 rearrangement events after SCRaMbLEing of a synthetic strain harboring 6 synthetic yeast chromosomes. Remarkably, it was found that the rearrangement events exhibited a specific landscape of rearrangement frequency. We further revealed that the landscape was shaped by coordinated effects of chromatin accessibility and spatial contact probability. The rearrangements tend to occur in 3D spatially proximal and chromatin-accessible regions. Enormous numbers of rearrangements by SCRaMbLE provide a driving force to potentiate directed genome evolution and investigation of the population genomic resource offer mechanistic insights into the dynamic genome evolution.

sentences: Post mortem validation of DSI compressed sensing with optical imaging While many useful microstructural indices, as well as orientation distribution functions, can be obtained from multi-shell dMRI data, there is growing interest in exploring the richer set of microstructural features that can be extracted from the full ensemble average propagator (EAP). The EAP can be readily computed from diffusion spectrum imaging (DSI) data, at the cost of a very lengthy acquisition. Compressed sensing (CS) has been used to make DSI more practical by reducing its acquisition time. CS applied to DSI (CS-DSI) attempts to reconstruct the EAP from significantly undersampled q-space data. We present a post mortem validation study where we evaluate the ability of CS-DSI to approximate not only fully sampled DSI but also multi-shell acquisitions with high fidelity. Human brain samples are imaged with high-resolution DSI at 9.4T and with polarization-sensitive optical coherence tomography (PSOCT). The latter provides direct measurements of axonal orientations at microscopic resolutions, allowing us to evaluate the mesoscopic orientation estimates obtained from diffusion MRI, in terms of their angular error and the presence of spurious peaks. We test two fast, dictionary-based, L2-regularized algorithms for CS-DSI reconstruction. We find that, for a CS acceleration factor of R=3, i.e., an acquisition with 171 gradient directions, one of these methods is able to achieve both low angular error and low number of spurious peaks. With a scan length similar to that of high angular resolution multi-shell acquisition schemes, this CS-DSI approach is able to approximate both fully sampled DSI and multi-shell data with high accuracy. Thus it is suitable for orientation reconstruction and microstructural modeling techniques that require either grid- or shell-based acquisitions. We find that the signal-to-noise ratio (SNR) of the training data used to construct the dictionary can have an impact on the accuracy of CS-DSI, but that there is substantial robustness to loss of SNR in the test data. Finally, we show that, as the CS acceleration factor increases beyond R=3, the accuracy of these reconstruction methods degrade, either in terms of the angular error, or in terms of the number of spurious peaks. Our results provide useful benchmarks for the future development of even more efficient q-space acceleration techniques.

sentences: Increased Activation in the Fusiform Face Area to Greebles is a Result of Expertise Training, Not by its Face-likeness In 2011, Brants, Wagemans, & Op de Beeck (JOCN 23:12, pp. 3949-3958) trained eight individuals to become Greeble experts, and found neuronal inversion effects [NIEs; i.e., higher Fusiform Face Area (FFA) activity for upright, rather than inverted Greebles]. These effects were also found for faces, both before and after training. By claiming to have replicated the seminal Greeble training study (i.e., Gauthier, Tarr, Anderson, Skudlarski, & Gore, 1999, Nat Neurosci, 2, 568-573), Brants et al. interpreted these results as participants viewing Greebles as faces throughout training, contrary to the original argument of subjects becoming Greeble experts only after training. However, such a claim presents two issues. First, the behavioral training results of Brants et al. did not replicate those of Gauthier et al (1999), raising concerns of whether the right training regime had been adopted. Second, both a literature review and meta-analysis of NIE in the FFA suggest its unreliability as an index of face(-like) processing. To empirically evaluate these issues, the present study compared two documented training paradigms (i.e., Gauthier & Tarr, 1997, Vision Res, 37, 1673-1682; and Gauthier, Williams, Tarr, & Tanaka, 1998, Vision Res, 38, 2401-2428) and explored their impact on the FFA. The results showed significant increases in the FFA for Greebles, and a clear neural "adaptation" (i.e., decreased activity for faces following Greebles, but not following non-face objects, in the FFA) both only in the Gauthier97 group, and only after training, reflecting clear modulation of expertise following "appropriate" training. In both groups, no clear NIE for faces nor Greebles were found. Collectively, these data invalidate the two assumptions behind the Brants et al. findings, and provide not only the updated support, but also the new evidence, for the perceptual expertise hypothesis of FFA.

sentences: MCPH1 inhibits condensin II during interphase by regulating its SMC2-kleisin interface. The dramatic change in morphology of chromosomal DNAs between interphase and mitosis is one of the defining features of the eukaryotic cell cycle. Two types of enzymes, namely cohesin and condensin confer the topology of chromosomal DNA by extruding DNA loops. While condensin normally configures chromosomes exclusively during mitosis, cohesin does so during interphase. The processivity of cohesins LE during interphase is limited by a regulatory factor called WAPL, which induces cohesin to dissociate from chromosomes via a mechanism that requires dissociation of its kleisin from the neck of SMC3. We show here that a related mechanism may be responsible for blocking condensin II from acting during interphase. Cells from patients carrying mutations in the Mcph1 gene undergo premature chromosome condensation but it has never been established for certain whether MCPH1 regulates condensin II directly. We show that deletion of Mcph1 in mouse embryonic stem cells unleashes an activity of condensin II that triggers formation of compact chromosomes in G1 and G2 phases, which is accompanied by enhanced mixing of A and B chromatin compartments, and that this occurs even in the absence of CDK1 activity. Crucially, inhibition of condensin II by MCPH1 depends on the binding of a short linear motif within MCPH1 to condensin IIs NCAPG2 subunit. We show that the activities of both Cohesin and Condensin II may be restricted during interphase by similar types of mechanisms as MCPH1s ability to block condensin IIs association with chromatin is abrogated by the fusion of SMC2 with NCAPH2. Remarkably, in the absence of both WAPL and MCPH1, cohesin and condensin II transform chromosomal DNAs of G2 cells into chromosomes with a solenoidal axis showing that both SMC complexes must be tightly regulated to adjust both the chromatids structure and their segregation.

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Increasing neural network robustness improves match to macaque V1 eigenspectrum, spatial frequency preference and predictivity Task-optimized convolutional neural networks (CNNs) show striking similarities to the ventral visual stream. However, human-imperceptible image perturbations can cause a CNN to make incorrect predictions. Here we provide insight into this brittleness by investigating the representations of models that are either robust or not robust to image perturbations. Theory suggests that the robustness of a system to these perturbations could be related to the power law exponent of the eigenspectrum of its set of neural responses, where power law exponents closer to and larger than one would indicate a system that is less susceptible to input perturbations. We show that neural responses in mouse and macaque primary visual cortex (V1) obey the predictions of this theory, where their eigenspectra have power law exponents of at least one. We also find that the eigenspectra of model representations decay slowly relative to those observed in neurophysiology and that robust models have eigenspectra that decay slightly faster and have higher power law exponents than those of non-robust models. The slow decay of the eigenspectra suggests that substantial variance in the model responses is related to the encoding of fine stimulus features. We therefore investigated the spatial frequency tuning of artificial neurons and found that a large proportion of them preferred high spatial frequencies and that robust models had preferred spatial frequency distributions more aligned with the measured spatial frequency distribution of macaque V1 cells. Furthermore, robust models were quantitatively better models of V1 than non-robust models. Our results are consistent with other findings that there is a misalignment between human and machine perception. They also suggest that it may be useful to penalize slow-decaying eigenspectra or to bias models to extract features of lower spatial frequencies during task-optimization in order to improve robustness and V1 neural response predictivity.

Author summaryConvolutional neural networks (CNNs) are the most quantitatively accurate models of multiple visual areas. In contrast to humans, however, their image classification behaviour can be modified drastically by human-imperceptible image perturbations. To provide insight as to why CNNs are so brittle, we investigated the image features extracted by models that are robust and not robust to these image perturbations. We found that CNNs had a preference for high spatial frequency image features, unlike primary visual cortex (V1) cells. Models that were more robust to image perturbations had a preference for image features more aligned with those extracted by V1 and also improved predictions of neural responses in V1. This suggests that the dependence on high-frequency image features for image classification may be related to the image perturbations affecting models but not humans. Our work is consistent with other findings that CNNs may be relying on image features not aligned with those used by humans for image classification and suggests possible optimization targets to improve the robustness of and the V1 correspondence of CNNs.

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MUCOSECRETORY LUNG DISEASE: DIFFERENT ASSEMBLIES OF JAG1 AND JAG2 DETERMINE TRACHEOBRONCHIAL CELL FATE Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study used primary human bronchial air-liquid- interface cultures, gamma secretase inhibition, and neutralizing antibodies to show: 1) JAG1 and JAG2 were necessary for secretory progenitor cell fate determination; 2) JAG2 suppressed squamous differentiation; and 3) pausing of the ciliated cell differentiation process after Notch inhibition. Histological, cell fractionation, cell surface biotinylation, and ubiquitination analyses demonstrated that all cells were JAG1 positive but that little JAG1 was present on the cell surface. In contrast, JAG2 was expressed in a positive-negative pattern and was abundant on the cell surface. Glycogen synthase kinase 3 (GSK3) and tankyrase inhibition studies showed that GSK3 regulated JAG2 trafficking, and that this mechanism was WNT-independent. Collectively, these data indicate that variation in JAG2 trafficking creates regions of high, medium, and low ligand expression. Thus, distinct assemblies of JAG1 and JAG2 may regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/478334v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@37bb51org.highwire.dtl.DTLVardef@eaeaf5org.highwire.dtl.DTLVardef@e791dcorg.highwire.dtl.DTLVardef@1193d73_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Different assemblies of JAG1 and JAG2 may determine Notch signal strength and cell fate within the tracheobronchial epithelium. A cell which interacts with JAG1+ cells (blue squares) receives a low Notch signal (light yellow square). A cell which interacts with a mixture of JAG1+ and JAG1+/JAG2+ cells (purple squares) receives a medium (med) Notch signal (medium yellow square). A cell which interacts with JAG1+/JAG2+ cells receives a high Notch signal (bright yellow square).

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Genome-scale metabolic modelling enables deciphering ethanol metabolism via the acrylate pathway in the propionate-producer Anaerotignum neopropionicum BackgroundMicrobial production of propionate from diluted streams of ethanol (e.g., deriving from syngas fermentation) is a sustainable alternative to the petrochemical production route. Yet, few ethanol-fermenting propionigenic bacteria are known, and understanding of their metabolism is limited. Anaerotignum neopropionicum is a propionate-producing bacterium that uses the acrylate pathway to ferment ethanol and CO2 to propionate and acetate. In this work, we used computational and experimental methods to study the metabolism of A. neopropionicum and, in particular, the pathway for conversion of ethanol into propionate.

ResultsOur work describes iANEO_SB607, the first genome-scale metabolic model (GEM) of A. neopropionicum. The model was built combining the use of automatic tools with an extensive manual curation process, and it was validated with experimental data from this and published studies. The model predicted growth of A. neopropionicum on ethanol, lactate, sugars and amino acids, matching observed phenotypes. In addition, the model was used to implement a dynamic flux balance analysis (dFBA) approach that accurately predicted the fermentation profile of A. neopropionicum during batch growth on ethanol. A systematic analysis of the metabolism of A. neopropionicum combined with model simulations shed light into the mechanism of ethanol fermentation via the acrylate pathway, and revealed the presence of the electron-transferring complexes NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) and acryloyl-CoA reductase-EtfAB, identified for the first time in this bacterium.

ConclusionsThe realisation of the GEM iANEO_SB607 is a stepping stone towards the understanding of the metabolism of the propionate-producer A. neopropionicum. With it, we have gained insight into the functioning of the acrylate pathway and energetic aspects of the cell, with focus on the fermentation of ethanol. Overall, this study provides a basis to further exploit the potential of propionigenic bacteria as microbial cell factories.

sentences: Multiple mechanisms of photoreceptor spectral tuning following loss of UV color vision in Heliconius butterflies Color vision modifications occur in animals via a process known as spectral tuning. In Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. In the H. melpomene and H. ismenius lineages, the UV2 receptor has been lost. Here we compare how loss of the UV2 photoreceptor has altered the visual system of these butterflies. We compare visual system evolution in three Heliconius butterfly species using a combination of intracellular recordings, ATAC-seq, and antibody staining. We identify several spectral tuning mechanisms including adaptive evolution of opsins, deployment of two types of filtering pigments, and co-expression of two distinct opsins in the same cell. Our data show that opsin gain and loss is driving rapid divergence in Heliconius visual systems via tuning of multiple spectral classes of photoreceptor in distinct lineages, potentially contributing to ongoing speciation in this genus.

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Fusion-oncogenes are associated with increased metastatic capacity and persistent disease in pediatric thyroid cancers BackgroundIn 2014, data from a comprehensive multiplatform analysis of 496 adult papillary thyroid cancer samples reported by The Cancer Genome Atlas project suggested that reclassification of thyroid cancer into molecular subtypes, RAS-like and BRAF-like, better reflects clinical behavior than sole reliance on pathological classification. The aim of this study was to categorize the common oncogenic variants in pediatric differentiated thyroid cancer and investigate if mutation subtype classification correlated with the risk of metastasis and response to initial therapy in pediatric DTC.

MethodsSomatic cancer gene panel analysis was completed on DTC from 131 pediatric patients. DTC were categorized into RAS-mutant (H-K-NRAS), BRAF-mutant (BRAF p.V600E) and RET/NTRK fusion (RET, NTRK1 and NTRK3 fusions) to determine differences between subtype classification in regard to pathological data (AJCC TNM) as well as response to therapy 1-year after initial treatment had been completed.

ResultsMutation-based subtype categories were significant in most variables, including age at diagnosis, metastatic behavior, and the likelihood of remission at 1-year. Patients with RET/NTRK fusions were significantly more likely to have advanced lymph node and distant metastasis and less likely to achieve remission at one year than patients within RAS- or BRAF-mut subgroups.

ConclusionsOur data supports that genetic subtyping of pediatric DTC more accurately reflects clinical behavior than sole reliance on pathological classification with patients with RET/NTRK fusions having worse outcomes than those with BRAF-mutant disease. Future trials should consider inclusion of molecular subtype into risk stratification.

sentences: Versatile, do-it-yourself, low-cost spinning disk confocal microscope Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (~400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers.

sentences: Immunometabolic hijacking of immune cells by a Pseudomonas aeruginosa quorum-sensing signal Macrophages utilize metabolic pathways to generate energy and metabolites that may be vulnerable to pathogen hijacking to favor pathogen survival and persistence. It is unclear how bacterial pathogens alter metabolic pathways in immune cells for their benefit and persistence in the infected host. We have shown that the Pseudomonas aeruginosa quorum sensing (QS) signal molecule 2-aminoacetophenone (2-AA) allows pathogen persistence in host tissues by triggering host tolerization via histone deacetylase (HDAC)1-mediated epigenetic reprogramming. Here, we provide strong evidence that 2-AA-meditated persistence is linked to specific metabolic pathway alterations that reduce energy availability and biosynthetic macromolecules involved in host immune responses. 2-AA promotes a Warburg-like metabolic reprogramming effect, thereby increasing levels of lactate, which repressed inflammatory signaling in macrophages. Moreover, it interferes with pyruvate translocation to mitochondria, reducing mitochondrial (mt)-oxidative phosphorylation (OXPHOS) due to down-regulation of estrogen-regulated receptor (ERR) and mitochondrial pyruvate carrier (MPC)-1. This metabolic reprogramming dampened energy production, reduced the acetyl-CoA pool, and generated an anti-inflammatory milieu that favors P. aeruginosa persistence. These findings provide evidence of first-in-class metabolic reprogramming in immune cells mediated by a QS signaling molecule. The specific metabolic programs affected provide insights that may guide the design and development of therapeutics and protective interventions against pathogen-induced immunometabolic alterations and persistence factors.

sentences: Analysing and simulating energy-based models in biology using BondGraphTools Like all physical systems, biological systems are constrained by the laws of physics. However, mathematical models of biochemistry frequently neglect the conservation of energy, leading to unrealistic behaviour. Energy-based models that are consistent with conservation of mass, charge and energy have the potential to aid the understanding of complex interactions between biological components, and are becoming easier to develop with recent advances in experimental measurements and databases. In this paper, we motivate the use of bond graphs (a modelling tool from engineering) for energy-based modelling and introduce, BondGraphTools, a Python library for constructing and analysing bond graph models. We use examples from biochemistry to illustrate how BondGraphTools can be used to automate model construction in systems biology while maintaining consistency with the laws of physics.

sentences: Motivational trade-offs in bumblebees Mammals can supress their nociceptive responses to prioritise other important responses via endogenous modulation from the brain. It is well established that insects display nociception, but not whether the insect brain can modulate nociceptive processing. To address this question, we investigated whether bumblebees (Bombus terrestris) attraction to higher sucrose solution concentrations reduces their avoidance of noxious heat. Bees were given the choice between either unheated or noxiously-heated (55{degrees}C) feeders with different sucrose concentrations. The feeders were associated with colour stimuli to act as conditioned cues. Bees fed more from higher sucrose concentration heated feeders than lower sucrose concentration unheated feeders. Further, bees "testing out" of feeders (landing but not feeding) reduced as the experiment progressed, demonstrating that conditioned colour cues informed the bees behaviour. Therefore, bees trade off competing conditioned motivational stimuli to modulate nocifensive behaviour, suggesting a form of pain perception.

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

sentences: Transcriptomic stability or lability explains sensitivity to climate stressors in coralline algae Crustose coralline algae (CCA) are a group of calcifying red macroalgae crucial to tropical coral reefs because they form crusts that cement together the reef framework1. Previous research into the responses of CCA to ocean warming (OW) and ocean acidification (OA) have found reductions in calcification rates and survival2,3, with magnitude of effect being species-specific. Responses of CCA to OW and OA could be linked to evolutionary divergence time and/or their underlying molecular biology, the role of either being unknown in CCA. Here we show Sporolithon durum, a species from an earlier diverged lineage that exhibits low sensitivity to climate stressors, had little change in metabolic performance and did not significantly alter the expression of any genes when exposed to temperature and pH perturbations. In contrast, Porolithon onkodes, a species from a recently diverged lineage, reduced photosynthetic rates and had over 400 significantly differentially expressed genes in response to experimental treatments, with differential regulation of genes relating to physiological processes. We suggest earlier diverged CCA may be resistant to OW and OA conditions predicted for 2100, whereas taxa from more recently diverged lineages with demonstrated high sensitivity to climate stressors may have limited ability for acclimatisation.

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Dynamic human liver proteome atlas reveals functional insights into disease pathways Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell-type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence-unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time-course of primary cell cultures and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serves as spectral library to characterize a human cohort of non-alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of stellate cell activation resembling liver cirrhosis and providing functional insights. We built a web-based dashboard application for the interactively exploration of our resource.

HighlightsO_LICell-type resolved liver proteome with copy numbers for 10,500 proteins
C_LIO_LITime-course of human liver primary cells uncovers functional proteome shifts
C_LIO_LIA human cohort study reveals liver proteome changes in NASH and cirrhosis
C_LIO_LIAn interactive web portal integrates the results for easy exploration
C_LI

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Adolescent rats extend help to outgroup members, highlighting a neural network for group identity categorization. Prosocial behavior, in particular helping others in need, occurs preferentially in response to the perceived distress of ones own group members, or ingroup. The development of neural mechanisms underlying social selectivity towards ingroup members are not well established. Here, we used a rat helping behavior test to explore the development and neural basis of ingroup bias for prosocial behavior in adolescent rats. We previously found that adult rats selectively help others from their own social group, and that this selectivity is associated with activation in reward and motivation circuits. Surprisingly, we found that adolescent rats helped both ingroup and outgroup members, evidence suggesting that ingroup bias emerges in adulthood. Analysis of brain-wide neural activity, indexed by expression of the early-immediate gene c-Fos, revealed increased activity for ingroup members across a broad set of regions, which was congruent for adults and adolescents. However, adolescents showed reduced hippocampal and insular activity, and increased orbitofrontal cortex activity compared to adults. Adolescent rats who did not help trapped others also demonstrated increased amygdala connectivity. Together, these findings demonstrate that biases for group-dependent prosocial behavior develop with age in rats and suggest that specific brain regions contribute to this prosocial selectivity, overall pointing to possible targets for the functional modulation of ingroup bias.

One Sentence SummaryProsocial selectivity increases with age in parallel with hippocampal and insular activation, providing insight into the neural classification of group membership.

sentences: Epigenetic Remodeling by Vitamin C Potentiates the Differentiation of Mouse and Human Plasma Cells Ascorbate (vitamin C) is an essential micronutrient in humans. The chronic severe deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. Here, from a micronutrient screen, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique, as other antioxidants failed to promote plasma cell differentiation. Ascorbate is critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. Consistent with its role as a cofactor for epigenetic enzymes, ascorbate potentiates plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translocation), the enzymes responsible for DNA demethylation by oxidizing 5-methylcytosines into 5-hydroxymethylcytosine (5hmC). Genome-wide 5hmC profiling identified ascorbate responsive elements (EAR) at the Prdm1 locus, including a distal element with a STAT3 motif overlapped with a CpG that was methylated and modified by TET in the presence of ascorbate. The results suggest that an adequate level of VC is required for antibody response and highlight how micronutrients regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

sentences: A ratchet-like apical constriction drives cell ingression during the mouse gastrulation EMT Epithelial-to-Mesenchymal Transition (EMT) is a hallmark of gastrulation, an evolutionarily conserved developmental process. In mammals epiblast cells lose pluripotency and ingress at the primitive streak to form mesoderm. Cell exit from the epiblast epithelial layer and the associated EMT are dynamically regulated processes involving a stereotypical sequence of cell behaviors. 3D time-lapse imaging combined with cell and tissue scale data analyses of gastrulating mouse embryos revealed the stochastic ingression of epiblast cells at the primitive streak. Ingressing cells asynchronously constrict their apical surfaces in a pulsed ratchet-like fashion through asynchronous shrinkage of apical junctions. Quantitative analyses of the distribution of apical proteins, revealed the anisotropic and complementary distribution of members of the actomyosin network and Crumbs2 complex, potential regulators of asynchronous shrinkage of cell junctions. Analysis of mutants revealed a requirement for Crumbs2 in Myosin2 localization and activity at apical junctions, and as a candidate for regulating actomyosin anisotropy.

sentences: Crossbill: an open access single objective light-sheet microscopy platform We present an open access scanned oblique plane microscopy platform Crossbill. It combines a new optical configuration, open hardware assembly, a systematic alignment protocol, and dedicated control software to provide a compact, versatile, high resolution single objective light-sheet microscopy platform. The demonstrated configuration yields the most affordable sub-micron resolution oblique plane microscopy system to date. We add galvanometer enabled tilt-invariant lateral scan for multi-plane, multi-Hz volumetric imaging capability. A precision translation stage extends stitched field of view to centimeter scale. The accompanying open software is optimized for Crossbill and can be easily extended to include alternative configurations. Using Crossbill, we demonstrate large volume structural fluorescence imaging with sub-micron lateral resolution in zebrafish and mouse brain sections. Crossbill is also capable of multiplane functional imaging, and time-lapse imaging. We suggest multiple alternative configurations to extend Crossbill to diverse microscopy applications.

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Development of white matter tracts between and within the dorsal and ventral streams The degree of interaction between the ventral and dorsal visual streams has been discussed in multiple scientific domains for decades. Recently, several white matter tracts that directly connect cortical regions associated with the dorsal and ventral streams have become possible to study due to advancements in automated and reproducible methods. The developmental trajectory of this set of tracts, here referred to as the posterior vertical pathway (PVP), has yet to be described. We propose an input-driven model of white matter development and provide evidence for the model by focusing on the development of the PVP. We used reproducible, cloud-computing methods and diffusion imaging from adults and children (ages 5-8 years) to compare PVP development to that of tracts within the ventral and dorsal pathways. PVP microstructure was more adult-like than dorsal stream microstructure, but less adult-like than ventral stream microstructure. Additionally, PVP microstructure was more similar to the microstructure of the ventral than the dorsal stream and was predicted by performance on a perceptual task in children. Overall, results suggest a potential role for the PVP in the development of the dorsal visual stream that may be related to its ability to facilitate interactions between ventral and dorsal streams during learning. Our results are consistent with the proposed model, suggesting that the microstructural development of major white matter pathways is related, at least in part, to the propagation of sensory information within the visual system.

Significance StatementUnderstanding white matter development is important to building predictive models that can inform interventions and targeted educational methods. We propose and provide evidence for an input-driven model of white matter development. We tested an uncharacterized aspect of human brain development. Namely, how the recently described posterior vertical white matter tracts develop. Our results suggest a developmental progression along the known, direct anatomical connections from posterior visual areas to anterior ventral and dorsal areas. Our results suggest fundamental biological mechanisms that clarify the role of white matter in predicting human learning and behavior.

sentences: Characterization and rescue by oxytocin of an atypical thermo-sensory reactivity in neonatal mice lacking the autism-associated gene Magel2. Atypical responses to sensory stimuli are considered as a core aspect and early life marker of autism spectrum disorders (ASD). Although recent findings performed in mouse ASD genetic models report sensory deficits, these were explored exclusively during juvenile or adult period. Whether sensory dysfunctions might be present at the early life stage and rescued by therapeutic strategy are fairly uninvestigated. Here we identified that neonatal mice lacking the autism-associated gene Magel2 fail to react to cool sensory stimuli, while autonomic thermoregulatory function is active. This neonatal deficit was mimicked in control neonates by chemogenetic inactivation of oxytocin neurons. Importantly, intranasal administration of oxytocin was able to rescue the phenotype and brain Erk signaling impairment in mutants. This preclinical study establishes for the first-time early life impairments in thermosensory integration and shows the therapeutic potential benefits of intranasal oxytocin treatment on neonatal atypical sensory reactivity.

sentences: Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples -- so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fuorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample -- a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

sentences: Circulating osteopontin released by injured kidneys causes pulmonary inflammation and edema Multiorgan failure is devastating, and its mechanisms and mediators are not clear. Tissue injury in one organ appears to trigger disease in remote organs. Kidney and lung are frequently affected, such as when acute kidney injury (AKI) causes acute lung injury (ALI), a frequent clinical condition with high mortality. Here we identify factors secreted from the injured kidney that cause acute lung injury. We developed a murine model mimicking the generation of respiratory failure following acute kidney injury. To identify interorgan crosstalk mediators involved, we performed scRNAseq of mouse kidneys and lungs after AKI. We then applied ligand-receptor (L-R) pairing analysis across cells residing in kidney (ligands) or lung (receptors) to identify kidney-released circulating osteopontin (OPN) as a novel mediator of AKI-induced ALI (AKI-ALI). OPN release very early after AKI largely from tubule cells triggered neutrophil and macrophage infiltration into lungs associated with endothelial leakage, interstitial edema, and functional impairment. Pharmacological or genetic inhibition of OPN prevented AKI-ALI. Transplantation of ischemic wt kidneys into wt mice caused AKI-ALI, while transplantation of ischemic OPN-global-knockout kidneys failed to induce lung endothelial leakage and AKI-ALI, identifying circulating kidney-released OPN as sufficient to cause AKI-ALI in vivo. We show that AKI in humans results in elevations in OPN levels in the serum. Increased serum OPN levels in patients with multiorgan failure have been shown to positively correlate with reduced kidney function, respiratory failure, and mortality. Thus, our results identifying OPN as a mediator of AKI-ALI may have important therapeutic implications in human AKI-ALI and multiorgan failure.

sentences: Short duration overnight cattle kraaling in natural rangelands: does time after kraal use affect their utilization by wildlife and above ground grass parameters? Short duration overnight cattle kraaling in natural rangelands creates nutrients hotspots attractive to a diverse suite of large mammalian herbivores. However, few studies have determined the use of these sites by large mammalian herbivores. We determined the number of animal sightings per day from camera traps and used them as proxies for use of these newly created nutrient hotspots of varying ages (1, 2, 3 and 4 years) and surrounding vegetation. Six mammalian herbivores of different sizes belonging to three feeding guilds viz. grazers (Burchells zebra Equus quagga burchelli and warthog Phacochoerus africanus), mixed feeders (impala Aepyceros melampus and African savanna elephant Loxodonta africana africana) and browsers (northern giraffe Giraffa camelopardalis giraffa and greater kudu Tragelaphus strepsiceros) frequently used these nutrient hotspots. The number of sightings per day of mammalian herbivores was determined during three periods of the year (January - wet season; June - early dry season; October - late dry season) to ascertain their use of these nutrient hotspots. In addition, above ground grass biomass and height was measured and related to grazer sightings. Furthermore, we tested if repeated grazing in the newly created nutrient hotspots stimulated grass compensatory growth. All the mammalian herbivores used the newly created nutrient hotspots similarly throughout the year, with impala the most active users. Grazer and browser use of nutrient hotspots was not influenced by their age, while mixed feeders mostly used the one year old sites. Grazer use of nutrient hotspots was not influenced by aboveground grass biomass and height. Repeated clipping (proxy for grazing) resulted in compensatory aboveground grass biomass growth in nutrient hotspots. Impala benefited the most and zebra the least from the creation of nutrient hotspots in natural rangelands. We conclude that creation of nutrients hotspots through short duration overnight kraaling results in rangeland heterogeneity that improves availability of herbivore foraging sites.

sentences: The oxytocin system regulates tearing Tears are an exocrine physiological fluid secreted onto the ocular surface from the lacrimal apparatus in all mammals. Limited research has been conducted on the functional neuronal circuitry of tear production. In particular, the neuronal mechanisms of emotional tearing, which is a physiological reaction harmonized with enhanced emotional arousal and assumed to be unique to humans, remain unclear. We identified that the oxytocin neurons in the paraventricular hypothalamus is functionally projected to the oxytocin receptor-expressing neurons in the lacrimation center of the superior salivatory nucleus. Optogenetic activation or inhibition of these neurons and/or receptors can modulate the superior salivatory nucleus dependent tear secretion mediated through oxytocin. Moreover, we identified that maternal behavior, nociceptive behavior stimulation, and aversive memory retrieval are linked to tearing in mice, and that these emotional linked tearing are suppressed by optogenetic inhibition of the corresponding oxytocin system. Thus, tearing could be regulated through functional connections between central oxytocin systems in the paraventricular hypothalamus and the superior salivatory nucleus.

sentences: A Unified Genomic Mechanism of Cell-Fate Change The purpose of our studies is to elucidate the nature of massive control of whole genome expression with a particular emphasis on cell-fate change. Whole genome expression is coordinated by the emergence of a critical point (CP: a peculiar set of bi-phasic genes) through the genome-engine. In response to stimuli, the genome expression self-organizes three critical states, each exhibiting distinct collective behaviors with its center of mass acting as a local attractor, coexisting with whole genome attractor (GA). Genome-engine mechanism accounts for local attractors interaction in phase space. The CP acts as the organizing center of cell-fate change, and its activation makes local perturbation spread over the genome affecting GA. The activation of CP is in turn elicited by  hot-spots, genes with elevated temporal variance, normally in charge to keep genome expression at pace with microenvironment fluctuations. When hot-spots oscillation exceeds a given threshold, the CP synchronizes with the GA driving genome expression state transition. The expression synchronization wave invading the entire genome depends on the power law fusion-bursting dynamics of silencing pericentromere-associated heterochromatin domains and the consequent folding-unfolding status of transcribing euchromatin domains. The proposed mechanism is a unified step toward a time-evolutional transition theory of biological regulation.

sentences: Bifurcating Neurons In The Anterior Thalamic Nuclei The anterior thalamic nuclei (ATN) form a nodal point within a distributed memory network. The conventional view of ATN function describes segregated efferents to different terminal regions. By contrast, we found that bifurcating neurons are common within the anteromedial nucleus (AM) of the ATN. A substantial proportion of AM neurons ([~]36% within a region of interest) showed collateral projections when one of two retrograde neurotracers, Cholera Toxin Subunit B (CTB) conjugated to either Alexa Fluor(R) 488 or 594, was placed in the medial prefrontal cortex (mPFC) or dorsal subiculum (dSub). A marked degree of collateralization ([~]20% AM neurons) was also found when neurotracers were placed in mPFC and caudal retrosplenial cortex (cRSC); about 10% showed collaterals when the cRSC was paired with either dSub or vHF; the fewest (6%) was found for mPFC paired with the ventral hippocampal formation (vHF). A generally similar range of percentages of bifurcating neurons was found in the adjacent nucleus reuniens (Re). Evidence that AM neurons project simultaneously to many distantly-located structures provides a new perspective on ATN function. These neurons would facilitate direct coordination among key neural structures to support memory and may explain the strong association between the ATN and diencephalic amnesia.

sentences: Evolution and systematics of the Aculeata and kin (Hymenoptera),with emphasis on the ants (Formicoidea: {dagger}@@@idae fam. nov., Formicidae). Fossils provide unique opportunity to understand the tempo and mode of evolution and are essential for modeling the history of lineage diversification. Here, we interrogate the Mesozoic fossil record of the Aculeata, with emphasis on the ants (Formicidae), and conduct an extended series of ancestral state estimation exercises on distributions of tip-dated combined-evidence phylogenies. We developed and illustrated from ground-up a series of 576 morphological characters which we scored for 144 extant and 431 fossil taxa, including all families of Aculeata, Trigonaloidea, Evanioidea, and {dagger}Ephialtitoidea. We used average posterior probability support to guide composition of a target matrix of 303 taxa, for which we integrated strongly filtered ultraconserved element (UCE) data for 115 living species. We also implemented reversible jump MCMC (rjMCMC) and hidden state methods to model complex behavioral characters to test hypotheses about the pathway to obligate eusociality. In addition to revising the higher classification of all sampled groups to family or subfamily level using estimated character polarities to diagnose nodes across the phylogeny, we find that the mid-Cretaceous genera {dagger}Camelomecia and {dagger}Camelosphecia form a clade which is robustly supported as sister to all living and fossil Formicidae. For this reason, we name this extinct clade as {dagger}@@@idae fam. nov. and provide a definition for the expanded Formicoidea. Based on our results, we recognize three major phases in the early evolution of the ants: (1) origin of Formicoidea as ground-adapted huntresses during the Late Jurassic in the "stinging aggressor" guild (Aculeata) among various lineages of "sneaking parasitoids" (non-aculeate Vespina); (2) the first formicoid radiation during the Early Cretaceous, by the end of which all major extant linages originated; and (3) turnover of the Formicoidea at the end-Cretaceous leading to the second formicoid radiation. We conclude with a concentrated series of considerations for future directions of study with this dataset and beyond.

sentences: Glucoregulation and coping behavior after chronic stress: sex differences across the lifespan Exposure to prolonged stress during adolescence taxes adaptive and homeostatic processes leading to deleterious behavioral and metabolic outcomes. Although previous pre-clinical studies found effects of early life stress on cognition and stress hormone reactivity, these studies largely focused on males. The purpose of the current study was to determine how biological sex shapes behavioral coping and metabolic health across the lifespan after chronic stress. We hypothesized that examining chronic stress-induced behavioral and endocrine outcomes would reveal sex differences in the biological basis of susceptibility. During the late adolescent period, male and female Sprague-Dawley rats experienced chronic variable stress (CVS). Following completion of CVS, all rats experienced a forced swim test (FST) followed 3 days later by a fasted glucose tolerance test (GTT). The FST was used to determine coping in response to a stressor. Endocrine metabolic function was evaluated in the GTT by measuring glucose and corticosterone, the primary rodent glucocorticoid. Animals then aged to 15 months when the FST and GTT were repeated. In young animals, chronically stressed females exhibited more passive coping and corticosterone release in the FST. Additionally, chronically stressed females had elevated corticosterone and impaired glucose clearance in the GTT. Aging affected all measurements as behavioral and endocrine outcomes were sex specific. Furthermore, regression analysis between hormonal and behavioral responses identified associations depending on sex and stress. Collectively, these data indicate female susceptibility to the effects of chronic stress during adolescence. Further, translational investigation of coping style and glucose homeostasis may identify biomarkers for stress-related disorders.

sentences: Dissecting the microenvironment around biosynthetic scaffolds in murine skin wound healing Structural properties of biomaterials play critical roles in guiding cell behaviors and influence the immune response against them. We fabricated electrospun membranes with three types of surface topography (Random, Aligned, and Latticed). The aligned membranes showed immunomodulatory ability, and led to faster wound healing, reduced fibrotic response and enhanced regeneration of cutaneous appendages when used in skin wound repair. Based on that, we performed single-cell RNA sequencing analysis on cells from wounded mouse skin in the presence or absence of the Aligned scaffold. Keratinocytes, fibroblasts, and immune cells including neutrophils, monocytes, macrophages, dendritic cells, and T cells showed diverse cellular heterogeneity. More hair follicle progenitor cells, inner root sheath cells (anagen-related) and fibroblast subsets were found in the Aligned group, which corresponded to the improved regeneration of hair follicles and faster wound closure in the presence of scaffold. Immune responses towards the biomaterial differed from that of control group. In aligned samples, infiltrated macrophages and neutrophils were reduced, whereas more effector T cells were recruited. The time course of immune response was possibly advanced towards an adaptive immunity-dominant stage by the scaffold. The microenvironment around scaffold involved intricate interplay of immune cells and cutaneous cells, and wound healing was the comprehensive results of numerous influencing factors working together.

sentences: Tracheal motile cilia in mice require CAMSAP3 for formation of central microtubule pair and coordinated beating Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a  transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

sentences: Diet-dependent Natriuretic Peptide Receptor C expression in adipose tissue is mediated by PPARγ via long-range distal enhancers In addition to their established role to maintain blood pressure and fluid volume, the cardiac natriuretic peptides (NPs) can stimulate adipocyte lipolysis and control the brown fat gene program of nonshivering thermogenesis. The NP "clearance" receptor C (NPRC) functions to clear NPs from the circulation via peptide internalization and degradation and thus is an important regulator of NP signaling and adipocyte metabolism. It is well appreciated that the Nprc gene is highly expressed in adipose tissue and is dynamically regulated with nutrition and environmental changes. However, the molecular basis for how Nprc gene expression is regulated is still poorly understood. Here we identified Peroxisome Proliferator-Activated Receptor gamma (PPAR{gamma}) as a transcriptional regulator of Nprc expression in mouse adipocytes. During 3T3-L1 adipocyte differentiation, levels of Nprc expression increase in parallel with PPAR{gamma} induction. Rosiglitazone, a classic PPAR{gamma} agonist, increases, while siRNA knockdown of PPAR{gamma} reduces, Nprc expression in 3T3-L1 adipocytes. We demonstrate that PPAR{gamma} controls Nprc gene expression in adipocytes through its long-range distal enhancers. Furthermore, the induction of Nprc expression in adipose tissue during high-fat diet feeding is associated with increased PPAR{gamma} enhancer activity. Our findings define PPAR{gamma} as a mediator of adipocyte Nprc gene expression and establish a new connection between PPAR{gamma} and the control of adipocyte NP signaling in obesity.

sentences: An environmentally responsive transcriptional state modulates cell identities during root development Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this cell state reveals new layers of cell identity that may underpin the adaptive potential of plant development.

sentences: High permeability of the CSF flow in the juvenile mouse brain by brain-wide DCE-MRI The vasculature system with a lymphatic function in the brain is manifested as meningeal lymphatic vessels and the glymphatic system, which drain waste products from cerebrospinal fluid (CSF) and produce interstitial fluid. Invasion of stimulated immune cells or inflammatory cytokines during the maturation is regarded as a sign of the disruption of brain functions through excessive immune stresses. However, it is unclear when the lymphatic system is functionally completed and in which parts of the organ the brain immunity is privileged (i.e., the blood-brain barrier is fully established). Here, we visualized the whole-head signal of gadolinium (Gd) contrast agents in mouse brains to investigate the CSF flow from the signals of Gd-contrast agents using magnetic resonance imaging (MRI) in the course of developmental stages. We found higher Gd-signals in the olfactory bulbs, prefrontal cortex, parietal surface regions of the neocortex along with dorsal meningeal lymphatic vessels, ventral midbrain, and a part of cerebellum, as well as in the basal brain regions, at the immature stage of postnatal (P) 4 weeks, compared to the P8-12 weeks. Our results suggest that the barrier of the vasculature system in mouse brains is still permeable until P1-month and small molecules can leak into the parenchyma.

sentences: Structured random receptive fields enable informative sensory encodings Brains must represent the outside world so that animals survive and thrive. In early sensory systems, neural populations have diverse receptive fields structured to detect important features in inputs, yet significant variability has been ignored in classical models of sensory neurons. We model neuronal receptive fields as random, variable samples from parametrized distributions in two sensory modalities, using data from insect mechanosensors and neurons of mammalian primary visual cortex. We show that these random feature neurons perform a randomized wavelet transform on inputs which removes high frequency noise and boosts the signal. Our result makes a significant theoretical connection between the foundational concepts of receptive fields in neuroscience and random features in artificial neural networks. Further, these random feature neurons enable learning from fewer training samples and with smaller networks in artificial tasks. This structured random model of receptive fields provides a unifying, mathematically tractable framework to understand sensory encodings across both spatial and temporal domains.

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Host exposure to symbionts and ecological drift generate divergence in parasite community assembly The initial colonization of a host by symbionts, ranging from parasites to mutualists, can generate priority effects that alter within-host interactions and the trajectory of parasite community assembly. At the same time, variation in parasite communities among hosts can also stem from stochastic processes. Community ecology theory posits that multiple processes (e.g. dispersal, selection and drift) interact to generate variation in community structure, but these processes are rarely considered simultaneously during community assembly. To test the role of these processes in a parasite community, we experimentally simulated dispersal of three symbionts by factorially inoculating individual plants of tall fescue with two foliar fungal parasites, Colletotrichum cereale and Rhizoctonia solani, and a hypothesized mutualist endophyte, Epichloe coenophiala. We then tracked parasite infections longitudinally in the field. After the initial inoculations, hosts were exposed to a common pool of parasites in the field, which we expected to cause parasite communities to converge towards a similar community state. To test for convergence, we analyzed individual hosts parasite community trajectories in multivariate space. In contrast to our expectation, there was no signal of convergence. Instead, parasite community trajectories generally diverged over time between treatment groups and the magnitude of divergence depended on the symbiont species inoculated. Parasite communities of hosts that were inoculated with only the mutualist, Epichloe, showed significant trends of divergence relative to all other symbiont inoculation treatments. In contrast, hosts inoculated with only Rhizoctonia did not exhibit clear trends of divergence when compared to other parasite inoculations. Further, co-inoculation with both parasite species resulted in faster rates of divergence and greater temporal change in parasite communities relative to hosts inoculated with only the parasite Colletotrichum. As predicted by existing theory, parasite communities showed evidence of drift during the beginning of the experiment, which contributed to among-host divergence in parasite community structure. Overall, these data provide evidence that initial dispersal of symbionts produced persistent changes in parasite community structure via ecological selection, that drift was important during the early stages of parasite community assembly, and together, dispersal, selection and drift resulted in parasite community divergence.

Open Research statementThe data and code that support the findings of this study are available through Zenodo at https://doi.org/10.5281/zenodo.5714452

sentences: The anti-immune dengue subgenomic flaviviral RNA is found in vesicles in mosquito saliva and associated with increased infectivity Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We show that salivary sfRNA is protected in detergent-sensitive, protease-resistant compartments. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in human cells. Since sfRNA potently inhibits innate immunity in human cells, we posit that sfRNA in mosquito saliva is present in extracellular vesicles that deliver it to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

sentences: Phenotypic plasticity explains apparent reverse evolution of fat synthesis in parasitoid insects Over the last few decades, numerous examples have been described where a trait that was once lost during the course of evolution had been regained. Here, we argue that such reverse evolution can also become apparent when trait expression is plastic in response to the environment. We tested this hypothesis for the loss and regain of fat synthesis in parasitic wasps. Wasps from lineages that supposedly regained lipogenic ability ~80 million years ago were grown under a fat-poor or fat-rich environment. In line with our hypothesis, it turned out that fat synthesis had not been lost and regained, but was only switched on in low-fat environments. Functional protein domains of key lipogenesis genes were also found in other parasitoid species, suggesting that plasticity of fat synthesis may be more widespread in parasitoids. Individual-based simulations then revealed that a switch for plastic expression can remain functional in the genome for thousands of generations, even if it is only used sporadically. The evolution of plasticity may thus also explain other examples of apparent reverse evolution.

sentences: The SKBR3 Cell-Membrane Proteome: Role in Aberrant Cancer Cell Proliferation and Resource for Precision Medicine Applications The plasma membrane proteome resides at the interface between the extra- and intra-cellular environment and through its various roles in signal transduction, immune recognition, nutrient transport, and cell-cell and cell-matrix interactions plays an absolutely critical role in determining the fate of a cell. Our work was aimed at exploring the landscape of the cancer cell-membrane proteome responsible for sustaining uncontrolled cell proliferation, and its intrinsic resources for enabling detection and therapeutic interventions. SKBR3/HER2+ breast cancer cells were used as a model system and mass spectrometry for characterizing the proteome. The number of identified cell-membrane proteins exceeded 2,000, with ~1,300 being matched by two or more unique peptides. Classification in four major categories, i.e., proteins with receptor or enzymatic activity, CD antigens, transporters, and cell adhesion proteins, uncovered overlapping roles in biological processes that drive cell growth, apoptosis, differentiation, immune response, adhesion and migration, as well as capabilities for signaling crosstalk and alternate pathways for proliferation. The large number of tumor markers (>50) and putative drug targets (>100) exposed a vast potential for yet unexplored detection and targeting opportunities, whereas the presence of 15 antigen immunological markers enabled an assessment of epithelial, mesenchymal or stemness characteristics. Serum-starved cells displayed altered processes related to mitochondrial OXPHOS/ATP synthesis, protein folding and localization, while serum-treated cells exhibited attributes that support tissue invasion and metastasis. Altogether, our findings advance the understanding of the biological triggers that sustain aberrant cancer cell proliferation, survival and development of resistance to therapeutic drugs, and reveal the vast innate opportunities for guiding immunological profiling and precision medicine applications aimed at target selection or drug discovery.

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Early life gut microbiome dynamics mediate maternal effects of infant growth in vervet monkeys BackgroundMaternal parity is associated with variation in infant growth across mammals, but the mechanisms underlying this relationship are unclear. Given emerging links between growth and the microbiome, and the importance of maternal microbiota in establishing this community, the assembly of the infant gut microbiome may be a mediator of parity effects on infant growth.

ResultsHere, we analyzed 118 fecal and milk samples from mother-infant vervet monkey dyads across the first 6 months postpartum in a population with high growth-associated infant mortality. Despite poorer milk production, infants born to low parity females were larger at 6 months of age than their counterparts and exhibited divergent patterns in gut microbiome assembly. Gut microbiome alpha diversity increased rapidly from the first days of life to 4 months old in all infants, but infants born to low parity females exhibited reduced gut microbiome alpha diversity during early life. At the taxonomic level, infants broadly exhibited a shift from Bacteroides fragilis to Prevotella dominance. Infants of low parity females housed more B. fragilis in their guts, and B. fragilis dominance drove reduced alpha diversity. Maternal vertical transmission to the infant gut was greater from milk than from the maternal gut, and was greatest among infants born to low parity females. B. fragilis was 15-fold more abundant in milk than in the maternal gut and was greater in the milk of low parity females, suggesting that milk may be the primary maternal reservoir of B. fragilis. Path analyses demonstrated that both infant gut alpha diversity and B. fragilis mediated parity effects on postnatal growth: infants were larger at 6 months old if they exhibited reduced alpha diversity and a greater relative abundance of B. fragilis during early life.

ConclusionThe first days of life are a critical period of infant gut microbiome organization during which the establishment of a less diverse, milk-oriented microbial community abundant in B. fragilis promotes growth among infants born to reproductively inexperienced females.

sentences: Effects of acute and repeated administration of the selective M4 PAM VU0152099 on cocaine vs. food choice in male rats AO_SCPLOWBSTRACTC_SCPLOWLigands that stimulate muscarinic acetylcholine receptors 1 and 4 (M1, M4) have shown promising effects as putative pharmacotherapy for cocaine use disorder in rodent assays. We have previously shown reductions in cocaine effects with acute M4 stimulation, as well as long-lasting, delayed, reductions in cocaine taking and cocaine seeking with combined M1/M4 receptor stimulation or with M1 stimulation alone. M4 stimulation opposes dopaminergic signaling acutely, but direct dopamine receptor antagonists have proved unhelpful in managing cocaine use disorder because they lose efficacy with long-term administration. It is therefore critical to determine whether M4 approaches themselves can remain effective with repeated or chronic dosing. We assessed the effects of repeated administration of the M4 positive allosteric modulator (PAM) VU0152099 in rats trained to choose between intravenous cocaine and a liquid food reinforcer, to obtain quantitative measurement of whether M4 stimulation could produce delayed and lasting reduction in cocaine taking. VU0152099 produced progressively augmenting suppression of cocaine choice and cocaine intake, but produced neither rebound nor lasting effects after treatment ended. To compare and contrast effects of M1 vs. M4 stimulation, we tested whether the M4 PAM VU0152100 suppressed cocaine self-administration in mice lacking CalDAG-GEFI signaling factor, required for M1-mediated suppression of cocaine self-administration. CalDAG-GEFI ablation had no effect on M4-mediated suppression of cocaine self-administration. These findings support the potential usefulness of M4 PAMs as pharmacotherapy to manage cocaine use disorder, alone or in combination with M1-selective ligands, and show that M1 and M4 stimulation modulate cocaine-taking behavior by distinct mechanisms.

sentences: Respiratory Alkalosis Provokes Spike-Wave Discharges in Seizure- Prone Rats Hyperventilation reliably provokes seizures in patients diagnosed with absence epilepsy. Despite this predictable patient response, the mechanisms that enable hyperventilation to powerfully activate absence seizure-generating circuits remain entirely unknown. Using the WAG/Rij rat, an established rodent model of absence epilepsy, we demonstrate that absence seizures are highly sensitive to arterial carbon dioxide, suggesting that seizure-generating circuits are sensitive to pH. Moreover, hyperventilation consistently activated neurons within the intralaminar nuclei of the thalamus, a structure implicated in seizure generation. We show that intralaminar thalamus also contains pH-sensitive neurons. Collectively, these observations suggest that hyperventilation activates pH-sensitive neurons of the intralaminar nuclei to provoke absence seizures.

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Reorganization of F-actin nanostructures is required for the late phases of SARS-CoV-2 replication in pulmonary cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is worldwide the main cause of the COVID-19 pandemic. After infection of human pulmonary cells, intracellular viral replication take place in different cellular compartments resulting in the destruction of the host cells and causing severe respiratory diseases. Although cellular trafficking of SARS-CoV-2 have been explored, little is known about the role of the cytoskeleton during viral replication in pulmonary cells. Here we show that SARS-CoV-2 infection induces dramatic changes of F-actin nanostructures overtime. Ring-like actin nanostructures are surrounding viral intracellular organelles, suggesting a functional interplay between F-actin and viral M clusters during particle assembly. Filopodia-like structures loaded with viruses to neighbour cells suggest these structures as mechanism for cell-to-cell virus transmission. Strikingly, gene expression profile analysis and PKN inhibitor treatments of infected pulmonary cells reveal a major role of alpha-actinins superfamily proteins in SARS-CoV-2 replication. Overall, our results highlight cell actors required for SARS-CoV2 replication that are promises for antiviral targets.

TeaserImpairing regulation of actin filaments inhibits SARS-CoV-2 particle production in human pulmonary cells.

sentences: Crustacean cardiac ganglion model reveals constraints on morphology and conductances The crustacean cardiac ganglion (CG) network coordinates the rhythmic contractions of the heart muscle to control the circulation of blood. The network consists of 9 cells, 5 large motor cells (LC1-5) and 4 small endogenous pacemaker cells (SCs). We report a new three-compartmental biophysical model of an LC that is morphologically realistic and includes provision for inputs from the SCs via a gap-junction coupled spike-initiation-zone (SIZ) compartments. To determine physiologically viable LC models in this realistic configuration, maximal conductances in three compartments of an LC are determined by random sampling from a biologically-characterized 9D-parameter space, followed by a three-stage rejection protocol that checks for conformity with electrophysiological features from single cell traces. LC models that pass the single cell rejection protocol are then incorporated into a network model which is then used in a final rejection protocol stage. Using disparate experimental data, the study provides hitherto unknown structure-function insights related to the crustacean cardiac ganglion large cell, including predictions about morphology including the role of its SIZ, and the differential roles of active conductances in the three compartments. Further, we extend analyses of emergent conductance relationships and correlations in model neurons relative to their biological counterparts, allowing us to make inferences both with respect to the biological system as well as the implications of the ability to detect such relationships in populations of model neurons going forward.

sentences: A Discriminative Characterization of Heschl's Gyrus Morphology using Spectral Graph Features Heschls Gyrus (HG), which hosts the primary auditory cortex, exhibits large variability not only in size but also in its gyrification patterns, within (i.e., between hemispheres) and between individuals. Conventional structural measures such as volume, surface area and thickness do not capture the full morphological complexity of HG, in particular, with regards to its shape. We present a method for characterizing the morphology of HG in terms of Laplacian eigenmodes of surface-based and volume-based graph representations of its structure, and derive a set of spectral graph features that can be used to discriminate HG subtypes. We applied this method to a dataset of 177 adults previously shown to display considerable variability in the shape of their HG, including data from amateur and professional musicians, as well as non-musicians. Results show the superiority of the proposed spectral graph features over conventional ones in differentiating HG subtypes, in particular, single HG versus Common Stem Duplications (CSDs). We anticipate the proposed shape features to be found beneficial in the domains of language, music and associated pathologies, in which variability of HG morphology has previously been established.

sentences: A meta-analysis of Microarray Data is Effective for Identifying Gravity-Sensitive Genes Gravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells to simulated microgravity using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin transporter gene SLCO2A1 decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of SLCO2A1, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses.

sentences: Phenotyping of Klf14 mouse white adipose tissue enabled by whole slide segmentation with deep neural networks White adipose tissue (WAT) plays a central role in metabolism, with multiple diseases and genetic mutations causing its remodeling. Quantitative analysis of white adipocyte size is of great interest to understand physiology and disease, but previous studies of H&E histology have been limited to a subsample of whole depot cross-sections. In this paper, we present the deep learning pipeline DeepCytometer, that can segment mouse and human whole slides ([~=]40,000 cells per mouse slide on average) using an adaptive tiling method, correct for cell overlap and reject non-white adipocytes from the segmentation. Using quantile colour maps we show intra- and inter-depot cell size heterogeneity with local correlation; quantile estimates also suggest significant differences in population estimates from 75 whole slides compared to smaller data sets. We propose three linked levels (body weight BW, depot weight DW and cell area quartiles) for exploratory analysis of mouse Klf14 phenotypes in gonadal and subcutaneous depots. We find a rich set of phenotypes when stratifying by sex, depot and three genotype strata: (1) WTs/Hets with a Het father (Controls), (2) WTs with a Het mother, and (3) Hets with a Het mother (functional KOs or FKOs). Namely, at BW level, mean difference testing suggests that female FKOs are similar to Controls, but WTs with a Het mother are significantly larger. At DW and cell levels, linear models with interaction terms and BW or DW covariates, respectively, reveal phenotypes not shown by difference of means tests. For example, at DW level, gonadal and subcutaneous models are similar, and female FKOs have lower fat percentage than Controls due to both an offset and the DW/BW slope in the linear model. Meanwhile, female WTs with a Het mother have on average similar fat percentage to Controls, but as their slopes are close to zero, their DWs are uncorrelated to BW, suggesting that larger female WTs with a Het mother have lower fat percentage than smaller ones. In contrast to depot level, at cell level female gonadal phenotypes diverge from subcutaneous ones. Furthermore, male Controls and FKOs have similar average area values in subcutaneous depots, but area~DW slope flattening in FKOs suggests that larger DWs could be caused by cell size increase in Controls and by cell count increase in FKOs. Thus, DeepCytometer and associated exploratory analysis reveal new insights into adipocyte heterogeneity and phenotyping.

sentences: Modeling progression of single cell populations through the cell cycle as a sequence of switches AO_SCPLOWBSTRACTC_SCPLOWCell cycle is the most fundamental biological process underlying the existence and propagation of life in time and space. It has been an object for mathematical modeling for long, with several alternative mechanistic modeling principles suggested, describing in more or less details the known molecular mechanisms. Recently, cell cycle has been investigated at single cell level in snapshots of unsynchronized cell populations, exploiting the new methods for transcriptomic and proteomic molecular profiling. This raises a need for simplified semi-phenomenological cell cycle models, in order to formalize the processes underlying the cell cycle, at a higher abstracted level. Here we suggest a modeling framework, recapitulating the most important properties of the cell cycle as a limit trajectory of a dynamical process characterized by several internal states with switches between them. In the simplest form, this leads to a limit cycle trajectory, composed by linear segments in logarithmic coordinates describing some extensive (depending on system size) cell properties. We prove a theorem connecting the effective embedding dimensionality of the cell cycle trajectory with the number of its linear segments. We also develop a simplified kinetic model with piecewise-constant kinetic rates describing the dynamics of lumps of genes involved in S-phase and G2/M phases. We show how the developed cell cycle models can be applied to analyze the available single cell datasets and simulate certain properties of the observed cell cycle trajectories. Based on our modeling, we can predict with good accuracy the cell line doubling time from the length of cell cycle trajectory.

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A central protein complex essential for Invasionin Toxoplasma gondii Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. During invasion, the unique secretory organelles (micronemes and rhoptries) are sequentially released and several micronemal proteins have been suggested to be required for host cell recognition and invasion. However, to date only few micronemal proteins have been demonstrated to be essential for invasion.

Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan specific proteins. In Toxoplasma gondii, two CRMPs are present in the genome. The Kringle domain containing protein (CRMPA) and the GCC2 GCC3 domain containing protein (CRMPB). Here we demonstrate that both proteins form a complex that contains additional micronemal proteins. Disruption of this complex results in a block of rhoptry secretion and parasites being unable to invade the host cell. In conclusion, this complex is a central invasion complex conserved in all apicomplexans.

sentences: Sequential sampling from memory underlies action selection during abstract decision making The study of perceptual decision making in monkeys has provided insights into the process by which sensory evidence is integrated towards a decision. When monkeys make decisions with the knowledge of the motor actions the decisions bear upon, the process of evidence integration is instantiated by neurons involved in the selection of said actions. It is less clear how monkeys make decisions when unaware of the actions required to communicate their choice--what we refer to as  abstract decisions. We investigated this by training monkeys to associate the direction of motion of a noisy random-dot display with the color of two targets. Crucially, the targets were displayed at unpredictable locations after the motion stimulus was extinguished. We found that monkeys postponed decision formation until the targets were revealed. Neurons in the parietal association area LIP represented the integration of evidence leading to a choice, but as the stimulus was no longer visible, the samples of evidence must have been retrieved from short-term memory. Our results imply that when decisions are temporally unyoked from the motor actions they bear upon, decision formation is protracted until they can be framed in terms of motor actions.

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Population-level asymmetry of the cerebral cortex: reproducibility, lifespan changes, heritability, and individual differences Cortical asymmetry is a ubiquitous feature of brain organization that is altered in neurodevelopmental disorders and aging. Achieving consensus on cortical asymmetries in humans is necessary to uncover the genetic-developmental mechanisms that shape them and factors moderating cortical lateralization. Here, we delineate population-level asymmetry in cortical thickness and surface area vertex-wise in 7 datasets and chart asymmetry trajectories across life (4-89 years; observations = 3937; 70% longitudinal). We reveal asymmetry interrelationships, heritability, and test associations in UK Biobank (N=~37,500). Cortical asymmetry was robust across datasets. Whereas areal asymmetry is predominantly stable across life, thickness asymmetry grows in development and declines in aging. Areal asymmetry correlates in specific regions, whereas thickness asymmetry is globally interrelated across cortex and suggests high directional variability in global thickness lateralization. Areal asymmetry is moderately heritable (max h2SNP ~19%), and phenotypic correlations are reflected by high genetic correlations, whereas heritability of thickness asymmetry is low. Finally, we detected an asymmetry association with cognition and confirm recently-reported handedness links. Results suggest areal asymmetry is developmentally stable and arises in early life, whereas developmental changes in thickness asymmetry may lead to directional variability of global thickness lateralization. Our results bear enough reproducibility to serve as a standard for future brain asymmetry studies.

SignificanceCortical asymmetry is reduced in neurodevelopmental disorders, yet we lack knowledge of how cortical asymmetry development proceeds across life in health. We provide a definitive reference for asymmetry in the cerebral cortex. We find areal asymmetry is stable from childhood to old age, and specific areal asymmetries are formed under common genetic-developmental influence. In contrast, thickness asymmetry shows developmental growth, and is globally interrelated in a pattern suggesting highly left-lateralized individuals tend towards left-lateralization also in right-asymmetric regions (and vice versa). Heritability mapping also supported a prenatal-postnatal developmental dichotomy for areal and thickness asymmetry, and we find reduced asymmetry in the most lateralized brain region associates with reduced cognition. Our results provide novel insights into normal brain organization and development.

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Does the posteromedial cortex play a primary role for the capacity for consciousness in rats? It remains unclear how specific cortical regions contribute to the brains overall capacity for consciousness. Clarifying this could help distinguish between theories of consciousness. Here, we investigate the association between markers of regionally specific (de)activation and the brains overall capacity for consciousness.

We recorded electroencephalographic (EEG) responses to cortical electrical stimulation in 6 rats, and computed Perturbational Complexity Index state-transition (PCIST), which has been extensively validated as an index of the capacity for consciousness in humans. We also estimated the balance between activation and inhibition of specific cortical areas with the ratio between high and low frequency power (HF/LF) from spontaneous EEG activity at each electrode. We repeated these measurements during wakefulness, and under the influence of ketamine anaesthesia at two doses: the minimal dose needed to induce behavioural unresponsiveness and twice this dose.

We found that PCIST was only slightly reduced from wakefulness to light ketamine anaesthesia, but dropped significantly down with deeper anaesthesia. The high-dose effect was selectively associated with reduced HF/LF ratio in the posteromedial cortex, which strongly correlated with PCIST. Conversely, behavioural unresponsiveness induced by light ketamine anaesthesia, was associated with similar spectral changes in frontal, but not posterior cortical regions.

These findings seem to support the claim that the posteromedial cortex may play a primary role for the capacity for consciousness. Such region-specific associations between cortical activation and the overall capacity for consciousness must be accounted for by theories of consciousness.

sentences: Increased 20-HETE signaling suppresses neurovascular coupling after ischemic stroke in regions beyond the infarct Neurovascular coupling, the process by which neuronal activity elicits increases in the local blood supply, is impaired in stroke patients in brain regions outside the infarct. Such impairment may contribute to neurological deterioration over time, but its mechanism is unknown. Using the middle cerebral artery occlusion (MCAO) model of stroke, we show that neuronal activity-evoked capillary dilation is reduced by [~]75% in the intact cortical tissue outside the infarct border. This decrease in capillary responsiveness was not explained by a decrease in local neuronal activity or a loss of vascular contractility. Inhibiting synthesis of the vasoconstrictive molecule 20-HETE, either by inhibiting its synthetic enzyme CYP450 {omega}-hydroxylases or by increasing nitric oxide (NO), which is a natural inhibitor of {omega}-hydroxylases, rescued activity-evoked capillary dilation. The capillary dilation unmasked by inhibiting 20-HETE was dependent on PGE2 activation of EP4 receptors, a vasodilatory pathway previously identified in healthy animals. Cortical 20-HETE levels were increased following MCAO, in agreement with data from stroke patients. Inhibition of {omega}-hydroxylases normalized 20-HETE levels in vivo and increased cerebral blood flow in the peri-infarct cortex. These data identify 20-HETE-dependent vasoconstriction as a mechanism underlying neurovascular coupling impairment after stroke. Our results suggest that the brains energy supply may be significantly reduced after stroke in regions previously believed to be asymptomatic and that {omega}-hydroxylase inhibition may restore healthy neurovascular coupling post-stroke.

sentences: Facilitating the design of combination therapy in cancer using multipartite network models: Emphasis on acute myeloid leukemia From the drug discovery perspective, combination therapy is recommended in cancer due to efficiency and safety compared to the common cytotoxic and single-targeted monotherapies. However, identifying effective drug combinations is time- and cost-consuming. Here, we offer a novel strategy of predicting potential drug combinations and patient subclasses by constructing multipartite networks using drug response data on patient samples. In the present study, we used Beat AML and GDSC, two comprehensive datasets based on patient-derived and cell line-based samples, to show the potential of multipartite network modeling in cancer combinatorial therapy. We used the median values of cell viability to compare drug potency and reconstruct a weighted bipartite network, which models the interaction of drugs and biological samples. Then, clusters of network communities were identified in two projected networks based on the topological structure of networks. Chemical structures, drug-target networks, protein-protein interactions, and signaling networks were used to corroborate the intra-cluster homogeneity. We further leveraged the community structures within the drug-based multipartite networks to discover effective multi-targeted drug combinations, and the synergy levels which were supported with more evidence using the DrugComb and the ALMANAC databases. Furthermore, we confirmed the potency of selective combinations of drugs against monotherapy in vitro experiment using three acute myeloid leukemia (AML) cell lines. Taken together, this study presents an innovative data-driven strategy based on multipartite networks to suggest potential drug combinations to improve treatment of AML.

sentences: Glioblastoma embryonic-like stem cells exhibit immune-evasive phenotype Glioma stem cells (GSCs) are a subset of cells with self-renewal and tumor-initiating capacities that are thought to participate in drug resistance and immune evasion mechanisms in glioblastoma (GBM). Given GBM heterogeneity, we hypothesized that GSCs might also display cellular hierarchies associated with different degrees of stemness. We evaluated a single-cell RNA-seq glioblastoma dataset (n = 28) and identified a stem cell population co-expressing high levels of embryonic pluripotency markers, named core glioma stem cells (c-GSCs). This embryonic-like population represents 4.22% {+/-} 0.59 of the tumor cell mass, and pathway analysis revealed an upregulation of stemness and downregulation of immune-associated pathways. Using induced pluripotent stem cell technology, we generated an in vitro model of c-GSCs by reprogramming glioblastoma patient-derived cells into induced c-GSCs (ic-GSCs). Immunostaining of ic-GSCs showed high expression of embryonic pluripotency markers and downregulation of antigen presentation HLA proteins, mimicking its tumoral counterpart. Transcriptomic analysis revealed a strong agreement of enriched biological pathways between tumor c-GSCs and in vitro ic-GSCs ({kappa} = 0.71). Integration of ic-GSC DNA methylation and gene expression with chromatin state analysis of epigenomic maps (n = 833) indicated that polycomb repressive marks downregulate HLA genes in stem-like phenotype. Together, we identified c-GSCs as a GBM cell population with embryonic signatures and poor immunogenicity. Genome-scale transcriptomic and epigenomic profiling provide a valuable resource for studying immune evasion mechanisms governing c-GSCs and identifying potential therapeutic targets for GBM immunotherapy.

sentences: Single amino-acid mutation in a Drosophila melanogaster ribosomal protein: an insight in uL11 transcriptional activity The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co- localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. RNA- seq analysis from wing imaginal discs shows enrichment in the GO categories "glutathione metabolism" for up-regulated genes in both uL11K3A and uL11K3Y mutants and "regulation of transcription" for down-regulated genes in uL11K3A only. Analysis of the cis-regulatory sequences of these genes suggests that uL11 might regulate transcription of target genes in concert with the couple of transcription factors Mad/Med that mediate response to the Bone Morphogenetic Protein (BMP) signaling pathway.

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ACDC: Analysis of Congruent Diversification Classes 1. Diversification rates inferred from phylogenies are not identifiable. There are infinitely many combinations of speciation and extinction rate functions that have the exact same likelihood score for a given phylogeny, building a congruence class. The specific shape and characteristics of such congruence classes have not yet been studied. Whether speciation and extinction rate functions within a congruence class share common features is also not known.

2. Instead of striving to make the diversification rates identifiable, we can embrace their inherent non-identifiable nature. We use two different approaches to explore a congruence class: (i) testing of specific alternative hypotheses, and (ii) randomly sampling alternative rate function within the congruence class.

3. Our methods are implemented in the open-source R package ACDC (https://github.com/afmagee/ACDC). ACDC provides a flexible approach to explore the congruence class and provides summaries of rate functions within a congruence class. The summaries can highlight common trends, i.e. increasing, flat or decreasing rates.

Although there are infinitely many equally likely diversification rate functions, these can share common features. ACDC can be used to assess if diversification rate patterns are robust despite non-identifiability. In our example, we clearly identify three phases of diversification rate changes that are common among all models in the congruence class. Thus, congruence classes are not necessarily a problem for studying historical patterns of biodiversity from phylogenies.

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Decoding the IGF1 Signaling Gene Regulatory Network Behind Alveologenesis from A Mouse Model of Bronchopulmonary Dysplasia Lung development is precisely controlled by underlying Gene Regulatory Networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD), a chronic lung disease in preterm infants with morbid and sometimes lethal consequences that is characterized by lung immaturity and reduced alveolarization.

Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchins development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a birds-eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking of WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD.

Our network view of alveologenesis is transforming our perspective to understand and treat BPD. The new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

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Hypereosinophilia causes progressive cardiac pathologies in mice Hypereosinophilic syndrome is a progressive disease with extensive eosinophilia that results in organ damage. Cardiac pathologies are the main reason for its high mortality rate. A better understanding of the mechanisms of eosinophil-mediated tissue damage would benefit therapeutic development. Here, we describe the cardiac pathologies that developed in a mouse model of hypereosinophilic syndrome. These IL-5 transgenic mice exhibited decreased left ventricular function at a young age which worsened with age. Mechanistically, we demonstrated infiltration of activated eosinophils into the heart tissue that led to an inflammatory environment. Gene expression signatures showed tissue damage as well as repair and remodeling processes. Cardiomyocytes from IL-5Tg mice exhibited significantly reduced contractility relative to WT controls. This impairment may result from the inflammatory stress experienced by the cardiomyocytes and suggest that dysregulation of contractility and Ca2+ reuptake in cardiomyocytes contributes to cardiac dysfunction at the whole organ level in hypereosinophilic mice.

TeaserToo many eosinophils cause inflammation in the heart and change cardiomyocyte contraction leading to poor heart function.

sentences: Impact of Norway spruce pre-degradation stages induced by G. trabeum on fungal and bacterial communities In forests ecosystems, fungi and bacteria are key actors in wood degradation. However, few studies have focused on the impact of fungal pre-degradationon bacterial and fungal communities. Coniferous forests are dominated by Brown rot fungi that are involved in earliest phase of lignocellulose breakdown, and therefore, influencing the second phase of microbial colonization. This study aimed to investigate the first microbial species colonizing after intermediate and advanced pre-degradation by the brown rot fungus Gloeophyllum trabeum. Using Illumina metabarcoding, bacterial and fungal communities were monitored after 70 days from Picea abies wood blocks placed between forest floor and topsoil. Chemical composition of the wood samples was determined for each of the pre-degradation stages. We observed significant changes in the bacterial and fungal communities associated with the pre-degradation of P. abies, and that wood substrate condition acted as a gatekeeper for both microbial communities. Our data also suggested that fungal and bacterial communities could interact and act in a synergistic way during the wood decomposition process.

sentences: Pyroptosis of syncytia formed by fusion of SARS-CoV-2 Spike and ACE2 expressing cells SARS-Cov-2 infected cells fused with the ACE2-positive neighboring cells forming syncytia. However, the effect of syncytia in disease development is largely unknown. We established an in vitro cell-cell fusion system and used it to mimic the fusion of SARS-CoV-2 infected cells with ACE2-expressing cells to form syncytia. We found that Caspase-9 was activated after syncytia formation, and Caspase-3/7 was activated downstream of Caspase-9, but it triggered GSDME-dependent pyroptosis rather than apoptosis. What is more, single cell RNA-sequencing data showed that both ACE2 and GSDME were expression in alveolar type 2 cells in human lung. We propose that pyroptosis is the fate of syncytia formed by SARS-CoV-2 infected host cells and ACE2-positive cells, which indicated that lytic death of syncytia may contribute to the excessive inflammatory responses in severe COVID-19 patients.

sentences: Stereochemistry of transient protein-protein interactions in a signaling hub: exploring G3BP1-mediated regulation of CFTR deubiquitination Intrinsically disordered proteins (IDPs) can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large, reversible protein networks. Among these, the IDP hub protein G3BP1 forms protein complexes with Caprin1 and USP10, and the resulting control of USP10 activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling. However, while the identities of protein interactors are known for many of these IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of the G3BP1 affinities, and show that substitution of G3BP1 residues F15 or F33 to tryptophan reduces affinity for both the USP10 and Caprin1 motif peptides. These same mutations significantly reduce formation of complexes by the full-length proteins. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with the USP10 and Caprin1 peptides. An amino-acid scan of the USP10 and Caprin1 motif peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific mutations with the potential to create higher affinity peptides. Taken together, these data show that small changes in 1:1 binding affinity can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.

sentences: Neural Networks for parameter estimation in microstructural MRI: a study with a high-dimensional diffusion-relaxation model of white matter microstructure Specific features of white-matter microstructure can be investigated by using biophysical models to interpret relaxation-diffusion MRI brain data. Although more intricate models have the potential to reveal more details of the tissue, they also incur time-consuming parameter estimation that may con-verge to inaccurate solutions due to a prevalence of local minima in a degenerate fitting landscape. Machine-learning fitting algorithms have been proposed to accelerate the parameter estimation and increase the robustness of the attained estimates. So far, learning-based fitting approaches have been restricted to lower-dimensional microstructural models where dense sets of training data are easy to generate. Moreover, the degree to which machine learning can alleviate the degeneracy problem is poorly understood. For conventional least-squares solvers, it has been shown that degeneracy can be avoided by acquisition with optimized relaxation-diffusion-correlation protocols that include tensor-valued diffusion encoding; whether machine-learning techniques can offset these acquisition require-ments remains to be tested. In this work, we employ deep neural networks to vastly accelerate the fitting of a recently introduced high-dimensional relaxation-diffusion model of tissue microstructure. We also develop strategies for assessing the accuracy and sensitivity of function fitting networks and use those strategies to explore the impact of acquisition protocol design on the performance of the network. The developed learning-based fitting pipelines were tested on relaxation-diffusion data acquired with optimized and sub-sampled acquisition protocols. We found no evidence that machine-learning algorithms can by themselves replace a careful design of the acquisition protocol or correct for a degenerate fitting landscape.

sentences: Binocular Benefit Following Monocular Subretinal AAV Injection in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa (adRP) Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing both an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than 8 months following injection. To confirm that this same vector could protect the retinas of a different species and bearing a different RHO mutation, we injected mice transgenic for human P23H RHO at postnatal day 30 in one eye. For nine months, we monitored their retinal structure using spectral- domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG). We compared these to P23H RHO transgenic mice injected with AAV-GFP. Though retinas continued to thin over time, compared to control injected eyes, AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and decreased ERG amplitudes in AAV-RHO820-shRNA820 eyes during the nine-month study period. Unexpectedly, we also observed preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots provided evidence that a low amount of vector from injected eyes was present in uninjected eyes.

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Natural Occurring Non-Synonymous Single Nucleotide Polymorphisms in Integrase and RNase H Regulate Assembly and Autoprocessing of HIV-1 Recently, a genome-wide association study using plasma HIV RNA reported that 14 naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) in HIV derived from anti-retrovirus naive patients were associated with virus load (VL). However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP using site-directed mutagenesis and examined their replicative abilities and biological properties. An HIV variant containing Met-to-Ile change at codon 50 in integrase (HIV(IN:M50I)) was found an impaired virus. Despite the mutation being in integrase, a quantification assay demonstrated that the virus release was significantly suppressed (P<0.001). Transmission electron microscopy analyses revealed that the accumulation of abnormal shapes of buds on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins in the HIV(IN:M50I) particles. Forster Resonance Energy Transfer (FRET) assay displayed that GagPol containing IN:M50I (GagPol(IN:M50I)) significantly increased the efficiency of homodimerization (P<0.05) and heterodimerization with Gag (P<0.001), compared to GagPol(WT). HIV replication assay using a series of variants of HIV(IN:M50I) elucidated that the C-terminus residues, Asn at codon 288, plays a key role in the defect and the impaired maturation and replication capability was rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser change at codon 79 in RNase H. These data demonstrate that Gag and GagPol assembly, virus release and autoprocessing are not only regulated by integrase but also RNase H.

ImportanceA nascent HIV-1 is noninfectious. To become an infectious virus, Gag and GagPol polyproteins in the particles need to be cleaved by mature HIV protease (PR). PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by a self-cleavage is called autoprocessing. Here, during the evaluation of roles of naturally emerging non-synonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase in GagPol which increases the efficiency of heterodimerization with Gag. This defect was recovered by co-existing of other SNPs: Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

sentences: Combined Nanopore and Single-Molecule Real-Time Sequencing Survey of Human Betaherpesvirus 5 Transcriptome Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

sentences: Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

sentences: A Miniaturized 3D-Printed Pressure Regulator (μPR) for Microfluidic Cell Culture Applications Controlled fluid flows are the hallmark feature of microfluidic culture systems and provide precise definition over the biophysical and biochemical microenvironment. Flow control is commonly achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-based techniques. These methods offer complex flow capabilities but can be challenging to integrate into incubators or other confined environments due to their large form factors and accompanying peripheral equipment. Since many microfluidic cell culture studies use a single controlled flow rate to maintain or stimulate cells, a portable flow control platform that fits easily into an incubator will benefit the microfluidic community. Here, we demonstrate that a tunable, 3D printed micro pressure regulator (PR), combined with a battery-powered miniature air pump, can operate as a stand-alone pneumatic flow control platform for microfluidic applications. We detail the design and fabrication of the PR and demonstrate: i) a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), ii) highlight dynamic control in a microfluidic network, and iii) maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment membrane-based culture device under continuous flow conditions. We anticipate that our 3D-printed fabrication approach and open access designs will allow other laboratories to rapidly customize PRs to support a broad range of applications.

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The PD-L1 and TLR7 dual-targeting nanobody-drug conjugate exerts potent antitumor efficacy by orchestrating innate and adaptive immune responses A variety of tumors are insensitive to immune checkpoint blockade (ICB) therapy. We propose that ICB therapy alone is insufficient to fully reactivate antitumor T cells, while effective mobilization of antigen-presenting cells (APCs) to assist adaptive immune cell activation can lead to potent antitumor effects with broad responsiveness. The Toll-like receptor 7 (TLR7) agonist SZU-101 we developed can induce the innate immune response against tumors and increase the immunogenicity of tumors. Interestingly, SZU-101-induced upregulation of programmed death ligand 1 (PD-L1) expression in tumor tissues can further enhance the response rate of the PD-L1 antibody. In addition, PD-L1 nanobodies have better solid tumor penetration ability, and because of this ability, they can be used to precisely deliver SZU-101 to tumor tissues. Therefore, a PD-L1 and TLR7 dual-targeting nanobody-drug conjugate (NDC), a novel drug molecule, was developed. We found that TLR7 agonists and PD-L1 nanobodies act synergistically and that NDC treatment reshapes the tumor immune microenvironment, activates both innate and adaptive immune cells, and exerts antitumor effects in both "hot" and "cold" tumors primarily through CD8+ T cells and natural killer (NK) cells. Our data show that a PD-L1 and TLR7 dual-targeting NDC can exhibit potent antitumor efficacy by orchestrating innate and adaptive immune responses and shows good prospects for clinical development.

One Sentence SummaryBased on results showing that TLR7 agonists and PD-L1 nanobodies exert synergistic antitumor effects, a PD-L1 and TLR7 dual-targeting nanobody-drug conjugate that we developed shows good prospects for clinical development because it can orchestrate innate and adaptive immune responses, reshape the tumor immune microenvironment, and exert potent antitumor effects against both "hot" and "cold" tumors.

sentences: Core and rod structures of a thermophilic cyanobacterial light-harvesting phycobilisome Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.

sentences: Oxytocin modulates neurocomputational mechanisms underlying prosocial reinforcement learning Humans often act in the best interests of others. However, how we learn which actions result in good outcomes for other people and the neurochemical systems that support this  prosocial learning remain poorly understood. Using computational models of reinforcement learning, functional magnetic resonance imaging and dynamic causal modelling, we examined how different doses of intranasal oxytocin, a neuropeptide linked to social cognition, impact how people learn to benefit others (prosocial learning) and whether this influence could be dissociated from how we learn to benefit ourselves (self-oriented learning). We show that a low dose of oxytocin prevented decreases in prosocial performance over time, despite no impact on self-oriented learning. Critically, oxytocin produced dose-dependent changes in the encoding of prediction errors (PE) in the midbrain-subgenual anterior cingulate cortex (sgACC) pathway specifically during prosocial learning. Our findings reveal a new role of oxytocin in prosocial learning by modulating computations of PEs in the midbrain-sgACC pathway.

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FiNN: A toolbox for neurophysiological network analysis In recent years, neuroscience has seen a shift from localist approaches to network-wide investigations of brain function. Neurophysiological signals across different spatial and temporal scales provide an informative insight into neural communication. However, additional methodological considerations arise when investigating network-wide brain dynamics rather than local effects. Specifically, larger amounts of data, investigated across a higher dimensional space, are necessary.

Here, we present FiNN (Find Neurophysiological Networks), a novel toolbox for the analysis of neurophysiological data with a focus on functional and effective connectivity. FiNN provides a wide range of data processing methods, introduces new methodological developments to acquire efficient and reliable connectivity estimates that build on already existing concepts, and statistical and visualization tools to facilitate inspection of connectivity estimates and the resulting metrics of brain dynamics. The toolbox is freely available in Python (https://github.com/neurophysiological-analysis/FiNN), and complemented by online documentation (https://neurophysiological-analysis.github.io/FiNN/).

To highlight the properties of our toolbox, we evaluated FiNN against a number of established frameworks on both a conceptual and an implementation level. We found FiNN to require much less processing time and memory than other toolboxes. In addition, FiNN adheres to a design philosophy of easy access and modifiability, while providing efficient data processing implementations. Since the investigation of network-level neural dynamics is experiencing increasing interest, we place FiNN at the disposal of the neuroscientific community as open-source software.

Highlights- Investigation of network-wide brain dynamics requires specialized software.
- We provide a toolbox to ease the pre-processing and analysis steps of the investigation.
- FiNN requires less processing time and memory in comparison to other toolboxes.
- Extensive documentation facilitates usage by users from various technical backgrounds.

sentences: 3D pose estimation enables virtual head-fixation in freely moving rats The impact of spontaneous movements on neuronal activity has created the need to quantify behavior. We present a versatile framework to directly capture the 3D motion of freely definable body points in a marker-free manner with high precision and reliability. Combining the tracking with neural recordings revealed multiplexing of information in the motor cortex neurons of freely moving rats. By integrating multiple behavioral variables into a model of the neural response, we derived a virtual head-fixation for which the influence of specific body movements was removed. This strategy enabled us to analyze the behavior of interest (e.g., front paw movements). Thus, we unveiled an unexpectedly large fraction of neurons in the motor cortex with tuning to the paw movements, which was previously masked by body posture tuning. Once established, our framework can be efficiently applied to large datasets while minimizing the experimental workload caused by animal training and manual labeling.

sentences: Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.

sentences: Tales of Two α-Carboxysomes: the Structure and Assembly of Cargo Rubisco Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. It encapsulates carbonic anhydrase and Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalysing carbon fixation inside a proteinaceous shell. How Rubisco packs into the carboxysomes is unknown. Using cryo-electron tomography and subtomogram averaging, we present 3D organization of Rubisco inside two types of native -carboxysomes from a marine -cyanobacterium Cyanobium sp. PCC 7001 and a chemoautotrophic bacterium Halothiobacillus neapolitanus. We determined the structures of Rubiscos within native Halothiobacillus and Cyanobium carboxysomes at 3.3 [A] and 3.8 [A] resolution respectively and further identified an associated CsoS2 segment. Interestingly, CsoS2 is only associated with a sub-population of Rubiscos that are close to the shell in Halothiobacillus, but with all Rubiscos throughout Cyanobium carboxysome. Moreover, Rubiscos in Cyanobium carboxysomes are organized in three concentric layers whereas Rubiscos in Halothiobacillus carboxysomes are arranged in spiral arrays. Calcium treatment induced a drastic re-organization of Rubiscos, converting these two distinct assemblies into ordered lattice arrays in both -carboxysomes. Our findings provide critical knowledge of the assembly principles of -carboxysomes, which may aid in rational design and repurpose of carboxysome structures for new functions.

sentences: A single cell atlas of human and mouse white adipose tissue White adipose tissue (WAT), once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic, heterogenous, and involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control, and host defense1. High fat feeding and other metabolic stressors cause dramatic changes in adipose morphology, physiology, and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia, and type 2 diabetes (T2D)2. Here, we provide detailed cellular atlases of human and murine subcutaneous and visceral white fat at single cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells (ASPCs), vascular, and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease, and we provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits, and cell types in the function of WAT across species, depots, and nutritional conditions.

sentences: The effect of Septoria glycines and fungicide application on the soybean phyllosphere mycobiome Septoria brown spot (caused by Septoria glycines) is the most prevalent soybean disease in Illinois. It is common to use a foliar fungicide to control Septoria brown spot and other foliar diseases. The effects of fungicide on non-target organisms in the phyllosphere are unknown. To study the effect of S. glycines and fungicide application on the soybean phyllosphere mycobiome we sequenced full-length ITS and partial LSU region using oxford nanopore technologies. Sequencing produced 3,342 operational taxonomic units (OTUs). The richness of the fungal community significantly increased with the developmental stage. The soybean lines significantly affected the mycobiome diversity at the early developmental stage but not at the reproductive stages. S. glycines did not significantly affect the alpha diversity but some significant changes were observed for the beta diversity. At the R5 stage, fungicide application significantly changed the composition of the fungal community. The fungicide treatment significantly decreased the proportion of several fungal reads, but it increased the proportion of Septoria. The core microbiome in soybean leaves was composed of Gibberella, Alternaria, Didymella, Cladosporium, Plectosphaerella, Colletotrichum, and Bipolaris. Network analysis identified significant interactions between Septoria reads and Diaporthe, Bipolaris and two other taxonomic units. In this study, we set Septoria as the target organism and demonstrated that metabarcoding could be a tool to quantify the effect of multiple treatments on the fungal phyllosphere community. Better understanding the dynamics of the phyllosphere microbiome is necessary to start untangling late-season diseases of soybean.

sentences: Doxorubicin Induces Senescence in Intestinal Epithelial Cells Doxorubicin treatment induces DNA damage and apoptosis in rapidly dividing cell types like intestinal epithelial cells. This has been demonstrated both in vivo and in vitro. In certain cell types some cells do not undergo DNA damage-induced apoptosis in response to doxorubicin but instead become senescent. Induction of senescence in these cells can lead to dysfunction and chronic inflammation, which can lead to more damage. We questioned whether a single dose of doxorubicin would be able to induce apoptosis and senescence in intestinal epithelial cells in vitro. For these studies, we exposed IEC-6 small intestinal epithelial cells to doxorubicin to evaluate whether senescence is induced in a relatively homogeneous population of intestinal epithelial cells. Although some cells underwent apoptosis, those that did not showed traits of senescence. Our studies showed that doxorubicin treatment increased cell size and increased expression of senescence-associated {beta}-galactosidase. Concomitantly, we observed increased mRNA expression of several genes associated with a senescence-associated secretory phenotype including IL-6, Ptges, Faim2, and Cdkn1a and decreased expression of Sirt1. We also observed release of HMGB1, a cellular alarmin, from treated cells. Together, these data suggest that doxorubicin induces senescence in intestinal epithelial cells. Furthermore, our data indicate that cellular responses to a DNA damaging agent, such as doxorubicin, can differ within a population of cells suggesting differing levels of sensitivity within a relatively homogenous cell population. Further studies are needed to delineate the mechanisms that determine whether a cell moves down an apoptotic or senescent pathway following DNA damage.

sentences: Functional Annotation of Human Cognitive States using Deep Graph Convolution A key goal in neuroscience is to understand brain mechanisms of cognitive functions. An emerging approach is "brain decoding", which consists of inferring a set of experimental conditions performed by a participant, using pattern classification of brain activity. Few works so far have attempted to train a brain decoding model that would generalize across many different cognitive tasks drawn from multiple cognitive domains. To tackle this problem, we proposed a multidomain brain decoder that automatically learns the spatiotemporal dynamics of brain response within a short time window using a deep learning approach. We evaluated the decoding model on a large population of 1200 participants, under 21 different experimental conditions spanning six different cognitive domains, acquired from the Human Connectome Project task-fMRI database. Using a 10s window of fMRI response, the 21 cognitive states were identified with a test accuracy of 90% (chance level 4.8%). Performance remained good when using a 6s window (82%). It was even feasible to decode cognitive states from a single fMRI volume (720ms), with the performance following the shape of the hemodynamic response. Moreover, a saliency map analysis demonstrated that the high decoding performance was driven by the response of biologically meaningful brain regions. Together, we provide an automated tool to annotate human brain activity with fine temporal resolution and fine cognitive granularity. Our model shows potential applications as a reference model for domain adaptation, possibly making contributions in a variety of domains, including neurological and psychiatric disorders.

sentences: Systematic classification error profoundly impacts inference in high-depth Whole Genome Shotgun Sequencing datasets There is little consensus in the literature as to which approach for classification of Whole Genome Shotgun (WGS) sequences is best. In this paper, we examine two of the most popular algorithms, Kraken2 and Metaphlan2 utilizing four publicly available datasets. As expected from previous literature, we found that Kraken2 reports more overall taxa while Metaphlan2 reports fewer taxa while classifying fewer overall reads. To our surprise, however, Kraken 2 reported not only more taxa but many more taxa that were significantly associated with metadata. This implies that either Kraken2 is more sensitive to taxa that are biologically relevant and are simply missed by Metaphlan2, or that Kraken2s classification errors are generated in such a way to impact inference. To discriminate between these two possibilities, we compared Spearman correlations coefficients of each taxa against each taxa with higher abundance from the same dataset. We found that Kraken2, but not Metaphlan2, showed a consistent pattern of classifying low abundance taxa that generated high correlation coefficients with higher abundance taxa. Neither Metaphlan2, nor 16S sequences that were available for two of our four datasets, showed this pattern. Simple simulations based on a variable Poisson error rate sampled from the uniform distribution with an average error rate of 0.0005 showed strikingly strong concordance with the observed correlation patterns from Kraken2. Our results suggest that Kraken2 consistently misclassifies high abundance taxa into the same erroneous low abundance taxa creating "phantom" taxa have a similar pattern of inference as the high abundance source. Because of the large sequencing depths of modern WGS cohorts, these "phantom" taxa will appear statistically significant in statistical models even with a low overall rate of classification error from Kraken. Our simulations suggest that this can occur with average error rates as low as 1 in 2,000 reads. These data suggest a novel metric for evaluating classifier accuracy and suggest that the pattern of classification errors should be considered in addition to overall classification error rate since consistent classification errors have a more profound impact on inference compared to classification errors that do not always result in assignment to the same erroneous taxa. This work highlights fundamental questions on how classifiers function and interact with large sequencing depth and statistical models that still need to be resolved for WGS, especially if correlation coefficients between taxa are to be used to build covariance networks. Our work also suggests that despite its limitations, 16S rRNA sequencing may still be useful as neither of the two most popular 16S classifiers showed these patterns of inflated correlation coefficients between taxa.

sentences: Identifying and correcting multiple sources of misspecification in GWAS summary statistics for polygenic scores Publicly available genome-wide association studies (GWAS) summary statistics exhibit uneven quality, which can impact the validity of follow-up analyses. First, we present an overview of possible misspecifications that come with GWAS summary statistics. Then, in both simulations and real data analyses, we show that additional information such as imputation INFO scores, allele frequencies, and per-variant sample sizes in GWAS summary statistics can be used to detect possible issues and correct for misspecifications in the GWAS summary statistics. One important motivation for us is to improve the predictive performance of polygenic scores built from these summary statistics. Unfortunately, due to the lack of reporting standards for GWAS summary statistics, this additional information is not systematically reported. We also show that using well-matched LD references can improve model fit and translate into more accurate prediction. Finally, we discuss how to make polygenic score methods such as lassosum and LDpred2 more robust to these misspecifications to improve their predictive power.

sentences: Allosteric inhibitors of Zika virus NS2B-NS3 protease targeting protease in super-open conformation Zika virus (ZIKV) is a member of the Flaviviridae family and is considered a major health threat causing cases of microcephaly in newborns and Guillain-Barre syndrome in adults. Here we targeted a transient deep and hydrophobic pocket of the super-open conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the orthosteric inhibitors. After virtual docking screening of approximately 7 million compounds against the novel allosteric site we selected the top seven candidates and tested them in an enzymatic assay. Six out of seven top candidates selected by the docking screen inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations, as well as suppressing viral replication. These six compounds, targeting the selected protease pocket conserved in ZIKV as well as several other Flaviviruses, have opened an opportunity for a new kind of drug candidate that might be useful to treat several flaviviral infections.

sentences: Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as -tubulin and {beta}-tubulin. Also, I immunolocalized acetylated and tyrosinated -tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.

sentences: Key Drivers of Ecological Assembly in the Hindgut of Atlantic Cod (Gadus morhua) when Fed with a Macroalgal Supplemented diet - How Robust Is the Gut to Taxonomic Perturbation? The microbiota in the gastrointestinal tract of any species is shaped by internal and external cues in addition to random events which can be difficult to disentangle from a range of interacting variables. Estimating ecological assembly processes can help elucidate these factors. In our study, farmed Atlantic cod (Gadus morhua) were fed a diet of 10% macroalgae supplement (Ulva rigida species [ULVA] or Ascophyllum nodosum [ASCO] or a non-amended control diet [CTRL]) over a 12-week period and the ecological assembly processes quantified. The CTRL samples showed selection (variable selection - divergence in communities related to selective environmental conditions) as a key assembly process, while dispersal limitation (limited movement results in divergent communities through stochastic processes) was a driver of the gut microbiome for fish fed the macroalgae supplemented diet at Week 12 (i.e., ASCO and ULVA). Fish from the ASCO grouping diverged into ASCO_N (normal) and ASCO_LG (lower growth), where ASCO_LG individuals found the diet unpalatable. The recruitment of new taxa over time was altered in the ASCO_LG fish, with the gut microbiome showing phylogenetic under dispersion (nepotistic recruitment of species). Finally, the gut microbiome (CTRL and ULVA only) showed increasing robustness to taxonomic disturbance over time and an increase in functional redundancy. This study advances our understanding of the ecological assembly and succession in the hindgut of juvenile Atlantic cod across dietary regimes. Understanding the processes driving ecological assembly in the gut microbiome, in fish research specifically, could allow us to manipulate the microbiome for improved health or resilience to disease for improved aquaculture production.

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Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks The natural product jasplakinolide is a widely used tool compound to stabilize F-actin and influence actin dynamics. We have previously introduced photoswitchable jasplakinolides (optojasps) that are activated with violet light and deactivated with blue light. Based on insights from cryo-electron microscopy and structure-activity relationship (SAR) studies, we now developed a new generation of functionally superior optojasps that are better suited for biological investigations. These compounds are procured through chemical total synthesis and feature rationally designed red-shifted azobenzene photoswitches. Our new optojasps can be activated with longer wavelengths in the visible range (e.g. 440-477 nm) and rapidly return to their inactive state through thermal relaxation. This has enabled the reversible control of F-actin dynamics, as shown through live-cell imaging and cell migration, as well as cell proliferation assays. Brief sub-cellular activation with blue-green light resulted in highly localized F-actin clusters that gradually dissolved in the dark. Our light-responsive tools can be useful in diverse fields to study actin dynamics with outstanding spatiotemporal precision.

Graphical Abstract

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sentences: A potential role for reinforcement learning in speech production Reinforcement learning, the ability to change motor behavior based on external reward, has been suggested to play a critical role in early stages of speech motor development and is widely used in clinical rehabilitation for speech motor disorders. However, no current evidence exists that demonstrates the capability of reinforcement to drive changes in human speech behavior. Speech provides a unique test of the universality of reinforcement learning across motor domains: speech is a complex, high-dimensional motor task whose goals do not specify a task to be performed in the environment but ultimately must be self-generated by each speaker such that they are understood by those around them. Across four experiments, we examine whether reinforcement learning alone is sufficient to drive changes in speech behavior and parametrically test two features known to affect reinforcement learning in reaching: how informative the reinforcement signal is as well as the availability of sensory feedback about the outcomes of ones motor behavior. We show that learning does occur and is more likely when participants receive auditory feedback that gives an implicit target for production, even though they do not explicitly imitate that target. Contrary to results from upper limb control, masking feedback about movement outcomes has no effect on speech learning. Together, our results suggest a potential role for reinforcement learning in speech but that it likely operates differently than in other motor domains.

sentences: Retroviral Infection of Human Neurospheres and Use of Stem Cell EVs to Repair Cellular Damage HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection.

sentences: Gal1 repression memory in budding yeast Cells must continuously adapt to changing environments and, thus, have evolved mechanisms allowing them to respond to repeated stimuli. For example, faster gene induction upon a repeated stimulus aids adaptation - a process known as reinduction memory. However, whether such a memory exists for gene repression is unclear. Here, we studied gene repression across repeated carbon source shifts in over 2,500 single Saccharomyces cerevisiae cells. By monitoring the expression of a carbon source-responsive gene, galactokinase 1 (Gal1), and mathematical modeling, we discovered repression memory at the population and single-cell level. Using a repressor model to estimate single-cell repression parameters, we show that repression memory is due to a shortened repression delay, the estimated time gap between carbon source shift and Gal1 expression termination, upon the repeated carbon source shift. Additionally, we show that cells lacking Elp6 display a gain-of-repression-memory phenotype characterized by a stronger decrease in repression delay between two consecutive carbon source shifts. Collectively, our study provides the first quantitative description of repression memory in single cells.

sentences: The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins - major histocompatibility complex II (MHCII), CD86 and CD97 - from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

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Regnase-1 deficiency restrains Klebsiella pneumoniae infection by regulation of a Type I interferon response Excessive inflammatory responses can cause collateral tissue damage or autoimmune inflammation, sometimes with severe morbidity or mortality. During host defense responses, numerous negative feedback mechanisms are established to prevent excessive unchecked inflammation. However, this restraint can sometimes come at the cost of suboptimal infection control, and we do not fully understand how this balance is maintained during different infection settings. The endoribonuclease Regnase-1 (Reg1, Zc3h12a, MCPIP1) is an RNA binding protein (RBP) that binds and degrades many target mRNA transcripts. Reg1 is a potent feedback regulator of IL-17 and LPS signal transduction, among other stimuli. Consequently, Reg1 deficiency exacerbates autoimmune inflammation in multiple mouse models, but on the other hand, reduced Reg1 improves immunity to fungal infection. To date, the role of Reg1 in bacterial immunity is poorly defined. Here, we show that mice deficient in Reg1 are more resistant to pulmonary Klebsiella pneumoniae (KP) infection. Unexpectedly, effects of Reg1 deficiency were not due to accelerated eradication of bacteria or increased pro-inflammatory cytokine expression. Rather, alveolar macrophages from Reg1-deficient mice showed enrichment of Type I IFN-related genes upon KP infection, accompanied by increased Ifnb1 expression. Surprisingly, the stability of Ifnb1 mRNA was not altered by Reg1-deficiency; rather, mRNA encoding its upstream regulator IRF7 appeared to be a more prominent target. Blockade of IFNR during KP infection reversed disease improvement. Thus, impaired Reg1 induces Type I IFN and enhances resistance to KP, raising the possibility that Reg1 could be a potential clinical target in acute bacterial infections.

Graphical abstract

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sentences: Comparison of multiple tractography methods for reconstruction of the retinogeniculate visual pathway using diffusion MRI The retinogeniculate visual pathway (RGVP) conveys visual information from the retina to the lateral geniculate nucleus. The RGVP has four subdivisions, including two decussating and two non-decussating pathways that cannot be identified on conventional structural magnetic resonance imaging (MRI). Diffusion MRI tractography has the potential to trace these subdivisions and is increasingly used to study the RGVP. However, it is not yet known which fiber tracking strategy is most suitable for RGVP reconstruction. In this study, four tractography methods are compared, including constrained spherical deconvolution (CSD) based probabilistic (iFOD1) and deterministic (SD-Stream) methods, and multi-fiber (UKF-2T) and single-fiber (UKF-1T) unscented Kalman filter (UKF) methods. Experiments use diffusion MRI data from 57 subjects in the Human Connectome Project. The RGVP is identified using regions of interest created by two clinical experts. Quantitative anatomical measurements and expert anatomical judgment are used to assess the advantages and limitations of the four tractography methods. Overall, we conclude that UKF-2T and iFOD1 produce the best RGVP reconstruction results. The iFOD1 method can better quantitatively estimate the percentage of decussating fibers, while the UKF-2T method produces reconstructed RGVPs that are judged to better correspond to the known anatomy and have the highest spatial overlap across subjects. Overall, we find that it is challenging for current tractography methods to both accurately track RGVP fibers that correspond to known anatomy and produce an approximately correct percentage of decussating fibers. We suggest that future algorithm development for RGVP tractography should take consideration of both of these two points.

sentences: A new method for the sampling and preservation of placental specimens in low-resource settings for histology and analysis of nucleic acids Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope(R) hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq.

sentences: Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

sentences: Cerebello-Basal Ganglia Network Global Efficiency Predicts Cortical Network Global Efficiency The cerebellum (CB) and basal ganglia (BG) each have topographically distinct functional subregions that are functionally and anatomically interconnected with cortical regions through discrete thalamic loops and with each other via disynaptic connections, with previous work detailing high levels of functional connectivity between these phylogenetically ancient regions. It was posited that this CB-BG network provides support for cortical systems processing, spanning cognitive, emotional, and motor domains, implying subcortical network measures are strongly related to cortical network measures (Bostan & Strick, 2018); however, it is currently unknown how network measures within distinct CB-BG networks relate to cortical network measures. Here, 122 regions of interest comprising cognitive and motor CB-BG networks and 7 canonical cortical resting-state were used to investigate whether the integration (quantified using global efficiency, GE) of cognitive CB-BG network (CCBN) nodes and their segregation from motor CB-BG network (MCBN) nodes is related to cortical network GE and segregation in 233 non-related, right- handed participants (Human Connectome Project-1200). CCBN GE positively correlated with GE in the default mode, motor, and auditory networks and MCBN GE positively correlated with GE in all networks except the default mode and emotional. MCBN segregation was related to MN segregation. These findings highlight the CB-BG networks role in executive function, task switching, and verbal working memory. This work has implications for understanding cortical network organization and cortical-subcortical interactions in healthy adults and may help in deciphering subcortical differences seen in disease states.

sentences: A conserved uORF impacts APOBEC3G translation and is essential for translational inhibition by the HIV-1 Vif protein The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation and protein degradation) that together reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5 untranslated region (5UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky-scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.

sentences: Meta-Transcriptome Detector (MTD): a novel pipeline for metatranscriptome analysis of bulk and single-cell RNAseq data RNA-seq data contains not only host transcriptomes but also non-host information that comprises transcripts from active microbiota in the host cells. Therefore, joint and integrative analyses of both host and meta-transcriptome can reveal gene expression of microbial community in a given sample as well as the correlative and interactive dynamics of host response to the microbiome. However, there are no convenient tools that can systemically analyze host-microbiota interactions by simultaneously quantifying host and meta-transcriptome in the same sample at the tissue and the single-cell level, which poses a challenge for interested researchers with a limited expertise in bioinformatics. Here, we developed a software pipeline that can comprehensively and synergistically analyze and correlate the host and meta-transcriptome in a single sample using bulk and single-cell RNA-seq data. This pipeline, named MTD, can extensively identify and quantify microbiome, including viruses, bacteria, protozoa, fungi, plasmids, and vectors in the host cells and correlate the microbiome with the host transcriptome. MTD is easy to install and run, involving only a few lines of simple commands. It empowers researchers with unique genomics insights into host immune responses to microorganisms.

sentences: Genetic, metabolic, and molecular insights into the diverse outcomes of diet-induced obesity in mice Overweight and obesity are increasingly common public health issues worldwide, leading to a wide range of diseases from metabolic syndrome to steatohepatitis and cardiovascular diseases. While the increase in the prevalence of obesity is partly attributable to changes in lifestyle (i.e. increased sedentarity and changes in eating behaviour), the metabolic and clinical impacts of these obesogenic conditions varies between sexes and genetic backgrounds. The conception of personalised treatments of obesity and its complications require a thorough understanding of the diversity of responses to conditions such as high-fat diet intake. By analysing nine genetically diverse mouse strains, we show that much like humans, mice respond to high-fat diet in a genetic- and sex-dependent manner. Physiological and molecular responses to high-fat diet are associated with expression of genes involved in immunity and mitochondrial function. Finally, we find that mitochondrial function may explain part of the diversity of physiological responses. By exploring the complex interactions between genetics and metabolic phenotypes via gene expression and molecular traits, we shed light on the importance of genetic background and sex in determining metabolic outcomes. In addition to providing the community with an extensive resource for optimizing future experiments, our work serves as an exemplary design for more generalizable translational studies.

sentences: Alterations in excitatory and inhibitory synaptic development within the mesolimbic dopamine pathway in a mouse model of prenatal drug exposure The rise in rates of opioid abuse in recent years has led to an increase in the incidence of neonatal abstinence syndrome (NAS). Despite having a greater understanding of NAS and its symptoms, there still remains a lack of information surrounding the long-term effects of prenatal exposure to drugs of abuse on neurological development. One potential outcome of prenatal drug exposure that has been increasingly explored is disruption in normal synaptogenesis within the central nervous system. Both opioids and gabapentin, an antiepileptic drug commonly co-abused by opioid abuse disorder patients, have been shown to interfere with the normal functioning of astrocytes, non-neuronal glial cells known to serve many functions, including regulation of synaptic development. The goal of this study was to investigate the effects of prenatal drug exposure on synaptogenesis within brain regions associated with the mesolimbic dopamine pathway, the primary reward pathway within the brain associated with drug abuse and addiction, in a pregnant mouse model. Immunohistochemistry (IHC) and confocal fluorescence microscopy imaging studies on the brains of postnatal day 21 (P21) mouse pups revealed a significant increase in the mean number of excitatory synapses within the anterior cingulate cortex (ACC), nucleus accumbens (NAc), and prefrontal cortex (PFC) in mice that were prenatally exposed to either the opioid drug buprenorphine or gabapentin. These studies also revealed a significant decrease in the mean number of inhibitory synapses within the NAc and PFC of mice treated with buprenorphine. This observed net increase in excitatory signaling capability within the developing mesolimbic dopamine pathway suggests that exposure to drugs of abuse in utero can trigger maladaptive neuronal connectivity that persists beyond the earliest stages of life.

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Accurate Virus Identification with Interpretable Raman Signatures by Machine Learning Rapid identification of newly emerging or circulating viruses is an important first step toward managing the public health response to potential outbreaks. A portable virus capture device coupled with label-free Raman Spectroscopy holds the promise of fast detection by rapidly obtaining the Raman signature of a virus followed by a machine learning approach applied to recognize the virus based on its Raman spectrum. In this paper, we present a machine learning analysis on Raman spectra of human and avian viruses. A Convolutional Neural Network (CNN) classifier specifically designed for spectral data achieves very high accuracy for a variety of virus type or subtype identification tasks. In particular, it achieves 99% accuracy for classifying influenza virus type A vs. type B, 96% accuracy for classifying four subtypes of influenza A, 95% accuracy for differentiating enveloped and non-enveloped viruses, and 99% for differentiating avian coronavirus (infectious bronchitis virus, IBV) from other avian viruses. Furthermore, interpretation of neural net responses in the trained CNN model using a full-gradient algorithm highlights Raman spectral ranges that are most important to virus identification. By correlating ML-selected salient Raman ranges with the signature ranges of known biomolecules and chemical functional groups (e.g. amide, amino acid, carboxylic acid) we verify that our ML model effectively recognizes the Raman signatures of proteins, lipids and other vital functional groups present in different viruses and uses a weighted combination of these signatures to identify viruses. The accurate and interpretable machine learning model developed for Raman virus identification presents promising potential in a real-time virus detection system.

Significance StatementA portable micro-fluidic platform for virus capture promises rapid enrichment and label-free optical identification of viruses by Raman spectroscopy. A large Raman dataset collected on a variety of viruses enables the training of machine learning (ML) models capable of highly accurate and sensitive virus identification. The trained ML models can then be integrated with the portable device to provide real-time virus detection and identification capability. We validate this conceptual framework by presenting highly accurate virus type and subtype identification results using a convolutional neural network to classify Raman spectra of viruses.

sentences: Sex and metabolic state interact to influence expression of passive avoidance memory in rats: Potential contribution of A2 noradrenergic neurons Competing motivational drives coordinate behaviors essential for survival. For example, interoceptive feedback from the body during a state of negative energy balance serves to suppress anxiety-like behaviors and promote exploratory behaviors in rats. Results from past research suggest that this shift in motivated behavior is linked to reduced activation of specific neural populations within the caudal nucleus of the solitary tract (cNTS). However, the potential impact of metabolic state and the potential role of cNTS neurons on conditioned avoidance behaviors has not been examined. The present study investigated these questions in male and female rats, using a task in which rats learn to avoid a context (i.e., a darkened chamber) after it is paired with a single mild footshock. When rats later were tested for passive avoidance of the shock-paired chamber, male rats tested in an overnight food-deprived state and female rats (regardless of feeding status) displayed significantly less avoidance compared to male rats that were fed ad libitum prior to testing. Based on prior evidence that prolactin-releasing peptide (PrRP)-positive noradrenergic neurons and glucagon-like peptide 1 (GLP1)-positive neurons within the cNTS are particularly sensitive to metabolic state, we examined whether these neural populations are activated in conditioned rats after re-exposure to the shock-paired chamber, and whether neural activation is modulated by metabolic state. Compared to the control condition, chamber re-exposure activated PrRP+ noradrenergic neurons and also activated neurons within the anterior ventrolateral bed nucleus of the stria terminalis (vlBNST), which receives dense input from PrRP+ terminals. In parallel with sex differences in passive avoidance behavior, PrRP+ neurons were less activated in female vs. male rats after chamber exposure. GLP1+ neurons were not activated in either sex. Overnight food deprivation before chamber re-exposure reduced activation of PrRP+ neurons, and also reduced vlBNST activation. Our results support the view that PrRP+ noradrenergic neurons and their inputs to the vlBNST contribute to the expression of passive avoidance memory, and that this contribution is modulated by metabolic state.

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Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations Mitochondria represent an attractive drug target in the treatment of many diseases. One of the most commonly used approaches to deliver therapeutics specifically into mitochondria is their conjugation to the triphenylphosphonium (TPP) moiety. While the TPP molecule is often regarded as biologically inert, there is evidence that the moiety itself has a significant impact on the activity of mitochondrial respiratory chain complexes.

We studied the impact of a subchronic exposure of C2C12 mouse myoblasts to a set of TPP derivatives. Our results show that the alkyl-TPP cause dose- and hydrophobicity-dependent alterations of mitochondrial morphology and a selective decrease in the amounts of mitochondrial inner membrane (but not outer membrane) proteins including structural subunits of the respiratory chain complexes (such as MT-CO1 of complex IV or NDUFB8 of complex I), as well as components of the mitochondrial calcium uniporter complex (MCUC). The treatment with alkyl-TPP additionally resulted in OPA1-cleavage. Both the structural and functional effects of alkyl-TPP were found to be reversible. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ. We further show that this effect on protein levels cannot be explained solely by a decrease in mitochondrial membrane potential.

We conclude that TPP derivatives negatively affect mitochondrial structure and function at least in part through their effect on selective mitochondrial membrane protein levels via a reversible controlled process.

sentences: Monomeric α-Synuclein activates the Plasma Membrane Calcium Pump Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well-known for pathological aggregation in neurodegeneration. The physiological function of aSN however is disputed. Pull-down experiments have pointed to plasma membrane Ca2+-ATPase (PMCA) as a potential interaction partner. From proximity ligation assays we find that aSN and PMCA colocalize at neuronal synapses, and that calcium expulsion is activated by aSN and PMCA. From PMCA activity studies we show that soluble, monomeric aSN activates PMCA at par with CaM, yet independent of the autoinhibitory domain of PMCA, but highly dependent on acidic phospholipids and membrane-anchoring of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop region connecting the cytosolic A domain and transmembrane segment 3. Our studies point towards a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

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Flanker: a tool for comparative genomics of gene flanking regions Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial resistance genes. Here, we present Flanker, a Python package which performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as  flank patterns, are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterised to finetune outputs by characterising upstream and downstream regions separately and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (blaOXA-48 and blaKPC-2/3) and show that it successfully identifies distinct clusters of flank patterns, including both known and previously uncharacterised structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterised using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking gene characterisation. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and blaOXA-48 upregulation and demonstrate 17 flank patterns (compared to the 5 previously identified). More generally the demonstration in this study that flank patterns are associated with to geographical regions and antibiotic susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.

Data SummaryNCBI accession numbers for all sequencing data used in this study is provided in Supplementary Table 1. The analysis performed in this manuscript can be reproduced in a binder environment provided on the Flanker Github page (https://github.com/wtmatlock/flanker).

sentences: Neuronal activity in the human amygdala and hippocampus enhances emotional memory encoding Emotional events comprise our strongest and most valuable memories, yet it is unknown how the brain prioritizes emotional information for storage. Here, we examined the neural basis of this prioritization using direct brain recording, deep brain stimulation, and psychometric assessment, with human subjects performing an episodic memory task in which they showed improved performance for emotional stimuli. During the task, high-frequency activity (HFA), a correlate of neuronal spiking activity, increased in both the hippocampus and amygdala when subjects successfully encoded emotionally arousing stimuli. Applying inhibitory electrical stimulation to these regions decreased HFA and specifically reversed the enhancement of memory for emotional stimuli, indicating that neuronal activity in the amygdalohippocampal circuit has a direct role in prioritizing emotional memories. Finally, we found abnormal patterns of amygdalohippocampal HFA in depressed individuals which correlated with a bias for negative memories in these subjects. Going forward, targeted modulation that upregulates neuronal excitation in the amygdalohippocampal circuit may have a causal and translational role in modulating emotional memory.

sentences: Long-term potentiation at pyramidal cell to somatostatin interneuron synapses controls hippocampal network plasticity and memory Hippocampal somatostatin (SOM) cells are dendrite-projecting inhibitory interneurons. CA1 SOM cells receive major excitatory inputs from pyramidal cells (PC-SOM synapses) which show mGluR1a- and mTORC1-mediated long-term potentiation (LTP). PC-SOM synapse LTP contributes to CA1 network metaplasticity and memory consolidation, but whether it is sufficient to regulate these processes remains unknown. Here we used optogenetic stimulation of CA1 pyramidal cells and whole cell recordings in slices to show that optogenetic theta burst stimulation (TBSopto) produces LTP at PC-SOM synapses. At the network level, we found that TBSopto differentially regulates metaplasticity of pyramidal cell inputs: enhancing LTP at Schaffer collateral synapses and depressing LTP at temporo-ammonic synapses. At the behavioral level, we uncovered that in vivo TBSopto regulates learning-induced LTP at PC-SOM synapses, as well as contextual fear memory. Thus, LTP of PC-SOM synapses is a long-term feedback mechanism controlling pyramidal cell synaptic plasticity, sufficient to regulate memory consolidation.

sentences: Shell shape as an indicator of phenotypic stocks of Tehuelche scallop (Aequipecten tehuelchus) in Northern Patagonia, Argentina Tehuelche scallop, Aequipecten tehuelchus, is a commercially exploited species in Northern Patagonia, Argentina. Without genetic differentiation at the species level, A. tehuelchus presents three morphotypes: tehuelchus, madrynensis, and a non-common variant felipponei. The main goal of this study was to analyze the shell shape variation of Tehuelche scallop to differentiate and identify the phenotypic stocks. The shape differences between and within the two main morphotypes (tehuelchus and madrynensis) were assessed using geometric morphometrics in nine localities. The shell shape presented variability at geographic scale, with the morphologic traits that maximized the differentiation among localities between the tehuelchus and madrynensis morphotypes. Scallops from madrynensis morphotype presented higher and circular shell discs with smaller auricles than those from tehuelchus morphotype. Morphometric differentiation was also detected among localities of each morphotype, wherein most of the variability was related to the disc circularity and the symmetry of the auricles. The presence of morphologic variation in San Matias and San Jose gulfs, wherein a single genetic pool is shared, evidenced the plastic nature of the species. Given the distribution of this resource in distinct provincial jurisdictions, the differentiation of phenotypic stocks has relevance in the context of fishery management, especially if zoning and rotational strategies are implemented.

sentences: Physiological and genetic drivers underpinning canopy development are associated with durum wheat yield in rainfed environments New durum wheat (Triticum turgidum L. ssp. Durum) cultivars with improved adaptation to variable rainfall environments are required to sustain productivity in the face of climate change. Physiological traits related to canopy development underpin the production of biomass and yield, as they interact with solar radiation and affect the timing of water use throughout the growing season. This study explored the temporal canopy dynamics of durum wheat using a nested-association mapping population evaluated for longitudinal normalized difference vegetation index (NDVI) measurements. Association mapping was performed to identify quantitative trait loci (QTL) for time-point NDVI and spline-smoothed NDVI trajectory traits. Yield effects associated with QTL for canopy development were investigated using data from four rainfed field trials. Four QTL associated with slower canopy closure, improved yield in specific environments, and notably, were not associated with a yield penalty in any environment. This was likely due to optimised timing of water-use and pleiotropic effects on yield component traits, including spike number and spike length. Overall, this study suggests that slower canopy closure is beneficial for durum wheat production in rainfed environments. Selection for traits or loci associated with canopy development may improve yield stability of durum wheat in water limited environments.

sentences: Human embryos arrest in a quiescent-like state characterized by metabolic and zygotic genome activation problems Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3-8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a quiescent-like state, marked by cell cycle arrest, the downregulation of ribosomes and histones and downregulation of MYC and p53 activity. Mechanistically, the arrested embryos can be divided into three types. Type I embryos fail to complete the maternal-zygotic transition, and type II/III embryos have erroneously low levels of glycolysis and variable levels of oxidative phosphorylation. Treatment with resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype. The mechanism of reactivation involves the upregulation of SIRT1, and activation of glycolysis and fatty acid oxidation which forces the embryos out of a quiescent state. Overall, our data reveal how human embryo arrest can be overcome by modulating metabolic pathways.

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Signaling from the RNA sensor RIG-I is regulated by ufmylation The RNA binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFN). This signal transduction occurs at endoplasmic reticulum (ER)-mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, UFL1, as one of the proteins recruited to membranes at ER-mitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We find that following RNA virus infection, UFL1 is recruited to the membrane targeting protein 14-3-3{varepsilon}, and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 14-3-3{varepsilon} has an increase in UFM1-conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 14-3-3{varepsilon} with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a post-translational control for IFN induction.

SignificanceThe viral RNA sensor RIG-I initiates the antiviral innate immune response by activating a signaling cascade that induces interferon. Activation of the RIG-I signaling pathway is highly regulated to quickly mount a protective immune response while preventing dysregulation that can lead to excessive inflammation or autoimmune disorders. Here, we characterize one such mechanism of regulation. We describe that UFL1, an E3 ligase for the ubiquitin-like modifier conjugation system called ufmylation, is important to promote RIG-I signaling. Using molecular approaches, we show that ufmylation promotes RIG-I interaction with the membrane targeting protein 14-3-3{varepsilon}. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor proteins MAVS for induction of interferon in response to RNA virus infection.

sentences: Unsupervised mining of HLA-I peptidomes reveals new binding motifs and substantial false positives in community database Modern vaccine designs and studies of human leukocyte antigen (HLA)-mediated immune responses rely heavily on the knowledge of HLA allele-specific binding motifs and computational prediction of HLA-peptide binding affinity. Breakthroughs in HLA peptidomics have considerably expanded the databases of natural HLA ligands and enabled detailed characterizations of HLA-peptide binding specificity. However, cautions must be made when analyzing HLA peptidomics data because identified peptides may be contaminants in mass spectrometry or may weakly bind to the HLA molecules. Here, a hybrid de novo peptide sequencing approach was applied to large-scale mono-allelic HLA peptidomics datasets to uncover new ligands and refine current knowledge of HLA binding motifs. Up to 12-40% of the peptidomics data were low-binding affinity peptides with an arginine or a lysine at the C-terminus and likely to be tryptic peptide contaminants. Thousands of these peptides have been reported in a community database as legitimate ligands and might be erroneously used for training prediction models. Furthermore, unsupervised clustering of identified ligands revealed additional binding motifs for several HLA class I alleles and effectively isolated outliers that were experimentally confirmed to be false positives. Overall, our findings expanded the knowledge of HLA binding specificity and advocated for more rigorous interpretation of HLA peptidomics data that will ensure the high validity of community HLA ligandome databases.

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Protein Domain-Based Prediction of Compound-Target Interactions and Experimental Validation on LIM Kinases Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network- based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins structure/function, and bias in system training datasets. Here, we propose a new computational method "DRUIDom" to predict bio- interactions between drug candidate compounds and target proteins by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying physical or functional interactions. As such, other proteins containing the mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including the ones mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting compound-target pairs ([~]2.9M data points), and used as training data for calculating parameters of compound- domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of [~]5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. The datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

Author SummaryDrug development comprises several interlinked steps from designing drug candidate molecules to running clinical trials, with the aim to bring a new drug to market. A critical yet costly and labor-intensive stage is drug discovery, in which drug candidate molecules that specifically interact with the intended biomolecular target (mostly proteins) are identified. Lately, data-centric computational methods have been proposed to aid experimental procedures in drug discovery. These methods have the ability to rapidly assess large molecule libraries and reduce the time and cost of the process; however, most of them suffer from problems related to producing reliable biologically relevant results, preventing them from gaining real-world usage. Here, we have developed a new method called DRUIDom to predict unknown interactions between drugs/drug candidate compounds and biological targets by utilizing the modular structure of proteins. For this, we identify the domains, i.e., the evolutionary and functional building blocks of proteins, where these potential drug compounds can bind, and utilize this information along with protein domain annotations to predict new drug targets. We have tested the biological relevance of DRUIDom on selected proteins that play critical roles in the progression of numerous types of cancer. Cell-based experimental results indicated that predicted inhibitors are effective even on drug-resistant cancer cells. Our results suggest that DRUIDom produces novel and biologically relevant results that can be directly used in the early steps of the drug discovery process.

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Integrated multi-omics investigations reveal the key role of synergistic microbial networks in removing plasticizer di-(2-ethylhexyl) phthalate from estuarine sediments Di-(2-ethylhexyl) phthalate (DEHP) is the most widely used plasticizer worldwide with an annual global production of over eight million tons. Because of its improper disposal, endocrine-disrupting DEHP often accumulates in estuarine sediments in industrialized countries at sub-millimolar levels, resulting in adverse effects on both ecosystems and human beings. The microbial degraders and biodegradation pathways of DEHP in O2-limited estuarine sediments remain elusive. Here, we employed an integrated meta-omics approach to identify the DEHP degradation pathway and major degraders in this ecosystem. Estuarine sediments were treated with DEHP or its derived metabolites, o-phthalic acid and benzoic acid. The rate of DEHP degradation in denitrifying mesocosms was two times slower than that of o-phthalic acid, suggesting that side-chain hydrolysis of DEHP is the rate-limiting step of anaerobic DEHP degradation. On the basis of microbial community structures, functional gene expression, and metabolite profile analysis, we proposed that DEHP biodegradation in estuarine sediments is mainly achieved through synergistic networks between denitrifying proteobacteria. Acidovorax and Sedimenticola are the major degraders of DEHP side-chains; the resulting o-phthalic acid is mainly degraded by Aestuariibacter through the UbiD-dependent benzoyl-CoA pathway. We isolated and characterized Acidovorax sp. strain 210-6 and its extracellular hydrolase, which hydrolyzes both alkyl side-chains of DEHP. Interestingly, genes encoding DEHP/MEHP hydrolase and phthaloyl-CoA decarboxylase--key enzymes for side-chain hydrolysis and o-phthalic acid degradation, respectively--are flanked by transposases in these proteobacterial genomes, indicating that DEHP degradation capacity is likely transferred horizontally in microbial communities.

ImportanceXenobiotic phthalate esters (PAE) have been produced on a considerably large scale for only 70 years. The occurrence of endocrine-disrupting di-(2-ethylhexyl) phthalate (DEHP) in environments has raised public concern, and estuarine sediments are major DEHP reservoirs. Our multi-omics analyses indicated that complete DEHP degradation in O2-limited estuarine sediments depends on synergistic microbial networks between diverse denitrifying proteobacteria and uncultured candidates. Our data also suggest that the side-chain hydrolysis of DEHP, rather than o-phthalic acid activation, is the rate-limiting step in DEHP biodegradation within O2-limited estuarine sediments. Therefore, deciphering the bacterial ecophysiology and related biochemical mechanisms can help facilitate the practice of bioremediation in O2-limited environments. Furthermore, the DEHP hydrolase genes of active DEHP degraders can be used as molecular markers to monitor environmental DEHP degradation. Finally, future studies on the directed evolution of identified DEHP/MEHP hydrolase would bring a more catalytically efficient DEHP/MEHP hydrolase into practice.

sentences: Homeostasis Equation: An Approach to Theoretical Medicine Homeostasis is kind of force that makes living organism to live. In this study, we suggest an integral equation that models homeostasis in living organism. We also showed that various situations can be modeled by homeostasis, and give mathematical interpretation of mechanism of living organism. With our proposed integral equation, one can handle homeostasis quantitatively, and this approach is expected to unveil various hidden properties of living organism.

sentences: Visualization of Arabidopsis root system architecture in 3D by refraction-contrast X-ray micro-computed tomography Plant roots change their morphological traits in order to adapt themselves to different environmental conditions, resulting in alteration of the root system architecture. To understand this mechanism, it is essential to visualize morphology of the entire root system. To reveal effects of long-term alteration of gravity environment on root system development, we have performed an experiment in the International Space Station using Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants and obtained dried root systems grown in rockwool slabs (mineral wool substrate). X-ray computer tomography (CT) technique using an industrial X-ray scanner has been introduced for the purpose to visualize root system architecture of crop species grown in soil in 3D non-invasively. In the case of the present study, however, root system of Arabidopsis is composed of finer roots compared with typical crop plants and rockwool is also composed of fibers having similar dimension to that of the roots. A higher spatial resolution imaging method is required for distinguishing roots from rockwool. Therefore, in the present study, we tested refraction-contrast X-ray micro-CT using coherent X-ray optics available at the beamline BL20B2 of the synchrotron radiation facility SPring-8. Using this technique, both the primary and the secondary roots were successfully identified in the tomographic slices, clearly distinguished from the individual rockwool fibers and resulting in successful tracing of these roots from their basal regions. This newly-developed technique should contribute to elucidate the effect of microgravity on Arabidopsis root system architecture in space.

sentences: distinct: a novel approach to differential distribution analyses We present distinct, a general method for differential analysis of full distributions that is well suited to applications on single-cell data, such as single-cell RNA sequencing and high-dimensional flow or mass cytometry data. High-throughput single-cell data reveal an unprecedented view of cell identity and allow complex variations between conditions to be discovered; nonetheless, most methods for differential expression target differences in the mean and struggle to identify changes where the mean is only marginally affected. distinct is based on a hierarchical non-parametric permutation approach and, by comparing empirical cumulative distribution functions, identifies both differential patterns involving changes in the mean, as well as more subtle variations that do not involve the mean. We performed extensive bench-marks across both simulated and experimental datasets from single-cell RNA sequencing and mass cytometry data, where distinct shows favourable performance, identifies more differential patterns than competitors, and displays good control of false positive and false discovery rates. distinct is available as a Bioconductor R package.

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Age-related decline of the unfolded protein response in the heart promotes protein misfolding and cardiac pathology Cardiac myocyte death during heart failure is particularly detrimental, given that cardiac muscle exhibits limited regenerative potential. Protein aggregation was previously observed in end-stage heart failure, suggesting protein-misfolding in cardiac myocytes as a contributor to the disease process. However, the relationship between protein-misfolding, cardiac myocyte death, and myocardial dysfunction is yet to be clearly established. Here, we showed that protein synthesis and the unfolded protein response (UPR) declined as a function of mammalian postnatal development, especially in tissues with low mitotic activity, such as the heart. A deeper examination in animals models showed that compared to neonatal cardiac myocytes, adult cardiac myocytes expressed lower levels of the adaptive UPR transcription factor, ATF6, as well as lower levels of numerous ATF6-regulated genes, which was associated with susceptibility to ER stress-induced cell death. Further reduction of the ATF6-dependent gene program in ATF6 knock-out mice led to the accumulation of misfolded proteins in the myocardium and impaired myocardial function in response to cardiac stress, indicating that ATF6 plays a critical adaptive role in the setting of cardiac disease. Thus, strategies to increase ATF6 aimed at balancing proteostasis in cardiac myocytes might be a fruitful avenue for the development of novel therapies for heart disease and other age-associated diseases.

HighlightsO_LIThe unfolded protein response (UPR) declines as a function of age in adult mammalian tissues with low mitotic activity, such as the heart
C_LIO_LIDecreases in the UPR in adult cardiac myocytes is associated with impaired survival during ER stress
C_LIO_LIATF6 loss of function in adult hearts increases protein misfolding and cardiac disfunction during stress
C_LI

sentences: A South American mouse morbillivirus sheds light into a clade of rodent-borne morbilliviruses Morbilliviruses are negative sense single stranded monosegmented RNA viruses in the family Paramyxoviridae (order Mononegavirales). Morbilliviruses infect diverse mammals including humans, dogs, cats, small ruminants, seals, and cetaceans, which serve as natural hosts. Here, I report the identification and characterization of novel viruses associated with South American long-haired and olive field mice. The divergent viruses dubbed Raton olivaceo morbillivirus (RoMV) detected in renal samples from mice collected from Chile and Argentina are characterized by an unusually large genome including long intergenic regions and the presence of an accessory protein between the F and H genes redounding in a genome architecture consisting in 3-N-P/V/C-M-F-hp-H-L-5. Structural and functional annotation, genetic distance and evolutionary insights suggest that RoMV is a member of a novel species within genus Morbillivirus tentatively dubbed South American mouse morbillivirus. Phylogenetic analysis suggests that this mouse morbillivirus is closely related to, and clusters into a monophyletic group of novel rodent-borne morbilliviruses. This subclade of divergent viruses expands the host range, redefines the genomic organization and provides insights on the evolutionary history of genus Morbillivirus.

sentences: Melt into the groupElectrophysiological Evidence of Gestalt Perception of Human Dyad It has been shown recently that the human brain has dedicated networks for perception of human bodies in synchronous motion or in situation of interaction. However, below motion and interaction, how does the brain process a simple plurality of humans in close positioning? We used EEG frequency tagging technique to investigate integration of human dyad elements in a global percept. We presented to participants images of two silhouettes, a man and a woman flickering at different frequencies (5.88 vs.7.14Hz). Clear response at these stimulation frequencies reflected response to dyad parts, both when the dyad was presented upright and inverted. However, an emerging intermodulation component (7.14 + 5.88 = 13.02 Hz), a nonlinear response regarded as an objective signature of holistic representation, was significantly enhanced in upright relatively to inverted position. Inversion effect was significant only for the intermodulation component as opposed to stimulation frequencies revealing that dyad configuration perception overrides structural properties of dyad elements. Inversion effect was not significant for a pair of non-human objects. Our results show that merely facing two humans in close positioning leads to perceptually bind them and suggest that the perception of individuals is of different nature when they form a plurality.

sentences: Mobility of nucleosomes is regulated by their binding partners Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.

sentences: Identification of druggable host targets needed for SARS-CoV-2 infection by combined pharmacological evaluation and cellular network directed prioritization both in vitro and in vivo. Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

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Genomic regions associate with major axes of variation in gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.) Stomata and leaf veins play an essential role in transpiration and the movement of water throughout leaves. These traits are thus thought to play a key role in the adaptation of plants to drought and a better understanding of the genetic basis of their variation and coordination could inform efforts to improve drought tolerance. Here, we explore patterns of variation and covariation in leaf anatomical traits and analyze their genetic architecture via genome-wide association (GWA) analyses in cultivated sunflower (Helianthus annuus L.). Traits related to stomatal density and morphology as well as lower order veins were manually measured from digital images while the density of minor veins was estimated using a novel deep learning approach. Leaf, stomatal, and vein traits exhibited numerous significant correlations that generally followed expectations based on functional relationships. Correlated suites of traits could further be separated along three major principal component (PC) axes that were heavily influenced by variation in traits related to gas exchange, leaf hydraulics, and leaf construction. While there was limited evidence of colocalization when individual traits were subjected to GWA analyses, major multivariate PC axes that were most strongly influenced by several traits related to gas exchange or leaf construction did exhibit significant genomic associations. These results provide insight into the genetic basis of leaf trait covariation and showcase potential targets for future efforts aimed at modifying leaf anatomical traits in sunflower.

Significance StatementUsing traditional and automated/high-throughput (using a novel deep learning approach) phenotyping methods we studied leaf anatomical variation in sunflower. Genome-wide association (GWA) analyses identified numerous genomic regions underlying individual trait variation and regions underlying major multivariate axes of phenotypic variation. These results illustrate the value of employing a multivariate approach to GWA analyses and shed light on the extent to which leaf trait (co-)variation can be genetically decoupled to explore novel phenotypic space.

sentences: A microfluidic platform to investigate the role of mechanical constraints on tissue reorganization Mechanical constraints have a high impact on development processes, and there is a need for new tools to investigate the role of mechanosensitive pathways in tissue reorganization during development. We present here experiments where embryonic cell aggregates are aspired through constrictions in microfluidic channels, generating heterogeneous flows and cell deformations that can be imaged using biphoton microscopy. This tool provides a way to measure in situ local viscoelastic properties of 3D tissues and connect them to intercellular and extracellular events such as cell shape changes and cell rear-rangements. Perspectives include applications on organoids to investigate and quantify rheological properties of tissues, and to understand how constraints affect development.

sentences: Chitinase 3-like-1 is a Therapeutic Target That Mediates the Effects of Aging in COVID-19 COVID-19 is caused by the SARS-CoV-2 (SC2) virus and is more prevalent and severe in the elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor ACE2 and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging and that anti-CHI3L1, kasugamycin and inhibitors of phosphorylation, abrogate these ACE2- and SPP-inductive events. Human studies also demonstrated that the levels of circulating CHI3L1 are increased in the elderly and patients with CM where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP; that this induction is a major mechanism contributing to the effects of aging during SC2 infection and that CHI3L1 coopts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.

sentences: Transcription and splicing dynamics during early Drosophila development Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

sentences: A comprehensive list of the replication promoters of 	Bunyavirales reveals a unique promoter structure in 	Nairoviridae differing from other virus families Bunyaviruses belong to the order Bunyavirales, the largest group of RNA viruses. They infect a wide variety of host species around the world, including plants, animals and humans, and pose a major threat to public health. Major families in the order Bunyavirales have tri-segmented negative-sense RNA genomes, the 5 and 3 ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral RNA-dependent RNA polymerase recognizes the promoter to initiates RNA synthesis is important for understanding viral replication and pathogenesis, and for developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. Here, by using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5 and 3 ends of the genome that form a specific complementary strand. We showed that among tri-segmented bunyaviruses, viruses belonging to the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure that differs from that of other bunyaviruses. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses. It is possible that the large genome architecture confers a pathogenic advantage. The promoter list provided in this report is expected to be useful for predicting virus family-specific replication mechanisms of segmented negative-sense RNA viruses.

sentences: Recovery of silver fir (Abies alba Mill.) seedlings from ungulate browsing mirrors soil nitrogen availability Abies alba has a high potential for mitigating climate change in European mountain forests, yet, its natural regeneration is severely limited by ungulate browsing. Here, we simulated browsing in a common garden experiment to study growth and physiological traits, measured from bulk needles, using a randomized block design with two levels of browsing severity and seedlings originating from 19 populations across Switzerland. Genetic factors explained most variation in growth (on average, 51.5%) and physiological traits (10.2%) under control conditions, while heavy browsing considerably reduced the genetic effects on growth (to 30%), but doubled those on physiological traits related to C storage. While browsing reduced seedling height, it also lowered seedlings water use efficiency (decreased{delta} 13C) and N supply by mycorrhizal fungi as indicated by an increase in{delta} 15N. Different populations reacted differently to browsing stress, and for Height, Starch and{delta} 15N, population differences appeared to be the result of natural selection. We found that the fastest growing populations, originating from the warmest regions, decreased their needle starch level the most as a reaction to heavy browsing, suggesting a potential genetic underpinning for a growth-storage trade-off. Further, we found that seedlings originating from mountain populations growing on steep slopes had a significantly lower N discrimination in the common garden than those originating from flat areas, indicating that they have been selected to grow on N poor, potentially drained, soils. This finding was corroborated by the fact that N concentration in adult needles was lower on steep slopes than on flat ground, strongly indicating that steep slopes are the most N poor environments. Seedlings from these poor environments generally had a low growth rate and high storage, thus might be slower to recover from browsing stress than fast growing provenances from the warm environments with developed soils, such as the Swiss plateau.

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Pioglitazone administration restores a PPARG-dependent transcriptional network and ATP levels within skeletal muscles of mice implanted with patient-derived breast tumors BackgroundFatigue is common in patents with breast cancer (BC), and can occur in patients with early stage disease and in the absence of muscle wasting (i.e. cachexia). We have reported transcriptional and proteomic alterations in muscles from BC patients, which are associated with fatigue. Mice implanted with human BC xenografts recapitulate the muscle molecular composition changes seen in patients, coupled with a greater rate of contraction-induced fatigue. Multiple bioinformatics platforms in both human and mouse muscles have identified peroxisome proliferator activated receptor gamma (PPARG) as central to this phenotype, with multiple PPARG target genes downregulated in response to tumor growth. The current study tested the hypothesis that the PPARG agonist pioglitazone (pio), a commonly prescribed diabetes drug, would rescue the transcriptional alterations observed in muscles of tumor-bearing mice.

MethodsSixteen female NSG mice were implanted with breast cancer patient-derived orthotopic xenografts (BC-PDOX) via transplantation of Her2/neu+ human tumor fragments. BC-PDOX mice were randomly assigned to a treatment group that received daily oral pio at 30 mg.kg-1 (n=8), or a control group that received a similar volume of vehicle (n=8). Treatment was initiated when tumors reached a volume of 600mm3, and lasted for 2-weeks. Hindlimb muscles were isolated from BC-PDOX and non-tumor bearing mice for RNA-sequencing, gene expression validation, and ATP quantification. Differentially expressed genes (DEGs) in muscles from BC-PDOX mice relative to non-tumor bearing controls were identified using DESeq2, and multiple bioinformatics platforms were employed to contextualize the DEGs.

ResultsWe found that the administration of pio restored the muscle gene expression patterns of BC-PDOX mice to a profile resembling muscles of non-tumor bearing NSG control mice. Validation of skeletal muscle gene expression by qPCR confirmed pio increased the expression of PPARG target genes in skeletal muscles. Isolated mitochondria from muscles of BC-PDOX mice treated with pio contained greater levels of ATP. There were no differences in body weights, muscle weights, or tumor volumes in pio vs. vehicle treated BC-PDOX mice.

ConclusionsThese data demonstrate that oral pio supplementation rescues the BC-associated downregulation of PPARG target genes in skeletal muscle. Additionally, muscles from BC-PDOX mice treated with pio had greater levels of ATP, which would be associated a more fatigue-resistant muscle phenotype. Therefore, we propose that the FDA-approved and generic diabetes drug, pio, be considered as a supportive therapy for the treatment of BC-associated muscle fatigue.

sentences: A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice Short tandem repeats (STRs) are a class of rapidly mutating genetic elements characterized by repeated units of 1 or more nucleotides. We leveraged whole genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family derived from C57BL/6J and DBA/2J mice to study the effects of genetic background on genome-wide patterns of new mutations at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and identified a locus on chromosome 13 associated with the propensity of STRs to expand. Several dozen genes lie in the QTL region, including Msh3, a known modifier of STR stability at pathogenic repeat expansions in mice and humans. Detailed analysis of the locus revealed a cluster of variants near the 5 end of Msh3, including multiple protein-coding variants within the DNA mismatch recognition domain of MSH3, and a retrotransposon insertion overlapping an annotated exon. Additionally, gene expression analysis demonstrates co-localization of this QTL with expression QTLs for multiple nearby genes, including Msh3. Our results suggest a novel role for Msh3 in regulating genome-wide patterns of germline STR mutations and demonstrate that inherited genetic variation can contribute to variability in accumulation of new mutations across individuals.

sentences: Multiscale mechanical models of DNA nanotubes: A theoretical basis for the design and tuning of DNA nanocarriers DNA nanostructures are one of potential candidates for drug carriers due to their good biocompatibility and non-specificity in vivo. A reliable prediction about mechanical properties of artificial DNA structures is desirable to improve the efficiency of DNA drug carriers, however there is only a handful of information on mechanical functionalities of DNA nanotubes (DNTs). This paper focuses on quantifying the multiscale correlations among DNT deformation, packaging conditions and surrounding factors to tune mechanical properties of DNTs. By combining WLC statistical mechanics model, Parsegians mesoscopic liquid crystal model and Eulers continuum beam theory, we developed a multiscale DNA-frame model; then theoretically characterize the initial packed states of DNTs for the first time, and reveal the diversity mechanism in mechanical properties of DNTs induced by interchain interactions and initial packed states. Moreover, the study of parameters, such as packaging conditions and environmental factors, provides a potential control strategy for tuning mechanical properties of DNTs. These conclusions provide a theoretical basis for accurately controlling the property and deformation of DNT in various DNT dynamic devices, such as DNA nanocarriers.

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T cell Aryl Hydrocarbon Receptor Activity Tunes the Gut Microenvironment to Sustain Autoimmunity. Multiple sclerosis (MS) is an incurable T cell driven autoimmune disease of the central nervous system. The contribution of the environment and, in particular, the gut flora to MS etiology is well documented but not well understood. The aryl hydrocarbon receptor (AHR) binds numerous molecules present in the environment and is a therapeutic target for MS: however, its precise function in T lymphocytes, the orchestrators of MS, has not been described. Here, we show that CD4 specific Ahr knockout drives recovery in EAE by a decreasing T cell fitness. At the mechanistic level, we show that the absence of AHR changes the gut microenvironment composition to generate metabolites, such as bile salts and short chain fatty acids, that impact T cell viability and influence disease recovery. Our study demonstrates a newly emerging role for AHR in mediating the interdependence between T lymphocytes and the microbiota while identifying new potential molecular targets for the treatment of MS and other autoimmune diseases.

One Sentence SummaryThe aryl hydrocarbon receptor modulates the gut microenvironment to promote autoimmunity in a mouse model of multiple sclerosis.

sentences: Quadripulse Stimulation: A Replication Study with A Newly Developed Stimulator Quadripulse stimulation (QPS) is a promising patterned repetitive transcranial magnetic stimulation protocol, which allows the modulation of brain activity for over one hour after the stimulation. Recently, Deymed Diagnostic developed a new stimulator specifically designed to deliver QPS (DuoMAG MP-Quad, https://deymed.com/duomag-qps). The properties of the after-effect with this new stimulator were expected to parallel those obtained with another stimulator, the Magstim stimulator, which is currently used in the psychological and clinical research but not commercially available anymore. Nevertheless, experimental validation of DuoMAG MP-Quad was still warranted. We thus studied the QPS after-effect induced by this stimulator. As a result, motor evoked potentials were found to be bidirectionally modulated by the QPS for more than one hour, consistent with previous studies using the Magstim stimulator. Moreover, the degree of the after-effect was comparable to the after-effect induced by the other stimulator. Taken together, we conclude that the newly developed QPS stimulator is as effective as the Magstim stimulator.

sentences: Molecular Evolution across Mouse Spermatogenesis Genes involved in spermatogenesis tend to evolve rapidly, but we still lack a clear understanding of how different components of molecular evolution vary across this complex developmental process. We used fluorescence activated cell sorting (FACS) to generate expression data for both early (meiotic) and late (postmeiotic) cell types across thirteen inbred strains of mice (Mus) spanning ~7 million years of evolution. We used these comparative developmental data to investigate the evolution of lineage-specific expression, protein-coding sequences, and expression levels. We found increased lineage specificity and more rapid protein-coding and expression divergence during late spermatogenesis, suggesting that signatures of rapid testis molecular evolution are punctuated across sperm development. Despite strong overall developmental parallels in these components of molecular evolution, protein and expression divergences were only weakly correlated across genes. We detected more rapid protein evolution on the X chromosome relative to the autosomes, while X-linked gene expression tended to be relatively more conserved likely reflecting chromosome-wide regulatory constraints. Using allele-specific FACS expression data from crosses between four strains, we found that the relative contributions of different regulatory mechanisms also differed between cell-types. Genes showing cis-regulatory changes were more common late in spermatogenesis, and tended to be associated with larger differences in expression levels and greater expression divergence between species. In contrast, genes with trans-acting changes were more common early and tended to be more conserved across species. Our findings advance understanding of gene evolution across spermatogenesis and underscore the fundamental importance of developmental context in molecular evolutionary studies.

sentences: Post-transcriptional regulation of Leishmania fitness gain The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network driving parasite fitness, with genome instability causing highly reproducible, gene dosage-dependent changes in protein abundance linked to post-transcriptional regulation. These in turn were associated with a gene dosage-independent reduction in abundance of flagellar transcripts and a coordinated increase in abundance of coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of fitness-adapted ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

sentences: O-methylated N-glycans Distinguish Mosses from Vascular Plants In the animal kingdom, a stunning variety of N-glycan structures has emerged with phylogenetic specificities of various kinds. In the plant kingdom, however, N-glycosylation appears as strictly conservative and uniform. From mosses to all kinds of gymno- and angiosperms, land plants mainly express structures with the common pentasaccharide core substituted with xylose, core 1,3-fucose, maybe terminal GlcNAc residues and Lewis A determinants. In contrast, green algae biosynthesize unique and unusual N-glycan structures with uncommon monosaccharides, a plethora of different structures and various kinds of O-methylation. Mosses, a group of plants that are separated by at least 400 million years of evolution from vascular plants, were hitherto seen as harbouring an N-glycosylation machinery identical to that of vascular plants. To challenge this view, we have analysed the N-glycomes of several moss species using MALDI-TOF/TOF, PGC-MS/MS and GC-MS. While all species contained the plant-typical heptasaccharide with no, one or two terminal GlcNAc residues (MMXF, MGnXF and GnGnXF, respectively), many species exhibited MS signals with 14.02 Da increments as characteristic for O-methylation. Throughout all analysed moss N-glycans the level of methylation differed strongly even in the same family. In some species, methylated glycans dominated, while others had no methylation at all. GC-MS revealed the main glycan from Funaria hygrometrica to contain 2,6-O-methylated terminal mannose. Some mosses additionally presented very large, likewise methylated complex-type N-glycans. This first finding of methylation of N-glycans in land plants mirrors the presumable phylogenetic relation of mosses to green algae, where O-methylation of mannose and many other monosaccharides is a common trait.

sentences: Neurodevelopmental clustering of gene expression identifies lipid metabolism genes associated with neuroprotection and neurodegeneration. APOE variants present the strongest association with sporadic Alzheimers Disease. APOE is also highly expressed during neurodevelopment in the Central Nervous System (CNS) and has been shown to be neuroprotective in infancy and gestation. We explored other lipid metabolism genes to determine whether they show a similar neurodevelopmental expression trajectory and are associated with neurodegeneration. APOE was by far the most highly expressed of the lipid metabolism genes in the CNS. Two other genes, Apolipoprotein C1 (APOC1) Glutamate-Ammonia Ligase (GLUL--also known as glutamine synthetase), co-clustered with Apolipoprotein E (APOE) in its developmental trajectory in late gestation through early childhood. These three genes highlight brain structures and developmental time-windows distinct from other lipid metabolism genes. In the CNS they are primarily expressed in astrocytes and are implicated in neuroprotection and neurodegeneration.

sentences: Individual variability of neural computations in the primate retina Variation in the neural code contributes to making each individual unique. We probed neural code variation using [~]100 neural population recordings from major ganglion cell types in the macaque retina, combined with an interpretable computational representation of individual variability using machine learning. This representation captured individual variation and covariation in properties such as nonlinearity, temporal dynamics, and spatial receptive field size, while preserving invariances, such as asymmetries between ON and OFF cells. The covariation of response properties in different cell types was associated with the proximity of lamination of their synaptic inputs. Surprisingly, male retinas exhibited higher firing rates and faster temporal integration than female retinas. Exploiting data from previously recorded macaque retinas enabled efficient characterization of a new macaque retina, and of a human retina. Simulations indicated that combining a vast dataset of healthy macaque recordings with behavioral feedback could be used to identify the neural code and thus improve retinal implants for vision restoration.

sentences: Muilla lordsburgana (Asparagaceae: Brodiaeoideae), a new species found north of Lordsburg, southwestern New Mexico. AO_SCPLOWBSTRACTC_SCPLOWMuilla lordsburgana P.J.Alexander sp. nov. is described from eastern Lordsburg Mesa in the northwestern fringe of the Chihuahuan Desert, southwestern New Mexico. It is very similar to Muilla coronata, a species known in the Mojave Desert of California and a small area of adjacent Nevada. Compared to Muilla coronata, the style and stigma combined are longer, the anthers are longer, the fruits are larger, and the seeds are larger. It has a short flowering period in March, and is difficult or impossible to find at other times of the year. It is narrowly distributed, limited to a band of deep, coarse, sandy soils derived primarily from granite. Several species that are otherwise uncommon in the area also occur in this habitat, including Logfia depressa, Pectocarya platycarpa, and Plagiobothrys arizonicus. Invasive species, especially Erodium cicutarium in the winter annual flora, are generally dominant. Muilla lordsburgana has probably been overlooked until now because it is not easy to observe and occurs in habitats that are not attractive to botanists.

sentences: Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naive and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naive people. Of these, 944 from naive and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naive people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naive people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.

sentences: Fungal taste for minerals: the ectomycorrhizal fungus Paxillus involutus triggers specific genes when extracting potassium from different silicates Silicates make up about 90% of the Earths crust and constitute the main source of mineral nutrients for microorganisms and plants. Fungi can actively weather silicates to extract nutrients. However, it is unclear whether they are able to obtain the same amounts of nutrients and use the same mechanisms when tapping into different mineral sources. We performed a microcosm experiment using the ectomycorrhizal basidiomycetes Paxillus involutus and the silicates K-vermiculite, muscovite and phlogopite as only potassium sources, as they show a different resistance for the removal of K cations from the mineral structure. A combination of transcriptomic, elemental and SEM analyses showed that different minerals stimulated specific weathering mechanisms and led to a change in fungal genes expression. The differential expression of the fungal genes generated alternative chemical attacks on the minerals, resulting in a tailored dissolution and selective uptake of chemical elements according to the leachability of K from the silicate mineral. The K uptake capacity of the fungus was highest with vermiculite in comparison to growth on phlogopite and muscovite. The findings provide new insights into fungal-mineral interactions that will help to interpret key processes for the homeostasis of soil environments.

sentences: High-performance GPCR optogenetics based on molecular properties of animal opsins, MosOpn3 and LamPP Optogenetics for GPCR signaling is highly valuable but still requires effective and versatile tools with performance evaluation from molecular properties. Here we investigated performance of two animal opsins, mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP) in optical manipulation in vivo by using C. elegans. MosOpn3 introduced in a nociceptor neurons induced avoidance responses light-dependently with a retinal isomer ubiquitously present in every tissue, like ChR2 and unlike canonical vertebrate opsins. Remarkably, the sensitivity is ~7000 times higher than the case of ChR2 in the light-induced behavior. LamPP introduced in motor neurons induced violet light-dependent stop and green light-dependent go, demonstrating color-dependent manipulation of behaviors using LamPP. Furthermore, our molecular engineering extended the usability of MosOpn3 and LamPP to different signaling cascades and kinetics. Current findings demonstrated that the availability of two animal opsins is equivalent to that of ChR2 in terms of retinal requirement, providing solid strategies for GPCR optogenetics.

sentences: Induction of Bdnf from promoter I following electroconvulsive seizures contributes to structural plasticity in neurons of the piriform cortex The efficacy of electroconvulsive therapy (ECT) as a treatment for psychiatric disorders, including major depressive disorder (MDD) is hypothesized to depend on induction of molecular and cellular events that trigger structural plasticity in neurons. Electroconvulsive seizures (ECS) in animal models can help to inform our understanding of how electroconvulsive therapy (ECT) impacts the brain. ECS induces structural plasticity in neuronal dendrites in many brain regions, including the piriform cortex, a highly epileptogenic region that has also been implicated in depression. ECS-induced structural plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF), but the functional significance of these transcripts in dendritic plasticity is not clear. Here, we demonstrate that different Bdnf isoforms are expressed non-stochastically across neurons of the piriform cortex following ECS. Specifically, cells expressing Bdnf exon 1-containing transcripts show a unique spatial recruitment pattern in response to ECS. We further demonstrate that Bdnf Ex1 expression in these cells is necessary for ECS-induced dendritic spine plasticity.

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Molecular Basis for the Interaction of Catalase with D-Penicillamine : Rationalization of some of its Deleterious Effects D-penicillamine (D-Pen) is a sulfur compound used in the management of rheumatoid arthritis, Wilsons disease (WD), and alcohol dependence. Many side effects are associated with its use, particularly after long-term treatment. However, the molecular bases for such side effects are poorly understood. Based on the well-known oxidase activity of hemoproteins, and the participation of catalase in cellular H2O2 redox signaling, we posit that D-Pen could inactivate catalase, thus disturbing H2O2 levels. Herein, we report on the molecular bases that could partly explain the side effects associated with this drug compound, and we demonstrate that it induces the formation of compound II, a temporarily inactive state of the enzyme, through two distinct mechanisms. Initially, D-Pen reacts with native catalase and/or iron metal ions, used to mimic non heme iron overload observed in long-term treated WD patients, to generate thiyl radicals. These partake into a futile redox cycling, thus producing superoxide radical anions O2*- and hydrogen peroxide H2O2. Then, either H2O2 unexpectedly reacts with native CAT-Fe(II) to produce compound II, or both aforementioned reactive oxygen species intervene into compound II generation through compound I formation then reduction. These findings support evidence that D-Pen could perturb H2O2 redox homeostasis through transient but recurring catalase inactivation, which may in part rationalize some deleterious effects observed with this therapeutic agent, as discussed.

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Activity-dependent organization of prefrontal hub-networks for associative learning and signal transformation Associative learning is crucial for adapting to environmental changes. The encoding of associative learning involves the dorso-medial prefrontal cortex (dmPFC), and is underpinned by interactions within the resident neuronal population. However, the nature of this population coding is poorly understood. Here we developed a pipeline for computational dissection and longitudinal two-photon imaging of neural population activities in the mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC topology. Through regularized regression methods and graphical modeling, we found fear conditioning organized neuronal ensembles encoding conditioned responses (CR), with enhancing their coactivity, functional connectivity, and association with conditioned stimuli (CS). This suggests that fear conditioning drives dmPFC reorganization to generate novel associative circuits for CS-to-CR transformation. Importantly, neurons strongly responding to unconditioned stimuli (US) during conditioning anterogradely became a hub of the CR ensemble. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on an activity-dependent hub-network formation within the dmPFC.

TeaserOptical and computational dissection uncovered how prefrontal cortical networks are rewired to encode new associative memory

Significance statementAnimals learn to adapt to changing environments. Associative learning is one of the simplest types of learning that has been intensively studied over the past century. Recent development in molecular, genetic, and optogenetic methods has enabled the identification of a neural population encoding the associative memory in the brain. However, it remains unclear how information is stored and processed by the neural population to encode and retrieve the associative memory. To investigate the nature of this population coding, we developed an optical and computational dissection method, demonstrating how associative learning drives reorganization of the neural network in the dorso-medial prefrontal cortex and generates novel circuits for associative memory and signal transformation.

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Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher.

In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2.

IMPORTANCEIn this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection.

The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.

sentences: Dynamics of vesicle reshaping and scission under osmotic shocks We study the effects of osmotic shocks on lipid vesicles via coarse-grained molecular dynamics simulations by explicitly considering the solute in the system. We find that depending on their nature (hypo- or hypertonic) such shocks can lead to bursting events or engulfing of external material into inner compartments, among other morphology transformations. We characterize the dynamics of these processes and observe a separation of time scales between the osmotic shock absorption and the shape relaxation. Our work consequently provides an insight into the dynamics of compartmentalization in vesicular systems as a result of osmotic shocks, which can be of interest in the context of early proto-cell development and proto-cell compartmentalisation.

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Reinstatement of Pavlovian responses to alcohol cues by stress RationaleStress may contribute to relapse to alcohol use in part by enhancing reactivity to cues previously paired with alcohol. Yet, standard models of stress-induced reinstatement generally use contingent presentations of alcohol-paired cues to reinforce instrumental behaviors, making it difficult to isolate the ability of cues to invigorate alcohol-seeking.

ObjectiveHere we sought to test the impact of stress on behavioral responses to alcohol-paired cues, using a model of stress-induced reinstatement of Pavlovian conditioned approach, inspired by Nadia Chaudhris work on context-induced reinstatement.

MethodsLong Evans rats were trained to associate one auditory cue with delivery of alcohol or sucrose and an alternative auditory cue with no reward. Following extinction training, rats were exposed to a stressor prior to being re-exposed to the cues under extinction conditions. We assessed the effects of yohimbine, intermittent footshock and olfactory cues paired with social defeat on responses to alcohol-paired cues, and the effects of yohimbine on responses to sucrose-paired cues.

ResultsThe pharmacological stressor, yohimbine, enhanced Pavlovian responses to both alcohol and sucrose cues, but intermittent footshock and social defeat cues did not.

ConclusionsWhile yohimbine elicited reinstatement of Pavlovian conditioned responses, these effects may be unrelated to activation of stress systems.

sentences: Hierarchical autoencoder-based integration improves performance in multi-omics cancer survival models through soft modality selection With decreasing costs of high-throughput sequencing, more and more datasets providing omics profiles of cancer patients become available. Thus, novel survival analysis approaches integrating these differently sized and heterogeneous molecular and clinical groups of variables start being developed. Due to the difficulty of the task of multi-omics data integration, the Cox Proportional-Hazards (PH) model using clinical data has remained one of the best-performing techniques, barely outperformed by models using molecular data modalities. There is therefore a need for methods that can successfully perform multi-omics integration in survival analysis and outperform the clinical Cox PH model. Moreover, while certain deep learning methods have been shown to provide state-of-the-art accuracy of cancer survival prediction, most of them show no benefit or even decay in performance when integrating a larger number of modalities, further motivating a need to investigate how modality-specific representations should be integrated when using neural networks for multi-omics integration. We benchmarked multiple integration techniques for a neural network architecture, revealing that hierarchical autoencoder-based integration of modality-specific representations outperformed other methods such as max-pooling and was comparable with state-of-the-art statistical approaches for multi-omics integration. Further, we showed that the hierarchical autoencoder-based integration of modality-specific representations achieved increased performance through a soft modality selection mechanism, focusing on the most informative modalities for each cancer. We thus framed multiomics integration as a partial group-wise feature selection problem, highlighting that only those models performed well that could adequately weight important modalities in the presence of the high noise imposed by less important modalities.

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Immunotherapy of glioblastoma explants induces interferon-γ responses and immune cell rearrangements in tumor center, but not periphery Recent therapeutic strategies for glioblastoma (GBM) aim at targeting immune tumor microenvironment (iTME) components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform for GBM would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of therapy response and resistance.

We cultured 47 GBM explants from tumor center and periphery from 7 patients in perfusion bioreactors to assess iTME responses to immunotherapy. Explants were exposed to antibodies blocking the immune checkpoints CD47, PD-1 or or their combination, and were analyzed by highly multiplexed microscopy (CODEX, co-detection by indexing) using an immune-focused 55-marker panel. Culture media were examined for changes of soluble factors including cytokines, chemokines and metabolites. CODEX enabled the spatially resolved identification and quantification of >850,000 single cells in explants, which were classified into 10 cell types by clustering. Explants from center and periphery differed significantly in their cell type composition, their levels of soluble factors, and their responses to immunotherapy. In a subset of explants, culture media displayed increased interferon-{gamma} levels, which correlated with shifts in immune cell composition within specific tissue compartments, including the enrichment of CD4+ and CD8+ T cells within an adaptive immune compartment. Furthermore, significant differences in the expression levels of functional molecules in innate and adaptive immune cell types were found between explants responding or not to immunotherapy. In non-responder explants, T cells showed higher expression of PD-1, LAG-3, TIM-3 and VISTA, whereas in responders, macrophages and microglia showed higher cathepsin D levels. Our study demonstrates that ex vivo immunotherapy of GBM explants enables an active antitumoral immune response within the tumor center in a subset of patients, and provides a framework for multidimensional personalized assessment of tumor response to immunotherapy.

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Graphene oxide/silver nanoparticle ink formulations rapidly inhibit influenza A virus and OC43 coronavirus infection in vitro Respiratory tract infections present a significant risk to the human population, both through seasonal circulation and novel introductions with pandemic potential. There is a strong need for antiviral compounds with broad antimicrobial activity that can be coated onto filtration systems and personal protective equipment to augment their ability to remove infectious particles from the environment. Graphene oxide and silver nanoparticles are both materials with documented antimicrobial properties. Here, we tested the in vitro antiviral properties of several graphene oxide-silver nanoparticle composite materials, which were prepared through three different methods: reduction with silver salt, direct addition of silver nanospheres, and direct addition of silver nanospheres to thiolized graphene. These materials were tested over short time scales for their antiviral activity against two enveloped RNA viruses, influenza A virus and OC43 coronavirus, by performing viral plaque assays after exposure of the viruses to each material. It was found that the graphene oxide - silver nanoparticle materials generated by direct addition of the silver nanospheres were able to completely inhibit plaque formation by both viruses within one minute of exposure. Materials generated by the other two methods had varying levels of efficacy against influenza A virus. These studies indicate that graphene oxide-silver nanoparticle composite materials can rapidly neutralize RNA viruses and demonstrate their potential for use in a wide range of applications.

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sentences: The MuvB Complex Binds and Stabilizes Nucleosomes Downstream of the Transcription Start Site of Cell-Cycle Dependent Genes The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.

sentences: Decrease of Functional Connectivity within the Default Mode Network by a Brief Training of Focused Attention on the Breath in Novices Experienced meditators reduce the activity of the default mode network (DMN), a brain system preferentially active when people are not engaged in specific tasks. However, the neural modulation of the DMN in novices remain largely unexplored. By using electroencephalography, we investigated the DMN functional connectivity in two groups of novices: the meditation group practiced six consecutive days of focused attention on the breath; the control group practiced only on the first and last days. After the brief training, results showed a decrease in the DMN functional connectivity between the ventromedial prefrontal cortex and the posterior cingulate cortex in theta and alpha bands during the focused attention condition, in the meditation group compared to the control group. The change in DMN functional connectivity was significantly correlated with the increase in state-level mindfulness scores. These data elucidate DMN modifications already arising at the initial stages of mindfulness meditation training in novices.

sentences: Relative stability of mRNA and protein severely limits inference of gene networks from single-cell mRNA measurements Inference of gene regulatory networks from single-cell expression data, such as single-cell RNA sequencing, is a popular problem in computational biology. Despite diverse methods spanning information theory, machine learning, and statistics, it is unsolved. This shortcoming can be attributed to measurement errors, lack of perturbation data, or difficulty in causal inference. Yet, it is not known if kinetic properties of gene expression also cause an issue. We show how the relative stability of mRNA and protein hampers inference. Available inference methods perform benchmarking on synthetic data lacking protein species, which is biologically incorrect. We use a simple model of gene expression, incorporating both mRNA and protein, to show that a more stable protein than mRNA can cause loss in correlation between the mRNA of a transcription factor and its target gene. This can also happen when mRNA and protein are on the same timescale. The relative difference in timescales affects true interactions more strongly than false positives, which may not be suppressed. Besides correlation, we find that information-theoretic nonlinear measures are also prone to this problem. Finally, we demonstrate these principles in real single-cell RNA sequencing data for over 1700 yeast genes.

sentences: Dorsal CA1 Hippocampal Place Cells Form a Multi-Scale Representation of Megaspace Spatially firing "place cells" within the hippocampal CA1 region form internal maps of the environment necessary for navigation and memory. In rodents, these neurons have been almost exclusively studied in small environments (<4 m2). It remains unclear how place cells encode a very large open 2D environment, which is more analogous to the natural environments experienced by rodents and other mammals. Such an ethologically realistic environment would require a more complex spatial representation, capable of simultaneously representing space at overlapping multiple fine to coarse informational scales. Here we show that in a  megaspace (18.6 m2), the majority of dorsal CA1 place cells exhibited multiple place subfields of different sizes, akin to those observed along the septo-temporal axis. Furthermore, the total area covered by the subfields of each cell was not correlated with the number of subfields, and this total area increased with the scale of the environment. The multiple different-sized subfields exhibited by place cells in the megaspace suggest that the ensemble population of subfields form a multi-scale representation of space within the dorsal hippocampus. Our findings point to a new dorsal hippocampus ensemble coding scheme that simultaneously supports navigational processes at both fine- and coarse-grained resolutions.

sentences: Cardioluminescence in transgenic zebrafish embryos: a calcium imaging tool to study drug effects and pathological modeling The zebrafish embryo has emerged as an excellent model in cardiovascular research. The existing techniques to monitor Ca2+ in the heart based on fluorescent Ca2+ biosensors are limited due to phototoxicity and photobleaching. To overcome these issues, we have used bioluminescence. We generated a transgenic line expressing GFP-Aequorin in the heart, Tg(cmlc2:GA), and optimized an in vivo aequorin reconstitution protocol to improve the luminescence capacity. This allowed imaging Ca2+ in long duration recordings in embryos of 3 to 5 days post-fertilization. The analogs diacetyl h-coelenterazine and f-coelenterazine enhanced the light output and signal-to-noise ratio from the embryos. With this cardioluminescence model, we monitored the time-averaged Ca2+ levels and beat-to-beat Ca2+ oscillations. Changes in Ca2+ levels were observed by incubation with BayK8644, an L-type Ca2+ channel agonist, the channel blocker nifedipine, and {beta}-adrenergic blocker propranolol. Treatment of zebrafish embryos with terfenadine for 24 hours has been proposed as a model of heart failure. Tg(cmlc2:GA) embryos treated with terfenadine showed a 2:1 atrioventricular block and a decrease in the ventricular Ca2+ levels.

sentences: Enhancer-promoter interactions and transcription are maintained upon acute loss of CTCF, cohesin, WAPL, and YY1 It remains unclear why acute depletion of CTCF and cohesin only marginally affects expression of most genes despite substantially perturbing 3D genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling to find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-hour) depletion of CTCF, cohesin, and WAPL. YY1 has been proposed to be a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription, and 3D genome folding. Strikingly, live-cell single-molecule imaging revealed that cohesin depletion reduced transcription factor binding to chromatin. Thus, although neither CTCF, cohesin, WAPL, nor YY1 are required for the short-term maintenance of most E-P interactions and gene expression, we propose that cohesin may serve as a "transcription factor binding platform" that facilitates transcription factor binding to chromatin.

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NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP  M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase.

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sentences: Forgetting Enhances Episodic Control with Structured Memories Forgetting is a normal process in healthy brains, and evidence suggests that the mammalian brain forgets more than is required based on limitations of mnemonic capacity. Episodic memories, in particular, are liable to be forgotten over time. Researchers have hypothesized that it may be beneficial for decision making to forget episodic memories over time. Reinforcement learning offers a normative framework in which to test such hypotheses. Here, we show that a reinforcement learning agent that uses an episodic memory cache to find rewards in maze environments can forget a large percentage of older memories without any performance impairments, if they utilize mnemonic representations that contain structural information about space. Moreover, we show that some forgetting can actually provide a benefit in performance compared to agents with unbounded memories. Our analyses of the agents show that forgetting reduces the influence of outdated information and states which are not frequently visited on the policies produced by the episodic control system. These results support the hypothesis that some degree of forgetting can be beneficial for decision making, which can help to explain why the brain forgets more than is required by capacity limitations.

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Modelling the synergistic effect of bacteriophage and antibiotics on bacteria: killers and drivers of resistance evolution Bacteriophage (phage) are bacterial predators which can also spread antimicrobial resistance (AMR) genes between bacteria by generalised transduction. Phage are often present alongside antibiotics in the environment, yet evidence of their joint killing effect on bacteria is conflicted, and the dynamics of transduction in such systems are unknown. Here, we combine in vitro data and mathematical modelling to identify conditions where phage and antibiotics act in synergy to remove bacteria or drive AMR evolution.

We adapt a published model of phage-bacteria dynamics, including transduction, to add the pharmacodynamics of erythromycin and tetracycline, parameterised from new in vitro data. We simulate a system where two strains of Staphylococcus aureus are present at stationary phase, each carrying either an erythromycin or tetracycline resistance gene, and where multidrug-resistant bacteria can be generated by transduction only. We determine rates of bacterial clearance and multidrug-resistant bacteria appearance, when either or both antibiotics and phage are present at varying timings and concentrations.

Although phage and antibiotics act in synergy to kill bacteria, by reducing bacterial growth antibiotics reduce phage production. A low concentration of phage introduced shortly after antibiotics fails to replicate and exert a strong killing pressure on bacteria, instead generating multidrug-resistant bacteria by transduction which are then selected for by the antibiotics. Multidrug-resistant bacteria numbers were highest when antibiotics and phage were introduced simultaneously.

The interaction between phage and antibiotics leads to a trade-off between a slower clearing rate of bacteria (if antibiotics are added before phage), and a higher risk of multidrug-resistance evolution (if phage are added before antibiotics), exacerbated by low concentrations of phage or antibiotics. Our results form hypotheses to guide future experimental and clinical work on the impact of phage on AMR evolution, notably for studies of phage therapy which should investigate varying timings and concentrations of phage and antibiotics.

sentences: Inference of polyploid origin and inheritance mode from population genomic data Summary/AbstractWhole-genome duplications yield varied chromosomal pairing patterns, ranging from strictly bivalent to multivalent, resulting in disomic and polysomic inheritance modes. In the bivalent case, homeologous chromosomes form pairs, where in a multivalent pattern all copies are homologous and are therefore free to pair and recombine. As sufficient sequencing data is more readily available than high-quality cytological assessments of meiotic behavior or population genetic assessment of allelic segregation, especially for non-model organisms, here we describe two bioinformatics approaches to infer origins and inheritance modes of polyploids using short-read sequencing data. The first approach is based on distributions of allelic read depth at the heterozygous sites within an individual, as the expectations of such distributions are different for disomic and polysomic inheritance modes. The second approach is more laborious and based on a phylogenetic assessment of partially phased haplotypes of a polyploid in comparison to the closest diploid relatives. We discuss the sources of deviations from expected inheritance patterns, advantages and pitfalls of both methods, effects of mating types on the performance of the methods, and possible future developments.

sentences: Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.

sentences: SARS-CoV-2 activates ER stress and Unfolded protein response Coronavirus disease-2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus infection is a major global public health concern affecting millions of people worldwide. The scientific community has joint efforts to provide effective and rapid solutions to this disease. Knowing the molecular, transmission and clinical features of this disease is of paramount importance to develop effective therapeutic and diagnostic tools. Here, we provide evidence that SARS-CoV-2 hijacks the glycosylation biosynthetic, ER-stress and UPR machineries for viral replication using a time-resolved (0-48 hours post infection, hpi) total, membrane as well as glycoproteome mapping and orthogonal validation. We found that SARS-CoV-2 induces ER stress and UPR is observed in Vero and Calu-3 cell lines with activation of the PERK-eIF2-ATF4-CHOP signaling pathway. ER-associated protein upregulation was detected in lung biopsies of COVID-19 patients and associated with survival. At later time points, cell death mechanisms are triggered. The data show that ER stress and UPR pathways are required for SARS-CoV-2 infection, therefore representing a potential target to develop/implement anti-CoVID-19 drugs.

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Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants Ethnopharmacological relevanceArtemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria.

Aim of the StudySARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2.

Materials and MethodsUsing Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated.

ResultsHot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 M, 0.01-0.14 g, and 23.4-57.4 g, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 M, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 M. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values.

ConclusionsA. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

List of compounds studiedAmodiaquine
Artemisinin
Artesunate
Artemether
Deoxyartemisinin
Dihydroartemisinin

HighlightsO_LIArtemisia annua is effective in stopping replication of SARS-CoV-2 including 2 new variants.
C_LIO_LIThe anti-viral effect does not correlate to artemisinin, nor to the total flavonoid content.
C_LIO_LIThe anti-viral mechanism does not appear to involve blockade virus entry into cell.
C_LIO_LIThe plant offers two additional benefits: a decreased inflammatory response and blunting of fibrosis.
C_LIO_LIA. annua may provide a safe, low-cost alternative for treating patients infected with SARS-CoV-2.
C_LI

sentences: Donut-like organization of inhibition underlies categorical neural responses in the midbrain Categorical neural responses underlie various forms of selection and decision-making. Such binary-like responses promote robust signaling of the winner in the presence of input ambiguity and neural noise. Here, we show that a  donut-like inhibitory mechanism in which each competing option suppresses all options except itself, is highly effective at generating categorical neural responses. It surpasses motifs of feedback inhibition, recurrent excitation, and divisive normalization invoked frequently in decision-making models. We demonstrate experimentally not only that this mechanism operates in the midbrain spatial selection network in barn owls, but also that it is necessary for categorical signaling by it. The functional pattern of neural inhibition in the midbrain forms an exquisitely structured  multi-holed donut consistent with this networks combinatorial inhibitory function for stimulus selection. Additionally, modeling reveals a generalizable neural implementation of the donut-like motif for categorical selection. Self-sparing inhibition may, therefore, be a powerful circuit module central to categorization.

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Evolution is exponentially more powerful with frequency-dependent selection Valiant [1] proposed to treat Darwinian evolution as a special kind of computational learning from statistical queries. The statistical queries represent a genotypes fitness over a distribution of challenges. And this distribution of challenges along with the best response to them specify a given abiotic environment or static fitness landscape. Valiants model distinguished families of environments that are "adaptable-to" from those that are not. But this model of evolution omits the vital ecological interactions between different evolving agents - it neglects the rich biotic environment that is central to the struggle for existence.

In this article, I extend algorithmic Darwinism to include the ecological dynamics of frequency-dependent selection as a population-dependent bias to the distribution of challenges that specify an environment. Thus, extended algorithmic Darwinism suggests extended statistical queries rather than just statistical queries as the appropriate model for eco-evo dynamics. This extended algorithmic Darwinism replaces simple invasion of wild-type by a mutant-type of higher scalar fitness with an evolutionary game between wild-type and mutant-type based on their frequency-dependent fitness function. To analyze this model, I develop a game landscape view of evolution, as a generalization of the classic fitness landscape approach.

I show that this model of eco-evo dynamics on game landscapes can provide an exponential speed-up over the purely evolutionary dynamics of the strict algorithmic Darwinism. In particular, I prove that the Parity environment - which is known to be not adaptable-to under strict algorithmic Darwinism - is adaptable-to by eco-evo dynamics. Thus, the ecology of frequency-dependent selection does not just increase the tempo of evolution, but fundamentally transforms its mode. This happens even if frequency-dependence is restricted to short-time scales - such short bursts of frequency-dependent selection can have a transformative effect on the ability of populations to adapt to their environments in the long-term.

Unlike typical learning algorithms, the eco-evo dynamic for adapting to the Parity environment does not rely on Gaussian elimination. Instead, the dynamics proceed by simple isotropic mutations and selection in finite populations of just two types (the resident wild-type and invading mutant). The resultant process has two stages: (1) a quick stage of point-mutations that moves the population to one of exponentially many local fitness peaks; followed by (2) a slower stage where each  step follows a double-mutation by a point-mutation. This second stage allows the population to hop between local fitness peaks to reach the unique global fitness peak in polynomial time. The evolutionary game dynamics of finite populations are essential for finding a short adaptive path to the global fitness peak during the second stage of the adaptation process. This highlights the rich interface between computational learning theory, analysis of algorithms, evolutionary games, and long-term evolution.

sentences: Source tracking and global distribution of the mobilized tigecycline resistant gene tet(X) The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X15) and tet(X16), that were resistant to tigecycline. The metagenomic analysis revealed these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. The transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer was primarily promoted by the mobile elements ISBf11 and IS4351. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant determinants tet(X15) and tet(X16). Further tracing these tet(X) in single bacterial isolate from public repository indicated that tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp.., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X) gene. Additionally, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes.

sentences: Perturbation of ACE2 structural ensembles by SARS-CoV-2 spike protein binding The human ACE2 enzyme serves as a critical first recognition point of coronaviruses, including SARS-CoV-2. In particular, the extracellular domain of ACE2 interacts directly with the S1 tailspike protein of the SARS-CoV-2 virion through a broad protein-protein interface. Although this interaction has been characterized by X-ray crystallography and Cryo-EM, these structures do not reveal significant differences in ACE2 structure upon S1 protein binding. In this work, using several all-atom molecular dynamics simulations, we show persistent differences in ACE2 structure upon binding. These differences are determined with the Linear Discriminant Analysis (LDA) machine learning method and validated using independent training and testing datasets, including long trajectories generated by D. E. Shaw Research on the Anton 2 supercomputer. In addition, long trajectories for 78 potent ACE2-binding compounds, also generated by D. E. Shaw Research, were projected onto the LDA classification vector in order to determine whether the ligand-bound ACE2 structures were compatible with S1 protein binding. This allows us to predict which compounds are "apo-like" vs "complex-like", as well as to pinpoint long-range ligand-induced allosteric changes of ACE2 structure.

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Flight-tone mediated circadian control of audibility in mating swarms of Anopheles mosquitoes Mating swarms of malaria mosquitoes form every day at sunset throughout the tropical world, they typically last less than 30 minutes. Activity patterns must thus be highly synchronized between the sexes. Moreover, males must be able to identify the few sporadically entering females by detecting the females faint flight tones. We here show that the Anopheles circadian clock ensures a tight synchrony of male and female activity and -importantly - also retunes the males acoustic detection system: by lifting their own flight tones at dusk, males actively enhance the audibility of females. The reported phenomenon of  harmonic convergence is a random by-product of the mosquitoes flight tone variance. Intriguingly, flight tones of individual mosquitoes occupy narrow -partly non-overlapping-frequency ranges, suggesting that the audibility of individual females varies across males.

One Sentence SummaryMale mosquitoes sharpen their hearing at sunset.

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

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

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

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

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

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

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

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

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

sentences: Novelty detection in early olfactory processing of the honey bee, Apis mellifera Animals are constantly bombarded with stimuli, which presents a fundamental problem of sorting among pervasive uninformative stimuli and novel, possibly meaningful stimuli. We evaluated novelty detection behaviorally in honey bees as they position their antennae differentially in an air stream carrying familiar or novel odors. We then characterized neuronal responses to familiar and novel odors in the first synaptic integration center in the brain - the antennal lobes. We found that the neurons that exhibited stronger initial responses to the odor that was to be familiarized are the same units that later distinguish familiar and novel odors, independently of chemical identities. These units, including both projection neurons and local neurons, showed a decreased response to the familiar odor but an increased response to the novel odor. Our results suggest that the antennal lobe may assign a category of familiarity or novelty to an odor stimulus in addition to its chemical identity code. Therefore, the mechanisms for novelty detection may be present in early sensory processing, either as a result of local synaptic interaction or via feedback from higher brain centers.

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Stage-specific, morphological and molecular markers of encystation in Giardia lamblia Differentiation into environmentally resistant cysts is required for transmission of the ubiquitous intestinal parasite Giardia lamblia. Encystation in Giardia requires the production, processing and transport of Cyst Wall Proteins (CWPs) in developmentally-induced, Golgi-like, Encystation Specific Vesicles (ESVs). Progress through this trafficking pathway can be followed by tracking CWP localization over time. However, there is no recognized system to distinguish the advancing stages of this process which can complete at variable rates depending how encystation is induced. Here we propose a staging system for encysting Giardia based on the morphology of CWP1-stained ESVs. We demonstrate the molecular distinctiveness of maturing ESVs at these stages by following GlRab GTPases through encystation. Previously, we established that Giardias sole Rho family GTPase, GlRac, associates with ESVs and has a role in regulating their maturation and the secretion of their cargo. As a proof of principle, we delineate the relationship between GlRac and ESV stages. Through proteomic studies, we identify putative interactors of GlRac that could be used as additional stage-specific ESV markers. This staging system provides a common descriptor of ESV maturation regardless of the source of encysting cells. Furthermore, the identified set of molecular markers for ESV stages will be a powerful tool for characterizing trafficking mutants that impair ESV maturation and morphology.

ImportanceGiardiasis is a diarrheal disease that affects 280 million people worldwide. It is caused by Giardia lamblia, a protozoan parasite which rely on differentiating from host-dwelling trophozoites to environmentally-resistant cysts for transmission and survival. This encystation process requires the transport of Cyst Wall Proteins (1-3) within membrane-bound compartments called Encystation Specific Vesicles (ESV) from the endoplasmic reticulum to the surface of the cell. The whole process takes 24 hours to complete and these compartments are the only recognizable equivalent of Golgi apparatus in this minimalistic organism. Progress of this trafficking pathway can be followed by localizing Cyst Wall Protein 1 over time post induction of encystation but this can be ambiguous when specific molecular events need to be specified. Here we propose a staging system that is based on ESV morphology changes by capitalizing on the secretory/processing events we already know they represent. We validate the molecular distinctiveness of these stages by following Giardia Rabs through the pathway and characterize putative interactors of an established regulator of encystation, GlRac, to provide additional stage-specific molecular markers. This staging system will provide a definitive, yet adaptable, framework to map out functions of yet-to-be discovered players of this important pathway.

sentences: Emergent Diversity and Persistent Turnover in Evolving Microbial Cross-Feeding Networks A distinguishing feature of many ecological networks in the microbial realm is the diversity of substrates that could potentially serve as energy sources for microbial consumers. The microorganisms are themselves the agents of compound diversification via metabolite excretion or overflow metabolism. It has been suggested that the emerging richness of different substrates is an important condition for the immense biological diversity in microbial ecosystems. In this work, we study how complex cross-feeding networks (CFN) of microbial species may develop from a simple initial community given some elemental evolutionary mechanisms of resource-dependent speciation and extinctions using a network flow model. We report results of several numerical experiments and report an in-depth analysis of the evolutionary dynamics. We find that even in stable environments, the system is subject to persisting turnover, indicating an ongoing co-evolution. Further, we compare the impact of different parameters, such as the ratio of mineralization, as well as the metabolic versatility and variability on the evolving community structure. The results imply that high microbial and molecular diversity is an emergent property of evolution in cross-feeding networks, which affects transformation and accumulation of substrates in natural systems, such as soils and oceans, with potential relevance to biotechnological applications.

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

sentences: Unveiling unexpected complexity and multipotentiality of early heart fields Complex organs are composed of a multitude of specialized cell types which assemble to form functional biological structures. How these cell types are created and organized remains to be elucidated for many organs including the heart, the first organ to form during embryogenesis. Here, we show the ontogeny of mammalian mesoderm at high-resolution single cell and genetic lineage/clonal analyses, which revealed an unexpected complexity of the contribution and multi-potentiality of mesodermal progenitors to cardiac lineages creating distinct cell types forming specific regions of the heart. Single-cell transcriptomics of Mesp1 lineage-traced cells during embryogenesis and corresponding trajectory analyses uncovered unanticipated developmental relationships between these progenitors and lineages including two mesodermal progenitor sources contributing to the first heart field (FHF), an intraembryonic and a previously uncharacterized extraembryonic-related source, that produce distinct cardiac lineages creating the left ventricle. Lineage-tracing studies revealed that these extraembryonic-related FHF progenitors reside at the extraembryonic-intraembryonic interface in gastrulating embryos and generate cardiac cell types that form the epicardium and the dorsolateral regions of the left ventricle and atrioventricular canal myocardium. Clonal analyses further showed that these progenitors are multi-potent, creating not only cardiomyocytes and epicardial cell types but also extraembryonic mesoderm. Overall, these results reveal unsuspected multiregional origins of the heart fields, and provide new insights into the relationship between intraembryonic cardiac lineages and extraembryonic tissues and the associations between congenital heart disease and placental insufficiency anomalies.

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Efficient gradient boosting for prognostic biomarker discovery MotivationGradient boosting decision tree (GBDT) is a powerful ensemble machine learning method that has the potential to accelerate biomarker discovery from high-dimensional molecular data. Recent algorithmic advances, such as Extreme Gradient Boosting (XGB) and Light Gradient Boosting (LGB), have rendered the GBDT training more efficient, scalable and accurate. These modern techniques, however, have not yet been widely adopted in biomarkers discovery based on patient survival data, which are key clinical outcomes or endpoints in cancer studies.

ResultsIn this paper, we present a new R package Xsurv as an integrated solution which applies two modern GBDT training framework namely, XGB and LGB, for the modeling of censored survival outcomes. Based on a comprehensive set of simulations, we benchmark the new approaches against traditional methods including the stepwise Cox regression model and the original gradient boosting function implemented in the package gbm. We also demonstrate the application of Xsurv in analyzing a melanoma methylation dataset. Together, these results suggest that Xsurv is a useful and computationally viable tool for screening a large number of prognostic candidate biomarkers, which may facilitate cancer translational and clinical research.

AvailabilityXsurv is freely available as an R package at: https://github.com/topycyao/Xsurv

Contactxuefeng.wang@moffitt.org

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: The Origin of Conductive-Pulse Sensing Inside a Nanopore and the Role of Electro-Hydrodynamics Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic reduction of ionic flux during molecular occupancy of a pore, particularly at high salt concentrations. However, conductive pulses are widely reported at low salt conditions wherein electroosmotic flow can be quite significant. Aside from transporting molecules like DNA, we investigated whether electroosmotic flow has other potential impacts on sensing attributes such current enhancements due to the analyte molecule. The overwhelming majority of literature reports counterions as the dominant mechanism of conductive events (a moleculecentric theory for conductive events). Conductive events are not well understood due to the complex interplay between (charged) nanopore walls, DNA grooves, ion mobility, and counterion clouds. Yet, the prevailing consensus of counterions being introduced into the pore by the molecule does not fit well with a growing number of experiments including the fact that proteins can generate conductive events despite having a heterogeneous surface charge. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e. a pore-centric theory). We believe these findings will stimulate a useful discussion on the nature of conductive events and their impact on molecular sensing in nanoscale pores.

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Beyond tracking climate: niche evolution during native range expansion and its implications for novel invasions AimAlthough ecological niche models have been instrumental in understanding the widespread species distribution shifts under global change, rapid niche shifts limit model transferability to novel locations or time periods. Niche shifts during range expansion have been studied extensively in invasive species, but may also occur in native populations tracking climate change. We compared niche shifts during both types of range expansion in a Mediterranean annual plant to ask (i) whether the species native range expansion tracked climate change, (ii) whether further range expansion was promoted by niche expansion, and (iii) how these results changed forecasts of two ongoing invasions in Australia and California.

LocationEurasian Holarctic, California and Australia

TaxonDittrichia graveolens (L.) Greuter (Asteraceae)

MethodsNiche shifts were quantified in both environmental and geographic space, using the framework of niche centroid shift, overlap, unfilling, and expansion (COUE) as well as Maximum Entropy modelling. We used the historic native distribution and climate data (1901-1930) to project the expected distribution in the present climate (1990-2019), and compared it to the observed current distribution of D. graveolens. Finally, we compared invasion forecasts based on the historic and present native niches.

ResultsWe found that D. graveolens expanded its native range well beyond what would be sufficient to track climate change, associated with a 5.5% niche expansion towards more temperate climates. In contrast, both invasions showed niche conservatism, and were (still) constrained to climatic areas matching the historic native niche.

Main conclusionsour results show that contrary to hypotheses in the literature, niche shifts are not necessarily more rapid in invasions than in native range expansions. We conclude that niche expansion during the process of climate tracking may cause further range expansion than expected based on climate change alone.

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The kidney protects against sepsis by enhancing the systemic release of Uromodulin to stimulate macrophage function Sepsis is a significant cause of mortality in hospitalized patients. Concomitant development of acute kidney injury (AKI) increases sepsis mortality through unclear mechanisms. While electrolyte disturbances and toxic metabolite buildup during AKI could be important, it is possible that the kidney produces a protective molecule lost during sepsis with AKI. We previously demonstrated that systemic Tamm-Horsfall Protein (THP, uromodulin), a kidney-derived protein with immunomodulatory properties, falls in AKI. Using a mouse sepsis model without severe kidney injury, we show that the kidney increases circulating THP by enhancing basolateral release of THP from medullary thick ascending limb cells. In sepsis patients, changes in circulating THP are positively associated with critical illness. THP is also found de novo in injured lungs. Genetic ablation of THP in mice leads to increased mortality and bacterial burden during sepsis. Consistent with the increased bacterial burden, the presence of THP in vitro and in vivo leads macrophages and monocytes to upregulate a transcriptional program promoting cell migration, phagocytosis and chemotaxis and treatment of macrophages with purified THP increases phagocytosis. Rescue of septic THP-/- mice with exogenous systemic THP improves survival. Together, these findings suggest that through releasing THP, the kidney modulates the immune response in sepsis by enhancing mononuclear phagocyte function and systemic THP has therapeutic potential in sepsis.

Significance StatementSepsis is a significant contributor to kidney injury as well as morbidity and mortality worldwide. Specific therapies to improve outcomes in sepsis with kidney injury have largely been limited to symptom management and infectious agent control, in part because it is unclear how kidney injury increases sepsis mortality. This paper describes the identification of Tamm-Horsfall protein, previously known to protect in ischemic models of AKI, as protective in preclinical models of sepsis. It demonstrates how the loss of THP leads to decreased mononuclear phagocyte function and diversity, increased pathogen burden and decreased survival. THP also increases in sepsis without severe kidney injury and concentrates in injured organs. Further study of THP in sepsis could lead to novel sepsis therapeutics.

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Gene prioritization based on random walks with restarts and absorbing states, to define gene sets regulating drug pharmacodynamics from single-cell analyses Prioritizing genes for their role in drug sensitivity, is an important step in understanding drugs mechanisms of action and discovering new molecular targets for co-treatment. To formalize this problem, we consider two sets of genes X and P respectively composing the predictive gene signature of sensitivity to a drug and the genes involved in its mechanism of action, as well as a protein interaction network (PPIN) containing the products of X and P as nodes. We introduce Genetrank, a method to prioritize the genes in X for their likelihood to regulate the genes in P.

Genetrank uses asymmetric random walks with restarts, absorbing states, and a suitable renormalization scheme. Using novel so-called saturation indices, we show that the conjunction of absorbing states and renormalization yields an exploration of the PPIN which is much more progressive than that afforded by random walks with restarts only. Using MINT as underlying network, we apply Genetrank to a predictive gene signature of cancer cells sensitivity to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), performed in single-cells. Our ranking provides biological insights on drug sensitivity and a gene set considerably enriched in genes regulating TRAIL pharmacodynamics when compared to the most significant differentially expressed genes obtained from a statistical analysis framework alone. We also introduce gene expression radars, a visualization tool to assess all pairwise interactions at a glance.

Genetrank is made available in the Structural Bioinformatics Library (https://sbl.inria.fr/doc/Genetrank-user-manual.html). It should prove useful for mining gene sets in conjunction with a signaling pathway, whenever other approaches yield relatively large sets of genes.

sentences: Atypical small GTPase RABL3 interacts with RAB11 to regulate early ciliogenesis in human cells Primary cilia are near-ubiquitously assembled on cells in the human body and are broadly associated with genetic diseases and cancers. In the early stage of ciliogenesis, the ciliary vesicle (CV) is formed on the mother centriole, which nucleates the primary cilium. However, the regulatory mechanisms underlying CV formation have not yet been fully elucidated. Here, we found that the atypical small GTPase RAB-Like 3 (RABL3) is necessary to assemble primary cilia in human cells. RABL3 directly interacts with RAB11, which is involved in CV formation. RABL3 localizes around the centrosome during early ciliogenesis, reminiscent of RAB11 dynamics. Furthermore, RABL3 positively controls the CV formation like RAB11. These findings suggest that RABL3 plays an important role, in cooperation with RAB11, in CV formation during early ciliogenesis.

sentences: A hymenopteran odorant alerts flies to bury eggs Ants are ubiquitous and consume insects at all life stages, presumably creating a strong selective pressure for ant avoidance behaviors across insects. The insect egg stage can be especially defenseless against predation given that eggs are usually immobile and unguarded, suggesting insect mothers may have evolved oviposition strategies to minimize the ant predation risk to their offspring. Given the lack of parental care in most insects, these oviposition strategies would likely be innate rather than learned, since insect mothers are not usually present to assess predation of their eggs. Here, we use the vinegar fly Drosophila melanogaster as a model system for examining parental defensive responses to ant presence. Flies usually lay eggs partially inserted into the food substrate, although some are laid on top of the food and a few are inserted deeply into the food. We found that exposure to ants significantly alters fly oviposition depth: the proportion of eggs on the food surface decreased while the proportion of buried eggs increased. Buried eggs survive ant foraging bouts better than surface eggs, showing that this oviposition depth behavior is adaptive. This induced behavior is conserved across the genus Drosophila and is dependent on the fly olfactory system: anosmic mutant flies fail to bury their eggs in the presence of ants, and ant odor extracts are sufficient to induce egg burying. To further delineate the ant lineages to which flies respond, we exposed flies to the odors from numerous species of ants and other insects. Surprisingly, flies buried their eggs in response to the odors of nearly all hymenopterans tested, including hymenopteran groups that flies rarely interact with in nature like bees and paper wasps. Our data suggest that hymenopterans possess a conserved and ancient odorant, and that drosophilids evolved a mechanism for sensing this odorant early in their evolution as a means of protecting their offspring from ant predation. This study sheds light on the ecology and mechanisms underlying a sscommon biotic interaction in nature, that between insect parents ands the ants that would consume their offspring.

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In vitro inhibition of H. pylori in a preferential manner using bioengineered L. lactis releasing guided Antimicrobial peptides ObjectivesTargeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. This is even more important when a pathogen like H. pylori resides in stomach, a sensitive microbial ecosystem. Using a probiotic like Lactococcus lactis and bioengineering it to release a guided Antimicrobial Peptide (AMP) targeted towards the pathogen offers a pathway to specifically knock-out the deleterious species and not disturbing the stomach microbiome.

ResultsThree AMPs, Alyteserin, CRAMP and Laterosporulin, were genetically fused to a guiding peptide MM1, which selectively binds to Vacuolating Toxin A (VacA) of H. pylori and cloned into an excretory vector pTKR inside L. lactis. When cultured together in vitro, the L. lactis bioengineered with guided AMPs selectively killed H. pylori when compared to E. coli or Lactobacillus plantarum, as determined by qPCR. Chemically synthesized Alyteserin and MM1-Alyteserin showed similar preferential inhibition of H. pylori when compared against E. coli, with the MIC of MM1-Alyteserin becoming significantly higher for E. coli than Alytserin whereas no such effet was observed against H. pylori.

ConclusionsProbiotics bioengineered to excrete guided AMPs can be a novel and useful approach for combating pathogens without endangering the natural microbial flora. Given the wealth of AMPs and guiding ligands, both natural and synthetic, this approach can be adapted to develop a diverse array of chimeric guided AMPs and can be cloned into probiotics to create a safe and effective alternative to conventional chemical antibiotics.

sentences: Evolution Without Variation and Selection Natural selection successfully explains how organisms accumulate adaptive change despite that traits acquired over a lifetime are eliminated at the end of each generation. However, in some domains that exhibit cumulative, adaptive change--e.g., cultural evolution, and earliest life--acquired traits are retained; these domains do not face the problem that Darwins theory was designed to solve. Lack of transmission of acquired traits occurs when germ cells are protected from environmental change, due to a self-assembly code used in two distinct ways: (i) actively interpreted during development to generate a soma, and (ii) passively copied without interpretation during reproduction to generate germ cells. Early life and cultural evolution appear not to involve a self-assembly code used in these two ways. We suggest that cumulative, adaptive change in these domains is due to a lower-fidelity evolutionary process, and model it using Reflexively Autocatalytic and Foodset-generated networks. We refer to this more primitive evolutionary process as Self-Other Reorganisation (SOR) because it involves internal self-organising and self-maintaining processes within entities, as well as interaction between entities. SOR encompasses learning but in general operates across groups. We discuss the relationship between SOR and Lamarckism, and illustrate a special case of SOR without variation.

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FiberSim: a flexible open-source model of myofilament-level contraction FiberSim is a flexible open-source model of myofilament-level contraction. The code uses a spatially explicit technique, meaning that it tracks the position and status of each contractile molecule within the lattice framework. This allows the model to simulate some of the mechanical effects modulated by myosin binding protein-C, as well as dose-dependence of myotropes and the effects of varying isoform expression levels. This paper provides a short introduction to FiberSim and presents simulations of tension-pCa curves with and without regulation of thick filament transitions by myosin-binding protein C. The software was designed to be flexible (the user can define their own model and/or protocol) and computationally efficient (simulations can be performed on a regular laptop). We hope that other investigators will use FiberSim to explore myofilament level mechanisms and to accelerate research focusing on the contractile properties of sarcomeres.

Statement of significanceMyotropes, such as omecamtiv mecarbil and mavacamten, are new therapeutics that bind directly to sarcomeric proteins. Their clinical development has reenergized interest in mechanistic understanding of sarcomere level function. FiberSim is an open-source spatially-explicit computer model that simulates myofilament level mechanics and can predict how modulating the function of a sarcomeric protein will impact contractility. The software was specifically designed to be flexible and user-friendly and may help to further accelerate myofilament-research.

sentences: Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence Trypanosoma brucei is responsible for lethal diseases in humans and cattle in Sub-Saharan Africa. These extracellular parasites extravasate from the blood circulation into several tissues. The importance of the vasculature in tissue tropism is poorly understood. Using intravital imaging and bioluminescence, we found that gonadal white adipose tissue and pancreas are the two main parasite reservoirs. We show that reservoir establishment happens before vascular permeability is compromised, suggesting that extravasation is an active mechanism. Blocking endothelial surface adhesion molecules (E-selectin, P-selectins, or ICAM2) significantly reduced extravascular parasite load in all organs and delayed host lethality. Remarkably, blocking CD36 had a specific effect on adipose tissue tropism that was sufficient to delay lethality, suggesting that establishment of the adipose tissue reservoir is necessary for parasite virulence. This works demonstrates the importance of the vasculature in a T. brucei infection and identifies organ-specific adhesion molecules as key players for tissue tropism.

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Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs Nonsense mutations or premature termination codons (PTCs) comprise [~]11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes.

One Sentence SummarySuppression of endogenous CFTR nonsense mutations by anticodon engineered tRNAs significantly increases mRNA expression and channel function.

sentences: DNA-controlled Spatio-temporal Patterning of Active Matter Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton; an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel comprised of microtubules: a DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties.

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Stronger net selection on males across animals Sexual selection is considered the major driver for the evolution of manifold sex differences. However, the eco-evolutionary dynamics of sexual selection and their role for a populations adaptive potential to respond to environmental change have only recently been explored. Theory predicts that sexual selection promotes adaptation at a low demographic cost only if net selection is stronger on males compared to females. We used a comparative approach to show that net selection is indeed stronger in males in species prone to intense sexual selection. Given that both sexes share the vast majority of their genes, our findings corroborate the notion that the genome is often confronted with a more stressful environment when expressed in males. Collectively, our study supports a long-standing key assumption required for sexual selection to bolster adaptation, and intense sexual selection may therefore enable some species to track environmental change more efficiently.

One sentence summaryComparative study finds support for stronger net selection in males.

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Synonymous mutations can alter protein dimerization through localized interface misfolding involving self-entanglements Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding affinities by more than half despite leaving the proteins amino acid sequence unaltered. Here, we use coarse-grain simulations of protein synthesis, ejection from the ribosome, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of the two E. coli homodimers oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs and calculate the ensemble-average interaction energy between the resulting dimers. We find, similar to experiments with other dimers, that oligoribonucleases dimerization is altered by synonymous mutations. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, whose probability of occurrence depends on translation speed. Translation of the fast- and slow-translating mRNAs of oligoribonuclease decreases the population of these misfolded states relative to wildtype. For ribonuclease T, however, these misfolded populations are insensitive to synonymous mutations. Entanglements cause altered dimerization energies for oligoribonuclease as there is a significant association (odds ratio: 50) between non-native self-entanglements and weak-binding dimer conformations. These conclusions are independent of model resolution, as entangled structures persist in long-time-scale all-atom simulations. Thus, non-native changes in entanglement is a mechanism through which oligomer structure and function can be altered.

SIGNIFICANCE STATEMENTSynonymous mutations affect a range of post-translational protein functions, including dimerization, without altering the amino acid sequence of the encoded protein. This suggests that proteins somehow retain a "memory" of their translation-elongation kinetics long after synthesis is complete. Here, we demonstrate that synonymous mutations can change the likelihood that nascent proteins misfold into self-entangled conformations. These self-entangled structures are similar to the native state but with key conformational perturbations that disrupt the dimer interface, reducing their ability to dimerize. Rearrangement of such self-entangled states to the native state is a slow process, offering a structural explanation for how translation-elongation kinetics can influence long-time-scale protein-protein binding affinities.

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Minos: variant adjudication and joint genotyping of cohorts of bacterial genomes Short-read variant calling for bacterial genomics is a mature field, and there are many widely-used software tools. Different underlying approaches (eg pileup, local or global assembly, paired-read use, haplotype use) lend each tool different strengths, especially when considering non-SNP (single nucleotide polymorphism) variation or potentially distant reference genomes. It would therefore be valuable to be able to integrate the results from multiple variant callers, using a robust statistical approach to "adjudicate" at loci where there is disagreement between callers. To this end, we present a tool, Minos, for variant adjudication by mapping reads to a genome graph of variant calls. Minos allows users to combine output from multiple variant callers without loss of precision. Minos also addresses a second problem of joint genotyping SNPs and indels in bacterial cohorts, which can also be framed as an adjudication problem.

We benchmark on 62 samples from 3 species (Mycobacterium tuberculosis, Staphylococcus aureus, Klebsiella pneumoniae) and an outbreak of 385 M. tuberculosis samples. Finally, we joint genotype a large M. tuberculosis cohort (N{approx}15k) for which the rifampicin phenotype is known. We build a map of non-synonymous variants in the RRDR (rifampicin resistance determining region) of the rpoB gene and extend current knowledge relating RRDR SNPs to heterogeneity in rifampicin resistance levels. We replicate this finding in a second M. tuberculosis cohort (N{approx}13k).

Minos is released under the MIT license, available at https://github.com/iqbal-lab-org/minos.

sentences: Use of viral motif mimicry improves the proteome-wide discovery of human linear motifs Linear motifs have an integral role in dynamic cell functions including cell signalling, the cell cycle and others. However, due to their small size, low complexity, degenerate nature, and frequent mutations, identifying novel functional motifs is a challenging task. Viral proteins rely extensively on the molecular mimicry of cellular linear motifs for modifying cell signalling and other processes in ways that favour viral infection. This study aims to discover human linear motifs convergently evolved also in disordered regions of viral proteins, under the hypothesis that these will result in enrichment in functional motif instances. We systematically apply computational motif prediction, combined with implementation of several functional and structural filters to the most recent publicly available human-viral and human-human protein interaction network. By limiting the search space to the sequences of viral proteins, we observed an increase in the sensitivity of motif prediction, as well as improved enrichment in known instances compared to the same analysis using only human protein interactions. We identified > 8,400 motif instances at various confidence levels, 105 of which were supported by all functional and structural filters applied. Overall, we provide a pipeline to improve the identification of functional linear motifs from interactomics datasets and a comprehensive catalogue of putative human motifs that can contribute to our understanding of the human domain-linear motif code and the mechanisms of viral interference with this.

sentences: Deconstructing replicative senescence heterogeneity of human mesenchymal stem cells at single cell resolution reveals therapeutically targetable senescent cell sub-populations Cellular senescence is characterised by a state of permanent cell cycle arrest. It is accompanied by often variable release of the so-called senescence-associated secretory phenotype (SASP) factors, and occurs in response to a variety of triggers such as persistent DNA damage, telomere dysfunction, or oncogene activation. While cellular senescence is a recognised driver of organismal ageing, the extent of heterogeneity within and between different senescent cell populations remains largely unclear. Elucidating the drivers and extent of variability in cellular senescence states is important for discovering novel targeted seno-therapeutics and for overcoming cell expansion constraints in the cell therapy industry. Here we combine cell biological and single cell RNA-sequencing approaches to investigate heterogeneity of replicative senescence in human ESC-derived mesenchymal stem cells (esMSCs) as MSCs are the cell type of choice for the majority of current stem cell therapies and senescence of MSC is a recognized driver of organismal ageing. Our data identify three senescent subpopulations in the senescing esMSC population that differ in SASP, oncogene expression, and escape from senescence. Uncovering and defining this heterogeneity of senescence states in cultured human esMSCs allowed us to identify potential drug targets that may delay the emergence of senescent MSCs in vitro and perhaps in vivo in the future.

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Design considerations for workflow management systems use in production genomics research and the clinic BackgroundThe changing landscape of genomics research and clinical practice has created a need for computational pipelines capable of efficiently orchestrating complex analysis stages while handling large volumes of data across heterogeneous computational environments. Workflow Management Systems (WfMSs) are the software components employed to fill this gap.

ResultsThis work provides an approach and systematic evaluation of key features of popular bioinformatics WfMSs in use today: Nextflow, CWL, and WDL and some of their executors, along with Swift/T, a workflow manager commonly used in high-scale physics applications. We employed two use cases: a variant-calling genomic pipeline and a scalability-testing framework, where both were run locally, on an HPC cluster, and in the cloud. This allowed for evaluation of those four WfMSs in terms of language expressiveness, modularity, scalability, robustness, reproducibility, interoperability, ease of development, along with adoption and usage in research labs and healthcare settings. This article is trying to answer, "which WfMS should be chosen for a given bioinformatics application regardless of analysis type?".

ConclusionsThe choice of a given WfMS is a function of both its intrinsic language and engine features. Within bioinformatics, where analysts are a mix of dry and wet lab scientists, the choice is also governed by collaborations and adoption within large consortia and technical support provided by the WfMS team/community. As the community and its needs continue to evolve along with computational infrastructure, WfMSs will also evolve, especially those with permissive licenses that allow commercial use. In much the same way as the dataflow paradigm and containerization are now well understood to be very useful in bioinformatics applications, we will continue to see innovations of tools and utilities for other purposes, like big data technologies, interoperability, and provenance.

sentences: Oxylipin metabolism is controlled by mitochondrial b-oxidation during bacterial inflammation. Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial {beta}-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin {beta}-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-{gamma}. Last, dampening oxylipin levels by {beta}-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial {beta}-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

sentences: Evolutionary features of a prolific subtype of avian influenza A virus in European waterfowl Avian influenza A virus (AIV) is ubiquitous in waterfowl, and detected annually at high prevalence in waterfowl during the Northern Hemipshere autumn. Some AIV subtypes are globally common in waterfowl, such as H3N8, H4N6, and H6N2, and are detected in the same populations at high frequency, annually. In order to investigate genetic features associated to the long-term maintenance of common subtypes in migratory ducks, we sequenced 248 H4 viruses isolated across 8 years (2002-2009) from Mallards (Anas platyrhynchos) sampled in southeast Sweden. Phylogenetic analyses showed that both H4 and N6 sequences fell into in three distinct lineages, structured by year of isolation. Specifically, across the eight years of the study, we observed lineage replacement, whereby a different HA lineage circulated in the population each year. Analysis of deduced amino acid sequences of the HA lineages illustrated key differences in regions of the globular head of hemagglutinin that overlap with established antigentic sites in homologous hemagglutinin H3, suggesting the possibility of antigenic differences among these HA lineages. Beyond HA, lineage replacement was common to all segments, such that novel genome constellations were detected across years. A dominant genome constellation would rapidly amplify in the duck population, followed by unlinking of gene segments as a result of reassortment within 2-3 weeks following introduction. These data help reveal the evolutionary dynamics exhibited by AIV on both annual and decadal scales in an important reservoir host.

sentences: Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan The adhesion receptor dystroglycan provides a critical mechanical link between the extracellular matrix (ECM) and the actin cytoskeleton to help muscle cells withstand contraction and neural cells maintain the blood brain barrier. Disrupting the link is associated cancer and muscular dystrophy. Proteolysis of dystroglycan by matrix metalloproteinases (MMPs) provides a mechanism to break the mechanical anchor and is amplified in several pathogenic states, yet how MMP cleavage is regulated is largely unexplored. We solved the crystal structure of the extracellular membrane-adjacent domains of dystroglycan containing the MMP site and find the tandem Ig-like and SEA-like domains structurally interact in an L-shape to facilitate a permissive conformation for /{beta} auto-catalytic processing. The MMP cleavage site is located in a structural motif C-terminal to the SEA-like domain that folds back and nestles into a pocket within the SEA-like domain to protect the cleavage site from MMPs. We then show while the intact proteolytic switch domain is resistant to MMP cleavage, structurally disruptive muscular dystrophy-related mutations sensitize dystroglycan to proteolysis in secreted constructs and the full-length receptor. Intriguingly, previously uncharacterized cancer-associated mutations that map to the proteolytic switch domain similarly lead to increases in proteolysis. Taken together, this study demonstrates dystroglycan proteolysis is conformationally regulated and mutations in the domain lead to enhanced proteolysis.

sentences: Timing of exposure is critical in a highly sensitive model of SARS-CoV-2 transmission Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing. However, contrary to expectation, trends observed with sub-optimal exposure timing suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

sentences:
Soil Microbial Composition and Structure Allow Assessment of Biological Product Effectiveness and Crop Yield Prediction Understanding the effectiveness and potential mechanism of action of agricultural biological products under different soil profiles and crops will allow more precise product recommendations based on local conditions and will ultimately result in increased crop yield. This study aimed to use bulk and rhizosphere soils microbial composition and structure to evaluate the effect of a Bacillus amyloliquefaciens strain QST713 inoculant on potatoes, and to explore its relationship with crop yield. We implemented NGS and bioinformatics approaches to assess the bacterial and fungal biodiversity in 185 soil samples, distributed over four different time points -from planting to harvest -from three different geographical regions in the United States.

In addition to variety, phenological stage of the potato plant and geography being important factors defining the microbiome composition and structure, the microbial inoculant applied as a treatment also had a significant effect. However, treatment preserved the native communities without causing a detectable long-lasting effect on the alpha- and beta-diversity patterns after harvest. Specific taxonomic groups, and most interestingly the structure of the fungal and bacterial communities (measured using co-occurrence and co-exclusion networks), changed after inoculation. Additionally, using information about the application of the microbial inoculant and considering microbiome composition and structure data we were able to train a Random Forest model to estimate if a bulk or rhizosphere soil sample came from a low or high yield block with relatively high accuracy, concluding that the structure of fungal communities is a better estimator of potato yield than the structure of bacterial communities.

IMPORTANCEThe manuscripts results reinforce the notion that each crop variety on each location recruits a unique microbial community and that these communities are modulated by the vegetative growth stage of the plant. Moreover, inoculation of a Bacillus amyloliquefaciens strain QST713-based product on potatoes also changed specific taxonomic groups and, most interestingly, the structure of local fungal and bacterial networks in bulk and rhizosphere soil. The data obtained, coming from in-field assays performed in three different geographical locations, allowed training a predictive model to estimate the yield of a certain block, identifying microbiome variables -especially those related to microbial community structure- with a higher predictive power than the variety and geography of the block. The methods described here can be replicated to fit new models predicting yield in any other crop, and to evaluate the effect of any Ag-input product in the composition and structure of the soil microbiome.

sentences: 4D Cell-Condensate Bioprinting 4D bioprinting techniques that facilitate formation of shape-changing scaffold-free cell condensates with prescribed geometries have yet been demonstrated. Here, a simple yet novel 4D bioprinting approach is presented that enables formation of a shape-morphing cell condensate-laden bilayer system comprised of an actuation layer and a cell condensate-supporting microgel (MG) layer. The strategy produces scaffold-free cell condensates which morph over time into predefined complex shapes. With a sequential printing (i.e., MG printing first onto the preformed actuation hydrogel layer and cell-only printing inside the pre-printed MG construct second), cell condensate-laden bilayers with specific geometries are readily fabricated and can be further UV-crosslinked to form strong interlayer adhesion. Since the bilayers have tunable deformability and MG degradation can be tailored, this enables controllable morphological transformations and on-demand liberation of cell condensates. With this system, large cell condensate-laden constructs with various complex shapes were obtained through predefined conformational conversions. As a proof-of-concept study, the formation of the letter "C" and helix-shaped robust cartilage-like tissues differentiated from human mesenchymal stem cells (hMSCs) was demonstrated. This new system brings about a new versatile 4D bioprinting platform idea that is anticipated to broaden and facilitate the applications of cell condensation-based 4D bioprinting.

sentences: 8 A structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI As one of the largest protein complexes in eukaryotes, the nuclear pore complex (NPC) forms a conduit regulating nucleocytoplasmic transport. Here, we determined 8 [A] resolution cryo-electron microscopic (cryo-EM) structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC. With the aid of AlphaFold2, we managed to build a most comprehensive and accurate pseudoatomic model of the CR to date, including the Y complexes and flanking components of Nup358, Nup214 complexes, Nup205 and Nup93. Comparing with previously reported CR model, the Y complex structure in our model exhibits much tighter interactions in the hub region mediated by -solenoid domain in Nup160 C-terminus. Five copies of Nup358 are identified in each CR subunit to provide rich interactions with other Nups in stem regions of Y complexes. Two copies of Nup214 complexes lay in a parallel pattern and attach to the short arm region of Y complexes towards the central channel of NPC. Besides, the structural details of two copies of Nup205 on the side of the short arm region and one copy of Nup93 on the stem region of Y complexes in each CR subunit are also revealed. These in-depth novel structural features represent a great advance in understanding the assembly of NPCs.

sentences: Controls on the isotopic composition of microbial methane Microbial methane production (methanogenesis) is responsible for more than half of the annual emission of this major greenhouse gas to the atmosphere. Though the stable isotopic composition of methane is often used to characterize its sources and sinks, strictly empirical descriptions of the isotopic signature of methanogenesis currently limit such attempts. We developed a biochemical-isotopic model of methanogenesis by CO2 reduction, which predicts carbon and hydrogen isotopic fractionations, and clumped isotopologue distributions, as functions of the cells environment. We mechanistically explain multiple-isotopic patterns in laboratory and natural settings and show that such patterns constrain the in-situ energetics of methanogenesis. Combining our model with environmental data, we infer that in almost all marine environments and gas deposits, energy-limited methanogenesis operates close to chemical and isotopic equilibrium.

sentences: Coupling of metataxonomics and culturing improves description of bacterial communities from a multi-contaminated waste sediment and identifies a novel Rhizorhapis sp. with metal resistance potential Long-term contaminated environments have been recognized as potential hotspots for bacterial discovery in taxonomic and functional terms for bioremediation purposes. Here, bacterial communities from waste sediment collected from a former industrial dumpsite and contaminated with petroleum hydrocarbon and heavy metals were investigated through the parallel application of culture-independent (16S rRNA gene amplicon sequencing) and -dependent (plate culturing followed by colony picking and isolates identification by 16S Sanger sequencing) approaches. The bacterial diversities retrieved by both approaches greatly differed. Bacteroidetes and Proteobacteria were dominant in the culture-independent community, while Firmicutes and Actinobacteria were the main culturable groups. Only 2.7% of OTUs (operational taxonomic units) in the culture-independent dataset were cultured. Most of the culturable OTUs were absent or in very low abundances in the culture-independent dataset, revealing that cultivation is a useful tool to study the rare bacterial biosphere. One culturable OTUs (comprising only the isolate SPR117) was identified as a potential new species in the genus Rhizorhapis (class Alphaproteobacteria) and was selected for further characterization. Phytopathogenicity tests showed that Rhizorhapis sp. strain SPR117 (ATCC TSD-228) is not pathogenic for lettuce, despite the only described species within this genus, Rhizorhapis suberifaciens, is causal agent of the lettuce corky root disease. The genome of the strain SPR117 was sequenced, assembled in 256 contigs with a length of 4,419,522 bp and a GC content of 59.9% and its further annotation revealed the presence of genes related to the resistance to arsenic, copper and iron, among other metals. Therefore, the coupling of metataxonomic and culturing is a useful tool to obtain not only an improved description of bacterial communities in contaminated environments, but also to isolate microorganisms with bioremediation potential.

sentences: Nuclear PHGDH promotes neutrophil recruitment to drive liver cancer progression Metabolic dysregulation and the communications between cancer and immune cells are emerging as two essential features of malignant tumors. In this study, we observed that nuclear localization of phosphoglycerate dehydrogenase (PHGDH) associates with poor prognosis of liver cancer patients, and Phgdh is required for liver cancer progression in a mouse model. Unexpectedly, the impairment of Phgdh enzyme activity exerts a slight effect on liver cancer model, indicating PHGDH contributes to liver cancer progression mainly depending on its non-metabolic roles with nuclear location. PHGDH uses its ACT domain to bind cMyc in nuclear and forms a transactivation axis "PHGDH/p300/cMyc/AF9" which drives CXCL1/8 gene expression. Chemokines CXCL1/8 promotes neutrophils recruitment and then supports tumor associated macrophages (TAMs) filtration in liver, thereby urging liver cancer into advanced stages. Forced cytosolic location of PHGDH or destruction of the PHGDH/cMyc interaction abolishes the oncogenic function of nuclear PHGDH. Collectively, this study reveals a non-metabolic role of PHGDH with altered cellular location in liver cancer, and suggests a promising drug target for liver cancer therapy by targeting the interaction between PHGDH and "undruggable" cMyc.

sentences: Modeling the variability of the electromotor commandsystem of pulse electric fish Mormyrids, a family of weakly electric fish, use electric pulses for communication and for extracting information from the environment (active electroreception). The electromotor system controls the timing of pulse generation. Ethological studies have described several sequences of pulse intervals (SPIs) which are related to distinct behaviors (e.g. mating or exploratory behaviors). Accelerations, scallops, rasps, and cessations are four SPI patterns reported in these fish, each showing characteristic temporal structures and large variability. This paper presents a computational model of the electromotor command circuit that reproduces SPI patterns as a function of the inputs to the model while keeping the same internal network configuration. The topology of the model is based on a simplified representation of the network with four neuron clusters (nuclei). An initial ad hoc tuned configuration (S-T) was built to reproduce nucleus characteristics and network topology as described by detailed morphological and electrophysiological studies. Then, a genetic algorithm (GA) was developed and applied to automatically tune the synaptic parameters of the model connectivity. Two different configurations obtained from the GA are presented here: one optimized to a set of synthetic examples of SPI patterns (S-GA) and another configuration adjusted to patterns recorded from freely-behaving Gnathonemus Petersii specimens (R-GA). Robustness analyses to input variability were performed to discard overfitting and assess validity. Results show that the set of SPI patterns are consistently reproduced, both for synthetic data and for recorded data. This model can be used as a tool to test novel hypotheses regarding temporal structure in electrogeneration.

sentences: Development of Web Application for the Comparison of Segment Variability with Sequence Evolution and Immunogenic Properties for Highly Variable Proteins: An Application to Viruses. High rate of mutation and structural flexibilities in viral proteins quickly make them resistant to the host immune system and existing antiviral strategies. For most of the pathogenic viruses, the key survival strategies lie in their ability to evolve rapidly through mutations that affects the protein structure and function. Along with the experimental research related to antiviral development, computational data mining also plays an important role in deciphering the molecular and genomic signatures of the viral adaptability. Uncovering conserved regions in viral proteins with diverse chemical and biological properties is an important area of research for developing antiviral therapeutics, though assigning those regions is not a trivial work. Advancement in protein structural information databases and repositories, made by experimental research accelerated the in-silico mining of the data to generate more integrative information. Despite of the huge effort on correlating the protein structural information with its sequence, it is still a challenge to defeat the high mutability and adaptability of the viral genomics structure. In this current study, the authors have developed a user-friendly web application interface that will allow users to study and visualize protein segment variabilities in viral proteins and may help to find antiviral strategies. The present work of web application development allows thorough mining of the surface properties and variabilities of viral proteins which in combination with immunogenicity and evolutionary properties make the visualization robust. In combination with previous research on 20-Dimensional Euclidian Geometry based sequence variability characterization algorithm, four other parameters has been considered for this platform: [1] predicted solvent accessibility information, [2] B-Cell epitopic potential, [3] T-Cell epitopic potential and [4] coevolving region of the viral protein. Uniqueness of this study lies in the fact that a protein sequence stretch is being characterized rather than single residue-based information, which helps to compare properties of protein segments with variability. In current work, as an example, beside presenting the web application platform, five proteins of SARS-CoV2 was presented with keeping focus on protein-S. Current web-application database contains 29 proteins from 7 viruses including a GitHub repository of the raw data used in this study. The web application is up and running in the following address: http://www.protsegvar.com.

sentences: Notch signaling via Hey1 and Id2b regulates Muller glia's regenerative response to retinal injury Zebrafish Muller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MGs injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MGs injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MGs injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MGs injury-response threshold and proliferation.

sentences: Assessment of temporal complexity in functional MRI between rest and task conditions Dynamic models of cortical activity, as measured by functional magnetic resonance imaging (fMRI), have recently brought out important insights into the organization of brain function. In terms of temporal complexity, these hemodynamic signals have been shown to exhibit critical behaviour at the edge between order and disorder. In this study, we aimed to revisit the properties and spatial distribution of temporal complexity in resting state and task fMRI of 100 unrelated subjects from the Human Connectome Project (HCP). First, we compared two common choices of complexity measures (i.e., Hurst exponent versus multiscale entropy) and reported high similarity between them. Second, we investigated the influence of experimental paradigms and found high task-specific complexity. We considered four mental tasks in the HCP database for the analysis: Emotion, Working memory, Social, and Language. Third, we tailored a recently-proposed statistical framework that incorporates the structural connectome, to assess the spatial distribution of complexity measures. These results highlight brain regions including parts of the default mode network and cingulate cortex with significantly stronger complex behaviour than the rest of the brain, irrespective of task. In sum, temporal complexity measures of fMRI are reliable markers of the cognitive status.

sentences: Genome-wide analysis illustrates the effect of polyploidization in Phragmites australis Polyploidization is a common event in plant evolution, and it plays an important role in plant speciation and adaptation. To address the role of polyploidization in grass diversification, we studied Phragmites australis, a species with intraspecific variation of chromosome numbers ranging from 2n=36 to 144. A combined analysis of genome structure, phylogeny and population genetics were used to study the evolution of P. australis. Whole-genome sequencing of three representative lineages revealed the allopolyploid origin of the species, with subgenome divergence dating back to approximately 29 million years ago, and the genomes showed hallmarks of relaxed selection associated with asexual propagation. Genome-wide analysis of 88 individuals from different populations around the world using restriction site associated DNA sequencing (RAD-seq) identified seven main intraspecific lineages with extensive genetic admixture. Each lineage was characterized by a distinct ploidy level, mostly tetraploid or octoploid, suggesting several polyploid events. Furthermore, we observed octoploid and hexaploid lineages at contact zones in Romania, Hungary and South Africa, suggestively due to genomic conflicts in allotetraploid parental lineages. Polyploidy may have evolved as a strategy to escape from the evolutionary dead-end of asexual propagation and the resulting decrease in genomic plasticity.

sentences: ENGINEERING, DECODING AND SYSTEMS-LEVEL CHARACTERIZATION OF CHIMPANZEE CYTOMEGALOVIRUS The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the transcriptome and proteome of infected cells and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMVs cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.

sentences: The genetic architecture of phenotypic diversity in the betta fish (Betta splendens) The Betta fish displays a remarkable variety of phenotypes selected during domestication. However, the genetic basis underlying these traits remain largely unexplored. Here, we report a high-quality genome assembly and re-sequencing of 727 individuals representing diverse morphologies of the betta fish. We show that current breeds have a complex domestication history with extensive introgression with wild species. Using GWAS, we identify the genetic basis of multiple traits, including several coloration phenotypes, sex-determination which we map to DMRT1, and the long-fin phenotype which maps to KCNJ15. We identify a polygenic signal related to aggression with many similarities to human psychiatric traits, involving genes such as CACNB2 and DISC1. Our study provides a resource for developing the Betta fish as a genetic model for morphology and behavior in vertebrates.

sentences: MP-NeRF: A Massively Parallel Method for Accelerating Protein Structure Reconstruction from Internal Coordinates The conversion of proteins between internal and cartesian coordinates is a limiting step in many pipelines, such as molecular dynamics simulations and machine learning models. This conversion is typically carried out by sequential or parallel applications of the Natural extension of Reference Frame (NeRF) algorithm. This work proposes a massively parallel NeRF implementation which, depending on the polymer length, achieves speedups between 400-1200x over the previous state-of-the-art. It accomplishes this by dividing the conversion into three main phases: parallel composition of the monomer backbone, assembly of backbone subunits, and parallel elongation of sidechains; and by batching these computations into a minimal number of efficient matrix operations. Special emphasis is placed on reusability and ease of use. We open source the code (available at https://github.com/EleutherAI/mp_nerf) and provide a corresponding python package.

sentences: Loss of Rnf43 accelerates Kras-mediated neoplasia and remodels the tumor immune microenvironment in pancreatic adenocarcinoma RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been sufficiently characterized. In this study we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 (KRC) in exocrine cells. We demonstrate that Rnf43 loss results in an increased incidence of high-grade cystic lesions of the pancreas and PDAC. Importantly, KRC mice have a significantly decreased survival compared to mice containing only an oncogenic Kras mutation. By use of single cell RNA sequencing we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the Kras/Tp53 driven PDAC GEMM. Furthermore, expression of the chemokine, CXCL5, was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. This GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.

sentences: Contact-tracing in cultural evolution: a Bayesian mixture model to detect geographic areas of language contact When speakers of two or more languages interact, they are likely to influence each other: contact leaves traces in the linguistic record, which in turn can reveal geographic areas of past human interaction and migration. However the complex, multi-dimensional nature of contact has hindered the development of a rigorous methodology for detecting its traces. Specifically, other factors may contribute to similarities between languages. Inheritance (a property is passed from an ancestor to several descendant languages), and universal preference (a property is universally preferred), may both overshadow contact signals. How can we find geographic contact areas in language data, while accounting for the confounding effects of inheritance and universal preference? We present sBayes, an algorithm for Bayesian clustering in the presence of confounding effects. The algorithm learns which similarities in a set of features are better accounted for by confounders, and which are due to contact effects. Contact areas are free to take any shape or size, but an explicit geographic prior ensures their spatial coherence. We test the clustering method on simulated data and apply it in two case studies to reveal language contact in South America and the Balkans. Our results are supported by --mostly qualitative-- findings from previous studies. While we focus on the specific problem of language contact, the method can also be used to uncover other traces of shared history in cultural evolution, and more generally, to reveal latent spatial clusters in the presence of confounders.

sentences:
Elevated anti-SARS-CoV-2 antibodies and IL-6, IL-8, MIP-1β, early predictors of severe COVID-19. BackgroundViral and host immune kinetics during acute COVID-19 and after remission of acute symptoms need better characterization.

MethodsSARS-CoV-2 RNA, anti-SARS-CoV-2 IgA, IgM, and IgG antibodies, and pro-inflammatory cytokines were measured in sequential samples among hospitalized COVID-19 patients during acute infection and 6 months following diagnosis.

Results24 laboratory-confirmed COVID-19 patients with mild/moderate and severe COVID-19 were included. Most were males 83%, median age of 61 years. 21% were admitted to the ICU and 8 of them (33.3%) met criteria for severe COVID-19 disease. A delay in SARS-CoV-2 levels decline during the first 6 days of follow-up and viral load persistence until month 3 were related with severe COVID-19, but not viral load levels at the diagnosis. Higher levels of anti-SARS-CoV-2 IgA, IgM, IgG and the cytokines IL-6, IL-8 and MIP-1{beta} at the diagnosis time were related with severe COVID-19 outcome. Higher levels of MIP-1{beta}, IL-1{beta}, MIP-1 and IFN-{gamma} were observed at month 1/3 during mild/moderate disease compared to severe COVID-19. IgG persisted at low levels after 6 months of diagnosis.

ConclusionsHigher concentrations of IgA, IgM, and IgG, and IL-6, IL-8 and MIP-1{beta} are identified as early predictors of COVID-19 severity, but not SARS-CoV-2 RNA levels at diagnosis.

sentences:
High-resolution mass spectrometry reveals environmentally relevant uptake, elimination, and metabolic alterations following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as the body burden produced by standard exposure paradigms is still unknown. We performed targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We found that 10 ppb, 1 ppb, and 50 ppt waterborne exposures during early embryogenesis produced environmentally relevant body burden of TCDD: 38 {+/-} 4.34, 26.6 {+/-} 1.2, and 8.53 {+/-} 0.341 pg/embryo, respectively, at 24 hours post fertilization. In addition, we discovered that TCDD exposure was associated with the dysregulation of several metabolic pathways that are critical for brain development and function including glutamate metabolism, chondroitin sulfate biosynthesis, and tyrosine metabolism pathways. Together, these data demonstrate that existing exposure paradigms produce environmentally relevant body burdens of TCDD in zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR activation.

HIGHLIGHTSO_LIHistorical TCDD exposure paradigms produce environmentally relevant body burdens in zebrafish embryos.
C_LIO_LITCDD elimination for high doses can be modeled using an exponential regression.
C_LIO_LIExposure to TCDD alters metabolic pathways that are essential for brain development and function.
C_LI

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sentences: Mapping morphological malformation to genetic dysfunction in blood vessel organoids with 22q11.2 Deletion Syndrome DiGeorge Syndrome, or 22q11.2 deletion syndrome (22q11.2 DS), is a genetic disorder caused by microdeletions in chromosome 22, impairing the function of endothelial cells (EC) and/or mural cells and leading to deficits in blood vessel development such as abnormal aortic arch morphology, tortuous retinal vessels, and tetralogy of Fallot. The mechanism by which dysfunctional endothelial cells and pericytes contribute to the vasculopathy, however, remains unknown. In this study, we used human blood vessel organoids (VOs) generated from iPSC of 22q11.2 DS patients to model the vascular malformations and genetic dysfunctions. We combined high-resolution lightsheet imaging and single-cell transcriptome analysis to link the genetic profile and vascular phenotype at the single-cell level. We developed a comprehensive analytical methodology by integrating deep learning-mediated blood vessel segmentation, network graph construction, and tessellation analysis for automated morphology characterization. We report that 22q11.2DS VOs demonstrate a smaller size with increased angiogenesis/sprouting, suggesting a less stable vascular network. Overall, clinical presentations of smaller vascular diameter, less connected vasculature, and increased branch points were recapitulated in 22q11.2DS VOs. Single-cell transcriptome profiling showed heterogeneity in both 22q11.2DS and control VOs, but the former demonstrated alterations in endothelial characteristics that are organ-specific and suggest a perturbation in the vascular developmental process. Intercellular communication analysis indicated that the vascular dysfunctions in 22q11.2 deletion were due to a lower cell-cell contact and upregulated extracellular matrix organization involving collagen and fibronectin. Voronoi diagram-based tessellation analysis also indicated that the colocalization of endothelial tubes and mural cells was different between control and 22q11.2 VOs, indicating that alterations in EC and mural interactions might contribute to the deficits in vascular network formation. This study illustrates the utility of VO in revealing the pathogenesis of 22q11.2DS vasculopathy.

sentences: Application of personalized differential expression analysis in human cancer proteome Owing to the recent technological advances, liquid chromatography-mass spectrometry (LC-MS)-based quantitative proteomics can measure expression of thousands of proteins from biological specimens. Currently, several studies have used the LC-MS-based proteomics to measure protein expression levels in human cancer. Identifying differentially expressed proteins (DEPs) between tumors and normal controls is a common way to investigate carcinogenesis mechanisms. However, most statistical methods used for DEPs analysis can only identify deregulated proteins at the population-level and ignore the heterogeneous differential expression of proteins in individual patients. Thus, to identify patient-specific molecular defects for personalized medicine, it is necessary to perform personalized differential analysis at the scale of a single sample. To date, there is a scarcity of systematic and easy-to-handle tool that could be used to evaluate the performance of individualized difference expression analysis algorithms in human cancer proteome. Herein, we developed a user-friendly tool kit, IDEP, to enable implementation and evaluation of personalized difference expression analysis algorithms. IDEP evaluates five rank-based tools (RankComp v1/v2, PENDA, Peng and Quantile) through classic computational and functional criteria in lung, gastric and liver cancer proteome. The results show that the within-sample relative expression orderings (REOs) of protein pairs in normal tissues were highly stable, which provided the basis for individual level DEPs analysis. Moreover, these individualized difference analysis tools could reach much higher efficiency in detecting sample-specific deregulated proteins than the group-based methods. Pathway enrichment and survival analysis results were dataset and analysis method dependent. In summary, IDEP has integrated necessary toolkits for individualized identification of DEPs and supported flexible methods evaluation analysis and visualization modules. It could provide a robust and scalable framework to extract personalized deregulation patterns and could also be used for the discovery of prognostic biomarkers for personalized medicine.

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Water-Only Look-Locker Inversion recovery (WOLLI) T1 mapping PurposeModified Look-Locker Inversion recovery (MOLLI) and Shortened MOLLI (ShMOLLI) T1 values can deviate substantially from the water T1 in voxels containing large amounts of water and fat. We introduce Water-Only Look-Locker Inversion recovery (WOLLI) to map water T1 in tissue containing fat.

TheoryWOLLI comprises adiabatic water-selective inversion and several balanced steady-state free precession (bSSFP) readouts, followed immediately by adiabatic fat-selective inversion and bSSFP readout(s). Data are fitted pixel-wise and an adapted Deichmann-Haase saturation-correction gives the water T1.

MethodsWe compared two WOLLI protocols (WOLLI-w7f1 and WOLLI-w7f2f1), ShMOLLI and single-voxel spectroscopy at 3T in: simulations, fat/water phantoms, and 12 subjects livers.

ResultsIn simulations with 0-40% fat fraction, T1 varied by: <0.5% (WOLLI-w7f1), <12% (WOLLI-w7f2f1), and >100% (MOLLI). In phantoms, the accuracy of the methods (i.e. worst-case T1w difference vs spectroscopy) was: WOLLI-w7f1 (best, maximum difference of 10.2%), WOLLI-w7f2f1 (max. 10.5%), and ShMOLLI (max. 81.5%). In the liver, the root-mean-squared deviations vs spectroscopy were: 374ms (WOLLI-w7f1), 645ms (WOLLI-w7f2f1), and 700ms (ShMOLLI).

ConclusionCompared to ShMOLLI, WOLLI is less influenced by fat, but more by B0-inhomogeneity. WOLLI was a clear improvement in phantoms but less so in vivo. WOLLI may be suited to scan organs with high fat content such as the liver.

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Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+ and ionic strength The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using {Delta}33 or {Delta}61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA[->]G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.

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sentences: Cross-sectional genomic perspective of epidemic waves of SARS-CoV-2: a pan India study COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. In the present study, we aim for a cross-sectional analysis since its first incidence up to 26th July 2021. We have performed the pan Indian evolutionary study using 20,086 high-quality complete genomes of SARS-CoV-2. Based on the number of cases reported and mutation rates, we could divide the Indian epidemic into seven different phases. First, three phases constituting the pre-first wave had a very less average mutation rate (<11), which increased in the first wave to 17 and then doubled in the second wave (~34). In accordance with the mutation rate, variants of concern (alpha, beta, gamma and delta) and interest (eta and kappa) also started appearing in the first wave (1.5% of the genomes), which dominated the second (~96% of genomes) and post-second wave (100% of genomes) phases. Whole genome-based phylogeny could demarcate the post-first wave isolates from previous ones by the point of diversification leading to incidences of VOCs and VOIs in India. Nation-wide mutational analysis depicted more than 0.5 million events with four major mutations in ~97% of the total 20,086 genomes in the study. These included two mutations in coding (spike (D614G) and NSP 12b (P314L) of RNA dependent RNA polymerase), one silent mutation (NSP3 F106F) and one extragenic mutation (5 UTR 241). Large scale genome-wide mutational analysis is crucial in expanding knowledge on evolution of deadly variants of SARS-CoV-2 and timely management of the pandemic.

sentences: CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration ([~]80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.

sentences: Reuse of a wing venation gene-regulatory network in patterning the eyespot rings of butterflies Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we propose that a conserved venation gene network, used in patterning insect wings, has been recruited to pattern the sharp and distinct rings of color in butterfly eyespot development. This network involves Decapentaplegic (Dpp), acting as a central morphogen, activating optix and spalt at different concentration thresholds. It also involves Spalt cross-regulating optix resulting in the formation of a sharp boundary between the two genes. First, using in situ hybridizations, we show that dpp is expressed in the eyespot centers during the early pupal stage and that CRISPR-Cas9 embryonic knockouts of dpp, optix, and spalt result in the loss of eyespots, loss of the orange ring, and loss of the black scales, respectively. Furthermore, using RNAi during the pupal stage, we show that dpp down-regulation results in the complete or partial loss of eyespots. Using CRISPR disruptions of optix or spalt, followed by immunostainings, we show that Spalt represses optix in the central region of the eyespot, limiting optix expression to a more peripheral ring. We further illustrate that optix disruptions alter scale pigmentation and scale ultrastructure as they transform orange to brown scales. Altogether, the regulatory interactions between dpp, spalt and optix mimic those found in earlier larval developmental stages, in the anterior compartment of an insects wing, where these genes play a role in venation patterning. The network similarities suggest that the venation GRN was co-opted to aid in the differentiation of the eyespot rings.

sentences: Transcriptomic Analysis of 3D Vasculature-On-A-Chip Reveals Paracrine Factors Affecting Vasculature Growth and Maturation In vitro models of vasculature are of great importance for modelling vascular physiology and pathology. However, there is usually a lack of proper spatial patterning of interacting heterotypic cells in conventional vasculature dish models, which might confound results between contact and non-contact interactions. We use a microfluidic platform with structurally defined separation between human microvasculature and fibroblasts to probe their dynamic, paracrine interactions. We also develop a novel, versatile technique to retrieve cells embedded in extracellular matrix from the microfluidic device for downstream transcriptomic analysis, and uncover growth factor and cytokine expression profiles associated with improved vasculature growth. Paired receptor-ligand analysis further reveals paracrine signaling molecules that could be supplemented into the medium for vasculatures models where fibroblast co-culture is undesirable or infeasible. These findings also provide deeper insights into the molecular cues for more physiologically relevant vascular mimicry and vascularized organoid model for clinical applications such as drug screening and disease modeling.

sentences: Olfactory coding in the bumble bee antennal lobe Sociality is classified as one of the major transitions in evolution, with the largest number of eusocial species found in the insect order Hymenoptera, including the Apini (honey bees) and the Bombini (bumble bees). Bumble bees and honey bees not only differ in their social organization and foraging strategies, but comparative analyses of their genomes demonstrated that bumble bees have a slightly less diverse family of olfactory receptors than honeybees, suggesting that their olfactory abilities have adapted to different social and/or ecological conditions. However, unfortunately, no precise comparison of olfactory coding has been performed so far between honey bees and bumble bees, and little is known about the rules underlying olfactory coding in the bumble bee brain. In this study, we used in vivo calcium imaging to study olfactory coding of a panel of floral odorants in the antennal lobe (AL) of the bumble bee Bombus terrestris. Our results show that odorants evoke consistent neuronal activity in the bumble bee antennal lobe. Each odorant evokes a different glomerular activity pattern revealing this molecules chemical structure, i.e. its carbon chain length and functional group. Response intensity as well as odor-similarity relationships were highly correlated to those measured in honey bees. This study thus suggests that bumble bees, like honey bees, are equipped to respond to odorants according to their chemical features.

sentences: A Combined Evidence Approach to Prioritize Nipah Virus Inhibitors Nipah Virus (NiV) came into limelight due to an outbreak in Kerala, India. NiV causes severe disease and death in people with over 75% case fatality rate. It is a public health concern and has the potential to become a global pandemic. Lack of treatment has forced the containment methods to be restricted to isolation and surveillance. WHOs  R&D Blueprint list of priority diseases (2018) indicates that there is an urgent need for accelerated research & development for addressing NiV. In the quest for druglike NiV inhibitors (NVIs) a thorough literature search followed by systematic data curation was conducted. Rigorous data analysis was done with curated NVIs for prioritizing druglike compounds. For the same, more than 1800 descriptors of NVIs were computed and comparative analysis was performed with the FDA approved small molecules and antivirals. These compounds were further evaluated through PAINS filter to study their toxicity profile. Simultaneously, compounds were also prioritized based on the robustness of the assays through which they were identified. Our efforts lead to the creation of a well-curated structured knowledgebase of 182 NVIs with 98 small molecule inhibitors. The reported IC50/EC50 values for some of these inhibitors are in the nanomolar range - as low as 0.47 nM. In order to prioritize these inhibitors, we performed several tests and applied filters to identify drug-like non-toxic compounds. Of 98, a few compounds passed DruLito & PAINS filters exhibiting drug-like properties and were also prioritized in an independent screen based only the assay robustness. The NVIs have diverse structural features and offer a wide spectrum of ways in which they can be developed further as druglike molecules. We report a knowledgebase for furthering the development of NVIs. The platform has a diverse set of 98 NVIs of which a few have been prioritized based on a combined evidence strategy. The platform has the provision to submit new inhibitors as and when reported by the community for further enhancement of NiV inhibitor landscape.

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COVID-19 vaccine booster induces a strong CD8+ T cell response against Omicron variant epitopes in HLA-A*02:01+ individuals The >30 mutated residues in the Omicron spike protein have led to its rapid classification as a new SARS-CoV-2 variant of concern. As a result, Omicron may escape from the immune system, decreasing the protection provided by COVID-19 vaccines. Preliminary data shows a weaker neutralizing antibody response to Omicron compared to the ancestral SARS-CoV-2 virus, which can be increased after a booster vaccine. Here, we report that CD8+ T cells can recognize Omicron variant epitopes presented by HLA-A*02:01 in both COVID-19 recovered and vaccinated individuals, even 6 months after infection or vaccination. Additionally, the T cell response was stronger for Omicron variant epitopes after the vaccine booster. Altogether, T cells can recognize Omicron variants, especially in vaccinated individuals after the vaccine booster.

One-Sentence SummaryCD8+ T cells response against Omicron variant epitopes is stronger after the vaccine booster.

sentences: Experience-dependent plasticity modulates ongoing activity in the antennal lobe and enhances odor representations Ongoing neural activity has been observed across several brain regions and thought to reflect the internal state of the brain. Yet, it is not fully understood how ongoing brain activity interacts with sensory experience and shape sensory representations. Here, we show that projection neurons of the fruit fly antennal lobe exhibit spatiotemporally organized ongoing activity in the absence of odor stimulation. Upon repeated exposure to odors, we observe a gradual and long-lasting decrease in the amplitude and frequency of spontaneous calcium events, as well as a reorganization of correlations between olfactory glomeruli during ongoing activity. Accompanying these plastic changes, we find that repeated odor experience reduces trial-to-trial variability and enhances the specificity of odor representations. Our results reveal a previously undescribed experience-dependent plasticity of ongoing and sensory driven activity at peripheral levels of the fruit fly olfactory system.

sentences: Multi-omics reveal intricate network of mitochondrial adaptations to training in human skeletal muscle Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings.

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The determinants of African Swine Fever Virus Virulence - the Georgia 2007/1 strain and the host macrophage response African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here we characterise the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study we applied Cap Analysis Gene Expression sequencing (CAGE-seq) to map the 5 ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulent-specific transcripts belonging to multigene families, including two predicted MGF 100 genes I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated.

ImportanceAfrican swine fever virus (ASFV) causes haemorrhagic fever in domestic pigs with case fatality rates approaching 100%, and no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. Then spreading through Eastern Europe, and more recently across Asia. We used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the non-virulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

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Factorization-based Imputation of Expression in Single-cell Transcriptomic Analysis (FIESTA) recovers Gene-Cell-State Relationships MotivationSingle cell RNA sequencing (scRNA-seq) is a powerful gene expression profiling technique that is presently revolutionizing the study of complex cellular systems in the biological sciences. Existing single-cell RNA-sequencing methods suffer from sub-optimal target recovery leading to inaccurate measurements including many false negatives. The resulting  zero-inflated data may confound data interpretation and visualization.

ResultsSince cells have coherent phenotypes defined by conserved molecular circuitries (i.e. multiple gene products working together) and since similar cells utilize similar circuits, information about each each expression value or  node in a multi-cell, multi-gene scRNA-Seq data set is expected to also be predictable from other nodes in the data set. Based on this logic, several approaches have been proposed to impute missing values by extracting information from non-zero measurements in a data set. In this study, we applied non-negative matrix factorization approaches to a selection of published scRNASeq data sets to recommend new values where original measurements are likely to be inaccurate and where  zero measurements are predicted to be false negatives. The resulting imputed data model predicts novel cell type markers and expression patterns more closely matching gene expression values from orthogonal measurements and/or predicted literature than the values obtained from other previously published imputation approaches.

Contactbenjamin.spike@hci.utah.edu

Availability and implementationFIESTA is written in R and is available at https://github.com/elnazmirzaei/FIESTA and https://github.com/TheSpikeLab/FIESTA.

sentences: Top-down modulation of the retinal code via histaminergic neurons of the hypothalamus The mammalian retina is considered an autonomous circuit, yet work dating back to Ramon y Cajal indicates that it receives inputs from the brain. How such inputs affect retinal processing has remained unknown. We identified brain-to-retina projections of histaminergic neurons from the mouse hypothalamus, which densely innervated the dorsal retina. Histamine application, or chemogenetic activation of histaminergic axons, altered spontaneous and light-evoked activity of various retinal ganglion cells (RGCs), including direction-selective RGCs. These cells exhibited broader directional tuning and gained responses to high motion velocities. Such changes could improve vision when objects move fast across the visual field (e.g. while running), which fits with the known increased activity of histaminergic neurons during arousal. In humans, an antihistamine drug non-uniformly modulated visual sensitivity across the visual field, indicating an evolutionary conserved function of the histaminergic system. Our findings expose a previously unappreciated role for brain-to-retina projections in modulating retinal function.

sentences: Child abuse associates with increased recruitment of perineuronal nets in the ventromedial prefrontal cortex: a possible implication of oligodendrocyte progenitor cells Child abuse (CA) is a strong predictor of psychopathologies and suicide, altering normal trajectories of brain development in areas closely linked to emotional responses such as the prefrontal cortex (PFC). Yet, the cellular underpinnings of these enduring effects are unclear. Childhood and adolescence are marked by the protracted formation of perineuronal nets (PNNs), which orchestrate the closure of developmental windows of cortical plasticity by regulating the functional integration of parvalbumin interneurons (PV) into neuronal circuits. Using well-characterized post-mortem brain samples, we show that a history of CA is specifically associated with increased densities and morphological complexity of WFL-labeled PNNs in the ventromedial PFC (BA11/12), possibly suggesting increased recruitment and maturation of PNNs. Through single-nucleus sequencing and fluorescent in-situ hybridization, we found that the expression of canonical components of PNNs is enriched in oligodendrocyte progenitor cells (OPCs), and that they are upregulated in CA victims. These correlational findings suggest that early-life adversity may lead to persistent patterns of maladaptive behaviors by reducing the neuroplasticity of cortical circuits through the enhancement of developmental OPC-mediated PNN formation.

sentences: Small-molecule inhibitors of the RNA m6A demethylase FTO potently support the survival of dopamine neurons The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signalling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 M (compound 2) and Kd = 337 nM; IC50 = 28.9 M (compound 3). Importantly, the treatment of mouse midbrain dopamine neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, these inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The compounds 2 and 3 protected dopamine neurons with greater potency than our recently developed alkylation repair homolog protein 5 (AlkBH5) m6A demethylase inhibitors. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study, can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.

sentences: The GASA Gene Family in Theobroma cacao: Genome Wide Identification and Expression Analyses The gibberellic acid-stimulated Arabidopsis (GASA/GAST) gene family is widely distributed in plants. The role of the GASA gene family has been reported previously in various physiological and biological processes, such as cell division, root and seed development, stem growth, and fruit ripening. These genes also provide resistance to abiotic and biotic stresses including antimicrobial, antiviral, and antifungal. Here, we report 17 tcGASA genes in Theobroma cacao L. distributed on six chromosomes. The gene structure, promoter-region sequences, protein structure, and biochemical properties, expression, and phylogenetics of all tcGASAs were analyzed. Phylogenetic analyses divided tcGASA proteins into five groups. The nine segmentally duplicating genes form four pairs and cluster together in phylogenetic tree. Purifying selection pressure was recorded on tcGASA, including duplicated genes. Several stress/hormone-responsive cis-regulatory elements were also recognized in the promoter region of tcGASAs. Differential expression analyses revealed that most of the tcGASA genes showed elevated expression in the seeds (cacao food), implying their role in seed development. The black rod disease of genus Phytophthora caused up to 20-25% loss (700,000 metric tons) in world cacao production. The role of tcGASA genes in conferring fungal resistance was also explored based on RNAseq data against Phytophthora megakarya. The differential expression of tcGASA genes was recorded between the tolerant and susceptible cultivars of cacao plants, which were inoculated with the fungus for 24h and 72h. This differential expression indicating possible role of tcGASA genes to fungal resistant in cacao. Our findings provide new insight into the function, evolution, and regulatory system of the GASA family genes in T. cacao and provide new target genes for development of fungi-resistant cacao varieties in breeding programs.

sentences: ChIP-AP, An Integrated ChIP-Seq Analysis Pipeline ChIP-Seq is a technique used to analyse protein-DNA interactions. The protein-DNA complex is pulled down using a protein antibody, after which sequencing and analysis of the bound DNA fragments is performed. A key bioinformatics analysis step is "peak" calling - identifying regions of enrichment. Benchmarking studies have consistently shown that no optimal peak caller exists. Peak callers have distinct selectivity and specificity characteristics which are often not additive and seldom completely overlap in many scenarios. In the absence of a universal peak caller, we rationalized one ought to utilize multiple peak-callers to 1) gauge peak confidence as determined through detection by multiple algorithms, and 2) more thoroughly survey the protein-bound landscape by capturing peaks not detected by individual peak callers owing to algorithmic limitations and biases. We therefore developed an integrated ChIP-Seq Analysis Pipeline (ChIP-AP) which performs all analysis steps from raw fastq files to final result, and utilizes four commonly used peak callers to more thoroughly and comprehensively analyse datasets. Results are integrated and presented in a single file enabling users to apply selectivity and sensitivity thresholds to select the consensus peak set, the union peak set, or any sub-set in-between to more confidently and comprehensively explore the protein-bound landscape. (https://github.com/JSuryatenggara/ChIP-AP).

sentences: Reversible fronto-occipitotemporal signaling complements task encoding and switching under ambiguous cues Adaptation to changing environments involves the appropriate extraction of environmental information to achieve a behavioral goal. It remains unclear how behavioral flexibility is guided under situations where the relevant behavior is ambiguous. Using functional brain mapping of machine-learning decoders and directional functional connectivity, we show that brain-wide reversible neural signaling underpins task encoding and behavioral flexibility in ambiguously changing environments. When relevant behavior is cued ambiguously during behavioral shifting, neural coding is attenuated in distributed cortical regions, but top-down signals from the prefrontal cortex complement the coding. When behavioral shifting is cued more explicitly, modality-specialized occipitotemporal regions implement distinct neural coding about relevant behavior, and bottom-up signals from the occipitotemporal region to the prefrontal cortex supplement the behavioral shift. These results suggest that our adaptation to an ever-changing world is orchestrated by the alternation of top-down and bottom-up signaling in the fronto-occipitotemporal circuit depending on the availability of environmental information.

sentences: Scale-dependent tipping points of bacterial colonization resistance Bacteria are efficient colonizers of a wide range of secluded micro-habitats, such as soil pores, skin follicles, or intestinal crypts. How the structural diversity of these habitats modulates microbial self-organization remains poorly understood, in part because of the challenge to specifically manipulate the physical structure of microbial environments. Using a microfluidic device to grow bacteria in crypt-like incubation chambers of systematically varied lengths, we show that small variations in the physical structure of the micro-habitat can drastically alter bacterial colonization success and resistance against invaders. Small crypts are un-colonizable, intermediately sized crypts can stably support dilute populations, while beyond a second critical lengthscale, populations phase-separate into a dilute and a jammed region. The jammed state is characterized by extreme colonization resistance, even if the resident strain is suppressed by an antibiotic. Combined with a flexible biophysical model, we demonstrate that colonization resistance and associated priority effects can be explained by a crowding-induced phase transition, which results from a competition between proliferation and density-dependent cell leakage. The emerging sensitivity to scale underscores the need to control for scale in microbial ecology experiments. Systematic flow-adjustable lengthscale variations may serve as a promising strategy to elucidate further scale-sensitive tipping points and to rationally modulate the stability and resilience of microbial colonizers.

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Force generation of KIF1C is impaired by pathogenic mutations Intracellular transport is essential for neuronal function and survival. The fastest neuronal transporter is the kinesin-3 KIF1C. Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. However, neither the force generation of the KIF1C motor protein, nor the biophysical and functional properties of pathogenic mutant proteins have been studied thus far.

Here we show that full length KIF1C is a processive motor that can generate forces up to 5.7 pN. We find that KIF1C single molecule processivity relies on its ability to slip and re-engage under load and that its slightly reduced stall force compared to kinesin-1 relates to its enhanced probability to backslip. Two pathogenic mutations P176L and R169W that cause hereditary spastic paraplegia in humans maintain fast, processive single molecule motility in vitro, but with decreased run length and slightly increased unloaded velocity compared to the wildtype motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles.

Our results establish a baseline for the single molecule mechanics of Kif1C and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.

sentences: Causal inference in environmental sound recognition Sound is caused by physical events in the world. Do humans infer these causes when recognizing sound sources? We tested whether the recognition of common environmental sounds depends on the inference of a basic physical variable - the source intensity (i.e., the power that produces a sound). A sources intensity can be inferred from the intensity it produces at the ear and its distance, which is normally conveyed by reverberation. Listeners could thus use intensity at the ear and reverberation to constrain recognition by inferring the underlying source intensity. Alternatively, listeners might separate these acoustic cues from their representation of a sounds identity in the interest of invariant recognition. We compared these two hypotheses by measuring recognition accuracy for sounds with typically low or high source intensity (e.g., pepper grinders vs. trucks) that were presented across a range of intensities at the ear or with reverberation cues to distance. The recognition of low-intensity sources (e.g., pepper grinders) was impaired by high presentation intensities or reverberation that conveyed distance, either of which imply high source intensity. Neither effect occurred for high-intensity sources. The results suggest that listeners implicitly use the intensity at the ear along with distance cues to infer a sources power and constrain its identity. The recognition of real-world sounds thus appears to depend upon the inference of their physical generative parameters, even generative parameters whose cues might otherwise be separated from the representation of a sounds identity.

sentences: Efficient, selectable marker free gene targeting in soybean using novel Ochrobactrum haywardense-mediated delivery We report robust selectable marker-free gene targeting (GT) system in soybean, one of the most economically important crops. A novel efficient Ochrobactrum haywardense-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants in 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with [~] 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without a need of a selectable marker within the template. High-resolution Southern by Sequencing (SbS) analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require selectable marker within the donor DNA, and generates non-chimeric plants with heritable GT.

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Callosal fiber length scales with brain size according to functional lateralization, evolution, and development Brain size significantly impacts the organization of white matter fibers. Fiber length scaling - the degree to which fiber length varies according to brain size - was overlooked. We investigated how fiber lengths within the corpus callosum, the most prominent white matter tract, vary according to brain size. The results showed substantial variation in length scaling among callosal fibers, replicated in two large healthy cohorts ([~]2000 individuals). The underscaled callosal fibers mainly connected the precentral gyrus and parietal cortices, whereas the overscaled callosal fibers mainly connected the prefrontal cortices. The variation in such length scaling was biologically meaningful: larger scaling corresponded to larger neurite density index but smaller fractional anisotropy values; cortical regions connected by the callosal fibers with larger scaling were more lateralized functionally as well as phylogenetically and ontogenetically more recent than their counterparts. These findings highlight an interaction between interhemispheric communication and organizational and adaptive principles underlying brain development and evolution.

Significance StatementBrain size varies across evolution, development, and individuals. Relative to small brains, the neural fiber length in large brains is inevitably increased, but the degree of such increase may differ between fiber tracts. Such a difference, if it exists, is valuable for understanding adaptive neural principles in large versus small brains during evolution and development. The present study showed a substantial difference in the length increase between the callosal fibers that connect the two hemispheres, replicated in two large healthy cohorts. Altogether, our study demonstrates that reorganization of interhemispheric fibers length according to brain size is intrinsically related to fiber composition, functional lateralization, cortical myelin content, evolutionary and developmental expansion.

sentences: Incorporation of data from multiple hypervariable regions when analyzing bacterial 16S rRNA sequencing data Short read 16S rRNA amplicon sequencing is a common technique used in microbiome research. However, inaccuracies in estimated bacterial community composition can occur due to amplification bias of the targeted hypervariable region. A potential solution is to sequence and assess multiple hypervariable regions in tandem, yet there is currently no consensus as to the appropriate method for analyzing this data. Additionally, there are many sequence analysis resources for data produced from the Illumina platform, but fewer open-source options available for data from the Ion Torrent platform. Herein, we present an analysis pipeline using an open-source analysis platform that integrates data from multiple hypervariable regions and is compatible with data produced from the Ion Torrent platform. We used the ThermoFisher Ion 16S Metagenomics Kit and a mock community of 20 bacterial strains to assess taxonomic classification of amplicons from 6 separate hypervariable regions (V2, V3, V4, V6-7, V8, V9) using our analysis pipeline. We report that different hypervariable regions have different specificities for taxonomic classification, which also had implications for global level analyses such as alpha and beta diversity. Finally, we utilize a generalized linear modeling approach to statistically integrate the results from multiple hypervariable regions and apply this methodology to data from a small clinical cohort. We conclude that scrutinizing sequencing results separately by hypervariable region provides a more granular view of the taxonomic classification achieved by each primer set as well as the concordance of results across hypervariable regions. However, the data across all hypervariable regions can be combined using generalized linear models to statistically evaluate overall differences in community structure and relatedness among sample groups.

sentences: Modulation of the IKS channel by PIP2 requires two binding sites per monomer The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. The latter, expressed in the myocardium membrane is complexed with its auxiliary subunits, KCNE1 to generate the so-called IKS current. We present here molecular models of transmembrane domain of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2 lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2 binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2 in KCNE1 modulation of IKS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels.

sentences: MorphVAE: Generating Neural Morphologies from 3D-Walks using a Variational Autoencoder with Spherical Latent Space For the past century, the anatomy of a neuron has been considered one of its defining features: The shape of a neurons dendrites and axon fundamentally determines what other neurons it can connect to. These neurites have been described using mathematical tools e.g. in the context of cell type classification, but generative models of these structures have only rarely been proposed and are often computationally inefficient. Here we propose MO_SCPLOWORPHC_SCPLOWVAE, a sequence-to-sequence variational autoencoder with spherical latent space as a generative model for neural morphologies. The model operates on walks within the tree structure of a neuron and can incorporate expert annotations on a subset of the data using semi-supervised learning. We develop our model on artificially generated toy data and evaluate its performance on dendrites of excitatory cells and axons of inhibitory cells of mouse motor cortex (M1) and dendrites of retinal ganglion cells. We show that the learned latent feature space allows for better cell type discrimination than other commonly used features. By sampling new walks from the latent space we can easily construct new morphologies with a specified degree of similarity to their reference neuron, providing an efficient generative model for neural morphologies.

sentences: Deep learning tools and modeling to estimate the temporal expression of cell cycle proteins from 2D still images Automatic characterization of fluorescent labeling in intact mammalian tissues remains a challenge due to the lack of quantifying techniques capable of segregating densely packed nuclei and intricate tissue patterns. Here, we describe a powerful deep learning-based approach that couples remarkably precise nuclear segmentation with quantitation of fluorescent labeling intensity within segmented nuclei, and then apply it to the analysis of cell cycle dependent protein concentration in mouse tissues using 2D fluorescent still images. First, several existing deep learning-based methods were evaluated to accurately segment nuclei using different imaging modalities with a small training dataset. Next, we developed a deep learning-based approach to identify and measure fluorescent labels within segmented nuclei, and created an ImageJ plugin to allow for efficient manual correction of nuclear segmentation and label identification. Lastly, using fluorescence intensity as a readout for protein concentration, a three-step global estimation method was applied to the characterization of the cell cycle dependent expression of E2F proteins in the developing mouse intestine.

sentences: Entrectinib - a SARS-CoV-2 inhibitor in Human Lung Tissue (HLT) cells Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify potential drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a promising antiviral drug. We found that part of the antiviral action of entrectinib is mediated by a non-specific mechanism, likely occurring at the viral membrane level. Such a profile could provide entrectinib with protection against the development of drug resistance by emerging SARS-CoV-2 variants.

sentences: Visible blue light inactivates SARS-CoV-2 variants and inhibits Delta replication in differentiated human airway epithelia The emergence of SARS-CoV-2 variants that evade host immune responses has prolonged the COVID-19 pandemic. Thus, the development of an efficacious, variant-agnostic therapeutic for the treatment of early SARS-CoV-2 infection would help reduce global health and economic burdens. Visible light therapy has the potential to fill these gaps. In this study, visible blue light centered around 425 nm efficiently inactivated SARS-CoV-2 variants in cell-free suspensions and in a translationally relevant well-differentiated tissue model of the human large airway. Specifically, 425 nm light inactivated cell-free SARS-CoV-2 variants Alpha, Beta, Delta, Gamma, Lambda, and Omicron by up to 99.99% in a dose-dependent manner, while the monoclonal antibody bamlanivimab did not neutralize the Beta, Delta, and Gamma variants. Further, we observed that 425 nm light reduced virus binding to host ACE-2 receptor and limited viral entry to host cells in vitro. Further, the twice daily administration of 32 J/cm2 of 425 nm light for three days reduced infectious SARS-CoV-2 Beta and Delta variants by >99.99% in human airway models when dosing began during the early stages of infection. In more established infections, logarithmic reductions of infectious Beta and Delta titers were observed using the same dosing regimen. Finally, we demonstrated that the 425 nm dosing regimen was well-tolerated by the large airway tissue model. Our results indicate that blue light therapy has the potential to lead to a well-tolerated and variant-agnostic countermeasure against COVID-19.

sentences: WNK1 Enhances Migration and Invasion in Breast Cancer Models Metastasis is the major cause of mortality in breast cancer patients. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase WNK1 (with no lysine (K) 1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

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

sentences: simATAC: A single-cell ATAC-seq simulation framework Single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) identifies regulated chromatin accessibility modules at the single-cell resolution. Robust evaluation is critical to the development of scATAC-seq pipelines, which calls for reproducible datasets for benchmarking. We hereby present the simATAC framework, an R package that generates scATAC-seq count matrices that highly resemble real scATAC-seq datasets in library size, sparsity, and chromatin accessibility signals. simATAC deploys statistical models derived from analyzing 90 real scATAC-seq cell groups. simATAC provides a robust and systematic approach to generate in silico scATAC-seq samples with known cell labels for assessing analytical pipelines.

sentences: Mechanisms of Coupling between Angiotensin Converting Enzyme 2 and Nicotinic Acetylcholine Receptors Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus encapsulated by a spike (S) glycoprotein envelope, binds with high affinity to angiotensin converting enzyme 2 (ACE2) during cell entry of a susceptible host. Recent studies suggest nicotinic acetylcholine receptors (nAChRs) play a role in functional ACE2 regulation and nicotine may contribute to the progression of coronavirus disease 2019 (COVID-19). Here, we present evidence for coupling between ACE2 and nAChR through bioinformatic analysis and cell culture experiments. Following molecular and structural protein comparison of over 250 ACE2 vertebrate orthologues, a region of human ACE2 at positions C542-L554 was identified to have sequence similarity to nAChR-binding neurotoxin and rabies virus glycoproteins (RBVG). Furthermore, experiments conducted in PC12 cells indicate a potential for physical interaction between ACE2 and 7 nAChR proteins. Our findings support a model of nAChR involvement in in COVID-19.

sentences: Robust temporal map of human in vitro myelopoiesis using single-cell genomics Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

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Surface residues and non-additive interactions stabilize a consensus homeodomain protein Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared to an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per-substitution contributions come from substitutions that maintain charge state, suggesting that although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from charge-charge interactions. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain.

Significance StatementProteins composed of consensus sequences from multiple sequence alignments are often more stable than extant proteins used to create them. Often about half the residues in a consensus protein differ from those of extant proteins. The contributions of these differences to stability are unknown. Here we substitute groups of residues with different properties (conservation, charge variation, solvent accessibility) to determine which substitutions lead to consensus stabilization. We find that surface and charge-conserving substitutions contribute to stability, that weakly-conserved substitutions make a significant collective contribution to stability, and that there is a significant non-additive contribution to stability in the consensus background. These results provide insights to the sequence origins of consensus stabilization and the evolutionary constraints that determine protein sequences.

sentences: Acai (Euterpe oleracea Mart) modulates oxidative stress and inflammation by NF-κB inactivation and Nrf2 up-regulation in experimental diabetes To evaluate the effects of acai extract (EA) on oxidative stress and inflammation induced by high glucose in cultured mouse immortalized mesangial cells (MiMC) and diabetic rats. MiMC cell viability and proliferation were determined by MTT. Extracellular and intracellular nitric oxide (NO) and intracellular ROS were also measured. The cell proteins were extracted for analysis of catalase, Nrf2, p-Nrf2, SOD-1, SOD-2, iNOS, NF-{kappa}B, p-NF-{kappa}B and TNF- expression. Male, adult Wistar rats were distributed into 3 groups: control (CTL) and diabetic (DM) rats who received water and DMEA and received 1 mL/day EA (200 mg/kg) via gavage for 8 consecutive weeks. After treatment with EA, metabolic profile, renal function and thiobarbituric acid reactives substances (TBARS) levels were evaluated, and kidneys were collected for qualitative histological analysis. EA maintained cell viability above 90% in all groups; it decreased proliferation in the HG group, both significant. NO levels, ROS generation, iNOS, NF-{kappa}B, p-NF-{kappa}B and TNF- expression were reduced significantly after 72 h of EA treatment, with significant increases for all antioxidants studied. DMEA vs DM showed a significant increase in body weight, improved kidney function and reduced TBARS excretion. EA treatment decreased proliferation, oxidative stress and inflammation in MiMC, and although acai did not decrease fasting glucose, it recovered the body weight and delayed the decline of renal function in the diabetic animals, suppressing the signaling of inflammatory mediators via NF-{kappa}B inactivation and increasing all antioxidants studied by upregulating the Nrf2 response pathway.

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Compaction of Drosophila histoblasts in a crowded epidermis is driven by buckling of their apical junctions In many proliferating epithelia, cells present a polygonal shape that results from tensile forces of the cytoskeletal cortex and from the packing geometry set by the cell cycle1, 2. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. Here we show that histoblasts, which are initially polygonal, undergo a dramatic morphological transition in the course of larval development. Histoblasts change from a tensed network configuration, with straight cell outlines at the level of adherens junctions, to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions is reminiscent of elastic buckling. In accordance, we show that folding of junctions results from an imbalance between the growth of the junctions and the increasing crowding of the epidermis. The process also correlates with a change in the junctional acto-myosin cortex and possibly mechanical properties.

We propose a model in which crowding of the epidermis imposes a compressive load on the growing junctions which induces their buckling. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work also indicates that in growing non-dividing cells, compressive forces, instead of tension, may drive cell morphology.

sentences: Somatic mutational profiles and germline polygenic risk scores in human cancer The mutational profile of a cancer reflects the activity of the mutagenic processes which have been operative throughout the lineage of the cancer cell. These processes leave characteristic profiles of somatic mutations called mutational signatures. Mutational signatures, including single-based substitution (SBS) signatures, may reflect the effects of exogenous or endogenous exposures. Here, we used polygenic risk score (PRS) as proxies for exposures and examined the association between somatic mutational profiles and germline PRS in 12 cancer types from The Cancer Genome Atlas project. We found 17 statistically significant associations after Bonferroni correction (p < 3.15x10-5), including positive associations between germline inflammatory bowel disease PRS and number of somatic mutations of signature SBS1 in prostate cancer and APOBEC-related signatures in breast cancer. The age at menarche PRS was inversely associated with mutation counts of SBS1 in prostate cancer. Our analysis suggests that there are robust associations between tumor somatic mutational profiles and germline PRS. These may reflect mechanisms through hormone regulation and immunological responses that contribute to cancer etiology and drive cancer progression.

sentences: Diversity, function and evolution of aquatic vertebrate genomes Aquatic vertebrates consist of jawed fish (cartilaginous fish and bony fish), aquatic mammals, reptiles and amphibians. Here, we present a comprehensive analysis of 630 aquatic vertebrate genomes to generate a standardized compendium of genomic data. We demonstrate its value by assessing their genome features as well as illuminating gene families related to the transition from water to land, such as Hox genes and olfactory receptor genes. We found that LINEs are the major transposable element (TE) type in cartilaginous fish and aquatic mammals, while DNA transposons are the dominate type in bony fish. To our surprise, TE types are not fixed in amphibians, the first group that transitioned to living on land. These results illustrate the value of a unified resource for comparative genomic analyses of aquatic vertebrates. Our data and strategy are likely to support all evolutionary and ecological research on vertebrates.

sentences: Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

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Structural covariance and heritability of the optic tract and primary visual cortex in living human brains Individual differences among human brains exist at many scales, spanning gene expression, white matter tissue properties, and the size and shape of cortical areas. One notable example is an approximately 3-fold range in the size of human primary visual cortex (V1), a much larger range than is found in overall brain size. A previous study (Andrews et al., 1997) reported a correlation between optic tract cross-section area and V1 size in post-mortem human brains, suggesting that there may be a common developmental mechanism for multiple components of the visual pathways. We evaluated the relationship between properties of the optic tract and V1 in a much larger sample of living human brains by analyzing the Human Connectome Project 7 Tesla Retinotopy Dataset. This dataset includes retinotopic maps measured with functional MRI (fMRI) and fiber tract data measured with diffusion MRI (dMRI). We found a negative correlation between optic tract fractional anisotropy and V1 surface area (r = -0.2). This correlation, though small, was consistent across multiple dMRI datasets differing in acquisition parameters. Further, we found that both V1 size and optic tract properties were correlated among twins, with higher correlations for monozygotic than dizygotic twins, indicating a high degree of heritability for both properties. Together, these results demonstrate covariation across individuals in properties of the retina (optic tract) and cortex (V1) and show that each is influenced by genetic factors.

Significance statementThe size of human primary visual cortex (V1) has large inter-individual differences. These differences cannot be explained by differences in overall brain size. A previous post-mortem study reported a correlation between the size of the human optic tract and V1. In this study, we evaluated the relationship between the optic tract and V1 in living humans by analyzing a neuroimaging dataset that included functional and diffusion MRI data. We found a small, but robust correlation between optic tract tissue properties and V1 size, supporting the existence of structural covariance between the optic tract and V1 in living humans. The results suggest that characteristics of retinal ganglion cells, reflected in optic tract measurements, are related to individual differences in human V1.

sentences: A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

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Tyrosine kinase inhibitors trigger lysosomal damage-associated cell lysis to activate the NLRP3 inflammasome Inflammasomes are intracellular protein complexes that control proteolytic maturation and secretion of inflammatory interleukin-1 (IL-1) family cytokines and are thus important in host defense. While some inflammasomes are activated simply by binding to pathogen-derived molecules, others, including those nucleated by NLRP3 and NLRP1, have more complex activation mechanisms that are not fully understood. We screened a library of small molecules to identify new inflammasome activators that might shed light on activation mechanisms. In addition to validating dipeptidyl peptidase (DPP) inhibitors as NLRP1 activators, we find that clinical tyrosine kinase inhibitors (TKIs) including imatinib and masitinib activate the NLRP3 inflammasome. Mechanistically, these TKIs cause lysosomal swelling and damage, leading to cathepsin-mediated destabilization of myeloid cell membranes and cell lysis. This is accompanied by potassium (K+) efflux, which activates NLRP3. Both lytic cell death and NLRP3 activation but not lysosomal damage induced by TKIs are prevented by the cytoprotectant high molecular weight polyethylene glycol (PEG). Our study establishes a screening method that can be expanded for inflammasome research and immunostimulatory drug development, and provides new insight into immunological off-targets that may contribute to efficacy or adverse effects of TKIs.

One Sentence SummaryA functional small molecule screen identifies imatinib, masitinib and other tyrosine kinase inhibitors that destabilize myeloid cell lysosomes, leading to cell lysis and K+ efflux-dependent NLRP3 inflammasome activation.

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IRGM/Irgm1 Aggravates Progression of Atherosclerosis by Inducing Macrophage Apoptosis through the MAPK Signaling Pathway AimsAtherosclerosis underlies most cardiovascular diseases, among which acute coronary syndrome (ACS) caused by plaque rupture (PR) often leads to death. Immune-related GTPases (IRGM/Irgm1) have been extensively studied in inflammatory diseases, but their role in atherosclerosis is unclear. Determining how IRGM/Irgm1 promotes atherosclerotic plaque vulnerability will provide information for new biomarkers and/or therapeutic targets.

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

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

Translation viewOur findings indicate that IRGM/Irgm1 contributes to formation and rupture of vulnerable plaques. It suggests that IRGM may provide a new target for the early treatment of ACS.

sentences: CST interacts with the cohesin complex and promotes chromosome cohesion Sister chromatid cohesion (SCC) is established during DNA replication by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. How SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress can lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST interacts with the cohesin complex. Unexpectedly, we did not find evidence of defective cohesion establishment or mitotic progression in the absence of CST. However, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we supposed that CST may be required to maintain SCC following fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

sentences: Biomaterials direct functional B cell response in a material specific manner B cells are an adaptive immune target of biomaterials development in vaccine research but despite their role in wound healing have not been studied in tissue engineering and regenerative medicine. We evaluated the B cell response to biomaterial scaffold materials implanted in a muscle wound; a biological extracellular matrix (ECM) and synthetic polyester polycaprolactone. In the local muscle tissue, small numbers of B cells are recruited in response to tissue injury and biomaterial implantation. ECM materials induced plasmablasts in lymph nodes and antigen presentation in the spleen while the synthetic PCL implants delayed B cell migration and induced an antigen presenting phenotype. In muMt- mice lacking B cells, the fibrotic response to the synthetic biomaterials decreased. Immunofluorescence confirmed antigen presenting B cells in fibrotic tissue surrounding silicone breast implants. In sum, the adaptive B cell immune response to biomaterial depends on composition and induces local, regional and systemic immunological changes.

sentences: aCPSF1 in synergy with terminator U-tract dictates archaeal transcription termination efficiency via the KH domains recognizing U-tract Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea, while the working mechanism remains elusive. This work, through analyzing transcript-3'end-sequencing data of Methanococcus maripaludis, found positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficiencies (TTEs) at the genome-wide level. In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding abilities, and in vivo assays demonstrated the two are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract in synergy determine high TTEs. The N-terminal KH domains equip aCPSF1 of specific binding to terminator U-tract and the in vivo aCPSF1-terminator U-tract synergism; aCPSF1s nuclease activity was also required for TTEs. aCPSF1 also functioned as back-up termination for transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract synergy in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in Archaea, which could be widely employed by archaeal phyla; using one factor recognizing U-rich terminator signal and cleaving transcript 3'-end, the archaeal aCPSF1-dependent transcription termination could display a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

sentences: Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisomes cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

sentences: DNA opening during transcription initiation by RNA polymerase II in atomic detail RNA polymerase II (RNAP II) is a macro-molecular complex that synthesizes RNA by reading the DNA code, a process called transcription. During transcription initiation, RNAP II opens the double-stranded DNA to expose the DNA template to the active site. The molecular interactions driving and controlling the DNA opening are not well understood. We used all-atom molecular dynamics (MD) simulations to obtain a continuous atomistic pathway for the DNA opening process in human RNAP II. To achieve such large-scale and highly nonlinear transition, we steered the MD simulations along a combination of collective variables involving a guided DNA rotation and a set of path collective variables. The simulations reveal extensive interactions of the DNA with three protein loops near the active site, namely the rudder, fork loop 1, and fork loop 2. According to the simulations, these DNA-protein interactions support DNA opening by attacking Watson-Crick hydrogen bonds, and they stabilize the open DNA bubble by the formation of a wide set of DNA-protein salt bridges.

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On the relationship between Pathogenic Potential and Infective Inoculum Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasites ability to cause disease in a host, given the variables of inoculum, symptomology, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction symptomatic (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no symptoms. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero symptoms allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease.

Author SummaryThe ability of a microbe, virus, or parasite to cause disease is dependent on multiple factors, virulence factors. host immune defenses, the infective inoculum, and the type of immune response. For many microbes their capacity for causing disease is highly dependent on the inoculum. The mathematical formula for Pathogenic Potential (PP) is a way to compare the ability of an organism to have a pathogenic effect, as measured by Fraction Symptomatic (Fs), mortality (M), and inoculum (I), and can include the median survival time of the host (T). Increasing inoculum of the fungus Cryptococcus neoformans for a moth host resulted in exponentially smaller pathogenic potential, and the Fs/T versus inoculum plot showed a logarithmic relationship. Together, these relationships show diminishing returns with increasing cryptococcal inoculum, in which each individual fungus plays a smaller role in pathogenicity. Literature data shows that other microbes, mostly bacteria, had linear Fs/T versus inoculum relationships, which indicate that each bacterium contributed an equal amount to pathogenicity. These differences in relationships can point to differences in host-microbe interactions and suggest new ways in which the organism causes disease.

sentences: miR-124 regulates Notch and NeuroD1 to mediate transition states of neuronal development MicroRNAs (miRNAs) regulate gene expression by destabilizing target mRNA and/or inhibiting translation in animal cells. The ability to mechanistically dissect the function of miR-124 during specification, differentiation, and maturation of neurons during development within a single system has not been accomplished. Using the sea urchin (Strongylocentrotus purpuratus) embryo, we take advantage of the manipulability of the embryo and its well-documented gene regulatory networks (GRNs). We incorporated NeuroD1 as part of the sea urchin neuronal GRN and determined that miR-124 inhibition resulted in decreased gut contractions, swimming velocity, and neuronal development. We further integrated post-transcriptional regulation of miR-124 into the neuronal GRN. Inhibition of miR-124 resulted in increased number of cells expressing transcription factors associated with progenitor neurons and a concurrent decrease of mature and functional neurons. Results revealed that miR-124 regulates undefined factors early in neurogenesis during neuronal specification and differentiation in the early blastula and gastrula stages. In the late gastrula and larval stages, miR-124 regulates Notch and NeuroD1. Specifically, miR-124 regulates the transition between neuronal differentiation and maturation, by directly suppressing NeuroD1. Removal of miR-124s suppression of NeuroD1 results in increased mature neurons with decreased Synaptagmin B-positive mature, functional neurons. By removing both miR-124 suppression of NeuroD1 and Notch, we were able to phenocopy miR-124 inhibitor induced defects. Overall, we have improved the neuronal GRN and identified miR-124 to play a prolific role in regulating various transitions of neuronal development.

sentences: Impacts of water quality on Acropora coral settlement: The relative importance of substrate quality and light Coral larval settlement patterns are influenced by a vast array of factors; however, the relative roles of individual factors are rarely tested in isolation, leading to confusion about which are most crucial for settlement. For example, direct effects of light environment are often cited as a major determinate of settlement patterns, yet this has not been demonstrated under environmentally realistic lighting regimes in the absence of confounding factors. Here we apply programmable multispectral lights to create realistic light spectra, while removing correlating (but not obvious) factors that are common in laboratory settlement experiments. Using two common species of Acropora - key framework builders of the Great Barrier Reef - we find little evidence that light intensity or changes in the spectral profile play a substantial role in larval settlement under most environmentally realistic settings but can under more extreme or artificial settings. We alternatively hypothesise and provide evidence that chronic conditions of light and recent sediment exposures that impact benthic substrates (e.g., crustose coralline algae) preceding settlement have a greater impact, with up to 74% decrease in settlement observed on substrates with prior exposure and poor water quality conditions. Management of water quality conditions that impact the quality of benthic-settlement substrates therefore should present a priority area of focus for improving coral recruitment.

sentences: A Methodological Framework to Assess the Accuracy of Virtual Reality Hand-Tracking Systems: A case study with the Oculus Quest 2 Optical marker-less hand-tracking systems incorporated into virtual reality (VR) headsets are transforming the ability to assess motor skills, including hand movements, in VR. This promises to have far-reaching implications for the increased applicability of VR across scientific, industrial and clinical settings. However, so far, there is little data regarding the accuracy, delay and overall performance of these types of hand-tracking systems. Here we present a novel methodological framework which can be easily applied to measure these systems absolute positional error, temporal delay and finger joint-angle accuracy. We used this framework to evaluate the Meta Quest 2 hand-tracking system. Our results showed an average fingertip positional error of 1.1cm, an average finger joint angle error of 9.6o and an average temporal delay of 38.0ms. Finally, a novel approach was developed to correct for these positional errors based on a lens distortion model. This methodological framework provides a powerful tool to ensure the reliability and validity of data originating from VR-based, marker-less hand-tracking systems.

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Hotspot ESR1 mutations are multimodal and contextual drivers of breast cancer metastasis Constitutively active estrogen receptor- (ER/ESR1) mutations have been identified in approximately one third of ER+ metastatic breast cancer. Although these mutations are known mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant, but not local recurrences. In concordance with transcriptomic profiling of ESR1 mutant tumors, genome-edited Y537S and D538G cell models have a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally confers enhanced cell-cell contacts while decreased cell-ECM adhesion. Context-dependent migratory phenotypes revealed co-targeting of Wnt and ER as vulnerability. Mutant ESR1 exhibits non-canonical regulation of several metastatic pathways including secondary transactivation and de novo FOXA1-driven chromatin remodeling. Collectively, our data supports evidence for ESR1 mutation-driven metastases and provides insight for future preclinical therapeutic strategies.

SignificanceContext and allele-dependent transcriptome and cistrome reprogramming in genome-edited ESR1 mutation cell models elicit diverse metastatic phenotypes, including but not limited to alterations in cell adhesion and migration. The gain-of-function mutations can be pharmacologically targeted, and thus may be key components of novel therapeutic treatment strategies for ER-mutant metastatic breast cancer.

sentences: Sodium accumulation in breast cancer predicts malignancy and treatment response Breast cancer is the leading cause of cancer-related death in women worldwide. Development of novel noninvasive diagnostic and predictive pathophysiological biomarkers would represent a significant clinical improvement. Here, we explored the utility of non-invasive 23Na MRI to profile tumour physiology using preclinical mouse models of breast cancer. We establish that tissue Na+ concentration ([Na+]) is elevated vs non-tumour regions across multiple different tumour models. Ex vivo SBFI fluorescence imaging corroborated that this elevation in tumour [Na+] is due to increased intracellular [Na+]. Effective treatment with cytotoxic chemotherapy reduced tumour tissue [Na+], but was not detected by 1H diffusion-weighted imaging (DWI). Moreover, combining 23Na MRI and DWI measurements enabled superior classification accuracy of tumour vs non-tumour regions compared to either parameter alone. Quantification of breast tumour tissue [Na+] using 23Na MRI thus represents a novel, accurate, non-invasive diagnostic and predictive imaging biomarker.

sentences: Mutational bias in spermatogonia impacts the anatomy of regulatory sites in the human genome Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell-type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9-binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (>=5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease.

sentences: Succinate is Broadly Tissue-Permeant and Uncouples Mitochondrial Respiration from ATP Synthesis Succinate is central in energy metabolism. It is thought to be made and consumed within mitochondria, and not to cross the plasma membrane. Contrary to the canonical role of succinate, it is produced in the retina and released into the extracellular milleau. It is unknown how this affects adjacent retinal pigment epithelium, within eyecup tissue. By measuring consumption of 13C4-succinate and O2 we probe metabolism of the eyecup, we find robust succinate consumption by eyecup but not retina tissue. Succinate-dependant stimulation of eyecup respiration is oligomycin resistant; indicating that in eyecup tissue succinate uncouples mitochondrial ATP synthesis from respiration.

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Neural dynamics of semantic categorization in semantic variant of Primary Progressive Aphasia. Semantic representations are processed along a posterior-to-anterior gradient reflecting a shift from perceptual (e.g., it has eight legs) to conceptual (e.g., venomous spiders are rare) information. One critical region is the anterior temporal lobe (ATL): patients with semantic variant primary progressive aphasia (svPPA), a clinical syndrome associated with ATL neurodegeneration, manifest a deep loss of semantic knowledge. We test the hypothesis that svPPA patients perform semantic tasks by over-recruiting areas implicated in perceptual processing. We compared MEG recordings of svPPA patients and healthy controls during a categorization task. While behavioral performance did not differ, svPPA patients showed indications of greater activation over bilateral occipital cortices and superior temporal gyrus, and inconsistent engagement of frontal regions. These findings suggest a pervasive reorganization of brain networks in response to ATL neurodegeneration: the loss of this critical hub leads to a dysregulated (semantic) control system, and defective semantic representations are seemingly compensated via enhanced perceptual processing.

Impact StatementFollowing anterior temporal lobe neurodegeneration, defective semantic representations are compensated via enhanced perceptual processing and associated with a dysregulation of the semantic control system.

sentences: Escherichia coli antimicrobial resistance; Phenotypic and genotypic characterization Improper use of the antimicrobials as E. coli giving the microorganism multi-resistance against many antimicrobials by gene mutation on integrons, transposons and plasmids. Therefore, our aim in this study is to 1)examine antibiotics resistance phenotype and genotype in Escherichia coli, 2) identifying the structure of bacterial resistance genes on whole-genome sequencing against multi-drug resistant of Escherichia coli in marketed poultry meat. Samples collected, prepared and Bacteriological examination, Antimicrobial sensitivity test performed, Serological identification of Escherichia coli isolates. Results declared that; the prevalence of E. coli from tested chicken meat samples of 100 chicken meat samples surveyed against E. coli the result declared that about 40%. Antimicrobial susceptibility was; antibiotics of choice against E. coli Sulfonamides, Cephalosporins,Tetracyclines, Quinolones. Serologically, STEC (O157:H7) 30%, ETEC (O142) 10%, EHEC (O26:H11). The subunit B of shiga-like toxin (SLT) gene appeared as a homogenous band. Heat-labile toxin (LT) gene was screened in both genomic DNA and plasmid preps in tested strains. Control STEC as it represents a danger to the poultry consumers. We recommended to increase the hygienic measures during slaughtering, processing and/or handling of chicken carcasses and avoidance unnecessary usage of any antimicrobials to avoid appearance of new antimicrobials resistant.

sentences: Taxonomically Restricted Genes are Associated with Responses to Biotic and Abiotic Stresses in Sugarcane (Saccharum spp.) Orphan genes (OGs) are protein-coding genes that are restricted to particular clades or species and lack homology with genes from other organisms, making their biological function difficult to predict. OGs can rapidly originate and become functional; consequently, they may support rapid adaptation to environmental changes. Extensive spread of mobile elements, and whole genome duplication, occurred in the Saccharum group, which may have contributed to the origin and diversification of OGs in the sugarcane genome. Here, we identified and characterized OGs in sugarcane, examined their expression profiles across tissues and genotypes, and investigated their regulation under varying conditions. We identified 319 OGs in the Saccharum spontaneum genome without detected homology to protein-coding genes in green plants, except those belonging to Saccharinae. Transcriptomic analysis showed 288 sugarcane OGs with detectable expression levels in at least one tissue or genotype. We observed similar expression patterns of OGs in sugarcane genotypes originating from the closest geographical locations. We also observed tissue-specific expression of some OGs, possibly indicating a complex regulatory process for maintaining diverse functional activity of these genes across sugarcane tissues and genotypes. Sixty-six OGs were differentially expressed under stress conditions, especially cold and osmotic stresses. Gene co-expression network and functional enrichment analyses suggested that sugarcane OGs may be involved in several biological mechanisms, including stimulus response and defence mechanisms. These findings provide a valuable genomic resource for sugarcane researchers, especially those interested in selecting stress-responsive genes.

sentences: Crowdsourced EEG Experiments: A proof of concept for remote EEG acquisition using EmotivPRO Builder and EmotivLABS Online research platforms have enabled mass data collection enabling representative samples for cognitive behavioural studies. However, the benefits of online data collection have not been available for cognitive neuroscience fields such as electroencephalography (EEG). In this study, we introduce an approach for remote EEG data collection. We demonstrate how an experiment can be built via the EmotivPRO Builder and deployed to the EmotivLABS website where it can be completed by participants who own EMOTIV EEG headsets. To demonstrate the data collection technique, we collected EEG while participants engaged in a resting state task where participants sat with their eyes open and then eyes closed for two minutes each. We observed a significant difference in alpha power between the two conditions thereby demonstrating the well-known alpha suppression effect. Thus, we demonstrate that EEG data collection, particularly for frequency domain analysis, can be successfully conducted online with remote users.

sentences: SWR1C catalyzes H2A.Z deposition by coupling ATPase activity to the nucleosome acidic patch The SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Numerous studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated an essential role for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the nucleosomal acidic patch in the H2A.Z exchange reaction. Nucleosomes lacking acidic patch residues retain the ability to stimulate the ATPase activity of SWR1C, implicating a role in coupling the energy of ATP hydrolysis to H2A/H2B dimer eviction. A conserved arginine-rich region within the Swc5 subunit is identified that interacts with the acidic patch and is found to be essential for dimer exchange activity. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

sentences: A mean-field approach for modeling the propagation of perturbations in biochemical reaction networks Often, the time evolution of a biochemical reaction network is crucial for determining the effects of combining multiple pharmaceuticals. Here we illustrate a mathematical framework for modeling the dominant temporal behaviour of a complicated molecular pathway or biochemical reaction network in response to an arbitrary perturbation, such as resulting from the administration of a therapeutic agent. The method enables the determination of the temporal evolution of a target protein as the perturbation propagates through its regulatory network. The mathematical approach is particularly useful when the experimental data that is available for characterizing or parameterizing the regulatory network is limited or incomplete. To illustrate the method, we consider the examples of the regulatory networks for the target proteins c-Myc and Chop, which play an important role in venetoclax resistance in acute myeloid leukemia. First we show how the networks that regulate each target protein can be reduced to a mean-field model by identifying the distinct effects that groups of proteins in the regulatory network have on the target protein. Then we show how limited proteinlevel data can be used to further simplify the mean-field model to pinpoint the dominant effects of the network perturbation on the target protein. This enables a further reduction in the number of parameters in the model. The result is an ordinary differential equation model that captures the temporal evolution of the expression of a target protein when one or more proteins in its regulatory network have been perturbed. Finally, we show how the dominant effects predicted by the mathematical model agree with RNA sequencing data for the regulatory proteins comprising the molecular network, despite the model not having a priori knowledge of this data. Thus, while the approach gives a simplified model for the expression of the target protein, it allows for the interpretation of the effects of the perturbation on the regulatory network itself. This method can be easily extended to sets of target proteins to model components of a larger systems biology model, and provides an approach for partially integrating RNA sequencing data and protein expression data. Moreover, it is a general approach that can be used to study drug effects on specific protein(s) in any disease or condition.

sentences: A Drosophila in vivo chemical screen reveals that combination drug treatment targeting MEK and DGKα mitigates Ras-driven polarity-impaired tumourigenesis. The RAS oncogene and upregulation of the RAS signalling pathway is highly prevalent in human cancer, and therefore, therapeutically targeting the RAS pathway is a common treatment in cancer. However, RAS pathway upregulation is not sufficient to drive malignant cancer, since senescence mechanisms prevent cancer progression. Thus, additional mutations, such as mutations that prevent senescence or alter the tissue architecture (cell polarity), are required for RAS-driven tumour progression. Moreover, targeting RAS-driven cancers with RAS pathway inhibitors can often lead to undesirable side-effects and to drug resistance. Thus, identifying compounds that synergise with RAS-pathway inhibitors would enable lower doses of the RAS pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a boutique chemical screen using a Drosophila model of Ras-driven cell polarity-impaired cancer, we have identified compounds that reduce tumour burden by synergising with subtherapeutic doses of the RAS pathway inhibitor, Trametinib, which inhibits mitogen-activated kinase kinase (MEK). Analysis of one of the hits from the screen, Ritanserin, which targets serotonin receptors and diacy glycerol kinase alpha (DGK), revealed that DGK was the critical target in its synergism with Trametinib. We show that human mammary epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene, SCRIB, are also sensitive to treatment with low doses of Trametinib and DGK inhibition. Mechanistically, DGK inhibition synergises with Trametinib by inhibiting MEK and mTOR activity. Altogether, our results provide evidence that targeting RAS-driven human cancers with RAS pathway and DGK inhibitors will be an effective combination therapy.

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Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping CYP2D6 is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants.

Author SummaryDuring the last decades, the usefulness of personalized medicine has become increasingly apparent. Directly linked to that is the need for accurate genotyping assays to determine the pharmacogenetic profile of patients. Continuing research has led to the development of genotyping assays that perform quite robustly. However, complex genes remain an issue when it comes to determining the complete sequence correctly. An example of such a complex but very important pharmacogene is CYP2D6. Therefore, we developed a genotyping assay in an attempt to generate complete allele-specific consensus sequences of CYP2D6, by optimizing a targeted amplification-free long-read sequencing method and developing a new analysis pipeline. In reference samples, we showed that our genotyping assay performed accurately and confirmed the presence of variants that go undetected by most current assays. However, the implementation of this assay in practice is still hampered as the selected enrichment strategies inherently lead to a low percentage of on-target reads, resulting in low on-target sequencing depths. Further optimization and validation of the assay is thus needed, but definitely worth considering for follow-up research as we already demonstrated the added value for generating more complete genotypes, which on its turn will result in more accurate gene function predictions.

sentences: The Dorsolateral Prefrontal Cortex Presents Structural Variations Associated with Empathic Capacity in Psychotherapists Empathic capacity has been shown to be correlated with brain structural variations, such as cortical thickness. Since psychotherapists have a constant demand to modulate their empathic response, in this study we compared cortical thickness between a group of psychotherapists and a control group at prefrontal and cingulate brain regions, and investigated how this is correlated with their empathic skills. Eighteen psychotherapists and eighteen healthy controls underwent 3-Tesla MRI scanning and completed empathy-related psychometric assessments. Cortical thickness (CT) measures were estimated for each participant. We evaluated how these measures differed between groups, and if they were associated with individual empathy-related scores in a series of regions of interest. Our analysis shows that psychotherapists display a significantly greater CT at a region in the left dorsolateral prefrontal cortex (dlPFC; p < 0.05, FDR corrected). Moreover, psychotherapists CT in this region is correlated with the tendency to feel empathically concerned for others (p < 0.01, FDR corrected). This finding is relevant because the dlPFC region participates strongly in the cognitive components of the empathic response, such as emotion regulation and perspective-taking processes. Thus, our findings support the idea that empathic capacity is reflected by brain structural variations while also studying for the first time a sample of subjects for whom empathic responding is crucial in their profession.

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Tonic engagement of nicotinic receptors bidirectionally controls striatal spiny projection neuron spike timing Striatal spiny projection neurons (SPNs) transform convergent excitatory corticostriatal inputs into an inhibitory signal that shapes basal ganglia output. This process is fine-tuned by striatal GABAergic interneurons (GINs), which receive overlapping cortical inputs and mediate rapid corticostriatal feedforward inhibition of SPNs. Adding another level of control, cholinergic interneurons (CINs), which are also vigorously activated by corticostriatal excitation, can disynaptically inhibit SPNs by activating 4{beta}2 nicotinic acetylcholine receptors (nAChRs) on various GINs. Measurements of this disynaptic inhibitory pathway, however, indicate that it is too slow to compete with direct GIN-mediated feed-forward inhibition. Moreover, functional nAChRs are also present on populations of GINs that respond only weakly to phasic activation of CINs, such as parvalbumin-positive fast-spiking interneurons (PV-FSIs), making the overall role of nAChRs in shaping striatal synaptic integration unclear. Using acute striatal slices we show that upon synchronous optogenetic activation of corticostriatal projections blockade of 4{beta}2 nAChRs shortened SPN spike latencies and increased postsynaptic depolarizations. The nAChR-dependent inhibition was mediated by downstream GABA release, and data suggest that the GABA source was not limited to GINs that respond strongly to phasic CIN activation. In particular, the observed decrease in spike latency caused by nAChR blockade was associated with a diminished frequency of spontaneous inhibitory postsynaptic currents in SPNs, a parallel hyperpolarization of PV-FSIs, and was occluded by pharmacologically preventing cortical activation of PV-FSIs. Taken together, we describe a role for tonic (as opposed to phasic) activation of nAChRs in striatal function. We conclude that tonic activation of nAChRs by CINs maintains a GABAergic brake on cortically-driven striatal output by "priming" feedforward inhibition, a process that may shape SPN spike timing, striatal processing and synaptic plasticity.

Impact StatementA novel mechanism describing how tonic activation of nicotinic acetylcholine receptors can modulate the timing of striatal output by priming feed forward inhibition.

sentences: Eco-evolutionary feedbacks in the human gut microbiome Gut microbiota can evolve within their hosts on human-relevant timescales. Yet little is known about how these changes influence (or are influenced by) the composition of their surrounding community. Here, by combining ecological and evolutionary analyses of a large cohort of human gut metagenomes, we show that the short-term evolution of the microbiota is linked with shifts in its ecological structure. These correlations are not simply explained by expansions of the evolving species, and often involve additional fluctuations in distantly related taxa. We show that similar feedbacks naturally emerge in simple resource competition models, even in the absence of cross-feeding or predation. These results suggest that the structure and function of host microbiota may be shaped by their local evolutionary history, with potential implications for personalized medicine and disease.

sentences: RatsPub: a webservice aided by deep learning to mine PubMed for addiction-related genes Interpreting and integrating results from omics studies typically requires a comprehensive and time consuming survey of extant literature. Here, we introduce GeneCup, an easy to use literature mining web service that searches all PubMed abstracts for user-provided gene symbols in conjunction with a set of custom keywords organized into a customized ontology, as well as results from human genome-wide association studies (GWAS). As an example, we organized over 300 keywords related to drug addiction into seven categories. The literature search is conducted by querying the NIH PubMed server using a programming interface, which is followed by retrieving abstracts from a local copy of the PubMed archive. The main results presented to the user are individual sentences containing the gene symbol, organized by the keywords they also contain. These sentences are presented through an interactive graphical interface or as tables. GWAS results are displayed using a similar method. All results are linked to the original abstract in PubMed. In addition, a convolutional neural network is employed to distinguish sentences describing systemic stress from those describing cellular stress. The automated and comprehensive search strategy provided by GeneCup facilitates the integration of new discoveries from omic studies with existing literature. GeneCup is free and open source software. The source code of GeneCup and the link to a running instance is available at https://github.com/hakangunturkun/GeneCup

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Single-orientation Quantitative Susceptibility Mapping identifies the Ventral Intermediate Nucleus of the Thalamus IntroductionThe ventral intermediate nucleus (VIM) represents the primary target in the treatment of tremor. Accurate localization is extremely important given its proximity to other thalamic nuclei. We utilized single orientation quantitative susceptibility mapping (QSM) at 3T to directly visualize the VIM.

MethodsFour adult volunteers, one adult cadaver, and an essential tremor patient were scanned on a 3T MRI using a multi-echo gradient echo sequence. QSM images were generated using the improved sparse linear equation and least-squares (iLSQR) algorithm. Two adult subjects underwent multiple head orientation imaging for multi-orientation QSM reconstruction. The VIM was prospectively identified with direct visualization as well as indirect landmark-based localization.

ResultsThe bilateral VIM was consistently identified as a hypointense structure within the lateral thalamus, appearing similar on multi-orientation and single-orientation QSM, corresponding to the myelinated dentatorubrothalamic tract (DRTT). The indirect method resulted in a comparatively inferomedial localization, at times missing the VIM and DRTT.

ConclusionSingle-orientation QSM offers a clinically feasible, non-invasive imaging-based approach to directly localize the VIM.

sentences: A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

sentences: Outbreeding depression as a selective force on mixed mating in the mangrove rivulus fish, Kryptolebias marmoratus Mixed mating, a reproduction strategy utilized by many plants and invertebrates, optimizes the cost to benefit ratio of a labile mating system. One type of mixed mating includes outcrossing with conspecifics and self-fertilizing ones own eggs. The mangrove rivulus fish (Kryptolebias marmoratus) is one of two vertebrates known to employ both self-fertilization (selfing) and outcrossing. Variation in rates of outcrossing and selfing within and among populations produces individuals with diverse levels of heterozygosity. I designed an experiment to explore the consequences of variable heterozygosity across four ecologically relevant conditions of salinity and water availability (10{per thousand}, 25{per thousand}, and 40{per thousand} salinity, and twice daily tide changes). I report a significant increase in mortality in the high salinity (40{per thousand}) treatment. I also report significant effects on fecundity measures with increasing heterozygosity. The odds of laying eggs decreased with increasing heterozygosity across all treatments, and the number of eggs laid decreased with increasing heterozygosity in the 10{per thousand} and 25{per thousand} treatments. Increasing heterozygosity also was associated with a reduction liver mass and body condition in all treatments. My results highlight the fitness challenges that accompany living in mangrove forests ecosystem and provide the first evidence for outbreeding depression on reproductive and condition-related traits.

sentences: Simultaneous Brain, Brainstem and Spinal Cord pharmacological-fMRI reveals multilevel opioidergic roles in attentional analgesia in humans Pain perception is decreased by shifting attentional focus away from a threatening event. This attentional analgesia engages parallel descending control pathways from anterior cingulate (ACC) to locus coeruleus, and ACC to periaqueductal grey (PAG) - rostral ventromedial medulla (RVM), indicating possible roles for noradrenergic or opioidergic neuromodulators. To determine which pathway modulates nociceptive activity in humans we used simultaneous whole brain-spinal cord pharmacological-fMRI (N=39) across three sessions. Noxious thermal forearm stimulation generated somatotopic-activation of dorsal horn (DH, C6 segment) whose activity mirrored attentional pain modulation. Activity in an adjacent cluster reported the interaction between task and noxious stimulus. Effective connectivity analysis revealed that ACC recruits PAG and RVM to modulate spinal cord activity. Blocking endogenous opioids with Naltrexone impairs attentional analgesia and disrupts RVM-DH and ACC-PAG connectivity. Noradrenergic augmentation with Reboxetine did not alter attentional analgesia. Cognitive pain modulation is mediated by opioidergic ACC-PAG-RVM descending control which supresses spinal nociceptive activity.

sentences: Focal fMRI signal enhancement with implantable inductively coupled detectors Despite extensive efforts to increase the signal-to-noise ratio (SNR) of fMRI images for brain-wide mapping, technical advances of focal brain signal enhancement are lacking, in particular, for animal brain imaging. Emerging studies have combined fMRI with fiber optic-based optogenetics to decipher circuit-specific neuromodulation from meso to macroscales. Acquiring fMRI signal with high spatiotemporal resolution is needed to bridge cross-scale functional dynamics, but SNR of targeted cortical regions is a limiting factor. We have developed a multi-modal fMRI imaging platform with an implanted inductive coil detector. This detector boosts the tSNR of MRI images, showing a 2-3 fold sensitivity gain over conventional coil configuration. In contrast to the cryoprobe or array coils with limited spaces for implanted brain interface, this setup offers a unique advantage to study brain circuit connectivity with optogenetic stimulation and can be further extended to other multi-modal fMRI mapping schemes.

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Sweet revenge: AtSWEET12 in plant defense against bacterial pathogens by apoplastic sucrose limitation Depriving bacterial pathogens of sugars is a potential plant defense strategy. The relevance of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS (SWEETs) in plant susceptibility to pathogens has been established, but their role in plant defense remains unknown. We identified Arabidopsis thaliana SWEETs (AtSWEETs) involved in defense against nonhost and host Pseudomonas syringae pathogens through reverse genetic screening of atsweet1-17 mutants. Double/triple mutant, complementation, and overexpression line analysis, and apoplastic sucrose estimation studies revealed that AtSWEET12 suppresses pathogen multiplication by limiting sucrose availability in the apoplast. Localization studies suggested that plant defense occurred via increased plasma membrane targeting of AtSWEET12 with concomitant AtSWEET11 protein reduction. Moreover, the heterooligomerization of AtSWEET11 and AtSWEET12 was involved in regulating sucrose transport. Our results highlight a PAMP-mediated defense strategy against foliar bacterial pathogens whereby plants control AtSWEET11-mediated sucrose efflux in the apoplast through AtSWEET12. We uncover a fascinating new mechanism of pathogen starvation as a broad-spectrum disease resistance mechanism in parallel with existing immune pathways.

One sentence summaryThe transporter AtSWEET12 restricts bacterial pathogen multiplication by regulating sucrose availability to pathogens in the apoplast.

sentences: Interest of phenomic prediction as an alternative to genomic prediction in grapevine Phenomic prediction has been defined as an alternative to genomic prediction by using spectra instead of molecular markers. A reflectance spectrum reflects the biochemical composition within a tissue, under genetic determinism. Thus, a relationship matrix built from spectra could potentially capture genetic signal. This new methodology has been successfully applied in several cereal species but little is known so far about its interest in perennial species. Besides, phenomic prediction has only been tested for a restricted set of traits, mainly related to yield or phenology. This study aims at applying phenomic prediction for the first time in grapevine, using spectra collected on two tissues and over two consecutive years, on two populations and for 15 traits. First, we characterized the genetic signal in spectra and under which condition it could be maximized, then phenomic predictive ability was compared to genomic predictive ability. We found that the co-inertia between spectra and genomic data was stable across tissues or years, but variable across populations, with co-inertia around 0.3 and 0.6 for diversity panel and half-diallel populations, respectively. Differences between populations were also observed for predictive ability of phenomic prediction, with an average of 0.27 for the diversity panel and 0.35 for the half-diallel. For both populations, there was a correlation across traits between predictive ability of genomic and phenomic prediction, with a slope around 1 and an intercept of -0.2, thus suggesting that phenomic prediction could be applied for any trait.

sentences: Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization, but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-{beta}-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and {gamma}-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.

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Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called  chains that are each comprised of 17 repeating protein units called  particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism.

IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

sentences: Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice Relaxin/insulin-like-family peptide receptor-4 (RXFP4), the cognate receptor for insulin-like peptide 5 (INSL5), has been implicated in feeding behaviour as Rxfp4 knockout mice display shorter meal durations and reduced high fat diet (HFD) intake. Here, we generated transgenic Rxfp4-Cre mice to explore Rxfp4 expression and physiology. Using this model, we identified Rxfp4 expression in the central nervous system, including in the ventromedial hypothalamus (VMH). Intra-VMH infusion of INSL5 increased HFD and highly palatable liquid meal intake (HPM) of ad libitum fed wildtype mice. Single-cell RNA-sequencing of VMH Rxfp4-expressing cells (RXFP4VMH) defined a cluster of Rxfp4-labelled neurons expressing Esr1, Tac1 and Oxtr, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to the bed nucleus of the stria terminalis, paraventricular hypothalamus, paraventricular thalamus and central nucleus of the amygdala. Utilising designer receptors exclusively activated by designer drugs (DREADDs), we found that whole body chemogenetic inhibition (Di) of Rxfp4-expressing cells, mimicking native INSL5-RXFP4 signalling, increased intake of HFD and HPM, whilst activation (Dq), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and altered food preference. Ablating VMH Rxfp4-expressing cells recapitulated the lower HFD intake phenotype of Rxfp4 knockout mice, resulting in reduced body weight. These findings identify a discrete Rxfp4-expressing neuronal population as a key regulator of food intake and preference and reveal hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

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A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of both EkMasc and EkMascB genes in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

Author summaryThe sex-determining cascade in the silkworm, Bombyx mori, differs greatly from those of other insects. In B. mori, female development is initiated by Fem piRNA expressed from the W chromosome during early embryogenesis. Fem piRNA silences Masculinizer (Masc) thereby blocking the male pathway resulting in female development. It is currently unknown whether this cascade is conserved across Lepidoptera. In the Mediterranean flour moth, Ephestia kuehniella, we identified an ortholog of Masc and discovered its functional duplication on the Z chromosome, which has not yet been found in any other lepidopteran species. We provide two lines of evidence that both the EkMasc and EkMascB genes play an essential role in masculinization: (i) they show a peak of expression during early embryogenesis in ZZ but not in WZ embryos and (ii) their silencing by RNAi results in female-specific splicing of the E. kuehniella doublesex gene (Ekdsx) in ZZ embryos and in a female-biased sex ratio. Our results suggest a conserved role of the duplicated Masc gene in sex determination of E. kuehniella.

sentences: The integrated role of resource memory and scent-mediated territoriality in the emergence of home-ranges Despite decades of animal movement research, an integrated understanding of the processes underlying the emergence of home-ranges remains inadequate. We explored the effects of integrating two key processes of home-ranging arising from individual movement decisions: (i) memory-based resource use; and (ii) scent-based territoriality. Both mechanisms together led to the formation of exclusive, resource- and population density-dependent home-ranges, which were responsive to perturbations in the conspecific environment (i.e., removing individuals). Home-range patterns (size, size skewness and overlap) were primarily influenced by memory and scent decay rate parameters, demonstrating that home-ranging is ultimately a balance between an animals inherent exploratory tendency and its desire to avoid conspecifics. Model application to a population of feral cats demonstrated that general space use patterns could be approximated through simulation, and replication of finer-scale space use patterns is plausible with further model development. Our modelling framework provides a foundation for sophisticated theoretical models of space use in interacting animals.

sentences: Loss of Usp22 enhances histone H2B monoubiquitination and stimulates intracellular and systemic interferon immunity Interferons protect from virus infections by inducing hundreds of interferon-stimulated genes (ISG) which orchestrate anti-viral adaptive and innate immunity. Upon viral infection or type I interferon (IFN) stimulation of cell lines, a histone modification, monoubiquitinated histone 2B (H2Bub1), increases at ISG loci, raising the possibility that a specific chromatin state can broadly stimulate IFN immunity in vivo. Here we show that, in the absence of virus infection or elevated interferon levels, mice lacking the relevant deubiquitinase, Usp22, in immune cells have elevated H2Bub1 levels. Hypermonoubiquitinated H2B is physically associated with dozens of ISG loci, and expression of large numbers of ISG is upregulated. This epigenetic state promotes intracellular and systemic immune phenotypes akin to adaptive and innate interferon immunity, and thereby identifies Usp22 as a negative regulator of interferon immunity.

sentences: A Genome-Phenome Association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Association (MetaGPA) study framework, which allows to link genetic information in metagenomes with a dedicated functional phenotype. We applied MetaGPA to identify enzymes associated with cytosine modifications in environmental samples. From the 2365 genes that met our significance criteria, we confirm known pathways for cytosine modifications and proposed novel cytosine-modifying mechanisms. Specifically, we characterized and identified a novel nucleic acid modifying enzyme, 5-hydroxymethylcytosine carbamoyltransferase, that catalyzes the formation of a previously unknown cytosine modification, 5-carbamoyloxymethylcytosine, in DNA and RNA. Our work introduces MetaGPA as a novel and versatile tool for advancing functional metagenomics.

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LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that redundant QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Author SummaryBacteria communicate and count their cell numbers using a process called quorum sensing (QS). In response to changes in cell density, QS bacteria alternate between acting as individuals and participating in collective behaviors. Vibrio harveyi is used as a model organism to understand QS-mediated communication. Five small RNAs lie at the heart of the V. harveyi QS system, and they regulate the target genes that underlie the QS response. The small RNAs function redundantly making it difficult to understand why V. harveyi requires five of them. Here, we discover a regulator, called LuxT, that exclusively represses the gene encoding one of the QS small RNAs. LuxT regulation of one QS small RNA enables unique control of a specific subset of QS target genes. LuxT is broadly conserved among Vibrionaceae. Our findings show how redundant regulatory components can possess both common and unique roles that provide bacteria with plasticity in their behaviors.

sentences: Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells. Rett Syndrome is a neurodevelopmental disorder in girls that is caused by heterozygous inactivation of the chromatin remodeler gene MECP2. Rett Syndrome may therefore be treated by reactivation of the wild type copy of MECP2 from the inactive X chromosome. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model system for monitoring Mecp2 reactivation that is more sensitive and versatile than any bioluminescent and fluorescent system currently available. The model consists of neural stem cells derived from female mice with a dual reporter system where MECP2 is fused to NanoLuciferase and TdTomato on the inactive X chromosome. We show by bioluminescence and fluorescence that Mecp2 is synergistically reactivated by 5-Aza treatment and Xist knockdown. As expected, other genes on the inactive X chromosome are also reactivated, the majority of which overlaps with genes reactivated early during reprogramming of mouse embryonic fibroblasts to iPSCs. Genetic and epigenetic features such as CpG density, SINE elements, distance to escapees and CTCF binding are consistent indicators of reactivation, whereas different higher order chromatin areas are either particularly prone or resistant to reactivation. Our MeCP2 reactivation monitoring system thereby suggests that genetic and epigenetic features on the inactive X chromosome affect reactivation of its genes, irrespective of cell type or procedure of reactivation.

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InterARTIC: an interactive web application for whole-genome nanopore sequencing analysis of SARS-CoV-2 and other viruses MotivationInterARTIC is an interactive web application for the analysis of viral whole-genome sequencing (WGS) data generated on Oxford Nanopore Technologies (ONT) devices. A graphical interface enables users with no bioinformatics expertise to analyse WGS experiments and reconstruct consensus genome sequences from individual isolates of viruses, such as SARS-CoV-2. InterARTIC is intended to facilitate widespread adoption and standardisation of ONT sequencing for viral surveillance and molecular epidemiology.

Worked exampleWe demonstrate the use of InterARTIC for the analysis of ONT viral WGS data from SARS-CoV-2 and Ebola virus, using a laptop computer or the internal computer on an ONT GridION sequencing device. We showcase the intuitive graphical interface, workflow customisation capabilities and job-scheduling system that facilitate execution of small- and large-scale WGS projects on any common virus.

ImplementationInterARTIC is a free, open-source web application implemented in Python. The application can be downloaded as a set of pre-compiled binaries that are compatible with all common Ubuntu distributions, or built from source. For further details please visit: https://github.com/Psy-Fer/interARTIC/.

sentences: Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. In a 3 year case-control study, we investigated typhoid among children aged <16 years (4,670 febrile cases and 8,549 age matched controls) living in an informal settlement, Nairobi, Kenya. 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1%. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype58), with the majority of each (76% and 88%) being multidrug-resistant strains in 3 sublineages of H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between carriers and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting.

sentences: No evidence of SARS-CoV-2 infection in Neotropical Primates sampled during COVID-19 pandemic in Minas Gerais and Rio Grande do Sul, Brazil. In 2019, a new coronavirus disease (COVID-19) was detected in China. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was capable to infect domestic and captive mammals like cats, tigers and minks. Due to genetic similarities, concern about the infection of Non-Human Primates (NHPs) and the establishment of a sylvatic cycle has grown in the Americas. In this study, neotropical primates (NP) were sampled in different areas from Brazil to investigate whether they were infected by SARS-CoV-2. A total of 89 samples from 51 NP of four species were examined. No positive samples were detected via RT-qPCR, regardless of the NHP species, tissue or habitat tested. This work provides the first report on the lack of evidence of circulation of SARS-CoV-2 in NP. The expand of wild animals sampling is necessary to understand their role in the epidemiology of SARS-CoV-2.

sentences: Causes and consequences of bacteriophage diversification via genetic exchanges across lifestyles and bacterial taxa Bacteriophages (phages) evolve rapidly by acquiring genes from other phages leading to mosaic genomes. Here, we identify numerous genetic transfers between distantly related phages and aim at understanding their frequency, consequences and the conditions favoring them. Gene flow tends to occur between phages that are enriched for recombinases, transposases and non-homologous end joining, suggesting that both homologous and illegitimate recombination contribute to gene flow. Phage family and host phyla are strong barriers to gene exchange, but phage lifestyle is not. We observe more exchanges between temperate phages even if they tend to have smaller genomes. These acquisitions often include transcription regulators and lysins. Yet, there is also extensive gene flow between temperate and virulent phages, or between the latter. These predominantly involve virulent phages with large genomes previously classed as low gene flux, and lead to the preferential transfer of genes encoding functions involved in cell energetics, nucleotide metabolism, DNA packaging and injection, and virion assembly. Such exchanges may explain the acquisition of genes in virulent phages, which tend to have the largest genomes. We used genetic transfers, which occur upon co-infection of a host, to compare phage host range. We found that virulent phages have broader host ranges and mediate genetic exchanges between narrow host range temperate phages infecting distant bacterial hosts, thus contributing to gene flow between virulent phages, as well as between temperate phages. This gene flow drastically expands the gene repertoires available for phage and bacterial evolution, including the transfer of functional innovations across taxa.

sentences: Earliest photic zone niches probed by ancestral microbial rhodopsins For billions of years, life has continuously adapted to dynamic physical conditions near the Earths surface. Fossils and other preserved biosignatures in the paleontological record are the most direct evidence for reconstructing the broad historical contours of this adaptive interplay. However, biosignatures dating to Earths earliest history are exceedingly rare. Here, we combine phylogenetic inference of primordial rhodopsin proteins with modeled spectral features of the Precambrian Earth environment to reconstruct the paleobiological history of this essential family of photoactive transmembrane proteins. Our results suggest that ancestral microbial rhodopsins likely acted as light-driven proton pumps and were spectrally tuned toward the absorption of green light, which would have enabled their hosts to occupy depths in a water column or biofilm where UV wavelengths were attenuated. Subsequent diversification of rhodopsin functions and peak absorption frequencies was enabled by the expansion of surface ecological niches induced by the accumulation of atmospheric oxygen. Inferred ancestors retain distinct associations between extant functions and peak absorption frequencies. Our findings suggest that novel information encoded by biomolecules can be used as "paleosensors" for conditions of ancient, inhabited niches of host organisms not represented elsewhere in the paleontological record. The coupling of functional diversification and spectral tuning of this taxonomically diverse protein family underscores the utility of rhodopsins as universal testbeds for inferring remotely detectable biosignatures on inhabited planetary bodies.

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Fusobacterium nucleatum metabolically integrates commensals and pathogens in oral biofilms Fusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii, where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when co-cultured with S. gordonii, F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Co-culture with Veillonella parvula, another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum. We confirmed that ArcD-dependent ornithine excretion by S. gordonii results in synergistic putrescine production, and mass spectrometry imaging revealed this metabolic capability creates a putrescine-rich microenvironment inside F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis, with putrescine being a potent stimulator of biofilm development and dispersal, and confirmed that F. nucleatum-mediated conversion of ornithine to putrescine enhances biofilm formation by P. gingivalis. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum. Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multi-species consortia, and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities.

Significance StatementPeriodontitis is caused by the pathogenic transition of subgingival microbiota ecosystems, which is accompanied by alterations to microbiome functions including metabolic systems and the establishment of metabolic cross-feeding. While Fusobacterium nucleatum is a major constituent of the periodontal microbiota, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum, creating polyamine-rich microenvironments. We further show that this trophic web results in enhancement of biofilm formation and dispersal of a periodontal pathogen, Porphyromonas gingivalis. This work provides mechanistic insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.

sentences: The molecular mechanism of cytoadherence to placenta or tumor cells through VAR2CSA from Plasmodium falciparum Pregnancy Associated Malaria (PAM) threatens more than one million pregnant women and their infants in endemic regions due to poor outcomes. VAR2CSA plays a vital role in the cytoadherence of infected erythrocytes (IEs) to placenta against immuno-clearance via binding to Chondroitin sulfate A (CSA), which is displayed mostly on the surface of placental or tumor cells. In this study, we determined the cryo-EM structures of VAR2CSA ectodomain and its complex with CSA at a resolution of 3.6 [A] and 3.4 [A], respectively and revealed that CSA binding induces significant conformational change for ligand accommodation. Beyond structural studies, we generated VAR2CSA fragments and mutation and evaluated their binding activity to either isolated CSA or placental and tumor cell line using multi-disciplinary techniques. The results showed that 9-site mutation in DBL2X abolished the CSA binding activity and also disrupted its binding to both placental and tumor cells. Overall, our work clearly elucidated the molecular mechanism of cytoadherence to placental or tumor cells through VAR2CSA. Which may facilitate new PAM vaccine development and improve the drug delivery systems targeting both placenta and tumors.

sentences: Ontogenetic Color Switching in Lizards as a by-Product of Guanine Cell Development Many animals undergo dramatic changes in colour during development1,2. Changes in predation risk during ontogeny are associated with spectacular switches in defensive colours, typically involving the replacement of skin or the production of new pigment cells3. Ontogenetic colour systems are ideal models for understanding the evolution and formation mechanisms of animal colour which remain largely enigmatic2. We show that defensive colour switching in lizards arises by reorganization of a single photonic system, as an incidental by-product of chromatophore maturation. The defensive blue tail colour of hatchling A. beershebensis lizards is produced by light scattering from premature guanine crystals in underdeveloped iridophore cells. Camouflaged adult tail colours emerge upon reorganization of the guanine crystals into a photonic reflector during chromatophore maturation. The substituent guanine crystals form by the attachment of individual nanoscopic plates, which coalesce during growth to form single crystals. Our results show that the blue colour of hatchlings is a fortuitous, but necessary, precursor to the development of adult colour. Striking functional colours in animals can thus arise not as distinct evolutionary innovations but via exploitation of the timing of naturally occurring changes in chromatophore cell development.

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Genome size evolution in the diverse insect order Trichoptera BackgroundGenome size is implicated in form, function, and ecological success of a species. Two principally different mechanisms are proposed as major drivers of eukaryotic genome evolution and diversity: Polyploidy (i.e., whole genome duplication: WGD) or smaller duplication events and bursts in the activity of repetitive elements (RE). Here, we generated de novo genome assemblies of 17 caddisflies covering all major lineages of Trichoptera. Using these and previously sequenced genomes, we use caddisflies as a model for understanding genome size evolution in diverse insect lineages.

ResultsWe detect a ~14-fold variation in genome size across the order Trichoptera. We find strong evidence that repetitive element (RE) expansions, particularly those of transposable elements (TEs), are important drivers of large caddisfly genome sizes. Using an innovative method to examine TEs associated with universal single copy orthologs (i.e., BUSCO genes), we find that TE expansions have a major impact on protein-coding gene regions, with TE-gene associations showing a linear relationship with increasing genome size. Intriguingly, we find that expanded genomes preferentially evolved in caddisfly clades with a higher ecological diversity (i.e., various feeding modes, diversification in variable, less stable environments).

ConclusionOur findings provide a platform to test hypotheses about the potential evolutionary roles of TE activity and TE-gene associations, particularly in groups with high species, ecological, and functional diversities.

sentences: Evaluating brain parcellations using the distance controlled boundary coefficient One important approach to human brain mapping is to define a set of distinct regions that can be linked to unique functions. Numerous brain parcellations have been proposed, using cytoarchitectonic, structural or functional Magnetic Resonance Imaging (fMRI) data. The intrinsic smoothness of brain data, however, poses a problem for current methods seeking to compare different parcellations. For example, criteria that simply compare within-parcel to between-parcel similarity provide even random parcellations with a high value. Furthermore, the evaluation is biased by the spatial scale of the parcellation. To address this problem, we propose the Distance Controlled Boundary Coefficient (DCBC), an unbiased criterion to evaluate discrete parcellations. We employ this new criterion to evaluate existing parcellations of the human neocortex in their power to predict functional boundaries for an fMRI data set with many different tasks, as well as for resting-state data. We find that common anatomical parcellations do not perform better than chance, suggesting that task-based functional boundaries do not align well with sulcal landmarks. Parcellations based on resting-state fMRI data perform well; in some cases, as well as a parcellation defined on the evaluation data itself. Finally, multi-modal parcellations that combine functional and anatomical criteria perform substantially worse than those based on functional data alone, indicating that functionally homogeneous regions often span major anatomical landmarks. Overall, the DCBC advances the field of functional brain mapping by providing an unbiased metric that compares the predictive ability of different brain parcellations to define brain regions that are functionally maximally distinct.

sentences: Extended longevity geometrically-inverted proximal tubule organoids for protein uptake studies While some in vitro platforms have been adapted to study proteinuric kidney disease, organoids have been challenging to study the disease. This is because apical access is historically difficult, and this is the surface on which megalin (LRP2), an endocytic receptor responsible for tubular reabsorption of filtered plasma proteins, resides. Based on our previous geometrically-inverted organoids, this study established high-throughput basal-in and apical-out proximal tubule organoids to study proteinuric kidney disease in a more physiologically consistent manner. Organoids successfully formed around a minimal Matrigel scaffold, and were maintained in culture for 90+ days, the longest reported hanging drop culture to date. The proximal tubule organoids exhibited good polarization, showed upregulation of maturity markers, such as aquaporin-1 and megalin, and experienced less epithelial-to-mesenchymal (EMT) transition compared to 2D cells. To assess protein uptake, fluorescent albumin was placed in the surrounding media, facing the apical surface, and organoids demonstrated functional protein uptake even at 90 days. To mimic proteinuric conditions, organoids were exposed to human serum albumin and released kidney injury molecule-1 (KIM-1), a common biomarker for kidney injury, in both dose- and time-dependent manners. While this study focuses on applications for modeling proteinuric kidney disease conditions, these organoids are envisioned to have broad utility where apical proximal tubule cell access is required.

sentences: Visualization of SARS-CoV-2 Infection Scenes by 'Zero-Shot' Enhancements of Electron Microscopy Images Electron microscopy (EM) recordings of infected tissues serve to diagnose a disease, and they can contribute to our understanding of infection processes. Consequently, a large number of EM images of the interaction of SARS-CoV-2 viruses with cells have been made available by numerous labs. However, due to EM recording techniques at high resolution, images of infection scenes are very noisy and they appear two dimensional ( flat). Current research consequently aims (A) at methods that can remove noise, and (B) at techniques that allow for recovering a 3D impression of the virus or its parts. Here we discuss a novel method which can recover a spatial impression of a whole infection scene at high resolution. In contrast to previous approaches which aim at the reconstruction of single spike proteins or a single virus, the here used method can be applied to a single noisy EM image of an infection scene. As one example image, we show a high resolution image of SARS-CoV-2 viruses in Vero cell cultures (Fig. 1). The method we use is based on probabilistic machine learning algorithms which can operate in a  zero-shot setting, i.e., in a setting when just one noisy image (and no large and clean image corpus) is available. The probabilistic method we apply can estimate non-noisy images by inferring first order statistics (pixel means) across image patches using a previously learned probabilistic image representation. Estimating higher order statistics and appropriately chosen probabilistic models then allow for the generation of images that enhance details and give a spatial impression of a full nanoscopic scene.

sentences: Asymmetric depth-filtration - a versatile and scalable approach for isolation and purification of extracellular vesicles A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

sentences: Estradiol and oxytocin modulate sex differences in hippocampal reactivity and episodic memory Considerable evidence supports sex differences in episodic memory, which may translate to heightened vulnerability to stress- and trauma-related disorders in women. The hormones estradiol and oxytocin both affect episodic memory, but possible underlying hormonal interactions have not been systemically tested in humans. To this end, healthy women (n = 111) and men (n = 115) received estradiol gel (2 mg) or placebo before the administration of intranasal oxytocin (24 IU) or placebo in a randomized, placebo-controlled, parallel-group functional magnetic resonance imaging (fMRI) study. In the fMRI session, participants viewed positive, neutral, and negative scenes. A surprise recognition task was conducted three days later. Under placebo, women showed a significantly better recognition memory and increased hippocampal responses to subsequently remembered items independent of the emotional valence compared to men. The separate treatments with either hormone significantly diminished this mnemonic sex difference and reversed the hippocampal activation pattern. However, the combined treatments led to a memory performance comparable to that of the placebo group. Collectively, the results suggest that both hormones play a crucial role in modulating sex differences in episodic memory. Furthermore, possible antagonistic interactions between estradiol and oxytocin could explain previously observed opposing hormonal effects in women and men.

sentences: Stress-sensitive inference of task controllability Estimating the controllability of the environment enables agents to better predict upcoming events and decide when to engage controlled action selection. How does the human brain estimate controllability? Trial-by-trial analysis of choices, decision times, and neural activity in an explore-and-predict task demonstrate that humans solve this problem by comparing the predictions of an "actor" model with those of a reduced "spectator" model of their environment. Neural BOLD responses within striatal and medial prefrontal areas tracked the instantaneous difference in the prediction errors generated by these two statistical learning models. BOLD activity in the posterior cingulate, temporoparietal, and prefrontal cortices covaried with changes in estimated controllability. Exposure to inescapable stressors biased controllability estimates downward and increased reliance on the spectator model in an anxiety-dependent fashion. Taken together, these findings provide a mechanistic account of controllability inference and its distortion by stress exposure.

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Population Dynamics and Resource Availability Drive Seasonal Shifts in the Consumptive and Competitive Impacts of Introduced House Mice (Mus musculus) on an Island Ecosystem BackgroundHouse mice (Mus musculus) are widespread and invasive on many islands where they can have both direct and indirect ecological impacts on native ecological communities. Given their opportunistic, omnivorous nature the consumptive and competitive impacts of house mice on islands have the potential to vary over time in concert with resource availability and mouse population dynamics.

MethodsWe examined the ecological niche of invasive house mice on Southeast Farallon Island, California, USA using a combination of mouse trapping, food resource surveys, and stable isotope analysis to better understand their trophic interactions with native flora and fauna.

ResultsWe found that plants were the important resource for house mice during the spring months when vegetation is abundant and mouse populations are low. However, plants decline in dietary importance throughout the summer and fall as mouse populations increase, and seabird and insect resources become relatively more available and consumed by house mice. Mouse abundance peaks and other resource availability are low in the fall months when the isotopic niches of house mice and salamanders overlap significantly indicating the potential for competition, most likely for insect prey.

DiscussionThese results indicate how seasonal shifts in both the mouse abundance and resource availability are key factors that mediate the consumptive and competitive impacts of introduced house mice on this island ecosystem.

sentences: In vitro and in vivo interaction of caspofungin with isavuconazole against Candida auris planktonic cells and biofilms The in vitro and in vivo efficacy of caspofungin was determined in combination with isavuconazole against Candida auris. Drug-drug interactions were assessed utilising the fractional inhibitory concentration indices (FICIs), the Bliss independence model and an immunocompromised mouse model. Median planktonic minimum inhibitory concentrations (pMICs) of 23 C. auris isolates were between 0.5 and 2 mg/L and between 0.015 and 4 mg/L for caspofungin and isavuconazole, respectively. Median pMICs for caspofungin and isavuconazole in combination showed 2-128-fold and 2-256-fold decreases, respectively. Caspofungin and isavuconazole showed synergism in 14 out of 23 planktonic isolates (FICI range 0.03-0.5; Bliss cumulative synergy volume range 0-4.83). Median sessile MICs (sMIC) of 14 biofilm-forming isolates were between 32 and >32 mg/L and between 0.5 and >2 mg/L for caspofungin and isavuconazole, respectively. Median sMICs for caspofungin and isavuconazole in combination showed 0-128-fold and 0-512-fold decreases, respectively. Caspofungin and isavuconazole showed synergistic interaction in 12 out of 14 sessile isolates (FICI range 0.023-0.5; Bliss cumulative synergy volume range 0.13-234.32). In line with the in vitro findings, synergistic interactions were confirmed by in vivo experiments. The fungal kidney burden decreases were more than 3 log volumes in mice treated with combination of 1 mg/kg caspofungin and 20 mg/kg isavuconazole daily; this difference was statistically significant compared with control mice (p<0.001). Despite the favourable effect of isavuconazole in combination with caspofungin, further studies are needed to confirm the therapeutic advantage of this combination when treating an infection caused by C. auris.

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Formylglycine-generating enzyme-like proteins constitute a novel family of widespread type VI secretion system immunity proteins Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which prevent effector toxicity by specifically binding their cognate effector and occluding its active site. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of the antibacterial effector of unknown function Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data shows that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not protect against Tse3-dependent bacterial killing. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be co-opted as a scaffold in multiple proteins to carry out diverse functions.

ImportanceDespite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

sentences: The ratio versus difference optimization and its implications for optimality theory Among the classical models of optimization, some models maximize the ratio of returns per investment, others maximize the difference between returns and investment. However, the question under what conditions use of the ratio model is appropriate and under what conditions a difference model should be used remained unaddressed until recently. The question is important because the strategies indicated by ratio optimum can be substantially different than the ones suggested by difference optimum. We make a general case here for the set of conditions for appropriate use of ratio versus difference optimum. When the investable amount is perceived as limiting but not the investment opportunities a ratio optimum is appropriate and when the investment opportunities are perceived to be limiting but not the investable amount, difference optimum is appropriate. Taking examples of Concorde fallacy, parental investment, r and K selection, nectar production, pollinator behavior, protein synthesis and stability, viral burst size and human economic behavior we show that the ratiodifference distinction in optimization models resolves many long standing debates and conundrums in evolution, behavior and economics.

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Adaptive anti-tumor immunity is orchestrated by a population of CCL5-producing tissue-resident NK cells T cell-directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the IFN{gamma}-IL-12 pathway relies on the ability of a population of tissue-resident NK (trNK) cells to orchestrate an anti-tumor microenvironment. Particularly, utilizing an engineered adenoviral platform, we show that paracrine IL-12 enhances functional DC-CD8 T cell interactions to generate adaptive anti-tumor immunity. This effect depends on the abundance of trNK cells and specifically their capacity to produce the cDC1-chemoattractant CCL5. Failure to respond to IL-12 and other IFN{gamma}-inducing therapies such as immune checkpoint blockade in tumors with low trNK cell infiltration could be overcome by intra-tumoral delivery of CCL5. Our findings reveal a novel barrier for T cell-focused therapies and offer mechanistic insights into how T cell-NK cell-DC crosstalk can be enhanced to promote anti-tumor immunity and overcome resistance.

SignificanceWe identified the lack of CCL5-producing, tissue-resident NK (trNK) cells as a barrier to T cell-focused therapies. While IL-12 induces anti-tumoral DC-T cell crosstalk in trNK cellrich tumors, resistance to IL-12 or anti-PD-1 in trNK cellpoor tumors can be overcome by the additional delivery of CCL5.

sentences: Biomechanical  characteristics of scapula and glenohumeral movements during pitching motion in injury-prone college baseball pitchers Coordination of glenohumeral and scapular movements plays an important role in the injury prevention of baseball pitchers. However, there is no objective data establishing the direct relationship between pitching injuries and associated glenohumeral and scapular movements. Therefore, the objectives of the present study were to demonstrate biomechanical differences in scapular and glenohumeral movements during pitching between injury-prone pitchers and healthy college baseball pitchers. Thirty collegiate baseball pitchers were classified into two groups according to their injury status: injury-prone group (N=15, 20.7{+/-}1.4 years, 180.1{+/-}6.5 cm, 78.9{+/-}5.4 kg) and control group (N=15, 20.9{+/-}1.1 years, 177.1{+/-}6.6 cm, 72.3{+/-}6.7 kg). We obtained the pitching motion data using the three-dimensional motion analysis technique with four high-speed cameras. The horizontal abduction angles of the glenohumeral joint during cocking and acceleration phases were significantly greater in injury-prone pitchers [19.0{degrees} (95% CI: 14.4-23.6) at foot contact, -4.0{degrees} (95% CI: -7.7 to -0.2) at maximum external rotation (MER), and -0.3{degrees} (95% CI: -4.8 to -4.2) at ball release] than in healthy controls [11.7 {degrees}(95%CI:7.1 to 16.3) at foot contact, -10.0{degrees}(95%CI: -13.7 to -6.3) at MER, and -6.9{degrees}(95%CI: -11.4 to -2.4)](p<0.01). In addition, the external rotation angle (ER) of the scapula at MER was significantly greater in the injury-prone group [-0.1{degrees} (95% CI: -5.0 to 4.8)] than in the control group [-12.3{degrees} (95% CI: -17.2 to -7.4)] (p<0.01), but there was no difference in the scapular ER during foot contact between the two groups. These results suggests that injury-prone pitchers have less internal rotation of the scapula and more horizontal abduction of the glenohumeral joint during cocking and acceleration phases. Therefore, sports medicine practitioners may need to pay more attention to coordination of scapular and glenohumeral movements during the cocking and acceleration phases of pitching for prevention of shoulder injuries.

sentences: Missingness Adapted Group Informed Clustered (MAGIC)-LASSO: A novel paradigm for prediction in data with widespread non-random missingness The availability of large-scale biobanks linking rich phenotypes and biological measures are a powerful opportunity for scientific discovery. However, real-world collections frequently have extensive non-random missing data. Machine learning methods are able to predict missing data but performance is significantly impaired by block-wise missingness inherent to many biobanks. To address this, we developed Missingness Adapted Group-wise Informed Clustered LASSO (MAGIC-LASSO) which performs hierarchical clustering of variables based on missingness followed by sequential Group LASSO within clusters. Variables are pre-filtered for missingness and balance between training and target sets with final models built using stepwise inclusion of features ranked by completeness. This research has been conducted using the UK Biobank (n>500k) to predict unmeasured Alcohol Use Disorders Identification Test (AUDIT.) The phenotypic correlation between measured and predicted total score was 0.67 while genetic correlations between independent subjects was >0.86, demonstrating the method has significant accuracy and utility.

sentences: Vegetation characteristics control sediment and nutrient retention on but not underneath vegetation in floodplain meadows Sediment and nutrient retention are essential ecosystem functions that floodplains provide and that improve river water quality. During floods, the floodplain vegetation retains sediment, which settles on plant surfaces and the soil underneath plants. Both sedimentation processes require that flow velocity is reduced, which may be caused by the topographic features and the vegetation structure of the floodplain. However, the relative importance of these two drivers and their key components have rarely been both quantified. In addition to topographic factors, we expect vegetation height and density, mean leaf size and pubescence, as well as species diversity of the floodplain vegetation to increase the floodplains capacity for sedimentation. To test this, we measured sediment and nutrients (carbon, nitrogen and phosphorus) both on the vegetation itself and on sediment traps underneath the vegetation after a flood at 24 sites along the River Mulde (Germany). Additionally, we measured biotic and topographic predictor variables. Sedimentation on the vegetation surface was positively driven by plant biomass and the height variation of the vegetation, and decreased with the hydrological distance (total R2=0.56). Sedimentation underneath the vegetation was not driven by any vegetation characteristics but decreased with hydrological distance (total R2=0.42). Carbon, nitrogen and phosphorus content in the sediment on the traps increased with the total amount of sediment (total R2=0.64, 0.62 and 0.84, respectively), while C, N and P on the vegetation additionally increased with hydrological distance (total R2=0.80, 0.79 and 0.92, respectively). This offers the potential to promote sediment and especially nutrient retention via vegetation management, such as adapted mowing. The pronounced signal of the hydrological distance to the river emphasises the importance of a laterally connected floodplain with abandoned meanders and morphological depressions. Our study improves our understanding of the locations where floodplain management has its most significant impact on sediment and nutrient retention to increase water purification processes.

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CovRadar: Continuously tracking and filtering SARS-CoV-2 mutations for molecular surveillance The SARS-CoV-2 pandemic underlined the importance of molecular surveillance to track the evolution of the virus and inform public health interventions. Fast analysis, easy visualization and convenient filtering of the latest virus sequences are essential for this purpose. However, access to computational resources, the lack of bioinformatics expertise, and the sheer volume of sequences in public databases complicate surveillance efforts. CovRadar combines an analytical pipeline and a web application designed for the molecular surveillance of the spike gene of SARS-CoV-2, an important vaccine target. The intuitive web front-end focuses on mutations rather than viral lineages and provides easy access to frequencies and spatio-temporal distributions from global sample collections. The data is regularly updated based on a scalable and reproducible analytical back-end. With this platform, we aim to give users, those with or without bioinformatics skills or sufficient computational resources, the possibility to track and explore mutational changes in the SARS-CoV-2 spike gene and to filter, download, and further analyze data that meet their questions and needs. Advanced computational users have the ability to apply the analytical pipeline and data visualization methods locally on their own data. CovRadar is freely accessible at https://covradar.net, source code is available at https://gitlab.com/dacs-hpi/covradar.

GRAPHICAL ABSTRACT

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sentences: The phylogenomic landscape of the genus Serratia The genus Serratia has been studied for over a century and includes clinically-important and diverse environmental members. Despite this, there is a paucity of genomic information across the genus and a robust whole genome-based phylogenetic framework is lacking. Here, we have assembled and analysed a representative set of 664 genomes from across the genus, including 215 historic isolates originally used in defining the genus. Phylogenomic analysis of the genus reveals a clearly-defined population structure which displays deep divisions and aligns with ecological niche, as well as striking congruence between historical biochemical phenotyping data and contemporary genomics data. We show that Serratia is a diverse genus which displays striking plasticity and ability to adapt to its environment, including a highly-varied portfolio of plasmids, and provide evidence of different patterns of gene flow across the genus. This work provides an essential platform for understanding the emergence of clinical and other lineages of Serratia.

sentences: Machine learning guided signal enrichment for ultrasensitive plasma tumor burden monitoring In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGE uses deep learning and a ctDNA-specific feature space to increase SNV signal to noise enrichment in WGS by 300X compared to our previous noise suppression platform MRDetect. MRD-EDGE also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1Gb to 200Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal to noise enrichment in MRD-EDGE enables de novo mutation calling in melanoma without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition.

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Anterior thalamic nuclei neurons sustain memory A hippocampal-diencephalic-cortical network supports memory function. The anterior thalamic nuclei (ATN) form a key anatomical hub within this system. Consistent with this, injury to the mammillary body-ATN axis is associated with examples of clinical amnesia. However, there is only limited and indirect support that the output of ATN neurons actively enhances memory. Here, in rats, we first showed that mammillothalamic tract (MTT) lesions caused a persistent impairment in spatial working memory. MTT lesions also reduced rhythmic electrical activity across the memory system. Next, we introduced 8.5 Hz optogenetic theta-burst stimulation of the ATN glutamatergic neurons. The exogenously-triggered, regular pattern of stimulation produced an acute and substantial improvement of spatial working memory in rats with MTT lesions and enhanced rhythmic electrical activity. Neither behaviour nor rhythmic activity was affected by endogenous stimulation derived from the dorsal hippocampus. Analysis of immediate early gene activity, after the rats foraged for food in an open field, showed that exogenously-triggered ATN stimulation also increased Zif268 expression across memory-related structures. These findings provide clear evidence that increased ATN neuronal activity supports memory. They suggest that ATN-focused gene therapy may be feasible to counter clinical amnesia associated with dysfunction in the mammillary body-ATN axis.

HighlightsO_LIThe mammillothalamic tract (MTT) supports neural activity in an extended memory system.
C_LIO_LIOptogenetic activation of neurons in the anterior thalamus acutely improves memory after MTT lesions.
C_LIO_LIRescued memory associates with system-wide neuronal activation and enhanced EEG.
C_LIO_LIAnterior thalamus actively sustains memory and is a feasible therapeutic target.
C_LI

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sentences: Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gram-negative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and infect the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and becomes partially unfolded. Studies with chemical reducing agents have previously suggested that the intramolecular disulfide bond of HdeA aids in the maintenance of residual structure at low pH; it is believed that this residual structure is important for clustering exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative cysteine to serine mutation of the disulfide. We found that, although residual structure is greatly diminished at pH 2 without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.

sentences: An in vitro and in vivo approach for the isolation of Omicron variant from human clinical specimens Due to failure of virus isolation of Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, we infected Syrian hamsters and then passage into Vero CCL-81 cells. The Omicron sequences were studied to assess if hamster could incorporate any mutation to changes its susceptibility. L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene and absence of V17I mutation in E gene was observed in sequences of hamster passage unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequence which suggests usefulness of these isolates in future studies.

sentences: High-resolution secretory timeline from vesicle formation at the Golgi to fusion at the plasma membrane in S. cerevisiae Most of the components in the yeast secretory pathway have been studied, yet a high resolution temporal timeline of their participation is lacking. Here we define the order of acquisition, lifetime, and release of critical components involved in late secretion from the Golgi to the plasma membrane. Of particular interest is the timing of the many reported effectors of the secretory vesicle Rab protein Sec4, including the myosin-V Myo2, the exocyst complex, the lgl homolog Sro7, and the small yeast-specific protein Mso1. At the trans-Golgi network (TGN) Sec4s GEF, Sec2, is recruited to Ypt31-positive compartments, quickly followed by Sec4 and Myo2 and vesicle formation. While transported to the bud tip, the entire exocyst complex, including Sec3, is assembled on to the vesicle. Before fusion, vesicles tether for 5s, during which the vesicle retains the exocyst complex and stimulates lateral recruitment of Rho3 on the plasma membrane. Sec2 and Myo2 are rapidly lost, followed by recruitment of cytosolic Sro7, and finally the SM protein Sec1, which appears for just 2 seconds prior to fusion. Perturbation experiments reveal an ordered and robust series of events during tethering that provide insights into the function of Sec4 and effector exchange.

sentences: The Chloroflexi supergroup is metabolically diverse and representatives have novel genes for non-photosynthesis based CO2 fixation The Chloroflexi superphylum have been investigated primarily from the perspective of reductive dehalogenation of toxic compounds, anaerobic photosynthesis and wastewater treatment, but remain relatively little studied compared to their close relatives within the larger Terrabacteria group, including Cyanobacteria, Actinobacteria, and Firmicutes. Here, we conducted a detailed phylogenetic analysis of the phylum Chloroflexota, the phylogenetically proximal candidate phylum Dormibacteraeota, and a newly defined sibling phylum proposed in the current study, Eulabeiota. These groups routinely root together in phylogenomic analyses, and constitute the Chloroflexi supergroup. Chemoautotrophy is widespread in Chloroflexi. Two Form I Rubisco ancestral subtypes that both lack the small subunit are prevalent in ca. Eulabeiota and Chloroflexota, suggesting that the predominant modern pathway for CO2 fixation evolved in these groups. The single subunit Form I Rubiscos are inferred to have evolved prior to oxygenation of the Earths atmosphere and now predominantly occur in anaerobes. Prevalent in both Chloroflexota and ca. Eulabeiota are capacities related to aerobic oxidation of gases, especially CO and H2. In fact, aerobic and anaerobic CO dehydrogenases are widespread throughout every class-level lineage, whereas traits such as denitrification and reductive dehalogenation are heterogeneously distributed across the supergroup. Interestingly, some Chloroflexota have a novel clade of group 3 NiFe hydrogenases that is phylogenetically distinct from previously reported groups. Overall, the analyses underline the very high level of metabolic diversity in the Chloroflexi supergroup, suggesting the ancestral metabolic platform for this group enabled highly varied adaptation to ecosystems that appeared in the aerobic world.

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In vitro infection of human lung tissue with SARS-CoV-2: Heterogeneity in host defense and therapeutic response Cell lines are the mainstay in understanding the biology of COVID-19 infection, but do not recapitulate many of the complexities of human infection. The use of human lung tissue is one solution for the study of such novel respiratory pathogens. We hypothesized that a cryopreserved bank of human lung tissue allows for the in vitro study of the inter-individual heterogeneity of host response to SARS-CoV-2 infection, thus providing a bridge between studies with cell lines and studies in animal models. We generated a cryobank of tissues from 16 donors, most of whom had risk factors for severe illness from COVID-19. Cryopreserved tissues preserved 90% of cell viability and contained heterogeneous populations of metabolically active epithelial, endothelial, and immune cell subsets of the human lung. Samples were readily infectible with HCoV-OC43 and SARS-CoV-2 coronavirus strains, and demonstrated comparable susceptibility to infection. In contrast, we observed a marked donor-dependent heterogeneity in the expression of IL-6, CXCL8 and IFN{beta} in response to SARS-CoV-2 infection. Treatment of tissues with dexamethasone and the experimental drug, N-hydroxycytidine, suppressed viral growth in all samples, whereas chloroquine and remdesivir had no detectable effect. Metformin and sirolimus, molecules with predicted antiviral activity, suppressed viral replication in tissues from a subset of donors. In summary, we developed a novel system for the in vitro study of human SARS-CoV-2 infection using primary human lung tissue from a library of donor tissues. This model may be useful for drug screening and for understanding basic mechanisms of COVID-19 pathogenesis.

ImportanceThe current biological systems for the study of COVID-19 are in vitro systems that differ from the human lung in many respects, and animal hosts to which the virus is not adapted. We developed another alternative for studying pathogenesis and drug susceptibility of SARS-CoV-2 in a cryopreserved bank of human lung tissues. We consider the importance of this work to relate to the practical use of this culture system as a repeatable and scalable approach that allows for the study of an important infection in relevant tissues.

The tissue bank highlights the heterogeneous response to SARS-CoV-2 infection and treatment, which allows researchers to investigate why treatments work in some donors but not others.

sentences: The effect of vascular health factors on white matter microstructure mediates age-related differences in executive function performance Even within healthy aging, vascular risk factors can detrimentally influence cognition, with executive functions (EF) particularly vulnerable. Fronto-parietal white matter (WM) connectivity in part, supports EF and may be particularly sensitive to vascular risk. Here, we utilized structural equation modeling in 184 healthy adults (aged 20-94 years of age) to test the hypotheses that: 1) fronto-parietal WM microstructure mediates age effects on EF; 2) higher blood pressure (BP) and white matter hyperintensity (WMH) burden influences this association. All participants underwent comprehensive cognitive and neuropsychological testing including tests of processing speed, executive function (with a focus on tasks that require switching and inhibition) and completed an MRI scanning session that included FLAIR imaging for semi-automated quantification of white matter hyperintensity burden and diffusion-weighted imaging for tractography. Structural equation models were specified with age (as a continuous variable) and blood pressure predicting within-tract WMH burden and fractional anisotropy predicting executive function and processing speed. Results indicated that fronto-parietal white matter of the genu of the corpus collosum, superior longitudinal fasciculus, and the inferior frontal occipital fasciculus (but not cortico-spinal tract) mediated the association between age and EF. Additionally, increased systolic blood pressure and white matter hyperintensity burden within these white matter tracts contribute to worsening white matter health and are important factors underlying age-brain-behavior associations. These findings suggest that aging brings about increases in both BP and WMH burden, which may be involved in the degradation of white matter connectivity and in turn, negatively impact executive functions as we age.

sentences: Health hazards posed by ionizing radiation to human space missions and newly developing methodologies to investigate them Ionizing radiation is one of the main threats to human space exploration beyond low Earth orbit (BLEO). It is thus of primary importance to determine safe career dose limits for astronauts involved in BLEO missions. In this work the average equivalent doses received by a hypothetical human crew are established using the data of several robotic missions to the Moon and to Mars. The probabilities of the occurrence of deterministic effects due to radiation that could impair the success of a mission or lower the life expectancy of astronauts are evaluated using statistical methods. The limits of the obtained results concerning deterministic and stochastic effects basing on statistical methods are discussed. The conclusion is that new strategies are necessary in order to predict the insurgence of stochastic effects. We argue that the use of the so-called 3D models or organoids could be a good candidate of such a strategy that will be complementary to in vitro 2D models and animal models. Organoids are in fact able to recapitulate several relevant features of the real human organs and their in vivo surroundings. Yet, the main problem of detecting the onset of cancer in the human body remains outstanding. To this purpose, we propose here to combine organoid techniques with those of precision oncology and molecular medicine. Exactly these disciplines have greatly contributed in the recent time to the understanding of the patterns of cancer formation in several cases of tumors. Precision oncology and molecular medicine provide powerful markers to track down the events leading to tumorigenesis following exposure to radiation in organoids.

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Seasonal variability in carbon:234thorium ratios of suspended and sinking particles in coastal Antarctic waters: Field data and modeling synthesis 238U-234Th disequilibrium is a powerful tool for investigating particle cycling and carbon export associated with the oceans biological carbon pump. However, the interpretation of this method is complicated by multiple processes that can modify carbon:thorium ratios over small spatial scales. We investigated seasonal variability in the thorium and carbon cycles at a coastal site in the Western Antarctic Peninsula. Throughout the ice-free summer season, we quantified carbon and 234Th vertical flux, total water column 234Th, particulate 234Th, and the C:234Th ratios of sinking material and bulk suspended material. Simultaneous identification and separation of fecal pellets from sinking material showed that fecal pellets (primarily from krill) contributed 56% of carbon flux and that as a result of lower C:234Th ratios than suspended particles, these fecal pellets were primary drivers of variability in the C:234Th ratios of sinking material. Bulk suspended particles had highly variable C:234Th ratios and were consistently elevated in the euphotic zone relative to deeper waters. The fraction of 234Th adsorbed onto particles was positively correlated with chlorophyll and particulate organic carbon (POC) concentrations. The C:234Th ratios of suspended particles were positively correlated with POC, although during the spring diatom bloom C:234Th ratios were lower than would have been predicted based on POC concentrations alone. We hypothesize that diatom production of transparent exopolymers may have led to enhanced rates of thorium adsorption during the bloom, thus decreasing the C:234Th ratios. We used a Bayesian model selection approach to develop and parameterize mechanistic models to simulate thorium sorption dynamics. The best model incorporated one slowly-sinking POC pool and rapidly-sinking fecal pellets, with second-order sorption kinetics. The model accurately simulated temporal patterns in the C:234Th ratios of sinking and suspended particles and the fraction of 234Th adsorbed to particles. However, it slightly over-estimated C:234Th ratios during the spring (diatom-dominated) bloom and underestimated C:234Th ratios during the fall (mixed-assemblage) bloom. Optimized model parameters for thorium sorption and desorption were 0.0047 {+/-} 0.0002 m3 mmol C-1 d-1 and 0.017 {+/-} 0.008 d-1, respectively. Our results highlight the important role that specific taxa can play in modifying the C:234Th ratio of sinking and suspended particles and provide guidance for future studies that use 234Th measurements to investigate the functional relationships driving the efficiency of the biological pump.

HIGHLIGHTSInvestigated thorium and carbon cycling over full ice-free season

C:234Th ratios of sinking particles were controlled by low C:234Th of fecal pellets

C:234Th ratios of suspended particles were correlated with chlorophyll and POC

Diatom abundance may have led to high particulate thorium during spring bloom

Second-order thorium sorption kinetics model accurately simulates C:234Th ratios

sentences: Complete sequences of six Major Histocompatibility Complex haplotypes, including all the major MHC class II structures Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina and ultra-long Nanopore sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (B38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.32% - 0.69% in a 1000 Genomes Project read re-mapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

sentences: Obesity disrupts innate-adaptive immune network patterning in adipose tissue Obesity drives significant changes in adipose tissue that precede development of tissue and systemic insulin resistance. Immune cell infiltration and inflammation are known contributors to these changes but there is limited understanding of their spatial context tissue-wide. We sought to identify spatial patterning in epididymal adipose tissue immune cells in a time course of diet-induced obesity in mice. Using spatial transcriptomics and single-cell RNA-sequencing, we identified dominant cell type signatures preserved in their anatomical context, quantified gene expression patterns at spots throughout adipose tissue, performed cell type network analysis, and investigated ligand-receptor colocalization. Our data support increased innate immune cells, including macrophages, monocytes, and innate lymphoid cells, with tissue-wide interspersion and dampened adaptive immune cell signatures with obesity. Network analysis identified increased heterogeneity in all major immune cell types, consistent with increased subtypes. To capture tissue dynamics at obesity onset, we draw on mathematical principles from linear algebra and spectral graph theory. We provide a framework for better understanding cell cooperation toward emergence of multicellular tissue function. Further, we adapt Turings mathematical theory on morphogenesis to show lapse of emergence in adipose tissue.

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Exploring functional protein covariation across single cells using nPOP Many biological processes, such as the cell division cycle, are reflected in protein covariation across single cells. This covariation can be quantified and interpreted by single-cell proteomics with sufficiently high throughput and accuracy. Toward this goal, we developed the nano-ProteOmic sample Preparation (nPOP) method for single-cell proteomics. nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a fluorocarbon-coated slide. This design enables simultaneous sample preparation of thousands of single cells, including lysing, digesting, and labeling individual cells in volumes below 20 nl. We used nPOP to prepare 1,888 single cells and 128 negative controls in a single batch. Their analysis enabled quantifying the covariation between thousands of proteins and cell-cycle protein markers. Many protein sets covaried with the cell cycle similarly across all cell types and states, reflecting cell-type independent cell cycle functions. However, the cell cycle covariation of other protein sets differed markedly between cell types, even within subpopulation of melanoma cells expressing markers for drug-resistance priming. The cells expressing these markers accumulated in the G1 phase of the cell cycle and exhibited different covariation of enzymes catabolizing glucose. These results demonstrate that protein covariation across single cells may reveal functionally concerted biological differences between closely related cell states.

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Genes identified in rodent studies of alcohol intake are enriched for heritability of human substance use BackgroundRodent paradigms and human genome-wide association studies (GWASs) on drug use have the potential to provide biological insight into the pathophysiology of addiction.

MethodsUsing GeneWeaver, we created rodent alcohol and nicotine gene-sets derived from 19 gene expression studies on alcohol and nicotine outcomes. We partitioned the SNP-heritability of these gene-sets using four large human GWASs: 1) alcoholic drinks per week, 2) problematic alcohol use, 3) cigarettes per day and 4) smoking cessation. We benchmarked our findings with curated human alcoholism and nicotine addiction gene-sets and performed specificity analyses using other rodent gene-sets (e.g., locomotor behavior) and other human GWASs (e.g., height).

ResultsThe rodent alcohol gene-set was enriched for heritability of drinks per week, cigarettes per day, and smoking cessation, but not problematic alcohol use. However, the rodent nicotine gene-set was not significantly associated with any of these traits. Both rodent gene-sets showed enrichment for several non-substance use GWASs, and the extent of this relationship tended to increase as a function of trait heritability. In general, larger gene-sets demonstrated more significant enrichment. Finally, when evaluating human traits with similar heritabilities, both rodent gene-sets showed greater enrichment for substance use traits.

ConclusionOur results suggest that rodent gene expression studies can help to identify genes that capture heritability of substance use traits in humans, yet the specificity to human substance use was less than expected due to various factors such as the genetic architecture of a trait. We outline various limitations, interpretations and considerations for future research.

sentences: A transcriptomic axis predicts state modulation of cortical interneurons Transcriptomics has revealed the exquisite diversity of cortical inhibitory neurons1-7, but it is not known whether these fine molecular subtypes have correspondingly diverse activity patterns in the living brain. Here, we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, but that this diversity is organized by a single factor: position along their main axis of transcriptomic variation. We combined in vivo 2-photon calcium imaging of mouse V1 with a novel transcriptomic method to identify mRNAs for 72 selected genes in ex vivo slices. We used transcriptomic clusters (t-types)4 to classify inhibitory neurons imaged in layers 1-3 using a three-level hierarchy of 5 Families, 11 Classes, and 35 t-types. Visual responses differed significantly only across Families, but modulation by brain state differed at all three hierarchical levels. Nevertheless, this diversity could be predicted from the first transcriptomic principal component, which predicted a cell types brain state modulation and correlations with simultaneously recorded cells. Inhibitory t-types with narrower spikes, lower input resistance, weaker adaptation, and less axon in layer 1 as determined in vitro8 fired more in resting, oscillatory brain states. Transcriptomic types with the opposite properties fired more during arousal. The former cells had more inhibitory cholinergic receptors, and the latter more excitatory receptors. Thus, despite the diversity of V1 inhibitory neurons, a simple principle determines how their joint activity shapes state-dependent cortical processing.

sentences: Sexual dimorphism and the effect of wild introgressions on recombination in cassava (Manihot esculenta Crantz) breeding germplasm Recombination has essential functions in meiosis, evolution, and breeding. The frequency and distribution of crossovers dictate the generation of new allele combinations and can vary across species and between sexes. Here, we examine recombination landscapes across the 18 chromosomes of cassava (Manihot esculenta Crantz) with respect to male and female meioses and known introgressions from the wild relative Manihot glaziovii. We used SHAPEIT2 and duoHMM to infer crossovers from genotyping-by-sequencing (GBS) data and a validated multi-generational pedigree from the International Institute of Tropical Agriculture (IITA) cassava breeding germplasm consisting of 7,020 informative meioses. We then constructed new genetic maps and compared them to an existing map previously constructed by the International Cassava Genetic Map Consortium (ICGMC). We observed higher recombination rates in females compared to males, and lower recombination rates in M. glaziovii introgression segments on chromosomes 1 and 4, with suppressed recombination along the entire length of the chromosome in the case of the chromosome 4 introgression. Finally, we discuss hypothesized mechanisms underlying our observations of heterochiasmy and crossover suppression and discuss the broader implications for plant breeding.

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Fine-scale adaptations to environmental variation and growth strategies drive phyllosphere Methylobacterium diversity. Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium was structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 {degrees}C or 30 {degrees}C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100km; between forests) and small geographical scales (<1.2km within forests), among host tree species, and was dynamic over seasons. By measuring growth of 79 isolates at different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities, in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus.

ImportanceMethylobacterium is a bacterial group tied to plants. Despite its ubiquity and importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial scales (within 1.2 km). Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains in lab conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasted growth strategies in response to environmental variations.

sentences: Three-dimensional synaptic organization of layer III of the human temporal neocortex In the present study, we have used Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) to perform a study of the synaptic organization of layer III of Brodmanns area 21 in human tissue samples obtained from autopsies and biopsies. We analyzed the synaptic density, 3D spatial distribution, and type (asymmetric/symmetric), as well as the size and shape of each synaptic junction of 4945 synapses that were fully reconstructed in 3D. Significant differences in the mean synaptic density between autopsy and biopsy samples were found (0.49 and 0.66 synapses/m3, respectively). However, in both types of samples (autopsy and biopsy), the asymmetric:symmetric ratio was similar (93:7) and most asymmetric synapses were established on dendritic spines (75%), while most symmetric synapses were established on dendritic shafts (85%). We also compared several electron microscopy methods and analysis tools to estimate the synaptic density in the same brain tissue. We have shown that FIB/SEM is much more reliable and robust than the majority of the other commonly used EM techniques. The present work constitutes a detailed description of the synaptic organization of cortical layer III. Further studies on the rest of the cortical layers are necessary to better understand the functional organization of this temporal cortical region.

sentences: Pneumonia Detection with Semantic Similarity Scores X-ray images have been widely used for medical diagnoses of cardiothoracic and pulmonary abnormalities due to its noninvasiveness. Advancement in computer-aided diagnostic technologies, such as deep supervised methods, can help radiologists with a reliable early treatment and reduce diagnosis time. Nevertheless, these methods are prone to the small number of labeled samples and are limited to a specific abnormality. In this paper we combined a self-supervised contrastive method with a Mahalanobis distance score to develope an abnormality detection method that uses only healthy images during the training procedure. We were able to outperform previous unsupervised methods for the task of Pneumonia detection. We show that representation learned by the self-supervised method improves the supervised tasks for Pneumonia detection.

sentences: Genomic evidence that governmentally produced Cannabis sativa poorly represents genetic variation available in state markets The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genome of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDAs varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDAs varieties lack diversity in the single copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDAs varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.

sentences: SC1: A Tool for Interactive Web-Based Single Cell RNA-Seq Data Analysis Single cell RNA-Seq (scRNA-Seq) is critical for studying cellular function and phenotypic heterogeneity as well as the development of tissues and tumors. Here, we present SC1 a web-based highly interactive scRNA-Seq data analysis tool publicly accessible at https://sc1.engr.uconn.edu. The tool presents an integrated workflow for scRNA-Seq analysis, implements a novel method of selecting informative genes based on Term-Frequency Inverse-Document-Frequency (TF-IDF) scores, and provides a broad range of methods for clustering, differential expression analysis, gene enrichment, interactive visualization, and cell cycle analysis. The tool integrates other single cell omics data modalities like TCR-Seq and supports several single cell sequencing technologies. In just a few steps, researchers can generate a comprehensive analysis and gain powerful insights from their scRNA-Seq data.

sentences: Neuro-molecular characterization of fish cleaning interactions Coral reef fish exhibit a large variety of behaviours crucial for fitness and survival. The cleaner wrasse Labroides dimidiatus displays cognitive abilities during interspecific interactions by providing services of ectoparasite cleaning, thus serving as a good example to understand the processes of complex social behaviour. However, little is known about the molecular underpinnings of cooperative behaviour between L. dimidiatus and a potential client fish (Acanthurus leucosternon). Therefore, we investigated the molecular mechanisms in three regions of the brain (fore-, mid-, and hindbrain) during the interaction of these fishes. Here we show, using transcriptomics, that most of the transcriptional response in both species was regulated in the hindbrain and forebrain regions and that the interacting behaviour responses of L. dimidiatus involved immediate early gene alteration, dopaminergic and glutamatergic pathways, the expression of neurohormones (such as isotocin) and steroids (e.g. progesterone and estrogen). In contrast, in the client, fewer molecular alterations were found, mostly involving pituitary hormone responses. The particular pathways found suggested learning and memory processes in the cleaner wrasse, while the client indicated stress relief.

sentences: A Network-level Test of the Role of the Co-activated Default Mode Network in Episodic Recall and Social Cognition Resting-state network research is extremely influential, yet the functions of many networks remain unknown. In part, this is due to typical (e.g., univariate) analyses testing the function of individual regions and not the full set of co-activated regions that form a network. Connectivity is dynamic and the function of a region may change based on its current connections. Therefore, determining the function of a network requires assessment at the network-level. Yet popular theories implicating the default mode network (DMN) in episodic memory and social cognition, rest principally upon analyses performed at the level of individual brain regions. Here we use independent component analysis to formally test the role of the DMN in episodic and social processing at the network level. As well as an episodic retrieval task, two independent datasets were employed to assess DMN function across the breadth of social cognition; a person knowledge judgement and a theory of mind task. Each task dataset was separated into networks of co-activated regions. In each, the co-activated DMN, was identified through comparison to an a priori template and its relation to the task model assessed. This co-activated DMN did not show greater activity in episodic or social tasks than high-level baseline conditions. Thus, no evidence was found to support hypotheses that the co-activated DMN is involved in explicit episodic or social processing tasks at a network-level. The networks associated with these processes are described. Implications for prior univariate findings and the functional significance of the co-activated DMN are considered.

sentences: Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimers disease Genetic association studies have identified multiple variants at the SPI1 locus that modify risk and age of onset for Alzheimers Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests altered levels of SPi1 may alter microglial phenotype potentially impacting AD. This study determined how the mouse microglial transcriptome was altered following modest changes to Spi1 expression in primary microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected by Spi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways. Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggest Spi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.

sentences: Relish plays a dynamic role in the niche to modulate Drosophila bloodprogenitor homeostasis in development and infection. Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-{kappa}B (NF-{kappa}B), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program towards precocious differentiation, thereby bolstering the cellular arm of the immune response.

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CLN5 and CLN3 function as a complex to regulate endolysosome function CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, Neuronal Ceroid Lipofuscinosis. We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in Neuronal Ceroid Lipofuscinosis, Rab7A, and a subset of Rab7A effectors. Our data supports a model where CLN3 and CLN5 function as an endolysosome complex regulating various functions.

Summary StatementWe have previously demonstrated that CLN3 is required for efficient endosome-to-trans Golgi Network (TGN) trafficking of sortilin by regulating retromer function. In this work, we show that CLN5, which interacts with CLN3, regulates retromer function by modulating key interactions between CLN3, Rab7A, retromer, and sortilin. Therefore, CLN3 and CLN5 serve as endosomal switch regulating the itinerary of the lysosomal sorting receptors.

sentences: Hierarchical Regulation of Autophagy During Adipocyte Differentiation We previously showed that some adipogenic transcription factors such as CEBPB and PPARG directly and indirectly regulate autophagy gene expression in adipogenesis. The order and the effect of these events are undetermined. In this study, we modeled the gene expression, DNA-binding of transcriptional regulators, and histone modifications during adipocyte differentiation and evaluated the effect of the regulators on gene expression in terms of direction and magnitude. Then, we identified the overlap of the transcription factors and co-factors binding sites and targets. Finally, we built a chromatin states model based on the histone marks and studied their relation with the factors binding. Adipogenic factors differentially regulated autophagy genes as part of the differentiation program. Co-regulators associated with specific transcription factors and preceded them to the regulatory regions. Transcription factors differed in the binding time and location, and their effect on expression was either localized or long-lasting. Adipogenic factors disproportionately targeted genes coding for autophagy-specific transcription factors. To sum, a hierarchical arrangement between adipogenic transcription factors and co-factors drives the regulation of autophagy during adipocyte differentiation.

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Effective infection with dengue virus in experimental neonate and adult mice through intranasal route As a member of the family Flaviviridae, the dengue virus is transmitted to humans through mosquito vectors. The virus causes dengue fever with flu-like symptoms, life-threatening dengue hemorrhagic fever, and dengue shock syndrome that affects four billion people in 128 countries. It can also be transmitted through atypical routes, such as needle stick injury, vertical transmission, and the receipt of contaminated blood or organs. Although sporadic cases cannot be classified as atypical transmission routes and raise respiratory exposure concerns, the risks remain unclear. Here, we analyzed the respiratory infectivity of the dengue virus in BALB/c suckling and A6 adult mice using the dengue virus serotype 2 and intranasal inoculation. The results showed that the clinical symptoms of intranasally infected mice included excitement, emaciation, malaise, and death, and histopathological changes and viremia dynamics were observed in multiple tissues, including the brain, liver, and spleen. Notably, the DENV serotype 2 showed clear brain tropism after intranasal infection, and viral loads peaked at seven days post-inoculation. Furthermore, the virus was isolated from mouse brains, and the sequence homology between the viral stock and dengue virus isolates was 99.99%. Similar results were observed in adult IFN-/{beta} receptor-deficient mice. Taken together, our results suggest that the DENV serotype 2 can infect suckling mice and adult immune-deficient mice via the nasal route. The results of our study broaden our perception of atypical dengue transmission routes and may help explain dengue virus infections that occur without the presence of mosquito vectors.

ImportanceDengue is transmitted mainly via mosquito vectors. Flu-like dengue fever or life-threatening severe dengue threatens four billion people in 128 countries. Non-vector dengue cases have occurred occasionally since 1990. These cases cannot be classified as atypical transmission routes and are suspected to have arisen via sexual or respiratory transmission. Recently, the sexual transmission of dengue has been confirmed in humans. The respiratory infectivity of other members of the Flaviviridae family, such as Zika and Japanese encephalitis viruses, has been determined under experimental conditions. The potential for respiratory infection with the dengue virus has not yet been confirmed. In this study, we analyzed the respiratory infectivity of the dengue virus in suckling mice and adult immunodeficient mice via intranasal inoculation, thereby exploring the potential for respiratory transmission in cases with non-vector dengue infection, especially for patients who were exposed to dengue virus contaminants.

sentences: Household transmission of SARS-CoV-2 from humans to dogs in Washington and Idaho: burden and risk factors SARS-CoV-2 is believed to have emerged from an animal reservoir; however, the frequency of and risk factors for inter-species transmission remain unclear. We carried out a community-based study of pets in households with one or more confirmed SARS-CoV-2 infection in humans. Among 119 dogs and 57 cats with completed surveys, clinical signs consistent with SARS-CoV-2 were reported in 20 dogs (21%) and 19 cats (39%). Out of 81 dogs and 32 cats sampled for testing, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive; this discordance may be due to delays in sampling. Respondents commonly reported close human-animal contact and willingness to take measures to prevent transmission to their pets. Reported preventative measures showed a slightly protective trend for both illness and seropositivity in pets, while sharing of beds and bowls had slight harmful effects.

sentences: High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy Multi-photon microscopy has become a powerful tool to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. Yet, tissue scattering, optical aberrations, and motion artifacts degrade the achievable image quality with depth. Here we developed a minimally invasive intravital imaging methodology by combining three-photon excitation, indirect adaptive optics correction, and active electrocardiogram gating to achieve near-diffraction limited resolution up to a depth of 1.2mm in the mouse brain. We demonstrate near-diffraction-limited imaging of deep cortical and sub-cortical dendrites and spines as well as of calcium transients in deep-layer astrocytes in vivo.

sentences: Spindle-slow oscillation coupling during sleep predicts sequence-based language learning Language is one of the most defining human capabilities, involving the coordination of brain networks that generalise the meaning of linguistic units of varying complexity. On a neural level, neocortical slow oscillations and thalamic spindles during sleep facilitate the reactivation of newly encoded memory traces, manifesting in distinct oscillatory activity during retrieval. However, it is currently unknown if the effect of sleep on memory extends to the generalisation of the mechanisms that subserve sentence comprehension. We address this question by analysing electroencephalogram data recorded from 35 participants during an artificial language learning task and an 8hr nocturnal sleep period. We found that a period of sleep was associated with increased alpha/beta power and improved behavioural performance. Phase amplitude coupling analyses also revealed that spindle-slow oscillation coupling predicted the consolidation of sequence-based word orders, which was associated with distinct patterns of oscillatory activity during sentence processing. Taken together, this study presents converging behavioural and neurophysiological evidence for a role of sleep in the consolidation of higher order language learning and associated oscillatory neural activity.

sentences: Morphological integration during postnatal ontogeny: implications for evolutionary biology How covariance patterns of phenotypes change during development is fundamental for a broader understanding of evolution. There is compelling evidence that mammalian skull covariance patterns change during ontogeny. However, it is unclear to what extent variation in covariance patterns during ontogeny can impact the response to selection. To tackle this question we explored: i) the extent to which covariance patterns change during postnatal ontogeny; ii) in which ontogenetic stages covariance patterns differ the most, and iii) the extent to which the phenotypic covariance pattern at different ontogenetic stages can be explained by the same processes determining additive genetic covariance. We sampled postnatal ontogenetic series for both marsupials, and placentals. Within each ontogenetic series, we compared covariance matrices (P-matrices) at different ontogenetic stages. Furthermore, we compared these P-matrices to two target matrices [adult P-matrix and an additive genetic covariance matrix (G-matrix)]. Our results show that for all ontogenetic series, covariance patterns from weaning onward are conserved and probably shaped by the same processes determining the G-matrix. We conclude that irrespective of eventual differences in how selection operates during most of postnatal ontogeny, the net response to such pressures will probably not be affected by ontogenetic differences in the covariance pattern.

sentences: Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

sentences: Using Biometric Data to Measure and Predict Emotional Engagement of Video Games Measurement and prediction of engagement in video games are very important because these are indicators to guide the development of game applications. Existing methods to predict game engagement are mostly based on subjective evaluations. This paper presents results correlating subjective and biometric data to show the potential of using biometric measures for assessment and prediction of engagement. Using three different games, we measured the biometric responses of participants and analyzed the responses using statistical and data mining methods. We compared the results with those obtained using subjective evaluations. Our results show that biometric measurements do correlate with subjective measures and better predict the self-reported engagement of a game.

sentences: Inter- and intra-specific variation in hair cortisol concentrations of Neotropical bats Quantifying hair cortisol has become popular in wildlife ecology for its practical advantages for evaluating health. Before hair cortisol levels can be reliably interpreted however, it is key to first understand the intrinsic factors explaining intra- and interspecific variation. Bats are an ecologically diverse group of mammals that allow studying such variation. Given that many bat species are threatened or have declining populations in parts of their range, non-invasive tools for monitoring colony health and identifying cryptic stressors are needed to efficiently direct conservation efforts. Here we describe intra- and interspecific sources of variation in hair cortisol levels in 18 Neotropical bat species from Mexico and Belize. We found that fecundity is an important ecological trait explaining interspecific variation in bat hair cortisol. Other ecological variables such as colony size, roost durability, and basal metabolic rate did not explain hair cortisol variation among species. At the individual level, females exhibited higher hair cortisol levels than males, and the effect of body mass varied among species. Overall, our findings help validate and accurately apply hair cortisol as a monitoring tool in free-ranging bats.

sentences: Learning interpretable cellular responses tocomplex perturbations in high-throughput screens Recent advances in multiplexed single-cell transcriptomics experiments are facilitating the high-throughput study of drug and genetic perturbations. However, an exhaustive exploration of the combinatorial perturbation space is experimentally unfeasible, so computational methods are needed to predict, interpret, and prioritize perturbations. Here, we present the compositional perturbation autoencoder (CPA), which combines the interpretability of linear models with the flexibility of deep-learning approaches for single-cell response modeling. CPA encodes and learns transcriptional drug responses across different cell type, dose, and drug combinations. The model produces easy-to-interpret embeddings for drugs and cell types, which enables drug similarity analysis and predictions for unseen dosage and drug combinations. We show that CPA accurately models single-cell perturbations across compounds, doses, species, and time. We further demonstrate that CPA predicts combinatorial genetic interactions of several types, implying that it captures features that distinguish different interaction programs. Finally, we demonstrate that CPA can generate in-silico 5,329 missing genetic combination perturbations (97.6% of all possibilities) with diverse genetic interactions. We envision our model will facilitate efficient experimental design and hypothesis generation by enabling in-silico response prediction at the single-cell level, and thus accelerate therapeutic applications using single-cell technologies.

sentences: Unique functions for Notch4 in murine embryonic lymphangiogenesis In mice, embryonic dermal lymphatic development is well-understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts, and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5, and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increased in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

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Multimodal profiling of lung granulomas reveals cellular correlates of tuberculosis control Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. There is also a temporal aspect to bacterial control, in that granulomas that arise later in infection (in the context of an established immune response) share the functional characteristics of restrictive granulomas and are more capable of killing Mtb. Taken together, our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

One-Sentence SummaryBacterial control in TB lung granulomas correlates with distinct cellular immune microenvironments and time of formation after infection.

sentences: Correcting Cardiorespiratory Noise in Resting-state Functional MRI Data Acquired in Critically Ill Patients Resting-state functional MRI (rs-fMRI) is being used to develop diagnostic, prognostic, and therapeutic biomarkers for critically ill patients with severe brain injuries. In studies of healthy volunteers and non-critically ill patients, prospective cardiorespiratory data are routinely collected to remove non-neuronal fluctuations in the rs-fMRI signal during analysis. However, the feasibility and utility of collecting cardiorespiratory data in critically ill patients on a clinical MRI scanner are unknown. We concurrently acquired rs-fMRI (TR=1250ms), cardiac and respiratory data in 23 critically ill patients with acute severe traumatic brain injury (TBI), and 12 healthy control subjects. We compared the functional connectivity results after denoising with cardiorespiratory data (i.e., RETROICOR) with the results obtained after standard bandpass filtering. Rs-fMRI data in 7 patients could not be analyzed due to imaging artifacts. In 6 of the remaining 16 patients (37.5%), cardiorespiratory data were either incomplete or corrupted. In both patients and control subjects, the functional connectivity corrected with RETROICOR did not significantly differ from that corrected with bandpass filtering of 0.008-0.125 Hz. Collectively, these findings suggest that there is a limited feasibility and utility to prospectively acquire high-quality cardiorespiratory data during rs-fMRI in critically ill patients with severe TBI for physiological correction.

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Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III-dependent promoters to enhance non-coding RNA expression RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses ({gamma}HVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during {gamma}HV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 ({gamma}HV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with {gamma}HV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis.

IMPORTANCEGammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

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In vivo imaging and quantification of carbon tracer dynamics in nodulated root systems of pea plants Legumes associate with root colonizing rhizobia that provide fixed nitrogen to its plant host in exchange for recently fixed carbon. There is a lack in understanding how individual plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. One reason is that most approaches are based on destructive sampling, making quantification of localized carbon allocation dynamics in the root system difficult. We established an experimental workflow for routinely using non-invasive Positron Emission Tomography (PET) to follow the allocation of leaf-supplied 11C tracer towards individual nodules in a three-dimensional (3D) root system of pea (Pisum sativum). Nitrate was used for triggering the shutdown of biological nitrogen fixation (BNF) expected to rapidly affect carbon allocation dynamics in the root-nodule system. This nitrate treatment lead to a reduction of 11C tracer allocation to nodules by 40% - 47% in 5 treated plants while the variation in control plants was less than 11%. The established experimental pipeline enabled for the first time that several plants could consistently be labelled and measured using 11C tracer in a PET approach to quantify C-allocation to individual nodules following a BNF shutdown. This demonstrates the strength of using 11C tracers in a PET approach for non-invasive quantification of dynamic carbon allocation in several growing plants over several days. A major advantage of the approach is the possibility to investigate carbon dynamics in small regions of interest in a 3D system such as nodules in comparison to whole plant development.

One sentence summaryPositron Emission Tomography for quantification of carbon allocation dynamics in individual nodules within a 3D root system revealed strong effect of nitrate on carbon allocation.

sentences: Sri Lankan Footprint in Global Aquatic Science Research: A Scientometric Analysis The present study summarizes the research productivity and international collaboration in aquatic studies conducted by Sri Lankan scholars during 2000-2019. The study was based on the SCOPUS(R) database. R programming language, package bibliometrix and Vosviewer software were employed in the analysis. Results of the present study indicate that increasing growth trend in the annual number of publications. A significant correlation (p<0.05) between the number of articles and per capita GDP (Gross Domestic Production) was also observed. Senior authors dominated in terms of the article count, citation count, h index, and other author productivity indices. Journal of the National Science Foundation of Sri Lanka had the highest article count (n=33). Aquatic studies in Sri Lanka were more locally funded. Sri Lanka had strong research collaborations with Japan, South Korea and Australia. During 2000-2019, the transition of aquatic studies from lacustrine field studies to molecular lab-based studies were observed. The findings of the present study may provide a comprehensive understanding on the current context and future directions of aquatic studies in Sri Lanka.

sentences: The influence of stereopsis on visual saliency in a proto-object based model of selective attention Some animals including humans use stereoscopic vision which reconstructs spatial information about the environment from the disparity between images captured by eyes in two separate adjacent locations. Like other sensory information, such stereoscopic information is expected to influence attentional selection. We develop a biologically plausible model of binocular vision to study its effect on bottom-up visual attention, i.e., visual saliency. In our model, the scene is organized in terms of proto-objects on which attention acts, rather than on unbound sets of elementary features. We show that taking into account the stereoscopic information improves the performance of the model in the prediction of human eye movements with statistically significant differences.

sentences: Cortical response variability is driven by local excitability changes with somatotopic organization Identical sensory stimuli can lead to different neural responses depending on the instantaneous brain state. Specifically, neural excitability in sensory areas may shape the brains response already from earliest cortical processing onwards. However, whether these dynamics affect sensory domains globally or occur on a spatially local level is largely unknown. We studied this in the somatosensory domain of 38 human participants with EEG, presenting stimuli to the median and tibial nerves alternatingly, and testing the co-variation of initial cortical responses in hand and foot areas, as well as their relation to pre-stimulus oscillatory states. We found that amplitude fluctuations of initial cortical responses to hand and foot stimulation - the N20 and P40 components of the somatosensory evoked potential (SEP), respectively - were not related, indicating local excitability changes in primary sensory regions. In addition, effects of pre-stimulus alpha (8-13 Hz) and beta (18-23 Hz) band amplitude on hand-related responses showed a robust somatotopic organization, thus further strengthening the notion of local excitability fluctuations. However, for foot-related responses, the spatial specificity of pre-stimulus effects was less consistent across frequency bands, with beta appearing to be more foot-specific than alpha. Connectivity analyses in source space suggested this to be due to a somatosensory alpha rhythm that is primarily driven by activity in hand regions while beta frequencies may operate in a more hand-region-independent manner. Altogether, our findings suggest spatially distinct excitability dynamics within the primary somatosensory cortex, yet with the caveat that frequency-specific processes in one sub-region may not readily generalize to other sub-regions.

sentences: Long-term conservation effects of protected areas in stochastic population dynamics Terrestrial and marine protected areas are essential tools in mitigating anthropogenic impacts and promoting population persistence and resource sustainability. Adequately implemented protected areas (PAs) promote long-term conservation effects. Stochasticity causes fluctuations in the conservation effects of PAs, and so it is important to investigate the variabilities of these conservation effects to inform their long-term conservation effects. To investigate long-term conservation effects, I develop and analyze new models of stochastic processes that encompass the fluctuations generated by demographic or environmental stochasticity in PAs management. The stochastic model is built upon individual processes. In the model, density-independent mortality, migration between PAs and non-PAs, and site preferences characterize the features of the PA. The effect of PAs size is also examined. The long-term conservation effects are quantified using the coefficient of variation (CV) of population size in PAs, where a lower CV indicates higher robustness in stochastic variations. Typically, the results from this study demonstrate that sufficiently reduced density-independent mortality in PAs and high site preference and immigration rate of PA are likely to decrease the CV. However, different types of stochasticity induce rather different consequences: under demographic stochasticity, the CV is always reduced when PAs increase the population size therein, but an increased population size by PAs does not always decrease the CV under environmental stochasticity. The deterministic dynamics of the model are investigated, facilitating effective management decisions.

sentences: A Multiple Regression Assessment of the Biomineral Urease Activity from Urine Drainpipes of California Rest Areas Clogging and odor is strongly associated with ureolytic biomineralization in waterless and low-flow urinal drainage systems in high usage settings. These blockages continue to hinder widespread waterless and low-flow urinal adoption due to subsequent high maintenance requirements and hygiene concerns. Through field observations, hypothesis testing, and multiple regression analysis, this study attempts to characterize, for the first time, the ureolytic activity of the biomineralization found in alternative technologies located at 9 State-owned restrooms. Multiple regression analysis (n = 55, df = 4, R2 = 0.665) suggests that intrasystem sampling location [Formula], annual users per rest area [Formula], and the organic/inorganic mass fraction [Formula], are statistically significant influencers of the ureolytic activity of biomineral samples (p < 0.05). Conversely, ureC gene abundance (p = 0.551), urinal type (p = 0.521) and sampling season (p = 0.956) are not significant predictors of biomineral ureolytic activity. We conclude that high concentrations of the urease alpha subunit, ureC, which can be interpreted as proxy measure of a strong, potentially ureolytic community, does not necessarily mean that the gene is being expressed. Future studies should assess ureC transcriptional activity to measure gene expression rather than gene abundance to assess the relationship between environmental conditions, their role in transcription, and urease activities. In sum, this study presents a method to characterize biomineral ureolysis and establishes baseline values for future ureolytic inhibition treatment studies that seek to improve the usability of urine collection and related source separation technologies.

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Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3, 5-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Author SummaryThe recalcitrance of Mycobacterium tuberculosis (Mtb) to conventional antibiotics has created a need to identify novel pharmacological mechanisms to inhibit Mtb pathogenesis. There is a growing understanding of the metabolic adaptations Mtb adopts during infection to support its survival and pathogenesis. This has generated interest in identifying small molecule compounds that effectively inhibit these in vivo metabolic adaptations, while overcoming challenges like poor pharmacokinetic properties or redundancy in target pathways. The Mtb cholesterol utilization pathway has repeatedly been speculated to be a desirable antibiotic target, but compounds that successfully inhibit this complex pathway and are suitable for use in vivo are lacking. Here, we establish that stimulating cAMP synthesis in Mtb is a mechanism that is sufficient to block cholesterol utilization by the bacterium, preventing the release of key metabolic intermediates that are derived from breakdown of the cholesterol molecule. For the first time, we identify small molecule agonists of the Mtb adenylyl cyclase Rv1625c that have promising pharmacological properties and are suitable for use during in vivo studies. These Rv1625c agonists increase cAMP synthesis, inhibit cholesterol utilization by Mtb, and disrupt Mtb pathogenesis in mouse models of chronic infection.

sentences: Wireless closed-loop smart bandage for chronic wound management and accelerated tissue regeneration Chronic non-healing wounds represent a major source of morbidity for patients and a significant economic burden. Current wound care treatments are generally passive and are unable to adapt to changes in the wound environment in real time. By integrating multimodal sensors and adding stimulators in a bandage, real-time physiological monitoring is possible and provides an opportunity for active intervention into the complex wound environment. Here, we develop a battery-free flexible bioelectronic system consisting of wirelessly powered, closed-loop sensing and stimulation circuits with tissue-interfacing tough conducting hydrogel electrodes for robust signal transduction, on-demand adhesion, and detachment. Using multiple pre-clinical models, we demonstrate the capability of our wound care system to continuously monitor skin impedance and temperature, to trigger directional electrical stimulation. The accelerated wound closure was confirmed to be due to the activation of pro-regenerative genes linked to accelerated wound closure, increased neovascularization, and enhanced dermal recovery.

sentences: Revealing Free Energy Landscape from MD Data via Conditional Angle Partition Tree Deciphering the free energy landscape of biomolecular structure space is crucial for understanding many complex molecular processes, such as protein-protein interaction, RNA folding, and protein folding. A major source of current dynamic structure data is Molecular Dynamics (MD) simulations. Several methods have been proposed to investigate the free energy landscape from MD data, but all of them rely on the assumption that kinetic similarity is associated with global geometric similarity, which may lead to unsatisfactory results. In this paper, we proposed a new method called Conditional Angle Partition Tree to reveal the hierarchical free energy landscape by correlating local geometric similarity with kinetic similarity. Its application on the benchmark alanine dipeptide MD data showed a much better performance than existing methods in exploring and understanding the free energy landscape. We also applied it to the MD data of Villin HP35. Our results are more reasonable on various aspects than those from other methods and very informative on the hierarchical structure of its energy landscape.

sentences: Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

sentences: Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1 Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

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Short-term and bystander effects of radiation on murine submandibular glands Many patients treated for head and neck cancers experience salivary gland hypofunction due to radiation damage. Understanding the mechanisms of cellular damage induced by radiation treatment is important in order to design methods of radioprotection. In addition, it is crucial to recognize the indirect effects of IR and the systemic responses that may alter saliva secretion. In this study, radiation was delivered to murine submandibular glands (SMG) bilaterally, using a 137Cs gamma ray irradiator, or unilaterally, using a small animal radiation research platform (SARRP). Analysis at 3, 24 and 48 hours showed dynamic changes in mRNA levels and protein abundance in SMG irradiated bilaterally. Unilateral irradiation using the SARRP caused similar changes in the irradiated SMG, as well as significant off-target, bystander effects in the non-irradiated contralateral SMG.

Summary statementRapid changes in gene expression occur in irradiated salivary glands within hours of irradiation, and in non-irradiated contralateral glands, due to bystander effects.

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Interneuronal dynamics facilitate the initiation of cortical spreading depression Cortical spreading depression (CSD) is thought to precede migraine attacks with aura and is characterized by a slowly traveling wave of inactivity through cortical pyramidal cells. During CSD, pyramidal cells experience hyperexcitation with rapidly increasing firing rates, major changes in electrochemistry, and ultimately spike block that propagates slowly across the cortex. While the identifying characteristic of CSD is the pyramidal cell hyperexcitation and subsequent spike block, it is currently unknown how the dynamics of the cortical microcircuits and inhibitory interneurons affect the initiation of CSD.

We tested the contribution of cortical inhibitory interneurons to the initiation of spike block using a cortical microcircuit model that takes into account changes in ion concentrations that result from neuronal firing. Our results show that interneuronal inhibition provides a wider dynamic range to the circuit and generally improves stability against spike block.

Despite these beneficial effects, strong interneuronal firing contributed to rapidly changing extracellular ion concentrations, which facilitated hyperexcitation and led to spike block first in the interneuron and then in the pyramidal cell. In all cases, a loss of interneuronal firing triggered pyramidal cell spike block. However, preventing interneuronal spike block was insufficient to rescue the pyramidal cell from spike block. Our data thus demonstrate that while the role of interneurons in cortical microcircuits is complex, they are critical to the initiation of pyramidal cell spike block and CSD. We discuss the implications that localized effects on cortical interneurons have beyond the isolated microcircuit.

sentences: University-wide chronotyping shows late-type students have lower grades, shorter sleep, poorer well-being, lower self-regulation, and more absenteeism A persons preferred timing of nocturnal sleep (chronotype) has important implications for cognitive performance. Students who prefer to sleep late may have a selective learning disadvantage for morning classes due to inadequate sleep and circadian desynchrony. Here, (1) we tested whether late-type students perform worse only for morning classes, and (2) we investigated factors that may contribute to their poorer academic achievement. Chronotype was determined objectively in 33,645 university students (early, n=3,965; intermediate, n=23,787; late, n=5,893) by analyzing the diurnal distribution of their logins on the universitys Learning Management System (LMS). Late-type students had lower grades than their peers for courses held at all different times of day, and during semesters when they had no morning classes. Actigraphy studies (n=261) confirmed LMS-derived chronotype was associated with students sleep patterns. Nocturnal sleep on school days was shortest in late-type students because they went to bed the latest and woke up early compared with non-school days. Surveys showed that late-type students had lower self-rated health and mood (n=357), and lower metacognitive self-regulation (n=752). Wi-Fi connection data for classrooms (n=17,356) revealed that late-type students had lower lecture attendance than their peers for classes held in both the morning and the afternoon. Our findings suggest that multiple factors converge to impair learning in late-type students. Shifting classes later can improve sleep and circadian synchrony in late-type students but is unlikely to eliminate the performance gap. Interventions that focus on improving students well-being and learning strategies may be important for addressing the late-type academic disadvantage.

sentences: S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair. The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

sentences: Cortical microtubule pulling forces contribute to the union of the parental genomes in the C. elegans zygote Previously, we reported that the Polo-like kinase PLK-1 phosphorylates the single C. elegans lamin (LMN-1) to trigger lamina depolymerization during mitosis. We showed that this event is required for the formation of a pronuclear envelopes scission event that removes membranes on the juxtaposed oocyte and sperm pronuclear envelopes in the zygote, allowing the parental chromosomes to merge in a single nucleus after segregation (Velez-Aguilera, 2020). Here we show that cortical microtubule pulling forces contribute to pronuclear envelopes scission by promoting mitotic spindle elongation. We also demonstrate that weakening of the pronuclear envelopes, via PLK-1-mediated lamina depolymerization, is a prerequisite for the astral microtubule pulling forces to trigger pronuclear membranes scission. Finally, we provide evidence that PLK-1 mainly acts via lamina depolymerization in this process. These observations thus indicate that temporal coordination between lamina depolymerization and mitotic spindle elongation facilitates pronuclear envelopes scission and parental genomes unification.

sentences: MCGA: a multi-strategy conditional gene-based association framework integrating with isoform-level expression profiles reveals new susceptible and druggable candidate genes of schizophrenia Linkage disequilibrium and disease-associated variants in non-coding regions make it difficult to distinguish truly associated genes from redundantly associated genes for complex diseases. In this study, we proposed a new conditional gene-based framework called MCGA that leveraged an improved effective chi-squared statistic to control the type I error rates and remove the redundant associations. MCGA initially integrated two conventional strategies to map genetic variants to genes, i.e., mapping a variant to its physically nearby gene and mapping a variant to a gene if the variant is a gene-level expression quantitative trait locus (eQTL) of the gene. We further performed a simulation study and demonstrated that the isoform-level eQTL was more powerful than the gene-level eQTL in the association analysis. Then the third strategy, i.e., mapping a variant to a gene if the variant is an isoform-level eQTL of the gene, was also integrated with MCGA. We applied MCGA to predict the potential susceptibility genes of schizophrenia and found that the potential susceptibility genes identified by MCGA were enriched with many neuronal or synaptic signaling-related terms in the Gene Ontology knowledgebase and antipsychotics-gene interaction terms in the drug-gene interaction database (DGIdb). More importantly, nine susceptibility genes were the target genes of multiple approved antipsychotics in DrugBank. Comparing the susceptibility genes identified by the above three strategies implied that strategy based on isoform-level eQTL could be an important supplement for the other two strategies and help predict more candidate susceptibility isoforms and genes for complex diseases in a multi-tissue context.

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Exosomes from COVID-19 patients carry tenascin-C and fibrinogen-β in triggering inflammatory signals in distant organ cells SARS-CoV-2 infection causes cytokine storm and overshoot immunity in humans; however, it remains to be determined whether genetic material of SARS-CoV-2 and/or virus induced soluble mediators from lung epithelial cells as natural host are carried out by macrophages or other vehicles at distant organs causing tissue damage. We speculated that exosomes as extracellular vesicles are secreted from SARS-CoV-2 infected cells may transport messages to other cells of distant organs leading to pathogenic consequences. For this, we took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and SARS-CoV-2 infected patients. Our results revealed that tenascin-C (TNC) and fibrinogen-{beta} (FGB) are highly abundant in exosomes from SARS-CoV-2 infected patients plasma as compared to that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via NF-{kappa}B pathway, we examined the status of TNF-, IL-6 and CCL5 expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase when compared with that from healthy subjects. Together, our results demonstrated that soluble mediators, like TNC and FGB, are transported through plasma exosomes in SARS-CoV-2 infected patients and trigger pro-inflammatory cytokine expression in cells of distant organs in COVID-19 patients.

ImportanceExosomes play an important role in intercellular communication by inducing physiological changes in recipient cells by transferring bioactive proteins. Little is known about exosomes from SARS-CoV-2 infected cells and their role in pathogenesis. Here, we have carefully examined and analyzed this aspect of SARS-CoV-2 infection. Our results uncovered the potential mechanisms by which SARS-CoV-2 communicates with other cells of distant organs and promotes pathogenesis. We expect to detect whether other factors are modulated in the presence of COVID-19 exosomes. Our exosomes related proteomic experiments prioritize after initial verification to further examine their role in SARS-CoV-2 associated other pathogenic mechanisms to target for therapeutic modalities.

sentences: Human interictal epileptiform discharges are bidirectional traveling waves echoing ictal discharges Interictal epileptiform discharges (IEDs), also known as interictal spikes, are large intermittent electrophysiological events observed between seizures in patients with epilepsy. Though they occur far more often than seizures, IEDs are less studied, and their relationship to seizures remains unclear. To better understand this relationship, we examined multi-day recordings of microelectrode arrays implanted in human epilepsy patients, allowing us to precisely observe the spatiotemporal propagation of IEDs, spontaneous seizures, and how they relate. These recordings showed that the majority of IEDs are traveling waves, traversing the same path as ictal discharges during seizures, and with a fixed direction relative to seizure propagation. Moreover, the majority of IEDs, like ictal discharges, were bidirectional, with one predominant and a second, less frequent antipodal direction. These results reveal a fundamental spatiotemporal similarity between IEDs and ictal discharges. These results also imply that most IEDs arise in brain tissue outside the site of seizure onset and propagate toward it, indicating that the propagation of IEDs provides useful information for localizing the seizure focus.

sentences: The atypical RNA-binding protein TAF15 regulates dorsoanterior neural development through diverse mechanisms in Xenopus tropicalis. The FET family of atypical RNA-binding proteins includes Fused in sarcoma (Fus), Ewings sarcoma (EWS), and the TATA-binding protein-associate factor 15 (TAF15). FET proteins are highly conserved, suggesting specialized requirements for each protein. Fus regulates splicing of transcripts required for mesoderm differentiation and cell adhesion in Xenopus, but roles that EWS and TAF15 play remain unknown. Here we analyze the roles of maternally deposited and zygotically transcribed TAF15, which is essential for the proper development of dorsoanterior neural tissues. By measuring changes in exon usage and transcript abundance from TAF15-depleted embryos we found TAF15 may regulate dorsoanterior neural development through fgfr4 and ventx2.1. TAF15 uses distinct mechanisms to downregulate FGFR4 expression: 1) retention of a single intron within fgfr4 when maternal and zygotic TAF15 is depleted, and 2) reduction of total fgfr4 transcript when zygotic TAF15 alone is depleted. The two mechanisms of gene regulation (post-transcriptional vs transcriptional) suggest TAF15-mediated gene regulation is target and cofactor-dependent, depending on the milieu of factors that are present at different times of development.

sentences: SARS-CoV-2 genome sequencing with Oxford Nanopore Technology and Rapid PCR Barcoding in Bolivia SARS-CoV-2 genomic surveillance has Illumina technology as the golden standard. However, Oxford Nanopore Technology (ONT) provides significant improvements in accessibility, turnaround time and portability. Characteristics that gives developing countries the opportunity to perform genome surveillance. The most used protocol to sequence SARS-CoV-2 with ONT is an amplicon-sequencing protocol provided by the ARTIC Network which requires DNA ligation. Ligation reagents can be difficult to obtain in countries like Bolivia. Thus, here we provide an alternative for library preparation using the rapid PCR barcoding kit (ONT). We mapped more than 3.9 million sequence reads that allowed us to sequence twelve SARS-CoV-2 genomes from three different Bolivian cities. The average sequencing depth was 324X and the average genome length was 29527 bp. Thus, we could cover in average a 98,7% of the reference genome. The twelve genomes were successfully assigned to four different nextstrain clades (20A, 20B, 20E and 20G) and we could observe two main lineages of SARS-CoV-2 circulating in Bolivia. Therefore, this alternative library preparation for SARS-CoV-2 genome sequencing is effective to identify SARS-CoV-2 variants with high accuracy and without the need of DNA ligation. Hence, providing another tool to perform SARS-CoV-2 genome surveillance in developing countries.

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Population replacement gene drive characteristics for malaria elimination in a range of seasonal transmission settings: a modeling study Genetically engineering mosquitoes is a promising new vector control strategy to reinvigorate the fight against malaria in Sub-Saharan Africa. Using an agent-based model of malaria transmission with vector genetics, we examine the impacts of releasing population-replacement gene drive mosquitoes on malaria transmission and quantify the gene drive system parameters required to achieve local elimination within a spatially-resolved, seasonal Sahelian setting. We evaluate the performance of two different gene drive systems: "classic" and "integral." Various transmission regimes (low, moderate, and high - corresponding to annual entomological inoculation rates of 10, 30, and 80 infectious bites per person) and other simultaneous interventions, including deployment of insecticide-treated nets (ITNs) and passive healthcare seeking, are also simulated. Local elimination probabilities decreased with pre-existing population target site resistance frequency, increased with transmission-blocking effectiveness of the introduced antiparasitic gene and drive efficiency, and were context dependent with respect to fitness costs associated with the introduced gene. Of the four parameters, transmission-blocking effectiveness may be the most important to focus on for improvements to future gene drive strains because a single release of classic gene drive mosquitoes is likely to locally eliminate malaria in low to moderate transmission settings only when transmission-blocking effectiveness is very high (above ~80-90%). However, simultaneously deploying ITNs and releasing integral rather than classic gene drive mosquitoes significantly boosts elimination probabilities, such that elimination remains highly likely in low to moderate transmission regimes down to transmission-blocking effectiveness values as low as ~50% and in high transmission regimes with transmission-blocking effectiveness values above ~80-90%. Thus, a single release of currently achievable population replacement gene drive mosquitoes, in combination with traditional forms of vector control, can likely locally eliminate malaria in low to moderate transmission regimes within the Sahel. In a high transmission regime, higher levels of transmission-blocking effectiveness than are currently available may be required.

Author summaryMalaria remains a significant health burden in Sub-Saharan Africa. The mass deployment of insecticide-treated nets and antimalarial drugs have drastically reduced malaria incidence, but insecticide and drug resistance threaten to stall these efforts. The genetic engineering of mosquito populations is a promising new vector control strategy to reinvigorate the fight against malaria. Releases of engineered gene drive mosquitoes that can spread introduced antimalarial genes quickly throughout the mosquito population may be a particularly effective new method for reducing malaria transmission. Important questions arise, however, about how well these gene drive systems must work in order to deliver substantial reductions in transmission. Here we use a spatial model of individual humans and vectors to simulate the effects of releasing gene drive mosquitoes with antimalarial properties on malaria transmission in a Sahelian setting. We quantify the gene drive system parameters required to achieve local elimination and find that when deployed in combination with traditional forms of vector control, a single release of gene drive mosquitoes with realistically achievable characteristics is highly likely to locally eliminate malaria in low to moderate transmission regimes. In a high transmission regime, improved strains of gene drive mosquitoes may be required. In all settings, releasing gene drive mosquitoes with antimalarial properties helps create a window of opportunity during which malaria prevalence is suppressed and other interventions can be ramped up to achieve elimination, even when a single gene drive mosquito release by itself cannot.

sentences: Automatic post-processing and merging of multiple spike-sorting analyses with Lussac The increasing site counts of multielectrodes used in extracellular recording preclude traditional manual spike-sorting and have led to the development of several new software suites with increased automation. It remains difficult, however, to find parameters enabling optimal processing of signals from all neuronal types simultaneously. Here, we describe a procedure for automatically post-processing and merging the outputs of multiple spike-sorting analyses, enabling the accumulation of results optimised for different activity features. This approach was motivated by and first applied to the analysis of cerebellar activity, in which the complex spikes challenge standard analyses. The core of our modular procedure involves the identification of cluster-fusion candidates and assurance that only fusions that increase a clearly defined cluster-quality metric are performed; important subsidiary steps that have also been automated include spike alignment and duplicate removal. The procedure was validated by recovery of injected spike waveforms. In tests combining analyses from Kilosort and MountainSort, our procedure yielded a 50 % increase in the detected number of Purkinje cell clusters with both simple and complex spikes. A further validation used the synthetic SpikeForest dataset approximating neocortical activity, in which our package also improved performance. Combining multiple analyses therefore offers a general method for improving spike-sorting, and furthermore Lussac reduces manual input and increases the objectivity of analyses.

sentences: Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML - a model pre-mRNA substrate - guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.

sentences: Significance of Oct-4 transcription factor as a pivotal therapeutic target for CD44+/24- mammary tumour initiating cells; aiming at the root of the recurrence Breast cancer (BC) remains one of the deadliest and frequently diagnosed metastatic cancers worldwide. Cancer stem cells (CSCs) are the cell population within the tumour niche, having an epithelial to mesenchymal (EMT) transition phenotype, high self-renewal, vigorous metastatic capacity, drug resistance, and tumour relapse. Identification of targets for induction of apoptosis is essential to provide novel therapeutic approaches in BC. Our earlier studies showed that Vitamin C induces apoptotic cell death by losing redox balance in TNBC CSCs. In this study, we have attempted to identify previously unrecognized CSC survival factors that can be used as druggable targets for bCSCs apoptosis regulators isolated from the TNBC line, MDA MB 468. After a thorough literature review, Oct-4 was identified as the most promising marker for its unique abundance in cancer and absence in normal cells and the contribution of Oct-4 to the sustenance of cancer cells. We then validated a very high expression of Oct-4 in the MDA MB 468 bCSCs population using flow-cytometry. The loss of Oct-4 was carried out using siRNA-mediated knockdown in the bCSCs, followed by assessing for cellular apoptosis. Our results indicated that Oct-4 knockdown induced cell death, changes in cellular morphology, inhibited mammosphere formation, and positive for Annexin-V expression, thereby indicating the role of Oct-4 in bCSC survival. Moreover, our findings also suggest the direct interaction between Oct-4 and Vitamin C using in silico docking. This data, hence, contributes towards novel information about Oct-4 highlighting this molecule as a novel survival factor in bCSCs.

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Genomic surveillance of Bacillus cereus sensu lato strains isolated from meat and poultry products in South Africa enables inter- and intra-national surveillance and source tracking Members of the Bacillus cereus sensu lato (s.l.) species complex, also known as the B. cereus group, vary in their ability to cause illness, but are frequently isolated from foods, including meat products; however, food safety surveillance efforts that employ whole-genome sequencing (WGS) often neglect these potential pathogens. Here, WGS was used to characterize B. cereus s.l. strains (n = 25) isolated during surveillance of meat products in South Africa. Strains were collected from beef, poultry, and mixed meat products obtained from (i) retail outlets, processing plants, and butcheries across six South African provinces (n = 15, 7, and 1, respectively), and (ii) imports in cold stores (n = 2). Strains were assigned to panC Groups IV, III, II, and V (n = 18, 5, 1, and 1, respectively) and spanned multiple genomospecies, regardless of the taxonomy used. All strains possessed diarrheal toxin-encoding genes, while one sequence type 26 (ST26) strain possessed cereulide (emetic toxin) synthetase-encoding genes. No strains harbored anthrax toxin- or capsule-encoding genes. The 25 strains were partitioned into 15 lineages via in silico seven-gene multi-locus sequence typing (MLST), six of which contained multiple strains sequenced in this study, which were identical or nearly identical at the whole-genome scale. Five MLST lineages contained (nearly) identical genomes collected from two or three South African provinces; one MLST lineage contained nearly identical genomes from two countries (South Africa and the Netherlands), indicating that B. cereus s.l. can spread intra- and inter-nationally via foodstuffs.

ImportanceNation-wide foodborne pathogen surveillance programs that employ high-resolution genomic methods have been shown to provide vast public health and economic benefits. However, B. cereus s.l. are often overlooked during large-scale, routine WGS efforts. Thus, to our knowledge, no studies to date have evaluated the potential utility of WGS for B. cereus s.l. surveillance and source tracking in foodstuffs. In this proof-of-concept study, we applied WGS to B. cereus s.l. strains collected via South Africas national surveillance program of domestic and imported meat products, and we provide strong evidence that B. cereus s.l. can be disseminated intra- and inter-nationally via the agro-food supply chain. Our results showcase that WGS can be used for source tracking of B. cereus s.l. in foods, although future WGS and isolate metadata collection efforts are needed to ensure that B. cereus s.l. surveillance initiatives are on par with those of other foodborne pathogens.

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G-Protein Coupled Receptor-Ligand Dissociation Rates and Mechanisms from {tau}RAMD Simulations There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of timescales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the {tau}-Random Acceleration Molecular Dynamics ({tau}RAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the {beta}2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.

TABLE OF CONTENTS GRAPHIC

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sentences: Caecilian genomes reveal molecular basis of adaptation and convergent evolution of limblessness in snakes and caecilians. We present genome sequences for Geotrypetes seraphini (3.8Gb) and Microcaecilia unicolor (4.7Gb) caecilians, a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. We identify signatures of positive selection unique to caecilians in 1,150 orthogroups, with enrichment of functions for olfaction and detection of chemical signals. All our caecilian genomes are missing the ZRS enhancer of Sonic Hedgehog, shown by in vivo deletions to be required for limb development in mice and also absent in snakes, thus revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.

sentences: Humans hastened the range collapse and extinction of woolly mammoth Pathways to extinction start long before the death of the last individual. However, causes of early-stage population declines and the susceptibility of small residual populations to extirpation are typically studied in isolation. Using validated process-explicit models, we disentangle the ecological mechanisms and threats that were integral in the initial decline and later extinction of the woolly mammoth. We show that reconciling ancient DNA data on woolly mammoth population decline with fossil evidence of location and timing of extinction requires process-explicit models with specific demographic and niche constraints, and a constrained synergy of climatic change and human impacts. Validated models needed humans to hasten climate-driven population declines by many millennia, and to allow woolly mammoths to persist in mainland Arctic refugia until the mid-Holocene. Our results show that the role of humans in the extinction dynamics of woolly mammoth began well before the Holocene, exerting lasting effects on the spatial pattern and timing of its range-wide extinction.

sentences: ProtFinder: finding subcellular locations of proteins using protein interaction networks AO_SCPLOWBSTRACTC_SCPLOWProtein subcellular localization prediction plays a crucial role in improving our understanding of different diseases and consequently assists in building drug targeting and drug development pipelines. Proteins are known to co-exist at multiple subcellular locations which make the task of prediction extremely challenging. A protein interaction network is a graph that captures interactions between different proteins. It is safe to assume that if two proteins are interacting, they must share some subcellular locations. With this regard, we propose ProtFinder - the first deep learning-based model that exclusively relies on protein interaction networks to predict the multiple subcellular locations of proteins. We also integrate biological priors like the cellular component of Gene Ontology to make ProtFinder a more biology-aware intelligent system. ProtFinder is trained and tested using the STRING and BioPlex databases whereas the annotations of proteins are obtained from the Human Protein Atlas. Our model obtained an AUC-ROC score of 90.00% and an MCC score of 83.42% on a held-out set of proteins. We also apply ProtFinder to annotate proteins that currently do not have confident location annotations. We observe that ProtFinder is able to confirm some of these unreliable location annotations, while in some cases complementing the existing databases with novel location annotations. The source code for ProtFinder is available at https://github.com/UCLouvain-CBIO/ProtFinder.

sentences: Continuous tracking of gravistimulated roots in a chambered coverslip by confocal microscopy allows first glimpse on mechanoadaptation of cell files during curvature initiation Mechanical responses of individual cells to plant internal and external stimuli modulate organ movement and ensure plant survival as sessile organism in a constantly changing environment. The root is a complex, three-dimensional object, which continuously modifies its growth path. Autonomous and paratonic root movements are both orchestrated by different signaling pathways, whereby auxin modulated directional growth adaptations, including gravitropic response, were already subject of manifold studies. But we still know very little about how cells adapt upon gravitropic stimulus to initiate curvature establishment, which is required to align root tip growth again along the gravitropic vector. This manuscript shows first insights into cell file movements upon gravitropic stimulus of Arabidopsis thaliana roots that initiate curvature establishment. The roots were grown shaded from light and without exogenous sucrose supplementation, both growth conditions that are known to negatively interfere with directed root growth, which allowed a more uniform tracking of root bending by using a confocal microscope with vertical stage.

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Rapid label-free identification of pathogenic bacteria species from a minute quantity 1 exploiting three-dimensional quantitative phase imaging and artificial neural network The healthcare industry is in dire need for rapid microbial identification techniques. Microbial infection is a major healthcare issue with significant prevalence and mortality, which can be treated effectively during the early stages using appropriate antibiotics. However, determining the appropriate antibiotics for the treatment of the early stages of infection remains a challenge, mainly due to the lack of rapid microbial identification techniques. Conventional culture-based identification and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy are the gold standard methods, but the sample amplification process is extremely time-consuming. Here, we propose an identification framework that can be used to measure minute quantities of microbes by incorporating artificial neural networks with three-dimensional quantitative phase imaging. We aimed to accurately identify the species of bacterial bloodstream infection pathogens based on a single colony-forming unit of the bacteria. The successful distinction between a total of 19 species, with the accuracy of 99.9% when ten bacteria were measured, suggests that our framework can serve as an effective advisory tool for clinicians during the initial antibiotic prescription.

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sentences: Modernizing the NEURON Simulator for Sustainability, Portability, and Performance The need for reproducible, credible, multiscale biological modeling has led to the development of standardized simulation platforms, such as the widely-used NEURON environment for computational neuroscience. Developing and maintaining NEURON over several decades has required attention to the competing needs of backwards compatibility, evolving computer architectures, the addition of new scales and physical processes, accessibility to new users, and efficiency and flexibility for specialists. In order to meet these challenges, we have now substantially modernized NEURON, providing continuous integration, an improved build system and release workflow, and better documentation. With the help of a new source-to-source compiler of the NMODL domain-specific language we have enhanced NEURONs ability to run efficiently, via the CoreNEURON simulation engine, on a variety of hardware platforms, including GPUs. Through the implementation of an optimized in-memory transfer mechanism this performance optimized backend is made easily accessible to users, providing training and model-development paths from laptop to workstation to supercomputer and cloud platform. Similarly, we have been able to accelerate NEURONs reaction-diffusion simulation performance through the use of just-in-time compilation. We show that these efforts have led to a growing developer base, a simpler and more robust software distribution, a wider range of supported computer architectures, a better integration of NEURON with other scientific workflows, and substantially improved performance for the simulation of biophysical and biochemical models.

sentences: Fine-tuning the Spike: Role of the nature and topology of the glycan shield in the structure and dynamics of SARS-CoV-2 S The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer; a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shields topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that N-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, potentially increases host cell surface localization.

sentences: Neonate personality affects early-life resource acquisition in a large social mammal Current debate in the field of animal personality revolves around whether personality is reflecting individual differences in resource allocation or acquisition. Despite the large body of literature, the proximate relationships between personality, resource allocation, and acquisition are still unclear, especially during early stages of development. Here we studied how among-individual differences in behaviour develop over the first 6 months of life, and their potential association with resource acquisition in a free-ranging population of fallow deer (Dama dama). We related proxies of neonate personality - i.e. neonate physiological (heart rate) and behavioural (latency to leave at release) anti-predator responses to human handling - to the proportion of time fawns allocated to scanning during their first summer and autumn of life. We then investigated whether there was a trade-off between scanning time and foraging time in these juveniles, and how it developed over their first 6 months of life. We found that neonates with longer latencies at capture (i.e. risk-takers) allocated less time scanning their environment, but that this relationship was only present when fawns were 3-6 months old during autumn, but not when fawns were only 1-2 months old during summer. We also found that time spent scanning was negatively related to time spent foraging - a relationship rarely tested in juveniles of large mammals - and that this relationship becomes stronger over time, as fawns gradually switch from a nutrition rich (milk) to a nutrition poor (grass) diet. Our results highlight a potential mechanistic pathway in which neonate personality may drive differences in early-life resource acquisition, through allocation, of a large social mammal.

sentences: Oligomer-to-Monomer Transition Underlies the Chaperone Function of AAGAB in AP1/AP2 Assembly Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homotetramer. AAGAB tetramerization is mediated by its C-terminal domain, which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the tetramerization domain (TD), revealing a dimer of dimer assembly. Interestingly, AAGAB uses the same TD to recognize and stabilize the {gamma} subunit in the AP1 complex and the  subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of TD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

sentences: Potential geographic distribution and ecological niche of New World dobsonflies (Megaloptera, Corydalidae): the case of the Neartic-Neotropical transition zone The dobsonflies (Megaloptera, Corydalidae) are an interesting group of insects, but among the New World dobsonflies, it is not known the effect of the Neartic-Neotropical transition zone on their biogeographic distribution. Here we studied at the species level, the records on the geographic range of the dobsonflies that occurred in and near the transition zone. We presented potential geographic distributions based on ecological niche models for several species of dobsonflies. Results suggested that the geographic range of dobsonflies in the transition zone is associated to mountainous formations and that most species favor warm climates with higher precipitation rates. Climate types tend to be important for species that show narrow geographic ranges, but precipitation tends to be the most important variable to explain species dispersion. Overall, our models support the dispersion of dobsonflies from the Neotropics to North America and explain the two endemic species in Mexico as the result of the formation of the transition zone.

sentences: The dopamine dip amplitude is a quantitative measure of disappointment Great expectations can lead to greater disappointment when the desired results are not achieved. Although it is well-documented that a better-than-expected reward is encoded quantitatively via midbrain dopaminergic (DA) activity, it has been less addressed experimentally whether disappointment, a worse-than-expected outcome is also expressed quantitatively by the DA signal. We show that the degree of disappointment is quantified by the magnitude of a transient decrease in the extracellular DA concentration (DA dip) in the ventral striatum of mice. We set up a lever press task on a fixed ratio (FR) schedule requiring five lever presses as an effort for a food reward (FR5). Mice occasionally checked the food magazine without a reward before completing the task. The percentage of this premature magazine entry (PME) increased as the number of lever presses approached five. This behavioral readout shows rising expectations with increasing proximity to task completion, leading to greater disappointment in the mice. Fiber photometry of the extracellular DA dynamics in the ventral striatum using a fluorescent protein (genetically encoded GPCR-activation-based-DA sensor: GRABDA2m) revealed that the amplitude of the DA dip following a PME was correlated with the percentage of the PME, demonstrating a monotonic relationship between the DA dip amplitude and the degree of disappointment. Computational modeling of the lever press task implementing temporal difference errors and state transitions replicated the positive correlation between the PME frequency and DA dip amplitude during improvement of the FR5 task. Taken together, these findings indicate that the degree of disappointment while making an effort toward a goal is represented monotonically by the magnitude of the DA dip in the ventral striatum, which may guide behavioral adjustment.

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The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread, meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for development of novel therapeutic strategies. In this study, we have produced in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (herein termed " P2RX7B"), in OS progression and metastasis. Our data shows that P2RX7B expression confers a survival advantage in TE85+P2RX7B and MNNG-HOS+ P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, suggesting a probable metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS+P2RX7B tumours exhibited ectopic bone formation that was abrogated with A740003 treatment. An increased metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of the tumour to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. In conclusion, our data indicates for the first time a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a potential therapeutic target in human OS.

Novelty and ImpactWe provide evidence for the pro-tumorigenic role of the B isoform of the P2RX7 purinergic receptor in osteosarcoma (OS). In addition to increasing proliferation, P2RX7B increases the cancerous properties of OS cells, reducing adhesion and increasing migration and invasion. In vivo, P2RX7B does not affect primary tumour growth, but does lead to an increased propensity to metastasize. RNA-seq revealed a new axis of oncogenic genes inhibited by the P2RX7 antagonist and this data could potentially lead to new targets for OS treatment.

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KIBRA Regulates AMPA Receptor Expression, Synaptic Plasticity, and Memory in an Age-Dependent Manner The biological mechanisms supporting age-dependent changes in learning and memory remain elusive. While a growing body of human literature implicates KIBRA in memory and neurodevelopmental disorders, KIBRAs molecular function and contribution to maturation of synaptic function and cognition remain poorly understood. Despite being expressed throughout early postnatal development, germline deletion of KIBRA impairs synaptic plasticity selectively in adult rodents. However, it is unclear whether KIBRA facilitates proper brain maturation necessary for adult plasticity or whether it plays a distinct role in plasticity in the adult brain. Here, using an inducible KIBRA knockout mouse, we demonstrate that acutely deleting KIBRA in adult forebrain neurons impairs both spatial memory and long-term potentiation (LTP). The deficits in LTP correlate with an adult-selective decrease in extrasynaptic AMPA receptors under basal conditions. We also identify a novel role for KIBRA in LTP-induced AMPAR upregulation. In contrast, acute deletion of KIBRA in juvenile forebrain neurons did not affect LTP and had minimal effects on basal AMPAR expression. These data suggest that KIBRA serves a unique role in adult hippocampal function through regulation of basal and activity-dependent AMPAR proteostasis that supports synaptic plasticity.

Significance StatementSynaptic plasticity supported by trafficking of postsynaptic AMPA receptors is a conserved mechanism underlying learning and memory. The nature and efficacy of learning and memory undergo substantial changes during childhood and adolescent development, but the mechanisms underlying this cognitive maturation remain poorly understood. Here, we demonstrate that the human memory- and neurodevelopmental disorder-associated gene KIBRA facilitates memory and hippocampal synaptic plasticity selectively in the adult hippocampus. Furthermore, we show that selective loss of KIBRA from adult but not juvenile neurons reduces expression of extrasynaptic AMPA receptors and prevents LTP-induced increases in AMPAR expression. Overall, our results suggest that KIBRA participates in cellular and molecular processes that become uniquely necessary for memory and synaptic plasticity in early adulthood.

sentences: Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis, Paramecium bursaria Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote-eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria-Chlorella spp. Using comparative genomics and transcriptomics supported by phylogentics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA, and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We then validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down paradox controls to rescue the effect u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating use of the P. bursaria-Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

sentences: Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dyneins function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the clusters size of ionotropic glutamate receptor (iGluR), and an increase in the amplitude and frequency of mEJPs. However, PI(4,5)P2 levels do not affect iGluR clustering and dynein does not affect the protein levels of iGluR subunits at the NMJ, suggesting a separate, transport independent function for dynein in iGluR cluster organization. As dynein punctae closely associate with iGluR clusters, we propose that dynein physically tethers iGluR clusters at the postsynaptic membrane to ensure proper synaptic transmission.

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NDR1/2 kinases regulate membrane trafficking, enable efficient autophagy and prevent neurodegeneration Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.

HighlightsO_LIDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration.
C_LIO_LIPhosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates.
C_LIO_LIRaph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis.
C_LIO_LINdr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy.
C_LIO_LINDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A.
C_LI

sentences: High throughput screen in a co-culture model to uncover therapeutic strategies to potentiate the cancer-inhibiting properties of the tumor-stroma in pancreatic cancer Pancreatic stellate cells (PSCs) differentiate into multiple subtypes of cancer associated fibroblasts (CAFs) that modulate disease progression in pancreatic ductal adenocarcinoma (PDAC). CAF subtypes demonstrate functional heterogeneity in tumor development, with conflicting consequences for disease progression. Here, we show that myofibroblastic CAFs (myCAFs), but not inflammatory CAFs (iCAFs), can act to restrain tumor cell growth in an in vitro PDAC model of tumor organoids co-cultured with PSCs. Inhibiting myCAF formation by TGF-{beta} pathway inhibition improved tumor organoid growth, indicating that manipulating the balance of CAF subtypes may be exploited as a therapeutic approach. We therefore conducted a high throughput screen of approximately 36,000 compounds on the co-culture model to find novel compounds to inhibit PDAC tumor cell growth via CAF manipulation. We identify a new role for GNF-5, a known Abelson tyrosine kinase (Abl) inhibitor, in the context of PDAC as a compound that inhibits tumor cell growth in co-culture; an effect that was accompanied by an induction of the myCAF phenotype around tumor organoids. This highlights the therapeutic potential of novel therapies targeting specific CAF subtypes in PDAC.

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A Self-Replicating Radiation-Shield for Human Deep-Space Exploration: Radiotrophic Fungi can Attenuate Ionizing Radiation aboard the International Space Station The greatest hazard for humans on deep-space exploration missions is radiation. To protect astronauts venturing out beyond Earths protective magnetosphere, advanced passive radiation protection is highly sought after. In search of innovative radiation-shields, biotechnology appeals with suitability for in-situ resource utilization (ISRU), self-regeneration, and adaptability.

Certain fungi thrive in high-radiation environments on Earth, such as the contamination radius of the Chernobyl Nuclear Power Plant. Analogous to photosynthesis, these organisms appear to perform radiosynthesis, utilizing ionizing radiation to generate chemical energy. It has been postulated that the absorption of radiation is attributable to the pigment melanin. It is further hypothesized that this phenomenon translates to radiation-shielding properties.

Here, growth of Cladosporium sphaerospermum and its capability to attenuate ionizing radiation, was studied aboard the International Space Station (ISS) over a period of 26 days, as an analog to habitation on the surface of Mars. At full maturity, radiation beneath a {approx} 1.7 mm thick lawn of the dematiaceous radiotrophic fungus was approx. 0.84% lower as compared to the negative-control. In addition, a growth advantage in Space of [~] 21% was observed, substantiating the thesis that the fungus radiotropism is extendable to Space radiation.

sentences: Identifying drivers of spatio-temporal variation in survival in four blue tit populations In a context of rapid climate change, the influence of large-scale and local climate on population demography is increasingly scrutinized, yet studies are usually focused on one population. Demographic parameters, including survival, can vary significantly across populations of a given species, depending on global or local climatic fluctuations but also on many other population-specific parameters such as breeding density, habitat naturalness, predation or parasitism. Such ecological differences between populations could lead to different paces-of-life (POL), whereby populations where individuals display higher reproductive investment and bolder behaviours would have lower survival probabilities. We use here long-term (19 to 38 years) monitoring datasets from four Mediterranean populations of blue tits (Cyanistes caeruleus) to investigate the effects of sex, age class, large-scale and local climate temporal variation and population breeding density, on adult survival, using Capture-Mark-Recapture modelling. Environment heterogeneity in these four populations (two in evergreen and two in deciduous forests) has been linked to strong multi-trait phenotypic variation, suggesting blue tits in deciduous forests display faster POL compared to their conspecifics in evergreen habitats. The present results show heterogeneity in average survival probabilities across the four populations, with, as predicted, lower survival in the  fast blue tits occupying deciduous habitats. Interestingly, the year-to-year variation in survival probabilities was synchronous among populations. This suggests that regional environmental conditions could drive survival fluctuations across populations. However, breeding densities were not correlated across populations, and we found no evidence that adult survival is correlated with either large-scale or local, climate temporal variation in these four blue tit populations. Finally, two of the focal populations displayed a linear temporal decrease in adult survival over the study period, calling for further investigation to explain this decline. Overall, this multi-site study shows that blue tit parental survival from one spring to the next can vary substantially across years, in a synchronous way across populations, yet the climate indices we used are not correlated with the temporal variation. This calls for further investigations in other potential drivers such as resource (in particular insect) abundance, predation or parasitism.

sentences: Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land.

sentences: The impact of whole genome duplications on the human gene regulatory networks This work studies the effects of the two rounds of Whole Genome Duplication (WGD) at the origin of the vertebrate lineage on the architecture of the human gene regulatory networks. We integrate information on transcriptional regulation, miRNA regulation, and protein-protein interactions to comparatively analyse the role of WGD and Small Scale Duplications (SSD) in the structural properties of the resulting multilayer network. We show that complex network motifs, such as combinations of feed-forward loops and bifan arrays, deriving from WGD events are specifically enriched in the network. Pairs of WGD-derived proteins display a strong tendency to interact both with each other and with common partners and WGD-derived transcription factors play a prominent role in the retention of a strong regulatory redundancy. Combinatorial regulation and synergy between different regulatory layers are in general enhanced by duplication events, but the two types of duplications contribute in different ways. Overall, our findings suggest that the two WGD events played a substantial role in increasing the multi-layer complexity of the vertebrate regulatory network by enhancing its combinatorial organization, with potential consequences on its overall robustness and ability to perform high-level functions like signal integration and noise control.

sentences: Aging affects KV7 channels and perivascular-adipose tissue-mediated vascular tone Aging is an independent risk factor for hypertension, cardiovascular morbidity, and mortality. However, detailed mechanisms linking aging to cardiovascular disease are unclear. We studied the aging effects on the role of perivascular adipose tissue and downstream vasoconstriction targets, voltage-dependent KV7 channels, and their pharmacological modulators (flupirtine, retigabine, QO58, QO58-lysine) in a murine model. We assessed vascular function of young and old mesenteric arteries in vitro using wire myography. We also performed bulk RNA sequencing and quantitative reverse transcription polymerase chain reaction tests in mesenteric arteries and perivascular adipose tissue to elucidate molecular underpinnings of age-related phenotypes. Results revealed impaired perivascular adipose tissue-mediated control of vascular tone particularly via KV7.3-5 channels with increased age through metabolic and inflammatory processes and release of perivascular adipose tissue-derived relaxation factors. Moreover, QO58 was identified as novel pharmacological vasodilator to activate XE991-sensitive KCNQ channels in old mesenteric arteries. Our data suggest that targeting inflammation and metabolism in perivascular adipose tissue could represent novel approaches to restore vascular function during aging. Furthermore, QO58 represents a novel tool for cardiovascular and hypertension research in aging.

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Knockdown of Dnmt1 links Gene body DNA methylation to regulation of gene expression and maternal-zygotic transition in the wasp Nasonia Gene body methylation (GBM) is an ancestral aspect of DNA methylation (Sarda, Zeng, Hunt, & Yi, 2012; Yi, 2012; Zemach, McDaniel, Silva, & Zilberman, 2010) whose role in development has been obscured by the more prominent roles of promoter and CpG island methylation. The wasp Nasonia has little promoter and CpG island methylation, yet retains strong GBM (Park et al., 2011; Wang et al., 2013; Werren et al., 2010), making it an excellent model for elucidating the role of GBM. Here we show that Nasonia DNA methyl transferase 1a (Nv-Dnmt1a) knockdown leads to failures in cellularization and gastrulation of the embryo. Both of these disrupted events are hallmarks of the maternal-zygotic transition (MZT) in insects. Analysis of the embryonic transcriptome and methylome revealed strong reduction of GBM and widespread disruption of gene expression during embryogenesis after Nv-Dnmt1a knockdown. There was a strong correlation between loss of GBM and reduced gene expression in thousands of methylated loci, while affected unmethylated genes tended to be upregulated. We propose that reduced GBM and subsequent lower expression levels of methylated genes was the direct effect of Nv-Dnmt1 knockdown, and that this disruption led to widespread downstream dysregulation of MZT, and manifesting in developmental failure at gastrulation.

Significance StatementThe importance of gene-body methylation (GBM) in development is unclear, due to the difficulty in teasing apart the effects of cis-regulatory methylation from those of GBM in vertebrate model systems. Unlike vertebrate models, the methylation machinery in the jewel wasp Nasonia vitripennis appears to exclusively mediate GBM, thus simplifying interpretation of the role of GBM in development. Knockdown of DNMT1 (Nv-Dnmt1a) in Nasonia leads to embryonic lethality, which we show is caused by a failure of cellularization and gastrulation. Nv-Dnmt1a knockdown resulted in a global loss of GBM in the embryo, which was strongly correlated with a down-regulation of gene expression. We propose that GBM facilitated by Nv-Dnmt1a is required for proper zygotic genome activation in the wasp.

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Inner hair cell dysfunction in Klhl18 mutant mice leads to low frequency progressive hearing loss Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency hearing impairment in two mutant mouse lines, carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Auditory brainstem response (ABR) thresholds (a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies. Klhl18 mutant mice exhibit an uncommon low frequency hearing impairment with physiological features consistent with Auditory Neuropathy Spectrum Disorder (ANSD).

SIGNIFICANCE STATEMENTWe describe a novel progressive hearing loss in Klhl18 mutant mice that affects the lower frequencies of its hearing range. Investigation of two mutant alleles of this gene revealed primary inner hair cell defects affecting the neural output of the cochlea while outer hair cell function appeared normal. The tallest stereocilia of inner hair cells showed an abnormal tapering shape, especially notable in the apical half of the cochlear duct corresponding to the low frequency hearing loss. Our finding of a primary inner hair cell defect associated with raised thresholds for auditory brainstem responses combined with normal outer hair cell function suggests that Klhl18 deficiency and inner hair cell pathology may contribute to Auditory Neuropathy Spectrum Disorder in humans.

sentences: Structural identification of individual helical amyloid filaments by integration of cryo-electron microscopy-derived maps in comparative morphometric atomic force microscopy image analysis The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297-391), termed  dGAE. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are closely related structurally to the paired helical filaments (PHFs) isolated from Alzheimers disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.

sentences: Central nervous system infusion of TrkB agonist, 7,8-dihydroxyflavone, is ineffective in promoting myelin repair in cuprizone and experimental autoimmune encephalomyelitis mouse models of multiple sclerosis Small molecular weight functional mimetics of brain-derived neurotrophic factor (BDNF) which act via the TrkB receptor have been developed to overcome the pharmacokinetic limitations of BDNF as a therapeutic agent for neurological disease. Activation of TrkB signalling on oligodendrocytes has been identified as a potential strategy for promoting myelin repair in demyelinating conditions such as Multiple Sclerosis (MS). Here, we tested the efficacy of intracerebroventricular infusion of TrkB agonist 7,8-dihydroxyflavone (DHF) to promote myelin repair in the cuprizone model of de- and remyelination and alter the course of experimental autoimmune encephalomyelitis (EAE), after the onset of clinical signs. In these two distinct, but common mouse models used for the preclinical testing of MS therapeutics, we found that DHF infusion increased the percentage of myelin basic protein and density of oligodendrocyte progenitor cells (OPCs) in the corpus callosum of female C57BL/6 mice after cuprizone demyelination. However, DHF did not alter the percentage of axons myelinated or increase the density of post-mitotic oligodendrocytes in this model. Direct central nervous system infusion of DHF infusion also had no effect on the clinical course of EAE in male and female C57BL/6 mice, and examination of the lumbar spinal cord after 21 days of treatment revealed extensive demyelination, with active phagocytosis of myelin debris by Iba1+ macrophages/microglia. These results indicate that direct central nervous system infusion of DHF is ineffective at promoting myelin repair in toxin-induced and inflammatory models of demyelination.

sentences: A drastic shift in the energetic landscape of toothed whale sperm cells Mammalia spermatozoa are a notable example of energetic compartmentalization. While mitochondrial oxidative phosphorylation is restricted to the midpiece, sperm-specific glycolysis operates in the flagellum. Consequently, these highly specialized cells exhibit a clear adaptability to fuel substrates. This plasticity is essential to ensure sperm motility, and is known to vary among species. Here we describe an extreme example of spermatozoa-energetics adaptation. We show that toothed whales exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that dolphin spermatozoa motility depends uniquely on endogenous fatty acid {beta}-oxidation, but not carbohydrates. Our findings substantiate the observation of large mitochondria in spermatozoa, possibly boosting ATP production from endogenous fatty acids. This unique energetic rewiring emphasizes the physiological body reorganisation imposed by the carbohydrate-depleted marine environment.

sentences: A convergent structure-function substrate of cognitive imbalances in autism Autism is a common neurodevelopmental condition characterized by substantial phenotypic heterogeneity, which hinders diagnosis, research, and intervention. A leading example can be found in marked imbalances in language and perceptual skills, where deficits in one domain often co-exist with normal or even superior performance in the other domain. The current work capitalized on multiple data analytics including data-driven subtyping and dimensional approaches to quantify cognitive imbalances in multi-site datasets of individuals diagnosed with autism spectrum disorder (ASD) and neurotypical controls, and assessed structural and functional brain network substrates. Studying cognitive dimensions as well as multimodal neuroimaging signatures in 155 ASD and 151 neurotypical individuals, we observed robust evidence for a structure-function substrate of cognitive imbalances in ASD. Specifically, ASD presented with marked imbalances in cognitive profiles relative to neurotypical controls, characterized by verbal to non-verbal intelligence discrepancy. Different analytical approaches including subtyping and dimensional regression methods converged in showing that these imbalances were reflected in atypical cortical thickening and functional integration of language networks, alongside with sensory and higher cognitive networks. Phenotypic findings could be replicated in an independent sample of 325 ASD and 569 neurotypical controls. Although verbal and non-verbal intelligence are currently considered as specifiers unrelated to the categorical diagnosis of autism, our results show that intelligence disparities are accentuated in ASD and relate to a consistent structure-function substrate affecting multiple brain networks. Our findings motivate the incorporation of cognitive imbalances in future autism research, which may help to parse the phenotypic heterogeneity of autism and potentially inform intervention-oriented subtyping.

sentences: Leveraging omic features with F3UTER enables identification of unannotated 3'UTRs for synaptic genes There is growing evidence for the importance of 3 untranslated region (3UTR) dependent regulatory processes. However, our current human 3UTR catalogue is incomplete. Here, we developed a machine learning-based framework, leveraging both genomic and tissue-specific transcriptomic features to predict previously unannotated 3UTRs. We identify unannotated 3UTRs associated with 1,513 genes across 39 human tissues, with the greatest abundance found in brain. These unannotated 3UTRs were significantly enriched for RNA binding protein (RBP) motifs and exhibited high human lineage-specificity. We found that brain-specific unannotated 3UTRs were enriched for the binding motifs of important neuronal RBPs such as TARDBP and RBFOX1, and their associated genes were involved in synaptic function and brain- related disorders. Our data is shared through an online resource F3UTER (https://astx.shinyapps.io/F3UTER/). Overall, our data improves 3UTR annotation and provides novel insights into the mRNA-RBP interactome in the human brain, with implications for our understanding of neurological and neurodevelopmental diseases.

sentences: Powering electronic implants by high frequency volume conduction: in human validation Wireless power transfer (WPT) is frequently used as an alternative to batteries to accomplish miniaturization in electronic medical implants (eMIs). However, established WPT methods require bulky parts within the implant or cumbersome external systems, hindering minimally invasive deployments and the development of networks of eMIs. As an alternative, we propose a WPT approach based on volume conduction of high frequency (HF) current bursts. These currents are applied through external textile electrodes and are collected by the eMIs through two electrodes at their opposite ends. This approach avoids bulky components to obtain power, making it possible to develop implants with a flexible threadlike conformation. In here we study in humans if HF (6.78 MHz) current bursts complying with safety standards and applied through two textile electrodes strapped around a limb can provide substantial powers from pairs of implanted electrodes. Time averaged electric powers obtained from needle electrodes (diameter = 0.4 mm, length = 3 mm, separation = 30 mm) inserted into arms and lower legs of five healthy participants were 5.9 {+/-} 0.7 mW and 2.4 {+/-} 0.3 mW respectively. We also report a procedure to characterize the coupling between the external system and the implants using two-port impedance models generated from medical images. The results demonstrate for the first time in humans that innocuous and imperceptible HF current bursts that flow through the tissues by volume conduction can be used to wirelessly power threadlike eMIs, overcoming the limitations of existing WPT methods in terms of minimal invasiveness and usability.

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The SAGA core module is critical during Drosophila oogenesis and is broadly recruited to promoters The Spt/Ada-Gcn5 Acetyltransferase (SAGA) coactivator complex has multiple modules with different enzymatic and non-enzymatic functions. How each module contributes to gene activation in specific biological contexts is not well understood. Here we analyzed the role of the non-enzymatic core module during Drosophila oogenesis. We show that depletion of several SAGA-specific subunits belonging to the core module blocked egg chamber development during mid-oogenesis stages, resulting in stronger phenotypes than those obtained after depletion of SAGAs histone acetyltransferase module or deubiquitination module. These results, as well as additional genetic analyses pointing to an interaction with TBP, suggested a differential role of SAGA modules at different promoter types. However, SAGA subunits co-occupied all promoter types of active genes in ChIP-seq and ChIP-nexus experiments. Thus, the SAGA complex appears to occupy promoters in its entirety, consistent with the strong biochemical integrity of the complex. The high-resolution genomic binding profiles are congruent with SAGA recruitment by activators upstream of the start site, and retention on chromatin by interactions with modified histones downstream of the start site. The stronger genetic requirement of the core module during oogenesis may therefore be explained through its interaction with TBP or its role in recruiting the enzymatic modules to the promoter. We propose the handyman principle, which posits that a distinct genetic requirement for specific components may conceal the fact that the entire complex is physically present.

Author SummaryEmbryonic development critically relies on the differential expression of genes in different tissues. This involves the dynamic interplay between DNA, sequence-specific transcription factors, coactivators and chromatin remodelers which guide the transcription machinery to the appropriate promoters for productive transcription. To understand how this happens at the molecular level, we need to understand when and how coactivator complexes such as SAGA function. SAGA consists of multiple modules with well characterized enzymatic functions. This study shows that the non-enzymatic core module of SAGA is required for Drosophila oogenesis, while the enzymatic functions are largely dispensable. Despite this differential requirement, SAGA subunits appear to be broadly recruited to all promoter types, consistent with the biochemical integrity of the complex. These results suggest that genetic requirements and physical organization do not always go hand in hand.

sentences: Genetic analysis of the contribution of the proprotein convertases to the in vivo regulation of FGF23 Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179{downarrow}, but whether they are fulfilling this function in vivo is currently unknown. To address this question we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1{beta} injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

sentences: Fine-scale spatial genetic structure across the species range reflects recent colonization of high elevation habitats in silver fir (Abies alba Mill.) Variation in genetic diversity across species ranges has long been recognized as highly informative for assessing populations resilience and adaptive potential. The spatial distribution of genetic diversity, referred to as fine-scale spatial genetic structure (FSGS), also carries information about recent demographic changes, yet it has rarely been connected to range scale processes. We studied eight silver fir (Abies alba Mill.) population pairs (sites), growing at high and low elevations, representative of the main genetic lineages of the species. A total of 1368 adult trees and 540 seedlings were genotyped using 137 and 116 single nucleotide polymorphisms (SNPs), respectively. Sites revealed a clear east-west isolation-by-distance pattern consistent with the post-glacial colonization history of the species. Genetic differentiation among sites (FCT=0.148) was an order of magnitude greater than between elevations within sites (FSC=0.031), nevertheless high elevation populations consistently exhibited a stronger FSGS. Structural equation modeling revealed that elevation and, to a lesser extent, post-glacial colonization history, but not climatic and habitat variables, were the best predictors of FGSG across populations. These results may suggest that high elevation habitats have been colonized more recently across the species range. Additionally, paternity analysis revealed a high reproductive skew among adults and a stronger FSGS in seedlings than in adults, suggesting that FSGS may conserve the signature of demographic changes for several generations. Our results emphasize that spatial patterns of genetic diversity within populations provide complementary information about demographic history and could be used for defining conservation priorities.

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miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections A liver-specific microRNA, miR-122, anneals to the HCV genomic 5 terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5 UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division.

IMPORTANCEThe mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as potential HCV therapy.

sentences: SUMOylation of ABCD3 restricts bile acid synthesis and regulates metabolic homeostasis Mitochondrial anchored protein ligase (MAPL) has been shown to function as both a SUMO and ubiquitin ligase with multiple roles in mitochondrial quality control, cell death pathways and inflammation. To examine the global function of MAPL we generated a knock-out mouse model and sought functional insight through unbiased BioID, transcriptomics and metabolic analysis. MAPL KO mice are lean and highly insulin sensitive, ultimately developing fully penetrant, spontaneous hepatocellular carcinoma after 18 months. BioID revealed the peroxisomal bile acid transporter ABCD3 as a primary MAPL interacting partner, which we show is SUMOylated in a MAPL-dependent manner. MAPL KO animals showed increased bile acid secretion in vivo and in isolated primary hepatocytes, along with robust compensatory changes in the expression of enzymes synthesizing and detoxifying bile acid. In addition, MAPL KO livers showed signs of ER stress and secreted high levels of Fgf21, the starvation hormone known to drive the reduction of white fat stores and promote insulin sensitivity. Lastly, during aging all MAPL KO mice developed hepatocellular carcinomas. These data reveal a major function for MAPL in the regulation of bile acid synthesis leading to profound changes in whole body metabolism and the ultimate generation of liver cancer when MAPL is lost.

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Gait-phase modulates alpha and beta oscillations in the pedunculopontine nucleus BackgroudThe pedunculopontine nucleus (PPN) is a reticular collection of neurons at the junction of the midbrain and pons, playing an important role in modulating posture and locomotion. Deep brain stimulation of the PPN has been proposed as an emerging treatment for patients with Parkinsons disease (PD) or multiple system atrophy (MSA) suffering gait-related atypical parkinsonian syndromes.

ObjectiveIn this study, we investigated PPN activities during gait to better understand its functional role in locomotion. Specifically, we investigated whether PPN activity is rhythmically modulated during locomotion.

MethodsPPN local field potential (LFP) activities were recorded from PD or MSA patients suffering from gait difficulties during stepping in place or free walking. Simultaneous measurements from force plates or accelerometers were used to determine the phase within each gait cycle at each time point.

ResultsOur results showed that activities in the alpha and beta frequency bands in the PPN LFPs were rhythmically modulated by the gait phase within gait cycles, with a higher modulation index when the stepping rhythm was more regular. Meanwhile, the PPN-cortical coherence was most prominent in the alpha band. Both gait-phase related modulation in the alpha/beta power and the PPN-cortical coherence in the alpha frequency band were spatially specific to the PPN and did not extend to surrounding regions.

ConclusionsThese results raise the possibility that alternating PPN stimulation in tandem with the gait rhythm may be more beneficial for gait control than continuous stimulation, although this remains to be established in future studies.

sentences: Signal processing capacity of the cellular sensory machinery regulates the accuracy of chemotaxis under complex cues Chemotaxis is ubiquitous in many biological processes, but it still remains elusive how cells sense and decipher multiple chemical cues. In this study, we postulate a hypothesis that the chemotactic performance of cells under complex cues is regulated by the signal processing capacity of the cellular sensory machinery. The underlying rationale is that cells in vivo should be able to sense and process multiple chemical cues, whose magnitude and compositions are entangled, to determine their migration direction. We experimentally show that the combination of TGF-{beta} and EGF suppresses the chemotactic performance of cancer cells using independent receptors to sense the two cues. Based on this observation, we develop a biophysical framework suggesting that the antagonism is caused by the saturation of the signal processing capacity, but not by the mutual repression. Our framework suggests the significance of the signal processing capacity in the cellular sensory machinery.

sentences: Overexpression of UCP4 in astrocytic mitochondria prevents multilevel dysfunctions in a mouse model of Alzheimer s disease Alzheimers disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenge as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxydative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.

sentences: LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the activity of the molecular motor cytoplasmic dynein. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it regulates the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the expression of extracellular matrix (ECM) and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post- transcriptional regulation underlying development and mechanosensitive processes.

sentences: γδ T cells are effectors of immune checkpoint blockade in mismatch repair-deficient colon cancers with antigen presentation defects DNA mismatch repair deficient (MMR-d) cancers present an abundance of neoantigens that likely underlies their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. However, MMR-d colon cancers that evade CD8+ T cells through loss of Human Leukocyte Antigen (HLA) class I-mediated antigen presentation3-6, frequently remain responsive to ICB7 suggesting the involvement of other immune effector cells. Here, we demonstrate that HLA class I-negative MMR-d cancers are highly infiltrated by {gamma}{delta} T cells. These {gamma}{delta} T cells are mainly composed of V{delta}1 and V{delta}3 subsets, and express high levels of PD-1, activation markers including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors (KIRs). In vitro, PD-1+ {gamma}{delta} T cells, isolated from MMR-d colon cancers, exhibited a cytolytic response towards HLA class I-negative MMR-d colon cancer cell lines and {beta}2-microglobulin (B2M)-knockout patient-derived tumor organoids (PDTOs), which was enhanced as compared to antigen presentation-proficient cells. This response was diminished after blocking the interaction between NKG2D and its ligands. By comparing paired tumor samples of MMR-d colorectal cancer patients obtained before and after dual PD-1 and CTLA-4 blockade, we found that ICB profoundly increased the intratumoral frequency of {gamma}{delta} T cells in HLA class I-negative cancers. Taken together, these data indicate that {gamma}{delta} T cells contribute to the response to ICB therapy in patients with HLA class I-negative, MMR-d colon cancers, and illustrate the potential of {gamma}{delta} T cells in cancer immunotherapy.

sentences: Massively parallel identification of zipcodes in primary cortical neurons Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

sentences: Dopamine depletion can be predicted by the aperiodic component of subthalamic local field potentials Electrophysiological biomarkers reflecting the pathological activities in the basal ganglia are essential to gain an etiological understanding of Parkinsons disease (PD) and develop a method of diagnosing and treating the disease. Previous studies that explored electrophysiological biomarkers in PD have focused mainly on oscillatory or periodic activities such as beta and gamma oscillations. Emerging evidence has suggested that the nonoscillatory, aperiodic component reflects the firing rate and synaptic current changes corresponding to cognitive and pathological states. Nevertheless, it has never been thoroughly examined whether the aperiodic component can be used as a biomarker that reflects pathological activities in the basal ganglia in PD. In this study, we examined the parameters of the aperiodic component and tested its practicality as an electrophysiological biomarker of pathological activity in PD. We found that a set of aperiodic parameters, aperiodic offset and exponent, were significantly decreased by the nigrostriatal lesion. To further prove the usefulness of the parameters as biomarkers, acute levodopa treatment reverted the aperiodic offset. We then compared the aperiodic parameters with a previously established periodic biomarker of PD, beta frequency oscillation. We found a significantly low negative correlation with beta power. We showed that the performance of the machine learning-based prediction of pathological activities in the basal ganglia can be improved by using the lowly correlated parameters, beta power and aperiodic component. We suggest that the aperiodic component will provide a more sensitive measurement to early diagnosis PD and have the potential to use as the feedback parameter for the adaptive deep brain stimulation.

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Chemically-induced epimutagenesis allows bypassing reproductive barriers in hybrid seeds The "triploid block" prevents interploidy hybridizations in flowering plants, and is characterized by failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed imprinted genes (PEGs) that are up-regulated in the tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We have identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of "epimutagenized" plants, which may allow the identification of epialleles involved in the triploid block response in future studies. Finally, we demonstrate that chemically-induced epimutagenesis allows bypassing hybridization barriers in crosses between different Capsella species, thus potentially emerging as a novel strategy for producing triploids and interspecific hybrids with high agronomical interest.

One sentence summaryGenome-wide loss of DNA methylation induced by 5-Azacytidine allows bypassing interploidy and interspecific hybridization barriers in Arabidopsis and Capsella.

sentences: The Effect of A1/A2 Reactive Astrocyte Expression on Hydrocephalus Shunt Failure Understanding the composition characteristics of the glial scar contributing to the high failure rate of neuroprosthetic devices implanted in the brain has been limited, to date, with the evaluation of cells, tissue, and biomarkers obstructing the implant. However, there remains a critical knowledge gap in gene expression profiles of the obstructing cells. This first-time study investigates the phenotypic expression specific to astrocyte scarring from those cells on hydrocephalus shunt surfaces at the time of failure, aimed at the development of therapeutic approaches to target reactive astrocytes for improved functional outcome. Recent evidence has indicated that the tissue obstructing shunts is over 80% inflammatory, with a more exaggerated astrocytic response. To understand how to mitigate the astrocyte immune response to shunts, we performed gene expression profiling of the C3 and EMP1 genes to quantify if astrocytes were classically activated and pro-inflammatory (A1) or alternatively activated and anti-inflammatory (A2), respectively. Shunt catheters were removed from patients at the time of failure and categorized by obstructed vs non-obstructed shunts. RNAscope fluorescent in situ hybridization and quantitative PCR analysis of the C3 and EMP1 expressed genes revealed that a heterogeneous mixed population of both the A1 and A2 reactive phenotype exist on the shunt surface. However, the number of A2 reactive astrocytes are significantly higher on obstructed shunts compared to A1 reactive astrocytes. ELISA data also confirmed higher levels of IL-6 for obstructed shunts involved in A2 reactive astrocyte proliferation and glial scar formation on the shunt surface. Since TNF- and IL-1{beta} propel resting astrocytes into an A2 reactive state, by simply blocking the secretion or action of these cytokines, astrocyte activation and attachment on obstructing shunts could be inhibited.

sentences: Genotype error biases trio-based estimates of haplotype phase accuracy Haplotypes can be estimated from unphased genotype data using statistical methods. When parent-offspring data are available for inferring true phase from Mendelian inheritance rules, the accuracy of statistical phasing is usually measured by the switch error rate, which is the proportion of pairs of consecutive heterozygotes that are incorrectly phased. We present a method for estimating the genotype error rate from parent-offspring trios and a method for estimating the bias in the observed switch error rate that is caused by genotype error. We apply these methods to 485,301 genotyped UK Biobank samples that include 899 White British trios and to 38,387 sequenced TOPMed samples that include 217 African Caribbean trios and 669 European American trios. We show that genotype error inflates the observed switch error rate and that the relative bias increases with sample size. For the UK Biobank White British trios, we estimate that the observed switch error rate in the trio offspring is 2.4 times larger than the true switch error rate (1.41 x 10-3 vs 5.79 x 10-4) and that the average distance between phase errors is 64 megabases.

sentences: Pyroptosis-associated molecular classification and prognostic signature in glioma Integrative analysis was performed in the Chinese Glioma Genome Atlas and The Cancer Genome Atlas to describe the pyroptosis-associated molecular classification and prognostic signature in glioma. Pyroptosis-related genes were used for consensus clustering and to develop a prognostic signature. The immune statuses, molecular alterations and clinical features of differentially expressed genes were analyzed among different subclasses and risk groups. A lncRNA-miRNA-mRNA network was built, and drug sensitivity analysis was used to identify small molecular drugs for the identified genes. Glioma can be divided into two subclasses using 30 pyroptosis-related genes. Cluster 1 displayed high immune signatures and poor prognosis as well as high immune-related function scores. A prognostic signature based on 15 pyroptosis-related genes of the CGGA cohort can predict the overall survival of glioma and was well validated in the TCGA cohort. Cluster 1 had higher risk scores. The high-risk group had high immune cell and function scores and low DNA methylation of pyroptosis-related genes. The differences in pyroptosis-related gene mutations and somatic copy numbers were significant between the high-risk and low-risk groups. The ceRNA regulatory network uncovered the regulatory patterns of different risk groups in glioma. Nine pairs of target genes and drugs were identified. In vitro, CASP8 promotes the progression of glioma cells. Pyroptosis-related genes can reflect the molecular biological and clinical features of glioma subclasses. The established prognostic signature can predict prognosis and distinguish molecular alterations in glioma patients. Our comprehensive analyses provide valuable guidelines for improving glioma patient management and individualized therapy.

sentences: SCRaPL: hierarchical Bayesian modelling of associations in single cell multi-omics data Single-cell multi-omics assays offer unprecedented opportunities to explore gene regulation at cellular level. However, high levels of technical noise and data sparsity frequently lead to a lack of statistical power in correlative analyses, identifying very few, if any, significant associations between different molecular layers. Here we propose SCRaPL, a novel computational tool that increases power by carefully modelling noise in the experimental systems. We show on real and simulated multi-omics single-cell data sets that SCRaPL achieves higher sensitivity and better robustness in identifying correlations, while maintaining a similar level of false positives as standard analyses based on Pearson correlation.

sentences: A Role for Visual Areas in Physics Simulations To engage with the world, we must regularly make predictions about the outcomes of physical scenes. How do we make these predictions? Recent evidence points to simulation - the idea that we can introspectively manipulate rich, mental models of the world - as one possible explanation for how such predictions are accomplished. While theories based on simulation are supported by computational models, neuroscientific evidence for simulation is lacking and many important questions remain. For instance, do simulations simply entail a series of abstract computations? Or are they supported by sensory representations of the objects that comprise the scene being simulated? We posit the latter and suggest that the process of simulating a sequence of physical interactions is likely to evoke an imagery-like envisioning of those interactions. Using functional magnetic resonance imaging, we demonstrate that when participants predict how a ball will fall through an obstacle-filled display, motion-sensitive brain regions are activated. We further demonstrate that this activity, which occurs even though no motion is being sensed, resembles activity patterns that arise while participants perceive the balls motion. This finding suggests that the process of simulating the balls movement is accompanied by a sensory representation of this movement. These data thus demonstrate that mental simulations recreate sensory depictions of how a physical scene is likely to unfold.

sentences: Structural snapshots of V/A-ATPase reveal a new paradigm for rotary catalysis V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 moiety rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here we have determined the atomic models of 18 catalytic intermediates of the V1 moiety of V/A-ATPase under different reaction conditions by single particle Cryo-EM, which revealed that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120{degrees} rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

sentences: MaxQuant and MSstats in Galaxy enable reproducible cloud-based analysis of quantitative proteomics experiments for everyone Quantitative mass spectrometry-based proteomics has become a high-throughput technology for the identification and quantification of thousands of proteins in complex biological samples. Two de facto standard tools, MaxQuant and MSstats, allow for the analysis of raw data and finding proteins with differential abundance between conditions of interest. To enable accessible and reproducible quantitative proteomics analyses in a cloud environment, we have integrated MaxQuant (including TMTpro 16/18plex), Proteomics Quality Control (PTXQC), MSstats and MSstatsTMT into the open-source Galaxy framework. This enables the web-based analysis of label-free and isobaric labeling proteomics experiments via Galaxys graphical user interface on public clouds. MaxQuant and MSstats in Galaxy can be applied in conjunction with thousands of existing Galaxy tools and integrated into standardized, sharable workflows. Galaxy tracks all metadata and intermediate results in analysis histories, which can be shared privately for collaborations or publicly, allowing full reproducibility and transparency of published analysis. To further increase accessibility, we provide detailed hands-on training materials. The integration of MaxQuant and MSstats into the Galaxy framework enables their usage in a reproducible way on accessible large computational infrastructures, hence realizing the foundation for high throughput proteomics data science for everyone.

sentences: Accurate sequence-dependent coarse-grained model for conformational and elastic properties of double-stranded DNA We introduce MADna, a sequence-dependent coarse-grained model of double-stranded DNA (dsDNA), where each nucleotide is described by three beads localized at the sugar and base moieties, and at the phosphate group. The sequence dependence is included by considering a step-dependent parameterization of the bonded interactions, which are tuned in order to reproduce the values of key observables obtained from exhaustive atomistic simulations from literature. The predictions of the model are benchmarked against an independent set of all-atom simulations, showing that it captures with high fidelity the sequence dependence of conformational and elastic features beyond the single step considered in its formulation. A remarkably good agreement with experiments is found for both sequence-averaged and sequence-dependent conformational and elastic features, including the stretching and torsion moduli, the twist-stretch and twist-bend couplings, the persistence length and the helical pitch. Overall, for the inspected quantities, the model has a precision comparable to atomistic simulations, hence providing a reliable coarse-grained description for the rationalization of singlemolecule experiments and the study of cellular processes involving dsDNA. Owing to the simplicity of its formulation, MADna can be straightforwardly included in common simulation engines.

sentences: Single-cell ATAC and RNA sequencing reveal pre-existing and persistent subpopulations of cells associated with relapse of prostate cancer Prostate cancer is profoundly heterogeneous and patients would benefit from methods that stratify clinically indolent from more aggressive forms of the disease. We employed single-cell assay for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identified pre-existing and treatment-persistent cell subpopulations that possess transcriptional stem-like features and regenerative potential when subjected to treatment. We found distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent stem-like cells were able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal hitherto unrecognized molecular predictors of treatment response. This suggests that high analytical resolution of pre-clinical models may powerfully inform clinical decision-making.

sentences: Negative regulation of IMD contributes to disease tolerance during systemic bacterial infection in Drosophila Disease tolerance is an infection phenotype where hosts show relatively high health despite harbouring elevated pathogen loads. Compared to the mechanisms of immune clearance our knowledge of the mechanisms underlying increased tolerance remains incomplete. Variation in the ability to reduce immunopathology may explain why some hosts can tolerate higher pathogen burdens with reduced pathology. Negative immune regulation would therefore appear to be a clear candidate for a mechanism underlying disease tolerance but this has not been tested directly for bacterial infections. Here, we examined how the negative regulation of the immune deficiency (IMD) pathway affects disease tolerance in Drosophila melanogaster when infected with the gram-negative bacterial pathogen Pseudomonas entomophila. We find that UASRNAi-mediated reduced expression of the negative regulators of IMD (pirk and caudal) severely reduced the ability to tolerate infection in both males and females across a wide range of infectious doses. While flies unable to regulate the IMD response exhibited higher expression of antimicrobial peptides and lower bacterial loads as expected, this was not accompanied by a proportional reduction in mortality. Instead, tolerance (measured as fly survival relative to its microbe load) was drastically reduced, likely due to the combination of increased immunopathology and cytotoxicity of elevated AMP expression. Our results therefore highlight that in addition to regulating an efficient pathogen clearance response, negative regulators of IMD also contribute to disease tolerance.

sentences: Brain dysfunction during warming is caused by oxygen limitation in larval zebrafish Understanding the physiological mechanisms that limit animal thermal tolerance is crucial in predicting how animals will respond to increasingly severe heatwaves. Despite its importance for understanding climate change impacts, these mechanisms underlying the upper thermal tolerance limits of animals are largely unknown. It has been hypothesised that the upper thermal tolerance in fish is limited by the thermal tolerance of the brain and that it is ultimately caused by a global brain depolarization. In this study, we developed methods for measuring the upper thermal limit (CTmax) in larval zebrafish (Danio rerio) with simultaneous recordings of brain activity using GCaMP6s calcium imaging in both free-swimming and agar-embedded fish. We discovered that during warming, CTmax precedes, and is therefore not caused by, a global brain depolarization. Instead, the CTmax coincides with a decline in spontaneous neural activity and a loss of neural response to visual stimuli. By manipulating water oxygen levels, we found that oxygen availability during heating affects both locomotor-related neural activity, the neural response to visual stimuli, and CTmax. Our results suggest that the mechanism limiting the upper thermal tolerance in zebrafish larvae is reduced oxygen availability causing impaired brain function.

sentences: Alterations in human gut microbiome composition and metabolism after exposure to glyphosate and Roundup and/or a spore-based formulation using the SHIME(R) technology Despite extensive research into the toxicology of the herbicide glyphosate, there are still major unknowns regarding its effects on the human gut microbiome. As a step in addressing this knowledge gap, we describe for the first time the effects of glyphosate and a Roundup glyphosate-based herbicide on infant gut microbiota using SHIME technology, which mimics the entire gastrointestinal tract. SHIME(R) microbiota culture was undertaken in the presence of a concentration of 100 mg/L (corresponding to a dose of 1.6 mg/kg/day) glyphosate and the same glyphosate equivalent concentration of Roundup, which is in the range of the US chronic reference dose, and subjected to molecular profiling techniques to assess outcomes. Roundup and to a lesser extent glyphosate caused an increase in fermentation activity, resulting in acidification of the microbial environment. This was also reflected by an increase in lactate and acetate production concomitant to a decrease in the levels of propionate, valerate, caproate and butyrate. Ammonium production reflecting proteolytic activities was increased by Roundup exposure. Global metabolomics revealed large scale disturbances in metabolite profiles, including an increased abundance of long chain polyunsaturated fatty acids (n3 and n6). Although changes in bacterial composition measured by qPCR and 16S rRNA sequencing were less clear, our results suggested that lactobacilli had their growth stimulated as a result of microenvironment acidification. Co-treatment with the spore-based probiotic formulation MegaSporeBiotic reverted some of the changes in short-chain fatty acid levels. Altogether, our results suggest that glyphosate can exert effects on human gut microbiota at permitted regulatory levels of exposure, highlighting the need for epidemiological studies aimed at evaluating the effects of glyphosate herbicides on human gut microbiome function.

sentences: Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

sentences: Trait plasticity and covariance along a continuous soil moisture gradient Water availability is perhaps the greatest environmental determinant of plant yield and fitness. However, our understanding of plant-water relations is limited because it is primarily informed by experiments considering soil moisture variability at two discrete levels - wet and dry - rather than as a continuously varying environmental gradient. Here we used experimental and statistical methods based on function-valued traits to explore responses to a continuous soil moisture gradient in physiological and morphological traits in two species and five genotypes each of the model grass Brachypodium. We find that most traits exhibit non-linear responses to soil moisture variability. We also observe differences in the shape of these non-linear responses between traits, species, and genotypes. Emergent phenomena arise from this variation including changes in trait correlations and evolutionary constraints as a function of soil moisture. These results point to the importance of considering non-linearity in plant-water relations to understand plastic and evolutionary responses to changing climates.

sentences: Building regulatory landscapes: enhancer recruits cohesin to create contact domains, engage CTCF sites and activate distant genes Developmental gene expression is often controlled by distal tissue-specific enhancers. Enhancer action is restricted to topological chromatin domains, typically formed by cohesin-mediated loop extrusion between CTCF-associated boundaries. To better understand how individual regulatory DNA elements form topological domains and control expression, we used a bottom-up approach, building active regulatory landscapes of different sizes in inactive chromatin. We demonstrate that transcriptional output and protection against gene silencing reduces with increased enhancer distance, but that enhancer contact frequencies alone do not dictate transcription activity. The enhancer recruits cohesin to stimulate the formation of local chromatin contact domains and activate flanking CTCF sites for engagement in chromatin looping. Small contact domains can support strong and stable expression of distant genes. The enhancer requires transcription factors and mediator to activate genes over all distance ranges, but relies on cohesin exclusively for the activation of distant genes. Our work supports a model that assigns two functions to enhancers: its classic role to stimulate transcription initiation and elongation from target gene promoters and a role to recruit cohesin for the creation of contact domains, the engagement of flanking CTCF sites in chromatin looping, and the activation of distal target genes.

sentences: Immunological findings in a group of individuals who were non-responders to standard two-dose SARS-CoV-2 vaccines Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic. The virus has infected more than 505 million people and caused more than 6 million deaths. However, data on non-responders to SARS-CoV-2 vaccines in the general population are limited. The objective of the study is to comprehensively compare the immunological characteristics of non-responders to SARS-CoV-2 vaccines in the 18-59 years with that in the 60 years and older using internationally recognized cutoff values. Participants included 627 individuals who received physical examinations and volunteered to participate in COVID-19 vaccination from the general population. The main outcome was an effective seroconversion characterized by anti-SARS-CoV-2 spike IgG level of at least 4-fold increase from baseline. Profiling of naive immune cells was analyzed prior to vaccination to demonstrate baseline immunity. Outcomes of effective seroconversion in the 18-59 years with that in the 60 years and older were compared. The quantitative level of the anti-spike IgG was significantly lower in the 60 years and older and in men among the 18-59 years. There were 7.5% of non-responders among the 18-59 years and 11.7% of non-responders in the 60 years and older using the 4-fold increase parameter. The effective seroconversion rate was significantly related to the level of certain immune cells before vaccination, such as CD4 cells, CD8 cells and B cells and the age. An individual with a titer of anti-SARS-CoV-2 spike IgG that is below 50 BAU/mL might be considered a non-responder between 14-90 days after the last vaccine dose. Booster vaccination or additional protective measures should be recommended for non-responders as soon as possible to reduce disease severity and mortality.

sentences: Genome sequencing of the bacteriophage CL31 and interaction with the host strain 	Corynebacterium glutamicum ATCC 13032 In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4 %). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage {Phi}16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the {Delta}resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

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Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms.

Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

sentences: Mapping vegetation types in Antarctic Peninsula and South Shetlands islands using Sentinel-2 images and Google Earth Engine cloud computing The Antarctic vegetation maps are usually made using very high-resolution images collected by orbital sensors or unmanned aerial vehicles, generating isolated maps with information valid only for the time of image acquisition. In the context of global environmental change, mapping the current Antarctic vegetation distribution on a regular basis is necessary for a better understanding of the changes in this fragile environment. This work aimed to generate validated vegetation maps for the North Antarctic Peninsula and South Shetlands Islands based on Sentinel-2 images using cloud processing. Sentinel-2 imagery level 1C, acquired between 2016 and 2021 (January-April), were used. Land pixels were masked with the minimum value composite image for the "water vapor" band. The NDVI maximum value composite image was sliced, and its classes were associated with the occurrence of algae (0.15 - 0.20), lichens (0.20 - 0.50), and mosses (0.50 - 0.80). The vegetation map was validated by comparing it with those from the literature. The present study showed that Sentinel-2 images allow building a vegetation type distribution map for Antarctica Peninsula and South Shetlands Islands.

sentences: Pre-metazoan origin of neuropeptide signalling Neuropeptides are a diverse class of signalling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments and gene-structure comparisons we show that both precursors are present in bilaterians, cnidarians, ctenophores and sponges. We also found phoenixin and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. Phoenixin in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays we question the originally proposed GPR-173 (SREB3) as a phoenixin receptor. Our findings indicate that signalling by secreted neuropeptide homologs has pre-metazoan origins and thus evolved before neurons.

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DNA damage response at telomeres boosts the transcription of SARS-CoV-2 receptor ACE2 during aging The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is known to be more common in the elderly, who show also more severe symptoms and a higher risk of hospitalization and death. Here we show that the expression of the Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV2 cell receptor, increases during aging in mouse and human lungs, and following telomere shortening or dysfunction in mammalian cells and in mouse models. This increase is regulated at the transcription level, and Ace2 promoter activity is DNA damage response (DDR)-dependent. Indeed, ATM inhibition or the selective inhibition of telomeric DDR, through the use of antisense oligonucleotides, prevents Ace2 upregulation following telomere damage, in cultured cells and in mice.

We propose that during aging telomeric shortening, by triggering DDR activation, causes the upregulation of ACE2, the SARS-CoV2 cell receptor, thus making the elderly likely more susceptible to the infection.

sentences: A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation cAMP signaling pathways are critical for both oncogenesis and tumor suppression. cAMP signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. We developed a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncovered a previously unappreciated nuclear cAMP microdomain that functionally activates a tumor suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397, export of YAP from the nucleus, without YAP protein degradation. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer, and since Hippo pathway deregulation can lead to oncogenesis and chemotherapeutic resistance, drugs directed at the nuclear cAMP microdomain may provide new avenues for the treatment of cancer.

sentences: Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2 and SATB1. They are organized convergently in genetically diverse subtypes of AML, and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

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RAD51AP1 and RAD54 underpin two distinct RAD51-dependent routes of DNA damage repair via homologous recombination Homologous recombination (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory sub-pathways.

In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that deletion of both RAD51AP1 and RAD54 synergistically sensitizes human cancer cell lines to treatment with a Poly(adenosine 5 s-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which stalls the progression of DNA replication forks. We infer that HR-directed anti-cancer treatment modalities shall consider this intra-pathway functional overlap, and we hypothesize that in cancerous cells the simultaneous inactivation of both RAD54 and RAD51AP1 will accentuate tumor kill.

sentences: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain repression of genes specific for other cell types and is essential for cell differentiation. In endometrial stromal sarcoma, the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. Here, we show that JAZF1-SUZ12 dysregulates PRC2 composition, recruitment, histone modification, gene expression and cell differentiation. The loss of the SUZ12 N-terminus in the fusion protein disrupted interaction with the PRC2 accessory factors JARID2, EPOP and PALI1 and prevented recruitment of PRC2 from RNA to chromatin. In undifferentiated cells, JAZF1-SUZ12 occupied PRC2 target genes but gained a JAZF1-like binding profile during cell differentiation. JAZF1-SUZ12 reduced H3K27me3 and increased H4Kac at PRC2 target genes, and this was associated with disruption in gene expression and cell differentiation programs. These results reveal the defects in chromatin regulation caused by JAZF1-SUZ12, which may underlie its role in oncogenesis.

sentences: Cued Naming and the left inferior frontal cortex Clinical studies have shown that naming can be behaviorally facilitated by priming, e.g., phonemic cues reduce anomia. Rehabilitation of language is argued to rely upon the same processes of priming in healthy speakers. Here we show, in healthy older adults, the immediate facilitatory behavioral and neural priming elicited by phonemic cues presented during an fMRI experiment of overt naming; thus, bridging the gap between lesion and neuroimaging studies. Four types of auditory cues were presented concurrently with an object picture (e.g., cat): (i) word (i.e., the target name (/kat/), (ii) initial phoneme segment (e.g., /ka/), (iii) final phoneme segment (/at/), or (iv) acoustic (noise) control cue. Naming was significantly faster with word, initial and final phonemic cues compared to noise; and word and initial cues compared to final cues, with no difference between word and initial cues. A neural priming effect - a significant decrease in neural activity - was observed in the left inferior frontal cortex (LIFC, pars triangularis, BA45) and the anterior insula bilaterally consistent with theories of primed articulatory encoding and post-lexical selection. The reverse contrast revealed increased activation in left posterior dorsal supramarginal gyrus for word cues that, we argue, may reflect integration of semantic and phonology processing during word rather than phonemic conditions. Taken together, these data from unimpaired speakers identified nodes within the naming network affected by phonemic cues. Activity within these regions may act as a possible biomarker to index anomic individuals responsiveness to phonemically cued anomia treatment.

sentences: The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5 ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

sentences: Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

sentences: The potyviral protein 6K2 from Turnip mosaic virus increases plant resilience to drought Drought is a major cause of yield loss for crops worldwide. Climate change is predicted to increase global crop losses due to drought through rising temperature and decreased water availability. Virus infection can increase drought tolerance of infected plants compared to non-infected plants; however, the mechanisms mediating virus-induced drought tolerance remain unclear. In this study, we demonstrate Turnip mosaic virus (TuMV) infection increases Arabidopsis thaliana survival under drought compared to uninfected plants. To determine if specific TuMV proteins mediate drought tolerance, we cloned the coding sequence for each of the major viral proteins and generated transgenic A. thaliana that constitutively express each protein. Three TuMV proteins, 6K1, 6K2, and NIa-Pro, enhanced drought tolerance of A. thaliana when expressed constitutively in plants compared to controls. Expression of 6K2 also increased plant biomass relative to controls, but had no impact on root biomass, trichome numbers, or on the number of stomata. While drought induced transcripts related to abscisic acid (ABA) biosynthesis and ABA levels in control plants, compared to under well-watered conditions, there were no changes in ABA or related transcripts in plants expressing 6K2 under drought conditions compared to well-watered. 6K2 expression also conveyed drought tolerance in another host plant, Nicotiana benthamiana, when expressed using a virus over expression construct derived from Foxtail mosaic virus (FoMV). Although the exact mechanisms are still unknown, these results suggest 6K2-induced drought tolerance is ABA-independent and that plant viruses may represent novel sources of drought tolerance for crop plants.

sentences: Elephant seal muscle cells adapt to sustained glucocorticoid exposure by shifting their metabolic phenotype Elephant seals experience natural periods of prolonged food deprivation while breeding, molting, and undergoing postnatal development. Prolonged food deprivation in elephant seals increases circulating glucocorticoids without inducing muscle atrophy, but the cellular mechanisms that allow elephant seals to cope with such conditions remain elusive. We generated a cellular model and conducted transcriptomic, metabolic, and morphological analyses to study how seal cells adapt to sustained glucocorticoid exposure. Seal muscle progenitor cells differentiate into contractile myotubes with a distinctive morphology, gene expression profile, and metabolic phenotype. Exposure to dexamethasone at three ascending concentrations for 48h modulated the expression of 6 clusters of genes related to structural constituents of muscle and pathways associated with energy metabolism and cell survival. Knockdown of the glucocorticoid receptor (GR) and downstream expression analyses corroborated that GR mediates the observed effects. Dexamethasone also decreased cellular respiration, shifted the metabolic phenotype towards glycolysis, and induced mitochondrial fission and dissociation of mitochondria-ER interactions without decreasing cell viability. Knockdown of DDIT4, a GR target involved in the dissociation of mitochondria-ER membranes, recovered respiration and modulated antioxidant gene expression. These results show that adaptation to sustained glucocorticoid exposure in elephant seal myotubes involves a metabolic shift toward glycolysis, which is supported by alterations in mitochondrial morphology and a reduction in mitochondria-ER interactions, resulting in decreased respiration without compromising cell survival.

sentences: Computer Vision and Deep Learning for Environment-Adaptive Control of Robotic Lower-Limb Exoskeletons Robotic exoskeletons require human control and decision making to switch between different locomotion modes, which can be inconvenient and cognitively demanding. To support the development of automated locomotion mode recognition systems (i.e., high-level controllers), we designed an environment recognition system using computer vision and deep learning. We collected over 5.6 million images of indoor and outdoor real-world walking environments using a wearable camera system, of which ~923,000 images were annotated using a 12-class hierarchical labelling architecture (called the ExoNet database). We then trained and tested the EfficientNetB0 convolutional neural network, designed for efficiency using neural architecture search, to predict the different walking environments. Our environment recognition system achieved ~73% image classification accuracy. While these preliminary results benchmark Efficient-NetB0 on the ExoNet database, further research is needed to compare different image classification algorithms to develop an accurate and real-time environment-adaptive locomotion mode recognition system for robotic exoskeleton control.

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Functional evaluation of the P681H mutation on the proteolytic activation the SARS-CoV-2 variant B.1.1.7 (Alpha) spike Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread.

HighlightsO_LISARS-CoV-2 B.1.1.7 VOC has a P681H mutation in the spike that is predicted to enhance viral infection
C_LIO_LIP681H does not significantly impact furin cleavage, viral entry or cell-cell spread
C_LIO_LIOther mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates
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Graphical abstract

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sentences: piRNAs of Caenorhabditis elegans broadly silence nonself sequences through functionally random targeting Small non-coding RNAs such as piRNAs serve as guides for Argonaute proteins, enabling sequence-specific, post-transcriptional regulation of gene expression. The piRNAs of Caenorhabditis elegans have been observed to bind targets with high mismatch tolerance, and appear to lack specific transposon targets, unlike piRNAs in Drosophila melanogaster and other organisms. These observations support a model in which C. elegans piRNAs provide a broad, indiscriminate net of silencing, acting in competition with siRNAs associated with the CSR-1 Argonaute that specifically protect self genes from silencing. However, the breadth of piRNA targeting has not been subject to in-depth quantitative analysis, nor has it been explained how piRNAs are distributed across sequence space to achieve complete coverage. Through a bioinformatic analysis of piRNA sequences, incorporating an original metric of piRNA-target distance, we demonstrate that C. elegans piRNAs are functionally random, in that their coverage of sequence space is comparable to that of random sequences. By possessing a sufficient number of distinct, essentially random piRNAs, C. elegans is able to target arbitrary nonself sequences with high probability. This result elucidates the mechanism by which newly transcribed mRNAs in C. elegans are classified as self or nonself, and has implications for piRNA evolution and biogenesis.

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A highly accurate model for screening prostate cancer using propensity index panel of ten genes Prostate-specific antigen (PSA) is a key biomarker, which is commonly used to screen patients of prostate cancer. There is a significant number of unnecessary biopsies that are performed every year, due to poor accuracy of PSA based biomarker. In this study, we identified alternate biomarkers based on gene expression that can be used to screen prostate cancer with high accuracy. All models were trained and test on gene expression profile of 500 prostate cancer and 51 normal samples. Numerous feature selection techniques have been used to identify potential biomarkers. These biomarkers have been used to develop various models using different machine learning techniques for predicting samples of prostate cancer. Our logistic regression-based model achieved highest AUROC 0.91 with accuracy 82.42% on validation dataset. We introduced a new approach called propensity index, where expression of gene is converted into propensity. Our propensitybased approach improved the performance of classification models significantly and achieved AUROC 0.99 with accuracy 96.36% on validation dataset. We also identified and ranked selected genes which can be used to discriminate prostate cancer patients from health individuals with high accuracy. It was observed that single gene-based biomarkers can only achieve accuracy around 90%. In this study, we got best performance using a panel of 10 genes; random forest model using propensity index.

HighlightsO_LIApplication of Machine learning techniques to identify Biomarkers for PRAD cancer.
C_LIO_LIHighly accurate models developed for classifying prostate cancer vs. normal sample.
C_LIO_LIIntroducing Propensity index concept for enhancing model performance.
C_LIO_LITop 10 genes identified using feature selection techniques.
C_LI

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Detecting change in interconnected ecosystem assets to inform indicators of condition in environmental economic accounts Environmental Economic Accounts track the condition of interconnected ecosystem assets, to inform environmental decision makers about its influence on the past and current status of ecosystem services. Indicators of condition need to be analysed to determine if they lead, are coincident with or lag environmental change, so that indicators are interpreted appropriately by decision makers. Here we conduct performance testing of ecological condition indicators developed with the United Nations System of Environmental-Economic Accounting - Ecosystem Accounting (SEEA-EA) framework. We chose a case-study of a catchment-to-coast system with strongly interconnected assets and high natural variability, which likely confounds our ability to track long-term change. We first quantified covariation among ecological indicators for pasture biomass, vegetation greenness and barramundi catch per unit effort. Covariation in the indicators was driven by river flow, with higher values of all indicators occurring in years with greater river flow. Barramundi catch per unit effort was most sensitive to changes in river flow, followed by a vegetation greenness and pasture biomass. We then defined reference bounds for each indicator that accounted for natural variation in river flow. We predicted the emergence times for each indicator, the time taken for each indicator to emerge from the background of natural variation. Emergence times were >10 years in all cases. Detecting change was more difficult where there were gaps in data. Ecosystem accounts can be used to compare ecological condition against performance objectives, but should consider natural variation in the system. National accounts are often used by decision makers to directly inform near-term actions, because economic indicators respond rapidly to new policies and the prevailing economic conditions. We found that ecological condition indicators in highly variable ecosystems are lagging indicators of change, and as such should be used to assess historical performance, not as leading indicators of future change.

Article impact statementEcological condition is a lagging indicator of environmental change and should only be used to inform on historical management performance.

sentences: Analysis of B-cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences Analyzing B cell receptor (BCR) repertoires is immensely useful in evaluating ones immunological status. Conventionally, repertoire analysis methods have focused on comprehensive assessments of clonal compositions, including V(D)J segment usage, nucleotide insertions/deletions, and amino acid distributions. Here, we introduce a novel computational approach that applies deep-learning-based protein embedding techniques to analyze BCR repertoires. By selecting the most frequently occurring BCR sequences in a given repertoire and computing the sum of the vector representations of these sequences, we represent an entire repertoire as a 100-dimensional vector and eventually as a single data point in vector space. We demonstrate that this new approach enables us to not only accurately cluster BCR repertoires of coronavirus disease 2019 (COVID-19) patients and healthy subjects but also efficiently track minute changes in immune status over time as patients undergo treatment. Furthermore, using the distributed representations, we successfully trained an XGBoost classification model that achieved a mean accuracy rate of over 87% given a repertoire of CDR3 sequences.

sentences: Diversity of cave Phlebotomines (Diptera: Psychodidae) from a Colombian cave Sandflies are vector species of Leishmania, among many other pathogens, with a global distribution and a variety of ecological niches. Previous samplings have found that karstic formations (i.e., caves and folds formed by the erosion of limestone) serve as a natural habitat to sandfly species. The majority of samplings of cave sandfly diversity have occurred in Brazil and to date none have studied the species composition in a cave in the Northern Andes. We collected sandflies in the Cave "Los Guacharos", in the state of Antioquia, Colombia. The sampling was carried out during two consecutive nights in September 2019. CDC-type light traps were installed inside the cavern and in other surrounding karst systems (caves and folds). In total, we identified 18 species of sandfly from the cave and surrounding karst systems, including three new records for Colombia (Bichromomyia olmeca nociva, Brumptomyia brumpti, and Warileya leponti), and provide the first karstic reports for four other species (Lutzomyia gomezi, Lutzomyia hartmanni, Pintomyia ovallesi, and Psychodopygus panamensis). We then used the results of our survey and published literature to test two hypotheses. First, that sandfly diversity in Neotropical caves is richest nearer to the equator and second that there is a phylogenetic signal of karstic habitat use in sandflies. Counter to our predictions, we found no evidence that diversity follows a latitudinal gradient. Further, we find no evidence of a phylogenetic signal of karstic habitat use, instead finding that the use of caves likely evolved multiple times across several genera. Our results highlight the importance of a wide sampling to understand the natural habitat of sandflies and other disease vectors.

sentences: Differential mutational profile of SARS-CoV-2 proteins across deceased and asymptomatic patients The SARS-CoV-2 infection spread at an alarming rate with many places showed multiple peaks in incidence. Present study involves a total of 332 SARS-CoV-2 sequences from 114 Asymptomatic and 218 Deceased patients from twenty-one different countries. The mining of mutations was done using the GISAID CoVSurver (www.gisaid.org/epiflu-applications/covsurver-mutations-app) with the reference sequence  hCoV-19/Wuhan/WIV04/2019 present in NCBI with Accession number NC-045512.2. The impact of the mutations on SARS-CoV-2 proteins mutation was predicted using PredictSNP1(loschmidt.chemi.muni.cz/predictsnp1) which is a meta-server integrating six predictor tools: SIFT, PhD-SNP, PolyPhen-1, PolyPhen-2, MAPP and SNAP. The iStable integrated server (predictor.nchu.edu.tw/iStable) was used to predict shifts in the protein stability due to mutations. A total of 372 variants were observed in the 332 SARS-CoV-2 sequences with several variants incident in multiple patients accounting for a total of 1596 incidences. Asymptomatic and Deceased specific mutants constituted 32% and 62% of the repertoire respectively indicating their exclusivity. However, the most prevalent mutations were those present in both. Though some parts of the genome are more variable than others but there was clear difference between incidence and prevalence. NSP3 with 68 variants had total occurrence of only 105 whereas Spike protein had 346 occurrences with just 66 variants. For Deleterious variants, NSP3 had the highest incidence of 25 followed by NSP2 (16), ORF3a (14) and N (14). Spike protein had just 7 Deleterious variants out of 66. Deceased patients have more Deleterious than Neutral variants as compared to the symptomatic ones. Further, it appears that the Deleterious variants which decrease protein stability are more significant in pathogenicity of SARS-CoV-2.

sentences: Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach  DNA-PAINT robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by  Exchange-PAINT that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 - 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.

sentences: Comparison of TIMS-PASEF quantitative proteomics data-analysis workflows using FragPipe, DIA-NN, and Spectronaut from a user's perspective Data-independent acquisition (DIA) methods have gained great popularity in bottom-up quantitative proteomics, as they overcome the irreproducibility and under-sampling limitations of data-dependent acquisition (DDA). diaPASEF, recently developed for the timsTOF Pro mass spectrometers, has brought improvements to DIA, providing additional ion separation (in the ion mobility dimension) and increasing sensitivity. Several studies have benchmarked different workflows for DIA quantitative proteomics, but mostly using instruments from Sciex and Thermo, and therefore, the results are not extrapolable to diaPASEF data. In this work, using a real-life sample set like the one that can be found in any proteomics experiment, we compared the results of analyzing PASEF data with different combinations of library-based and library-free analysis, combining the tools of the FragPipe suite, DIA-NN and including MS1-level LFQ with DDA-PASEF data, and also comparing with the workflows possible in Spectronaut. We verified that library-independent workflows, not so efficient not so long ago, have greatly improved in the recent versions of the software tools, and now perform as well or even better than librarybased ones. We report here information so that the user who is going to conduct a relative quantitative proteomics study using a timsTOF Pro mass spectrometer can make an informed decision on how to acquire (diaPASEF for DIA analysis, or DDA-PASEF for MS1-level LFQ) the samples, and what can be expected depending on the data analysis tool used, among the different alternatives offered by the recently optimized tools for TIMS-PASEF data analysis.

sentences: Intrinsic Degradation of the Type-II Antitoxin ParD from Pseudomonas aeruginosa Type-II Toxin Antitoxin (TA) systems are regulated by differential half-lives of the resulting non-secreted proteins, such that the neutralizing antitoxin undergoes continual degradation and replenishment to maintain neutralization of its cognate toxin. Antitoxin proteins are widely reported as labile, including upon purification and in vitro storage. During the course of studies on a ParDE TA system we noted a prevalent in vitro degradation of the ParD antitoxin. In efforts to combat this for practical use in assays, we characterized parameters impacting the degradation as well as the resulting products. These revealed a mechanism likely mediated by a serine or metal-dependent protease. Using Direct Infusion Mass Spectrometry, the cleavage products were identified as an essentially intact DNA binding region of the antitoxin and with the toxin binding domain completely removed. No other species were identified in the solution, such as a contaminant that may mediate such cleavage. Therefore, while our studies revealed viable strategies to mitigate the in vitro degradation they did not identify any protease, leaving open the possibility of a potential auto-catalytic proteolytic activity of the antitoxin proteins.

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Population-scale detection of non-reference sequence variants using colored de Bruijn Graphs MotivationWith the increasing throughput of sequencing technologies, structural variant (SV) detection has become possible across tens of thousands of genomes. Non-reference sequence (NRS) variants have drawn less attention compared to other types of SVs due to the computational complexity of detecting them. When using short-read data, the detection of NRS variants inevitably involves a de novo assembly which requires high-quality sequence data at high coverage. Previous studies have demonstrated how sequence data of multiple genomes can be combined for the reliable detection of NRS variants. However, the algorithms proposed in these studies have limited scalability to larger sets of genomes.

ResultsWe introduce PopIns2, a tool to discover and characterize NRS variants in many genomes, which scales to considerably larger numbers of genomes than its predecessor PopIns. In this article, we briefly outline the PopIns2 workflow and highlight our novel algorithmic contributions. We developed an entirely new approach for merging contig assemblies of unaligned reads from many genomes into a single set of NRS using a colored de Bruijn graph. Our tests on simulated data indicate that the new merging algorithm ranks among the best approaches in terms of quality and reliability and that PopIns2 shows the best precision for a growing number of genomes processed. Results on the Polaris Diversity Cohort and a set of 1000 Icelandic human genomes demonstrate unmatched scalability for the application on population-scale datasets.

AvailabilityThe source code of PopIns2 is available from https://github.com/kehrlab/PopIns2.

Contactthomas.krannich@bihealth.de or birte.kehr@klinik.uni-regensburg.de

sentences: A cell-free assay for rapid screening of inhibitors of hACE2-receptor - SARS-CoV-2-Spike binding We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently-labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle - hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the non-inhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to a known inhibitor. Finally, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.

sentences: miR-2 contributes to WSSV infection by targeting Caspase 2 in mud crab (Scylla paramamosain) As we known, Caspase 2 is widely studied for its apoptosis regulatory function in mammals. However, despite the fundamental role of apoptosis during the anti-viral immune response, the relationship between Caspase 2 and virus infection has not been extensively explored in invertebrates, whether miRNAs are involved in this process also remains unclear. To address this issue, the miRNA-mediated regulation of Caspase 2 in mud crab Scylla paramamosain was characterized in this study. The results suggested that Sp-Caspase 2 could suppress white spot syndrome virus (WSSV) infection via apoptosis induction. The further data showed that Caspase 2 was directly targeted by miR-2 in mud crab. Silencing or overexpression of miR-2 could affect apoptosis and WSSV replication through regulating the expression level of Caspase 2. Taken together, all these results demonstrated the crucial role of miR-2-Caspase 2 pathway in the innate immunity of mud crab and revealed a novel mechanism during anti-viral immune response in marine invertebrates.

sentences: Latching dynamics as a basis for short-term recall We discuss simple models for the transient storage in short-term memory of cortical patterns of activity, all based on the notion that their recall exploits the natural tendency of the cortex to hop from state to state - latching dynamics. We show that in one such model, and in simple spatial memory tasks we have given to human subjects, short-term memory can be limited to similar low capacity by interference effects, in tasks terminated by errors, and can exhibit similar sublinear scaling, when errors are overlooked. The same mechanism can drive serial recall if combined with weak order-encoding plasticity. Finally, even when storing randomly correlated patterns of activity the network demonstrates correlation-driven latching waves, which are reflected at the outer extremes of pattern space.

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Optimising passive surveillance of a neglected tropical disease in the era of elimination: A modelling study BackgroundSurveillance is an essential component of global programs to eliminate infectious diseases and avert epidemics of (re-)emerging diseases. As the numbers of cases decline, costs of treatment and control diminish but those for surveillance remain high even after the  last case. Reducing surveillance may risk missing persistent or (re-)emerging foci of disease. Here, we use a simulation-based approach to determine the minimal number of passive surveillance sites required to ensure maximum coverage of a population at-risk (PAR) of an infectious disease.

Methodology and Principal FindingsFor this study, we use Gambian human African trypanosomiasis (g-HAT) in north-western Uganda, a neglected tropical disease (NTD) which has been reduced to historically low levels (<1000 cases/year globally), as an example. To quantify travel time to diagnostic facilities, a proxy for surveillance coverage, we produced a high spatial-resolution resistance surface and performed cost-distance analyses. We simulated travel time for the PAR with different numbers (1-170) and locations (170,000 total placement combinations) of diagnostic facilities, quantifying the percentage of the PAR within 1h and 5h travel of the facilities, as per in-country targets. Our simulations indicate that a 70% reduction (51/170) in diagnostic centres still exceeded minimal targets of coverage even for remote populations, with >95% of a total PAR of ~3million individuals living [≤]1h from a diagnostic centre, and we demonstrate an approach to best place these facilities, informing a minimal impact scale back.

ConclusionsOur results highlight that surveillance of g-HAT in north-western Uganda can be scaled back without substantially reducing coverage of the PAR. The methodology described can contribute to cost-effective and equable strategies for the surveillance of NTDs and other infectious diseases approaching elimination or (re-)emergence.

Author SummaryDisease surveillance systems are an essential component of public health practice and are often considered the first line in averting epidemics for (re-)emerging diseases. Regular evaluation of surveillance systems ensures that they remain operating at maximum efficiency; systems that survey diseases of low incidence, such as those within elimination settings, should be simplified to reduce the reporting burden. A lack of guidance on how to optimise disease surveillance in an elimination setting may result in added expense, and/or the underreporting of disease. Here, we propose a framework methodology to determine systematically the optimal number and placement of surveillance sites for the surveillance of infectious diseases approaching elimination. By utilising estimates of geographic accessibility, through the construction of a resistance surface and a simulation approach, we identify that the number of operational diagnostic facilities for Gambian human African trypanosomiasis in north-western Uganda can be reduced by 70% with a minimal reduction in existing coverage, and identify the minimum number of facilities required to meet coverage targets. Our analysis can be used to inform the number and positioning of surveillance sites for diseases within an elimination setting. Passive surveillance becomes increasingly important as cases decline and active surveillance becomes less cost-effective; methods to evaluate how best to engage this passive surveillance capacity given facility capacity and geographic distribution are pertinent for several NTDs where diagnosis is complex. Not only is this a complicated research area for diseases approaching elimination, a well-designed surveillance system is essential for the detection of emerging diseases, with this work being topical in a climate where emerging pathogens are becoming more commonplace.

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Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c / Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signaling is well established, the function of their transmembrane (TM) regions remains unknown. Here we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 [A] resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

One-Sentence SummaryStructure of M. tuberculosis membrane adenylyl cyclase Cya

sentences: Insights into long non-coding RNA regulation of anthocyanin carrot root pigmentation Carrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript (NAT) linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

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Transcription factor retention through multiple polyploidisation steps in wheat O_LIWhole genome duplication (WGD) is widespread in plant evolutionary history, but the mechanisms of non-random gene loss after WGD are debated. The gene balance hypothesis proposes that dosage-sensitive genes such as regulatory genes are retained in polyploids. To test this hypothesis, we analysed the retention of transcription factors (TFs) in the recent allohexaploid bread wheat (Triticum aestivum).
C_LIO_LIWe annotated TFs in hexaploid, tetraploid and diploid wheats; compared the co-expression of homoeologous TF and non-TF triads; and analysed single nucleotide variation in TFs across cultivars.
C_LIO_LIWe found that, following each of two hybridisation and WGD events, the proportion of TFs in the genome increased. TFs were preferentially retained over other genes as homoeologous groups in tetraploid and hexaploid wheat. Across cultivars, TF triads contain fewer deleterious missense mutations than non-TFs.
C_LIO_LITFs are preferentially retained as three functional homoeologs in hexaploid wheat, in support of the gene balance hypothesis. High co-expression between TF homoeologs suggests that neo- and sub-functionalisation are not major drivers of TF retention in this young polyploid. Knocking out one TF homoeolog to alter gene dosage, using TILLING or CRISPR, could be a way to further test the gene balance hypothesis and generate new phenotypes for wheat breeding.
C_LI

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xcore: an R package for inference of gene expression regulators Elucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is one of the most common questions asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types. Given the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that the modeling can be done using ChiP-seq "signatures" directly, effectively skipping the motif finding and TFBS prediction steps. We present xcore, an R package that allows Transcription Factor activity modeling based on their ChiP-seq signatures and users gene expression data. We also provide xcoredata a companion data package that provides a collection of preprocessed ChiP-seq signatures. We demonstrate that xcore leads to biologically relevant predictions using TGF-beta induced epithelial-mesenchymal transition and rinderpest infection time-course CAGE data as examples.

Availability and Implementationxcore and xcoredata R packages are freely available at www.github.com/bkaczkowski/xcore and www.github.com/mcjmigdal/xcoredata.

xcore user guide is available www.bkaczkowski.github.io/xcore/articles/xcore_vignette.html.

Contactb.kaczkowski@gmail.com

sentences: Elevated fear responses to threatening cues in rats with early life stress is associated with greater excitability and loss of gamma oscillations in ventral-medial prefrontal cortex Stress experienced early in development can have profound influences on developmental trajectories and ultimately behaviors in adulthood. Potent stressors during brain maturation can profoundly disrupt prefrontal cortical areas in particular, which can set the stage for prefrontal-dependent alterations in fear regulation and risk of drug abuse in adulthood. Despite these observations, few studies have investigated in vivo signaling in prefrontal signals in animals with a history of early life stress (ELS). Here, rats with ELS experienced during the first post-natal week were then tested on a conditioned suppression paradigm during adulthood. During conditioned suppression, electrophysiological recordings were made in the ventral medial prefrontal cortex (vmPFC) during presentations of a fear-associated cues that resolved both single-unit activity and local field potentials (LFPs). Relative to unstressed controls, ELS-experienced rats showed greater fear-related suppression of lever pressing. During presentations of the fear-associated cue (CS+), neurons in the vmPFC of ELS animals showed a significant increase in the probability of excitatory encoding relative to controls, and excitatory phasic responses in the ELS animals were reliably of higher magnitude than Controls. In contrast, vmPFC neurons in ELS subjects better discriminated between the shock-associated CS+ and the neutral ("safe") CS-cue than Controls. LFPs recorded in the same locations revealed that high gamma band (65-95 Hz) oscillations were strongly potentiated in Controls during presentation of the fear-associated CS+ cue, but this potentiation was abolished in ELS subjects. Notably, no other LFP spectra differed between ELS and Controls for either the CS+ or CS-. Collectively, these data suggest that ELS experience alters the neurobehavioral functions of PFC in adulthood that are critical for processing fear regulation. As such, these alterations may also provide insight into to increased susceptibility to other PFC-dependent processes such as risk-based choice, motivation, and regulation of drug use and relapse in ELS populations.

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Single Prolonged Stress Reduces Intrinsic Excitability and Excitatory Synaptic Drive onto Pyramidal Neurons in the Infralimbic Prefrontal Cortex of Adult Rats Post-traumatic stress disorder (PTSD) is a chronic, debilitating mental illness marked by abnormal fear responses and deficits in extinction of fear memories. The pathophysiology of PTSD is linked to decreased activation of the ventromedial prefrontal cortex (vmPFC). This study aims to investigate underlying functional changes in synaptic drive and intrinsic excitability of pyramidal neurons in the rodent homolog of the vmPFC, the infralimbic cortex (IL), following exposure to single prolonged stress (SPS), a paradigm that mimics core symptoms of PTSD in rats. Rats were exposed to SPS and allowed one week of recovery following which brain slices containing the PFC were prepared for whole-cell patch clamp recordings from layer V pyramidal neurons in the IL. Our results indicate that SPS reduces spontaneous excitatory synaptic drive to pyramidal neurons. In addition, SPS decreases the intrinsic membrane excitability of IL PFC pyramidal cells, as indicated by an increase in rheobase, decrease in input resistance, hyperpolarization of resting membrane potential, and a reduction in repetitive firing rate. Our results suggest that SPS causes a lasting reduction in PFC activity, supporting a body of evidence linking traumatic stress with prefrontal hypoactivity.

Graphical AbstractSPS causes a decrease in excitatory synaptic drive and intrinsic excitability of IL pyramidal neurons.

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sentences: A data compendium of Mycobacterium tuberculosis antibiotic resistance The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a compendium of 15,211 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole genome sequencing (WGS) and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay. It is the largest matched phenotypic and genotypic dataset for M. tuberculosis to date. Here, we provide a summary detailing the breadth of data collected, along with a description of how the isolates were collected and uniformly processed in CRyPTIC partner laboratories across 23 countries. The compendium contains 6,814 isolates resistant to at least one drug, including 2,129 samples that fully satisfy the clinical definitions of rifampicin resistant (RR), multi-drug resistant (MDR), pre-extensively drug resistant (pre-XDR) or extensively drug resistant (XDR). Accurate prediction of resistance status (sensitive/resistant) to eight antitubercular drugs by using a genetic mutation catalogue is presented along with the presence of suspected resistance-conferring mutations for isolates resistant to the newly introduced drugs bedaquiline, clofazimine, delamanid and linezolid. Finally, a case study of rifampicin mono-resistance demonstrates how this compendium could be used to advance our genetic understanding of rare resistance phenotypes. The compendium is fully open-source and it is hoped that the dataset will facilitate and inspire future research for years to come.

sentences: Mixed Origins: HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, [~]7 months after the final vaccination. The majority of the pre-immune lineage members included IgM, however IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells.

sentences: Specialized protrusions coordinate migratory border cell cluster cohesion via Scribble, Cdep, and Rac Apicobasal polarity is a defining characteristic of epithelial cells and its disruption is a cancer hallmark. Distinct apical and basolateral protein modules antagonize each other to establish separate membrane domains. These modules interact with dozens of potential effector proteins. Here we describe polarity protein localization and function within a migrating epithelial cell cluster and identify a functionally significant effector protein. In Drosophila egg chambers, border cells delaminate from the follicular epithelium and migrate collectively. We report that the basolateral protein Scribble is required for border cell cluster cohesion and migration. The basolateral module localizes the Rac guanine nucleotide exchange factor Cdep to membranes, and Cdep knockdown phenocopies Scribble cluster cohesion defects. Remarkably, membrane targeting of Cdep is sufficient to partially suppress multiple Scribble phenotypes. We describe specialized basolateral protrusions that promote cluster cohesion. Scribble restricts these protrusions from encroaching onto the apical domain. Thus, a major function of the basolateral module is to localize Cdep, promoting specialized protrusions, cluster cohesion, and collective migration.

sentences: Accurate prediction of functional states of cis-regulatory modules reveals the universal epigenetic code in mammals We proposed a two-step approach for predicting active cis-regulatory modules (CRMs) in a cell/tissue type. We first predict a map of CRM loci in the genome using all available transcription factor binding data in the organism, and then predict functional states of all the putative CRMs in any cell/tissue type using few epigenetic marks. We have recently developed a pipeline dePCRM2 for the first step, and now presented machine-learning methods for the second step. Our approach substantially outperforms existing methods. Our results suggest common epigenetic rules for defining functional states of CRMs in various cell/tissue types in humans and mice.

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Synaptic and intrinsic mechanisms impair reticular thalamus and thalamocortical neuron function in a Dravet syndrome mouse model Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced intrinsic excitability in nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced ascending glutamatergic sensory input to VPL neurons, but not VPM neurons. In addition, glutamatergic cortical input to nRT neurons was reduced in Scn1a heterozygous mice, whereas cortical input to VPL and VPM neurons remained unchanged. These findings introduce input-specific alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in Dravet syndrome, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in Dravet syndrome and identifies new components of corticothalamic circuitry as potential therapeutic targets.

HIGHLIGHTSO_LIGABAergic reticular thalamus neurons have impaired tonic and burst firing properties in a NaV1.1 haploinsufficiency mouse model of Dravet syndrome.
C_LIO_LINaV1.1 haploinsufficiency has opposing effects on spike firing in two distinct glutamatergic thalamocortical neuron populations.
C_LIO_LINaV1.1 haploinsufficiency alters glutamatergic synaptic connectivity in an input-specific manner in the thalamus.
C_LIO_LIDysregulation of both intrinsic and synaptic mechanisms contribute to imbalanced thalamic excitation and inhibition in this Dravet syndrome mouse model.
C_LI

sentences: CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7 V-ATPases are rotary proton pumps that serve as signaling hubs with numerous proposed binding partners in cells. We used cryoEM combined with exhaustive focused classification to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 shows that mEAK-7s TLDc domain interacts with V-ATPases stator while its C-terminal  helix binds V-ATPases rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike inhibition of yeast V-ATPase by the TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK-7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. Together, these results show that differences in V-ATPase binding by TLDc domain-containing proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzymes activity.

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Lack of sex differences in gonadotropin-releasing hormone (GnRH) neuron potassium currents and excitability Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast (IA) and slow (IK) inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed in most potassium current properties, input resistance or capacitance and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells.

Significance StatementThe pattern of activity of gonadotropin-releasing hormone (GnRH) neurons is crucial to reproductive success in both males and females. Direct comparison of GnRH neurons from mice of both sexes during negative feedback and after gonadectomy revealed few differences in potassium currents, excitability, and action potential properties. These results support the hypothesis that neurons presynaptic to GnRH neurons communicate negative feedback to these cells in a manner that does not alter their intrinsic biophysical properties.

sentences: Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis Lactoferrin binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin, and in providing protection against the cationic antimicrobial peptide lactoferricin. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-lactoferrin, forming a 1:1 complex and confirmed by SEC-SAXS. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of lactoferrin. Our structures provides insight into LbpBs preference towards holo-lactoferrin, and our mutagenesis and binding studies show that lactoferrin and lactoferricin bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve lactoferrin in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.

sentences: Evaluating test-retest reliability and sex/age-related effects on temporal clustering coefficient of dynamic functional brain networks The multilayer dynamic network model has been proposed as an effective method to understand how the brain functions dynamically. Specially, derived from the definition of clustering coefficient in static networks, the temporal clustering coefficient provides a direct measure of topological stability of dynamic brain networks and shows potential in predicting altered brain functions in both normal and pathological conditions. However, test-retest reliability and demographic-related effects on this measure remain to be evaluated. Using a publicly available dataset from the Human Connectome Project consisting of 337 young healthy adults (157 males/180 females; 22 to 37 years old), the present study investigated: (1) the test-retest reliability of temporal clustering coefficient across four repeated resting-state functional magnetic resonance imaging scans as measured by intraclass correlation coefficient (ICC); and (2) sex- and age-related effects on temporal clustering coefficient. The results showed that (1) the temporal clustering coefficient had overall moderate test-retest reliability (ICC > 0.40 over a wide range of densities) at both global and subnetwork levels; (2) female subjects showed significantly higher temporal clustering coefficient than males at both global and subnetwork levels, in particular within the default-mode and subcortical regions; (3) temporal clustering coefficient of the subcortical subnetwork was negatively correlated with age in young adults. Our findings suggest that temporal clustering coefficient is a reliable and reproducible approach for the identification of individual differences in brain function, and provide evidence for sex and age effects on human brain dynamic connectome.

sentences: Pan-cancer analysis of mRNA stability for decoding tumour post-transcriptional programs RNA stability is a crucial and often overlooked determinant of gene expression. Some of the regulators of mRNA stability are long known as key oncogenic or tumour suppressor factors. Nonetheless, the extent to which mRNA stability contributes to transcriptome remodeling in cancer is unknown, and the factors that modulate mRNA stability during cancer development and progression are largely uncharacterized. Here, by decoupling transcriptional and post-transcriptional effects in RNA-seq data of 7760 samples from 18 cancer types, we present a pan-cancer view of the mRNA stability changes that accompany tumour development and progression. We show that thousands of genes are dysregulated at the mRNA stability level, and identify the potential factors that drive these changes, including >80 RNA-binding proteins (RBPs) and microRNAs (miRNAs). Most RBPs and miRNAs have cancer type-specific activities, but a few show recurrent inactivation across multiple cancers, including the RBFOX family of RBPs and miR-29. Analysis of cell lines with phenotypic activation or inhibition of RBFOX1 and miR-29 confirms their role in modulation of genes that are dysregulated across multiple cancers, with functions in calcium signaling, extracellular matrix organization, and stemness. Overall, our study highlights the critical role of mRNA stability in shaping the tumour transcriptome, with recurrent post-transcriptional changes that are ~30% as frequent as transcriptional events. These results provide a resource for systematic interrogation of cancer-associated stability drivers and pathways.

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Carbon Dot-based Fluorescent Antibody Nanoprobes as Brain Tumour Glioblastoma Diagnostics. The development of efficient and sensitive tools for the detection of brain cancer in patients is of the utmost importance particularly because many of these tumours go undiagnosed until the disease has advanced and when treatment is less effective. Current strategies employ antibodies (Abs) to detect Glial Fibrillary Acid Protein (GFAP) in tissue samples, since GFAP is unique to the brain and not present in normal peripheral blood, and it relies on fluorescent reporters.

Herein we describe a low cost, practical and general method for the labelling of proteins and antibodies with fluorescent carbon dots (CD) to generate diagnostic probes that are robust, photostable and applicable to the clinical setting. The two-step protocol relies on the conjugation of a dibenzocyclooctyne (DBCO)-functionalised CD with azide functionalised proteins by combining amide conjugation and strain promoted alkyne-azide cycloaddition (SPAAC) ligation chemistry. The new class of Ab-CD conjugates developed using this strategy was successfully used for the immunohistochemical staining of human brain tissues of patients with glioblastoma (GBM) validating the approach. Overall, these novel fluorescent probes offer a promising and versatile strategy in terms of costs, photostability and applicability which can be extended to other Abs and protein systems.

sentences: Nuclear envelope reshaping around the vacuole determines the morphology of the ribosomal DNA The ribosomal DNA array (rDNA) of Saccharomyces cerevisiae has served as a model to address chromosome organization. In cells arrested before anaphase (mid-M), the rDNA acquires a highly structured chromosomal organization referred to as the rDNA loop, whose length can double the cell diameter. Previous works established that complexes such as condensin and cohesin are essential to attain this structure. Here, we report that the rDNA loop adopts distinct morphologies that arise as spatial adaptations to changes in the nuclear morphology triggered during the mid-M arrests. Interestingly, the formation of the rDNA loop results in the appearance of a space under the loop (SUL) which is devoid of any nuclear component yet colocalizes with the vacuole. We finally show that the formation and maintenance of the rDNA loop and the SUL require TORC1 and membrane synthesis. We propose that the rDNA-associated nuclear envelope (NE) reshapes into a loop to accommodate the vacuole, with the nucleus becoming bilobed.

sentences: LptB2FG is an ABC transporter with Adenylate Kinase activity regulated by LptC/A recruitment Lipopolysaccharide (LPS) is an essential glycolipid covering the surface of gram-negative bacteria. Its transport involves a dedicated 7 protein transporter system, the Lpt machinery, that physically spans the entire cell envelope. LptB2FG complex is an ABC transporter that hydrolyses Adenosine Triphosphate (ATP) to extract LPS from the inner membrane (IM). LptB2FG was extracted directly from IM with its original lipid environment by Styrene-Maleic acids polymers(SMA). SMA-LptB2FG in nanodiscs displays ATPase activity and a previously uncharacterized Adenylate Kinase (AK) activity. It catalyzes phosphotransfer between two ADP molecules to generate ATP and AMP. ATPase and AK activities of LptB2FG are both stimulated by the interaction on the periplasmic side with LptC and LptA partners and inhibited by the presence of LptC transmembrane helix. Isolated ATPase module (LptB) has weak AK activity in absence of LptF and LptG, and one mutation, that weakens affinity for ADP, has AK activity similar to that of fully assembled complex. LptB2FG is thus capable of producing ATP from ADP depending on the assembly of the Lpt bridge and the AK activity might be important to ensure efficient LPS transport in fully assembled Lpt system.

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Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

Author summaryThe mitotic spindle is a microtubule (MT)-based molecular machine that mediates precise chromosome segregation during cell division. Both Drosophila and human cells assemble their spindles exploiting two main classes of MTs: MTs nucleated by the centrosomes (MT nucleating organelles) and MTs generated at or near the kinetochores (the chromosome-associated structures that bind the spindle MTs). Cells of both species can assemble a functional mitotic spindle in the complete absence of centrosomes, but the mechanisms underlying this process are still poorly understood. We used Drosophila S2 cells as model system to analyze spindle reassembly following colcemid-induced MT depolymerization. MT regrowth (MTR) after colcemid treatment was particularly informative as this drug disrupts the MT nucleating ability of the centrosomes but allows kinetochore-driven MTR (KDMTR). We analyzed KDMTR in normal cells and in cells subjected to RNA interference (RNAi)-mediated depletion of 8 different evolutionarily conserved proteins involved in spindle assembly, and identified proteins that either promote or delay KDMTR. These results coupled with the analysis of proteins localization during spindle reassembly allowed us to integrate the current model on the role of kinetochore-driven MT growth in spindle formation.

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Interleukin 7 receptor drives Early T lineage Progenitor expansion Interleukin 7 (IL-7) and IL-7 receptor (IL-7r) are essential for T lymphocyte differentiation, by driving proliferation and survival of specific developmental stages. While early T lineage progenitors (ETP), the most immature thymocyte population known, have a history of IL-7r expression, it is unclear whether IL-7r is required at this stage. Here, we show that mice lacking IL-7 or IL-7r have a marked loss of ETPs that results mostly from a cell-autonomous defect in proliferation and survival, although no changes were detected in Bcl2 protein levels. Further, a fraction of ETPs responded to IL-7 stimulation ex vivo by phosphorylating Stat5, and IL-7r was enriched in the most immature Flt3+Ccr9+ ETPs. Consistently, IL-7 promoted the expansion of Flt3+ but not Flt3-ETPs on OP9-DLL4 cocultures, without affecting differentiation at either stage. Taken together, our data show that IL-7/IL-7r is necessary following thymus seeding, by promoting proliferation and survival of the most immature thymocytes.

SummaryPaiva et al. show that IL-7/IL-7r signaling upon thymus seeding is essential for proliferation and survival of the most immature early T lineage progenitors (ETP), thereby determining the physiological ETP cellularity.

sentences: Synthetic memory circuits for programmable cell reconfiguration in plants Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the ability to integrate multiple customizable input signals through a processing unit constructed from biological parts, to produce a predictable and programmable output. Here, we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Through utilization of genetic recombination these circuits create stable long-term changes in expression and recording of past stimuli. This highly-compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealised traits into plants.

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Fluximplied: A novel approach integrates rate limiting steps and differential expression for pathway analysis BackgroundMany tools exist to perform a rigorous pathway analysis, though traditional gene set enrichment analysis remains among the most common. While useful for many applications, one common situation where it is less so is the metabolic profiling of bulk omics datasets. Rate limiting steps in more linear pathways are the main determinant of flux through these pathways, but differential expression of the enzymes that catalyze these steps is usually not differentially weighted in pathway analysis. Fluximplied was built to perform pathway analysis with rate limiting steps in mind to assess the implied flux through a number of well validated metabolic pathways.

ResultsA database of rate limiting steps and their associated pathway was constructed. Using publicly available human RNA sequencing data from liver, putamen, and adipose tissue, fluximplied generally corroborated the pathway analysis. When comparing two CD8 T cell subsets from mouse lung, our algorithm confirmed previous findings that were not found previously.

ConclusionFluximplied is an accessible tool for pathway analysis which is intended to assist the user with hypothesis generation. Unlike traditional approaches to pathway analysis, it specifically queries a database of rate limiting steps in order to infer flux through canonical metabolic pathways.

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Molecular phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros gracilis and Lhcq/Lhcf diversity Diatoms adapt to various aquatic light environments and play major roles in the global carbon cycle using their unique light-harvesting system, i.e., fucoxanthin chlorophyll a/c binding proteins (FCPs). Structural analyses of photosystem II (PSII)-FCPII and photosystem I (PSI)-FCPI complexes from the diatom Chaetoceros gracilis have revealed the localization and interactions of many FCPs; however, the entire set of FCPs has not been characterized. Here, we identified 46 FCPs in the newly assembled genome and transcriptome of C. gracilis. Phylogenetic analyses suggested that these FCPs could be classified into five subfamilies: Lhcr, Lhcf, Lhcx, Lhcz, and novel Lhcq, in addition to a distinct type of Lhcr, CgLhcr9. The FCPs in Lhcr, including CgLhcr9 and some Lhcqs, had orthologous proteins in other diatoms, particularly those found in the PSI-FCPI structure. By contrast, the Lhcf subfamily, some of which were found in the PSII-FCPII complex, seemed to be diversified in each diatom species, and the number of Lhcqs differed among species, indicating that their diversification may contribute to species-specific adaptations to light. Further phylogenetic analyses of FCPs/light-harvesting complex (LHC) proteins using genome data and assembled transcriptomes of other diatoms and microalgae in public databases suggest that our proposed classification of FCPs was common among various red-lineage algae derived from secondary endosymbiosis of red algae, including Haptophyta. These results provided insights into the loss and gain of FCP/LHC subfamilies during the evolutionary history of the red algal lineage.

One sentence summaryPhylogenetic analysis of fucoxanthin-chlorophyll a/c proteins in C. gracilis revealed five major subfamilies and one minor subfamily, providing insights into the diversification of light-harvesting systems in red algae.

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The mitogen-activated protein kinase kinase kinase, ILK5, regulates plant purinergic receptor-mediated, innate immunity Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to elevated defense responses in Arabidopsis. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we identified Raf-like MAPKKK ILK5 as a downstream substrate of the P2K1 kinase. P2K1 phosphorylates ILK5 on serine 192. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses, such as wounding. ILK5 interacts with both MKK4 and MKK5, but only MKK5 is phosphorylated by ILK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type. The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to wild-type plants. Plants expressing only the mutant ILK5S192A protein, lacking kinase activity, did not activate the MAPK cascade upon ATP addition. Taken together, the results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MAPK3 and 6 associated with an elevated innate immunity response.

Significance statementPathogens invasion and subsequent wound stress activates extracellular ATP-mediated purinergic signaling cascades, a danger associated molecular pattern (DAMP) signal, which induces phosphorylation of MAPKs. Previous studies revealed that the P2K1 purinergic receptor increases MPK3/6 phosphorylation in response to eATP signaling cascades in Arabidopsis. However, the specific mechanism by which this occurs remains unknown. Here, we describe the isolation and characterization of Raf-like MAPKKK ILK5 (Integrin-linked Kinase 5) as a downstream substrate of P2K1 kinase activity. Initiation of an eATP-dependent signaling pathway by phosphorylation of ILK5 with subsequent activation of MKK5, leading to activation of MPK3/6 and downstream events is crucial to the plant innate immunity response.

sentences: Effects of α-crystallin gene knockout on zebrafish lens development The -crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role the -crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three -crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of A-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either Ba- or Bb-crystallin produced no significant lens defects. Mutation of each -crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for A-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.

sentences: Hippocampal CA1 represents action and reward events instantly compared to the superficial and deep layers of the lateral entorhinal cortex The entorhinal cortex (EC) is the main interface between the hippocampus and the neocortex. The EC plays a critical role in learning and memory. We investigated the neuronal representation of behavioral events during operant learning in the hippocampal-entorhinal circuit of head-fixed rats. Both CA1 and lateral entorhinal cortex (LEC) neurons develop task-related activities after learning. Among diverse task-related activities, we compared the transient peak activities that represent action and reward and found a distinct difference in the timing of behavioral event representation between CA1 and LEC. CA1 represents action and reward events in close to real-time, whereas both the superficial and deep layers of the LEC showed delayed representation of those events. Our results suggest that subpopulations exist within which CA1 and LEC neurons process the information in a different order from the anatomically defined hippocampal-entorhinal circuit.

sentences: Sex Differences in Cortical Morphometry during Ageing: Examining the Interplay between Lifestyle and Reproductive Factors Sex differences in neurodegenerative disorder prevalence have been attributed to life expectancy, modifiable risk factors related to lifestyle and the impact of changes in sex hormones and the reproductive system. Although these factors are known to interact with one another, they are often studied in isolation. Here, we used a multivariate approach to investigate how lifestyle, along with menopause and the number of children, interacts with cortical thickness (CT) in healthy adults. Using CT measures from T1-weighted scans (MPRAGE, 1 mm3 voxels; 124 participants; 67 females; 40-70 years old) from the Cam-CAN dataset. Using a partial least squares decomposition, we identified patterns of covariance between CT and lifestyle factors, menopause and the number of children. In women, we identified significant patterns that linked education, socioeconomic status, social contact and length of reproductive period to CT in the left prefrontal cortex, as well as alcohol consumption, physical activity and menopausal status to CT in the frontal poles. Contrastingly, the results in men were driven by education and anxiety, and involved increased CT in the temporal poles. Our findings suggests that sex differences in cortical anatomy during brain ageing might be driven by interactions between contrasting lifestyles and the female-specific endocrine environment.

sentences: The genetic basis of structural colour variation in mimetic Heliconius butterflies Structural colours, produced by the reflection of light from ultrastructures, have evolved multiple times in butterflies. Unlike pigmentary colours and patterns, little is known about the genetic basis of these colours. Reflective structures on wing-scale ridges are responsible for iridescent structural colour in many butterflies, including the Mullerian mimics Heliconius erato and Heliconius melpomene. Here we quantify aspects of scale ultrastructure variation and colour in crosses between iridescent and non-iridescent subspecies of both of these species and perform quantitative trait locus (QTL) mapping. We show that iridescent structural colour has a complex genetic basis in both species, with offspring from crosses having wide variation in blue colour (both hue and brightness) and scale structure measurements. We detect two different genomic regions in each species that explain modest amounts of this variation, with a sex-linked QTL in H. erato but not H. melpomene. We also find differences between species in the relationships between structure and colour, overall suggesting that these species have followed different evolutionary trajectories in their evolution of structural colour. We then identify genes within the QTL intervals that are differentially expressed between subspecies and/or wing regions, revealing likely candidates for genes controlling structural colour formation.

sentences: TAOK2 is an ER-localized Kinase that Catalyzes the Dynamic Tethering of ER to Microtubules The endoplasmic reticulum (ER) depends on extensive association with the microtubule cytoskeleton for its structure, function and mitotic inheritance. The identity of molecular tethers that mediate ER-microtubule coupling, and mechanisms through which dynamic tethering is regulated are poorly understood. Here, we identify, Thousand And One amino acid Kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to microtubules. We show that TAOK2 is a unique multipass membrane spanning serine/threonine kinase localized in distinct ER domains via four transmembrane and amphipathic helices. Using in vitro and cellular assays, we find that TAOK2 directly binds microtubules with high affinity. We define the minimal TAOK2 determinants that induce ER-microtubule tethering, and delineate the mechanism for its autoregulation. While ER membrane dynamics are increased in TAOK2 knockout cells, the movement of ER along growing microtubule plus-ends is disrupted. We show that ER-microtubule tethering is tightly regulated by catalytic activity of TAOK2 in both interphase and mitotic cells, perturbation of which leads to profound defects in ER morphology and cell division. Our study identifies TAOK2 as an ER-microtubule tether, and reveals a kinase-regulated mechanism for control of ER dynamics critical for cell growth and division.

sentences: Evolution of lower levels of inter-locus sexual conflict in D. melanogaster populations under strong selection for rapid development. D. melanogaster laboratory populations subjected to selection for rapid development and early reproduction have been found to have evolved reduced adult body size and lower levels of inter-locus sexual conflict compared to their ancestral controls. We tested the contribution of a smaller body to the evolution of reduced sexual conflict in these populations, since body size differences are known to affect sexual conflict levels in this species. We cultured larvae from the control populations at high density to obtain flies as small as those from the selected populations. The effect of body size reduction on sexual conflict was asymmetric, with smaller body size resulting in reduced male manipulative ability but not female resistance to mating-induced harm. These results were not due to differences in behavioural patterns of smaller flies, such as differences in overall mating exposure of females to different types of males. We hypothesize that evolution for rapid development and the correlated reduction in body size has resulted in lower male manipulative ability, and sexually antagonistic co-evolution has lowered female resistance to such manipulations. These populations have also evolved incipient reproductive isolation from their controls, likely through sexual conflict (reported earlier), and our results support the view that this is an outcome of strong, directional selection for rapid development.

sentences: Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15 Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 minutes, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-sites were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 Ser5, PAK2 Ser141, and STK17B Ser12. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.

sentences: The isoforms of pyruvate kinase act as nutrient sensors for the β-cell KATP channel Insulin release from pancreatic {beta}-cells is triggered by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels. Here, we used isoform-specific deletion of pyruvate kinase (PK) to identify the unique roles of PKm1 and PKm2 in the {beta}-cell nutrient response. In the absence of PK activity, cytosolic ATP/ADP was insufficient to close KATP channels. Both PK isoforms were found on the plasma membrane, where they endow KATP channels with nutrient sensitivity and specificity. Although PKm1 accounts for >90% of cellular activity, PKm2 controls glucosedependent KATP closure. Amino acids, which generate mitochondrial phosphoenolpyruvate (PEP) without allosterically activating PKm2, depend on PKm1 to raise ATP/ADP and close KATP channels. Shifting the {beta}-cell from PKm2 to PKm1 correspondingly increased amino acid sensitivity and boosted secretory output. In addition to these membrane depolarizing "on-switches," {beta}-cell deletion of PCK2 exposed a mitochondrial PEP-dependent "off-switch" that facilitates Ca2+ extrusion. Restricting PCK2 prolonged Ca2+ influx and increased insulin secretion. In summary, it is not the KATP channel alone, but the PK-KATP microcompartment that confers nutrient sensitivity to the {beta}-cell plasma membrane. The differential response of the PK isoforms to the glycolytic and mitochondrial sources of PEP influences {beta}-cell nutrient preference, and controls the oscillatory cycle regulating insulin secretion.

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High levels of cyclic diguanylate interfere with beneficial bacterial colonization During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a transcriptional regulatory interaction between two key polysaccharides, cellulose and Syp. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates.

IMPORTANCEThere is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing Syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.

sentences: Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates Malformations of or injuries to the developing lung are associated with perinatal morbidity and mortality with lifelong consequences for subsequent pulmonary health. One fetal exposure linked with poor health outcomes is chorioamnionitis, which impacts up to 25-40% of preterm births. Severe chorioamnionitis with prematurity is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may significantly alter developmental ontogeny of the lung. To test this hypothesis, we used intra-amniotic lipopolysaccharide (LPS, endotoxin) to generate experimental chorioamnionitis in prenatal Rhesus macaque (Macaca mulatta), a model which shares critical structural and temporal aspects of human lung development. Inflammatory injury directly disrupts the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis which was extensively disrupted by perinatal inflammation, leading to loss of gas exchange surface and alveolar simplification similar to that found in chronic lung disease of newborns. Blockade of IL1{beta} and TNF ameliorated endotoxin-induced inflammatory lung injury by blunting stromal response to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

sentences: Double mutant of chymotrypsin inhibitor 2 stabilized through increased conformational entropy The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side-chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: A reduction in conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

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Probiotic consortia improve anti-viral immunity to SARS-CoV-2 in ferrets Probiotics have been suggested as one solution to counter detrimental health effects by SARS-CoV-2, however, data so far is scarce. We tested the effect of two probiotic consortia, OL-1 and OL-2, against SARS-CoV-2 in ferrets and assessed their effect on cytokine production and transcriptome in a human monocyte-derived macrophage (Mf) and dendritic cell (DC) model.

The results showed that the consortia significantly reduced the viral load, modulated immune response, and regulated viral receptor expression in ferrets compared to placebo. In human Mf and DC model, OL-1 and OL-2 induced cytokine production and genes related to SARS-CoV-2 anti-viral immunity.

The study results indicate that probiotic stimulation of the ferret immune system leads to improved anti-viral immunity against SARS-COV-2 and that critical genes and cytokines for anti-SARS-CoV-2 immunity are stimulated in human immune cells in vitro. The effect of the consortia against SARS-CoV-2 warrants further investigations in human clinical trials.

sentences: NDR1 and the Arabidopsis Plasma Membrane ATPase AHA5 are Required for Processes that Converge on Drought Tolerance and Immunity NON-RACE-SPECIFIC DISEASE RISISTANCE1 (NDR1) is a key component of plant immune signaling, required for defense against the bacterial pathogen Pseudomonas syringae. Plant stress responses have overlapping molecular, physiological, and cell biology signatures, and given the central role of NDR1 during biotic stress perception and signaling, we hypothesized that NDR1 also functions in abiotic stress responses, including in a role that mediates signaling at the plasma membrane (PM) - cell wall (CW) continuum. Here, we demonstrate that NDR1 is required for the induction of drought stress responses in plants, a role that couples stress signaling in an abscisic acid-dependent manner. We show that NDR1 physically associates with the PM-localized H+-ATPases AHA1, AHA2, and AHA5 and is required for proper regulation of H+-ATPase activity and stomatal guard cell dynamics, providing a mechanistic function of NDR1 during drought responses. In the current study, we demonstrate that NDR1 functions in signaling processes associated with both biotic and abiotic stress response pathways, a function we hypothesize represents NDR1s role in the maintenance of cellular homeostasis during stress. We propose a role for NDR1 as a core transducer of signaling between cell membrane processes and intercellular stress response activation.

sentences: Modulating the left inferior frontal cortex by task domain, cognitive challenge and tDCS The left inferior frontal cortex (LIFC) is a key region for spoken language processing, but its neurocognitive architecture remains controversial. Here we assess the domain-generality vs. domain-specificity of the LIFC from behavioural, functional neuroimaging and neuromodulation data. Using concurrent fMRI and transcranial direct current stimulation (tDCS) delivered to the LIFC, we investigated how brain activity and behavioural performance are modulated by task domain (naming vs. non-naming), cognitive challenge (low vs. high), and tDCS (anodal vs. sham). The data revealed: (1) co-existence of neural signatures both common and distinct across tasks within the LIFC; (2) domain-preferential effects of task (naming); (3) significant tDCS modulations of activity in a LIFC sub-region selectively during high-challenge naming. The presence of both domain-specific and domain-general signals, and the existence of a gradient of activation where naming relied more on sub-regions within the LIFC, may help reconcile both perspectives on spoken language processing.

sentences: GPX4-associated Sedaghatian Type Spondylometaphyseal Dysplasia: A Protein Interactome Perspective Spondylometaphyseal dysplasia, Sedaghatian type (SMDS) is a rare and lethal skeletal dysplasia inherited in an autosomal recessive manner and caused by mutations in GPX4. In order to expand the functional landscape of this poorly studied disorder and accelerate the discovery of biologically insightful and clinically actionable targets, we constructed SMDS-centric and GPX4-centric protein-protein interaction (PPI) networks, augmented with novel protein interactors predicted by our HiPPIP algorithm. The SMDS-centric networks included those that showed the interconnections of GPX4 with other putative SMDS-associated genes and genes associated with other skeletal dysplasias. The GPX4-centric network showed the interconnections of GPX4 with genes whose perturbation has been known to affect GPX4 expression. We discovered that these networks either contained or were enriched with genes associated with specific SMDS pathophenotypes, tissue-naive/fetus-specific functional modules and genes showing elevated expression in brain and/or testis similar to GPX4. We identified 7 proteins as novel interactors of GPX4 (APBA3, EGR4, FUT5, GAMT, GTF2F1, MATK and ZNF197) and showed their potential biological relevance to GPX4 or SMDS. Comparative transcriptome analysis of expression profiles associated with chondroplasia and immune-osseous dysplasia versus drug-induced profiles revealed 11 drugs that targeted the neighborhood network of GPX4 and other putative SMDS-associated genes. Additionally, resveratrol, which is currently being tested against a skeletal dysplasia in a clinical trial, was identified as another potential candidate based on the proximity of its targets to GPX4.

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Altered neurogenic pathways in experimental rat models of ischemic cerebral stroke: A transcriptome based meta-analysis 1)ObjectivesPromoting neurogenesis mediated recovery is one of the most sought after strategies in recovery after cerebral stroke. In this paper we elucidate how neurogenesis related genes are altered in the early stroke environment, to hint at potential pathways for therapeutic recovery.

Materials and MethodsAround 97 microarray datasets derived from stroke affected rat brains were collected from NCBI-GEO. Datasets were normalized and subjected to meta-analysis in Network Analyst to identify differentially expressed genes. Gene enrichment analyses were carried out using GSEA, and WebGestalt, and results were visualized using Cytoscape Enrichment mapping.

ResultsNearly 939 differentially expressing genes were identified in the cerebral stroke group. Among them 30 neurogenesis related genes were identified through enrichment mapping analysis, and 35 genes through Protein- Protein Interaction analysis. Highest upregulated neurogenesis genes were found to be TSPO, GFAP, VIM, and TGFB1. Highest Downregulated neurogenesis genes were found to be THY1, NR1D1, CDK5, STX1B, and NOG.

ConclusionsThrough this study, we have identified that during the acute time frame after stroke, majority of the neurogenesis genes related to neural proliferation and neural differentiation are downregulated, while the majority of the genes related to neuronal migration were upregulated. Single or combined therapeutic approach against the identified dysregulated genes could greatly aid neural restoration and functional recovery during the postischemic stage.

sentences: Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.

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conST: an interpretable multi-modal contrastive learning framework for spatial transcriptomics MotivationSpatially resolved transcriptomics (SRT) shows its impressive power in yielding biological insights into neuroscience, disease study, and even plant biology. However, current methods do not sufficiently explore the expressiveness of the multi-modal SRT data, leaving a large room for improvement of performance. Moreover, the current deep learning based methods lack interpretability due to the "black box" nature, impeding its further applications in the areas that require explanation.

ResultsWe propose conST, a powerful and flexible SRT data analysis framework utilizing contrastive learning techniques. conST can learn low-dimensional embeddings by effectively integrating multi-modal SRT data, i.e. gene expression, spatial information, and morphology (if applicable). The learned embeddings can be then used for various downstream tasks, including clustering, trajectory and pseudotime inference, cell-to-cell interaction, etc. Extensive experiments in various datasets have been conducted to demonstrate the effectiveness and robustness of the proposed conST, achieving up to 10% improvement in clustering ARI in the commonly used benchmark dataset. We also show that the learned embedding can be used in complicated scenarios, such as predicting cancer progression by analyzing the tumour microenvironment and cell-to-cell interaction (CCI) of breast cancer. Our framework is interpretable in that it is able to find the correlated spots that support the clustering, which matches the CCI interaction pairs as well, providing more confidence to clinicians when making clinical decisions.

sentences: Tumor-Antagonizing Fibroblasts Secrete Prolargin as Tumor Suppressor in Hepatocellular Carcinoma Systemic treatment of hepatocellular carcinoma (HCC) targets the tumor microenvironment (TME) by combining immunotherapy with angiogenesis inhibitors. Cancer-associated fibroblasts (CAF) are important components of the TME, however their targeting in clinics remains challenging. To this end, the existence of tumor supressing CAF and their poor characterisation is a major conundrum. Starting from proteomics and single cell analysis, we outline CAF heterogeneity in HCC and describe a subtype of CAF that express a novel tumor-related protein prolargin. Upon secretion prolargin is deposited in the TME where its levels positively correlate with patient outcome (HR=0.37; p=0.01). In vivo, tumors with lower prolargin expression display faster progression (5-fold; p=0.01) and stronger angiogenesis. Mechanistically, aggressive HCC cells degrade prolargin using matrix metalloprotease 3 (MMP3). We show that prolargin binds and inhibits several growth factors, key to tumor progression. Inhibiting prolargin degradation combined with sorafenib, demonstrated superior tumor control compared to sorafenib treatment alone. In conclusion, prolargin-expressing CAF have tumor-antagonizing properties. Stabilizing prolargin tumoral levels should be considered for systemic therapy of HCC, involving CAF in existing TME targeting.

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HSP90.6 is involved in grain filling via carbon and nitrogen metabolism in maize. Carbon and nitrogen are the two most abundant nutrients in all living things, and their metabolism maintains normal plant growth. However, the molecular mechanism underlying carbon and nitrogen metabolism remains largely unknown. Here, we found that HSP90.6 is involved in the metabolism of carbon and nitrogen. We performed gene cloning and functional characterization of a maize EMS mutant ehsp90.6, whose kernels were small. HSP90.6 encodes heat shock protein 90.6, which has a single-amino acid mutation within its HATPase_c domain. Transcriptome profiling showed that the expression of amino acid biosynthesis- and carbon metabolism-related genes was significantly downregulated in hsp90.6. HSP90.6 is involved in the 26S proteasome degradation pathway, which affects nitrogen recycling to regulate amino acid synthesis; this occurs by interactions between HSP90.6 and the 26S proteasome subunits RPN6 and PBD2 (PRC2). The loss of HSP90.6 significantly reduced the activity of the 26S proteasome, resulting in the accumulation of ubiquitinated proteins and defects in nitrogen recycling. Moreover, HSP90.6 interacted with the 14-3-3 protein GF14-6 to participate in carbon metabolism. Together, these findings revealed that HSP90.6 regulates nutrient metabolism in maize seeds by affecting 26S proteasome-mediated nitrogen recycling and GF14-6-mediated carbon metabolism.

One sentence summaryHSP90.6 is involved in nutrient metabolism via 26S proteasome-mediated protein degradation to promote nitrogen recycling and GF14-6 protein-mediated carbon metabolism.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is Weibin Song (songwb@cau.edu.cn).

HighlightsO_LIHATPase_c is necessary for HSP90.6 to regulate maize kernel development.
C_LIO_LIHSP90.6 is involved in nitrogen recycling via the 26S proteasome degradation pathway.
C_LIO_LIHSP90.6 interacts with the 14-3-3 protein GF14-6 to affect carbon metabolism.
C_LI

IN A NUTSHELLO_ST_ABSBackgroundC_ST_ABSSeeds are the main harvested organs of maize. Understanding the regulatory mechanism of grain filling is helpful to cultivate high-quality and high-yield maize. In the past few years, the regulatory network of grain filling has been explored through multiple means, including transcriptomic, proteomic and functional genomic techniques. Many genes that control grain filling through different mechanisms have been cloned, such as CTLP1 (Choline Transporter-like Protein 1), OS1 (Opaque Endosperm and Small Germ 1), and MN6 (Miniature Seed6). To identify new genes involved in maize grain filling, ethyl methanesulfonate (EMS) was used to induce mutations, and the ehsp90.6 mutant, which exhibited abnormal kernel development, was isolated by bulked segregant analysis RNA sequencing (BSR).

QuestionWhy does the single-amino acid mutation of HSP90.6 affect grain size, and how does the loss of HSP90.6 affect grain filling?

FindingsA single-amino acid mutant (ehsp90.6) and knockout mutant (hsp90.6) were obtained. We found that HSP90-6 was involved in the regulation of maize grain filling. A single-single amino acid mutation in the HATPase_c domain reduced the ATPase activity of HSP90.6, resulting in smaller grains. The functional loss of HSP90.6 resulted in the expression of amino acid biosynthesis- and carbon metabolism-related genes being significantly downregulated in hsp90.6. We indicated that HSP90.6 is involved in the 26S proteasome degradation pathway, which affects nitrogen recycling to regulate amino acid synthesis by interacting with the 26S proteasome subunits RPN6 and PBD2 (PRC2). Moreover, HSP90.6 was found to interact with the 14-3-3 protein GF14-6 to participate in carbon metabolism.

Next stepsTo further verify that the interaction between HSP90.6 and 26S proteasome subunits and GF14-6 affects grain filling, knockout validation of RPN6, PBD2 (PRC2) and GF14-6 will be performed. In addition, since GF14-6 interacts with the phosphorylated proteins, we will determine the phosphorylation site of HSP90.6. Due to the important role of HSP90 family proteins in plant development, there are other regulatory pathways that need to be explored.

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SARS-CoV-2 mutations affect proteasome processing to alter CD8+ T cell responses Viral CD8+ epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8+ epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs.

One Sentence SummaryNatural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8+ T cell responses leading to partial escape.

sentences: Astrocytes regulate inhibition in Fragile X Syndrome Fragile X syndrome (FXS) is a leading genetic cause of autism-like symptoms that include sensory hypersensitivity and cortical hyperexcitability. Recent observations in humans and Fmr1 knockout (KO) animal models of FXS suggest abnormal GABAergic signaling. As most studies focused on neuron-centered mechanisms, astrocytes contribution to abnormal inhibition is largely unknown. Here we propose a non-neuronal mechanism of abnormal inhibitory circuit development in FXS. Astrocyte-specific deletion of Fmr1 during postnatal period leads to increased astrocytic GABA levels, but negatively impacts synaptic GABAA receptors and parvalbumin (PV) cell development. Developmental deletion of Fmr1 from astrocytes also affects communications between excitatory neurons and PV cells, impairing sound-evoked gamma synchronization in the cortex, while enhancing baseline and on-going sound-evoked EEG power, and leading to increased locomotor activity and altered social behaviors in adult mice. These results demonstrate a profound role of astrocytic FMRP in the development of inhibitory circuits and shaping normal inhibitory responses.

sentences: Systematic in vivo interrogation identifies novel enhancers and silencers associated to Atrial Fibrillation Cis-regulatory elements control gene expression in time and space and their disruption can lead to pathologies. Reporter assays allow the functional validation of enhancers and other regulatory elements, and such assays by means of the generation of transgenic mice provide a powerful tool to study gene regulation in development and disease. However, these experiments are time-consuming and, thus, their performance is very limited. Here, we increase the throughput of in vivo mouse reporter assays by using a piggyBac transposon-based system, and use it to decode the regulatory landscape of atrial fibrillation, a prevalent cardiac arrhythmia. We systematically interrogated ten human loci associated to atrial fibrillation in the search for regulatory elements. We found five new cardiac-specific enhancers and implicated novel genes in arrhythmia through genome editing and three-dimensional chromatin analysis by 4C-seq. Of note, functional dissection of the 7q31 locus identified a bivalent regulatory element in the second intron of the CAV1 gene differentially acting upon four genes. Our system also detected negative regulatory elements thanks to which we identified a ubiquitous silencer in the 16q22 locus that regulates ZFHX3 and can outcompete heart enhancers. Our study characterizes the function of new genetic elements that might be of relevance for the better understanding of gene regulation in cardiac arrhythmias. Thus, we have .established a new framework for the efficient dissection of the genetic contribution to common human diseases.

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Conserved mechanism for perception of root-active CLE peptides Precision in sensing the environmental cues and adjusting the growth and the physiology of the root system are necessary for plant robustness. Plants achieve their phenotypic plasticity by tightly controlling and buffering developmental decisions. In addition to the classical plant hormones that mediate plant development and stress responses, the CLE peptides constitute an additional crucial level of regulation. While the CLV3-CLV1 module appears to be highly conserved to control the proliferation of the shoot apical meristem stem cells, we do not yet fully understand the function of the additional CLE genes and whether they act in a similar way across the plant species, including tomato. Due to the small gene size and high sequence variability, it is extremely difficult to precisely annotate CLE genes in plant genomes. Here we present our analysis of the CLE family in tomato, based on a combination of iterative tBLASTn and Hidden-Markov-Model (HMM), which allowed us to identify thirty-seven new SlCLEs in addition to the fifteen reported previously. We could confirm the biological activities of selected SlCLEs in suppressing root meristematic cell divisions. We show that root response is mediated by SlCLAVATA2, indicating the conservation of CLE perception mechanism.

One-sentence summaryUsing a combination of iterative tBLASTn and Hidden-Markov-Model approaches, we uncovered 37 new tomato CLE genes predominantly expressed in roots, and we showed a conserved effect on root meristem arrest, that was SlCLAVATA2-dependent.

HighlightsO_LIWe applied a combined approach of iterative tBLASTn and Hidden-Markov-Model to identify fifty-two tomato SlCLE genes, including thirty-seven new genes
C_LIO_LIAll identified genes encode for pre-propeptides with a single CLE-domain containing conserved residues similar to Arabidopsis
C_LIO_LIAnalyzing the publicly available RNAseq datasets, we could confirm the expression of SlCLE genes that was often associated specifically with root or shoot, a certain developmental stage of the fruit, or with drought stress conditions
C_LIO_LIRemarkably, the majority of SlCLE genes are predominantly expressed in the root tissues
C_LIO_LIWe showed the conserved inhibitory effect on the root meristem and columella cells division for the selected SlCLE peptides that were SlCLAVATA2-dependent.
C_LI

sentences: Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring Aquatic environmental DNA (eDNA) surveys are transforming how we monitor marine ecosystems. The time-consuming pre-processing step of active filtration, however, remains a bottleneck. Hence, new approaches omitting active filtration are in great demand. One exciting prospect is to use the filtering power of invertebrates to collect eDNA. While proof-of-concept has been achieved, comparative studies between aquatic and filter feeder eDNA signals are lacking. Here, we investigated the differences among four eDNA sources (water; bivalves; sponges; and ethanol in which filter-feeding organisms were stored) along a vertical transect in Doubtful Sound, New Zealand using three metabarcoding primers (fish (16S); MiFish-E/U). While concurrent SCUBA diver observations validated eDNA results, laboratory trials corroborated in-field bivalve eDNA detection results. Combined, eDNA sources detected 59 vertebrates, while divers observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was detected in ethanol, although only a reduced number of species were detected. Bivalves failed to reliably detect eDNA in both field and mesocosm experiments. While additional research into filter feeder eDNA accumulation efficiency is essential, our results provide strong evidence for the potential of incorporating sponges into eDNA surveys.

sentences: A synthetic RNA-mediated evolution system in yeast Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in bakers yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.

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Synergistic TOR and ERK inhibition mitigates the hereditary haemorrhagic telangiectasia-like phenotype and excess kugel formation in endoglin mutant zebrafish RationaleHereditary haemorrhagic telangiectasia (HHT) is an inherited bleeding disorder characterised by arteriovenous malformations (AVMs). Such AVMs affect lungs, liver and brain, whilst telangiectases in mucocutaneous tissues are prone to haemorrhage. HHT type I is caused by loss-of-function endoglin (ENG) mutations. Evidence suggests AVMs result from abnormal responses to VEGF signalling.

ObjectiveWe therefore characterised the vascular abnormalities in eng mutant zebrafish and investigated whether these are prevented by inhibiting different pathways downstream of VEGF signalling.

Methods and ResultsWe used light sheet fluorescence microscopy to visualise the vasculature in engmu130 mutant zebrafish. In addition to previously described significantly enlarged dorsal aorta and posterior cardinal vein at 3d post fertilisation, engmu130 embryos had an enlarged basilar artery (BA), and increased formation of endothelial "kugeln" on cerebral vessels. Adult engmu130 fish developed skin AVMs, retinal vascular abnormalities, and an enlarged heart. Tivozanib (AV951), a VEGF receptor tyrosine kinase inhibitor, prevented development of the abnormally enlarged major vessels and normalised the number of kugeln in engmu130 embryos. Inhibiting discrete signalling pathways downstream of VEGFR2 in engmu130 embryos gave further insights. Inhibiting TOR or MEK prevented the abnormal trunk and cerebral vasculature phenotype, whilst targeting NOS and MAPK had no effect. Combining subtherapeutic TOR and MEK inhibition prevented the vascular phenotype, suggesting synergy between TOR and MEK/ERK signalling pathways.

ConclusionsThese results indicate the HHT-like phenotype in zebrafish endoglin mutants can be mitigated through modulation of VEGF signalling, and implicate combination low dose ERK and TOR pathway inhibitors as a therapeutic strategy in HHT.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Recurrent emergence of carbapenem resistance in Klebsiella pneumoniae mediated by an inhibitory ompK36 mRNA secondary structure Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae (KP), modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. Analysis of large KP genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine to thymine transition at position 25 (25c>t) in ompK36. We show that the 25c>t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates KP in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c>t transition tips the balance towards treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c>t transition mediates an intramolecular mRNA interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

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A gap-filling algorithm for prediction of metabolic interactions in microbial communities The study of microbial communities and their interactions has attracted the interest of the scientific community, because of their potential for applications in biotechnology, ecology and medicine. The complexity of interspecies interactions, which are key for the macroscopic behavior of microbial communities, cannot be studied easily experimentally. For this reason, the modeling of microbial communities has begun to leverage the knowledge of established constraint-based methods, which have long been used for studying and analyzing the microbial metabolism of individual species based on genome-scale metabolic reconstructions of microorganisms. A main problem of genome-scale metabolic reconstructions is that they usually contain metabolic gaps due to genome misannotations and unknown enzyme functions. This problem is traditionally solved by using gap-filling algorithms that add biochemical reactions from external databases to the metabolic reconstruction, in order to restore model growth. However, gap-filling algorithms could evolve by taking into account metabolic interactions among species that coexist in microbial communities. In this work, a gap-filling method that resolves metabolic gaps at the community level was developed. The efficacy of the algorithm was tested by analyzing its ability to resolve metabolic gaps on a synthetic community of auxotrophic Escherichia coli strains. Subsequently, the algorithm was applied to resolve metabolic gaps and predict metabolic interactions in a community of Bifidobacterium adolescentis and Faecalibacterium prausnitzii, two species present in the human gut microbiota, and in an experimentally studied community of Dehalobacter and Bacteroidales species of the ACT-3 community. The community gap-filling method can facilitate the improvement of metabolic models and the identification of metabolic interactions that are difficult to identify experimentally in microbial communities.

Author summaryMicrobes compose the most abundant form of life on our planet and they are almost never found in isolation, as they live in close association with one another and with other organisms. The metabolic capacity of individual microbial species dictates their ways of interaction with other species as well as with their environment. The metabolic interactions among microorganisms have been recognised as the driving force for the emergence of the properties of microbial communities. For this reason, understanding the effect of microbial metabolism on interspecies metabolic interactions is detrimental for the study of microbial communities. This study can be benefited by metabolic modeling and the insights offered by constraint-based methods which have been developed for interrogating metabolic models. In this paper, we present an algorithm that predicts cooperative and competitive metabolic interactions between species, while it resolves metabolic gaps in their metabolic models in a computationally efficient way. We use our community gap-filling algorithm to study microbial communities with interesting environmental and health-related applications.

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Host transcriptional response to SARS-CoV-2 infection in COVID-19 patients BackgroundOne of the most perplexing aspects of infection with the SARS-CoV-2 virus has been the variable response elicited in its human hosts. Investigating the transcriptional changes in individuals affected by COVID-19 can help understand and predict the degree of illness and guide clinical outcomes in diverse backgrounds.

MethodsAnalysis of host transcriptome variations via RNA sequencing from naso/oropharyngeal swabs of COVID-19 patients.

ResultsWe report strong upregulation of the innate immune response, especially type I interferon pathway, upon SARS-CoV-2 infection. Upregulated genes were subjected to a comparative meta-analysis using global datasets to identify a common network of interferon stimulated and viral response genes that mediate the host response and resolution of infection. A large proportion of mis-regulated genes showed a reduction in expression level, suggesting an overall decrease in host mRNA production. Significantly downregulated genes included those encoding olfactory, taste and neuro-sensory receptors. Many pro-inflammatory markers and cytokines were also downregulated or remained unchanged in the COVID-19 patients. Finally, a large number of non-coding RNAs were identified as down-regulated, with a few of the lncRNAs associated with functional roles in directing the response to viral infection.

ConclusionsSARS-CoV-2 infection results in the robust activation of the bodys innate immunity. Reduction of gene expression is well correlated with the clinical manifestations and symptoms of COVID-19 such as the loss of smell and taste, and myocardial and neurological complications. This study provides a critical dataset of genes that will enhance our understanding of the nature and prognosis of COVID-19.

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Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation.

Author summaryTick-borne encephalitis virus is an important human pathogen that can cause severe infection often resulting in life-long neurological complications or even death. As with other viruses, it fully relies on the host cells, and any successful infection starts with interactions between the viral structural proteins and cellular surface proteins. Mapping these interactions is essential both for the fundamental understanding of viral entry mechanisms, and for guiding the design of new antiviral drugs and vaccines. Here, we stabilise the interactions between tick-borne encephalitis virus and human proteins by chemical cross-linking. We then detect the interactions using mass spectrometry and analyse the data to identify protein-protein complexes. We demonstrate that we can visualise the protein interaction interfaces by mapping the cross-linked sites onto the host and viral protein structures. We reveal that there are eight distinct sites on the outer surface of the viral envelope protein that interact with host. Using this approach, we mapped interactions between the tick-borne encephalitis virus envelope protein, and 59 host proteins, identifying a possible new virus receptor. These results highlight the potential of chemical cross-linking coupled with mass spectrometry to identify and map interactions between viral and host proteins.

sentences: The Arabidopsis F-box protein FBW2 degrades AGO1 to avoid spurious loading of illegitimate small RNA RNA silencing is a conserved mechanism in eukaryotes and is involved in development, heterochromatin maintenance and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA (miRNA) and small interfering RNA (siRNA)-directed silencing and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 targets proteolysis of AGO1 by a CDC48-mediated mechanism. We found that FBW2 assembles an SCF complex that recognizes the MID-PIWI domain of AGO1 and requires its C-terminal domain containing a GW motif for AGO1 turnover. We showed that FBW2 prefers the unloaded and some mutated forms of AGO1 protein. While FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA silencing activity. Interestingly, under conditions in which small RNA production or accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Hence, the non-degradable AGO1 protein assembles high molecular weight complexes and binds illegitimate small RNA leading to the cleavage of new target genes that belong to stress responses and cellular metabolic processes. Therefore, the control of AGO1 homeostasis by ubiquitin ligases plays an important role in quality control to avoid off-target cleavage.

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Learning Rates Are Not All the Same: The Interpretation of Computational Model Parameters Depends on the Context Reinforcement Learning (RL) models have revolutionized the cognitive and brain sciences, promising to explain behavior from simple conditioning to complex problem solving, to shed light on developmental and individual differences, and to anchor cognitive processes in specific brain mechanisms. However, the growing RL literature shows increasingly contradictory results, potentially casting doubt on the usefulness of the method. We hypothesized that the observed contradictions instead arise from two fundamental assumptions about computational model parameters that are overly strong: That parameters generalize between contexts (e.g., tasks, models) and that they capture interpretable neurocognitive processes. If these assumptions are corrected, many contradictions dissolve. To test this idea, we asked 291 participants aged 8-30 years to complete three learning tasks during a single experimental session, and fitted their behavior using state-of-the-art RL models. As predicted, we found less-than-perfect parameter generalization between tasks (with better results for RL decision noise/exploration than learning rates) and a fundamental lack of interpretability (especially for learning rates), with a potential role of task similarity for generalization. We conclude that the systematic study of context (including task features and model parameterization) will enhance the generalizability of computational cognitive models.

HighlightsO_LIMany efforts in computational cognitive modeling are based on two implicit assumptions: 1) Model parameters generalize between studies and models. 2) Parameters are interpretable, i.e., neurally, cognitively, and/or mechanistically specific.
C_LIO_LIWe empirically investigate these assumptions in Reinforcement Learning (RL), using a large developmental dataset featuring three different learning tasks in a within-participant design.
C_LIO_LIWe find that RL decision noise/exploration parameters generalize between tasks in terms of captured between-participant variation, but not in terms of absolute parameter values. RL learning rates and Forgetting parameters fail to generalize. All parameters interpretability is low.
C_LIO_LIThese findings are in accordance with previous claims that decision noise/exploration decreases during development, but suggest that claims about the development of learning rates or Forgetting should not be generalized between tasks: parameters are unlikely to capture the same neuro-cognitive processes.
C_LIO_LIWe suggest ways forward to improve the generalizability and interpretability of model parameters, in particular by focusing more on task context.
C_LI

sentences: Deep behavioral phenotyping of mouse autism models using open-field behavior AO_SCPLOWBSTRACTC_SCPLOWAutism is noted for both its genotypic and phenotypic diversity. Repetitive action, resistance to environmental change, and motor disruptions vary from individual to individual. In animal models, conventional behavioral phenotyping captures such fine-scale variations incompletely. Here we use advances in computer vision and deep learning to develop a framework for characterizing mouse behavior on multiple time scales using a single popular behavioral assay, the open field test. We observed male and female C57BL/6J mice to develop a dynamic baseline of adaptive behavior over multiple days. We then examined two rodent models of autism, a cerebellum-specific model, L7-Tsc1, and a whole-brain knockout model, Cntnap2. Both Cntnap2 knockout and L7-Tsc1 mutants showed forelimb lag during gait. L7-Tsc1 mutants showed complex defects in multi-day adaptation, lacking the tendency of wild-type mice to spend progressively more time in corners of the arena. In L7-Tsc1 mutant mice, failure-to-adapt took the form of maintained ambling, turning, and locomotion, and an overall decrease in grooming. Adaptation in Cntnap2 knockout mice more broadly resembled that of wild-type. L7-Tsc1 mutant and Cntnap2 knockout mouse models showed different patterns of behavioral state occupancy. Our automated pipeline for deep phenotyping successfully captures model-specific deviations in adaptation and movement as well as differences in the detailed structure of behavioral dynamics.

sentences: Plasmodium falciparum Acetyl-CoA Synthetase is essential for parasite intraerythrocytic development and chromatin modification The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS) which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation or ribozyme induced transcript decay were created. Additionally, an ACS inhibitor was tested for its effectiveness and specificity in interfering with PfACS. The knockdown of PfACS or its inhibition led to impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its significant differences to human ACS, PfACS seems an interesting target for drug development.

sentences: Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

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Connectome Spatial Smoothing (CSS): concepts, methods, and evaluation Structural connectomes are increasingly mapped at high spatial resolutions comprising many hundreds--if not thousands--of network nodes. However, high-resolution connectomes are particularly susceptible to image registration misalignment, tractography artifacts, and noise, all of which can lead to reductions in connectome accuracy and test-retest reliability. We investigate a network analogue of image smoothing to address these key challenges. Connectome Spatial Smoothing (CSS) involves jointly applying a carefully chosen smoothing kernel to the two endpoints of each tractography streamline, yielding a spatially smoothed connectivity matrix. We develop computationally efficient methods to perform CSS using a matrix congruence transformation and evaluate a range of different smoothing kernel choices on CSS performance. We find that smoothing substantially improves the identifiability, sensitivity, and test-retest reliability of high-resolution connectivity maps, though at a cost of increasing storage burden. For atlas-based connectomes (i.e. low-resolution connectivity maps), we show that CSS marginally improves the statistical power to detect associations between connectivity and cognitive performance, particularly for connectomes mapped using probabilistic tractography. CSS was also found to enable more reliable statistical inference compared to connectomes without any smoothing. We provide recommendations on optimal smoothing kernel parameters for connectomes mapped using both deterministic and probabilistic tractography. We conclude that spatial smoothing is particularly important for the reliability of high-resolution connectomes, but can also provide benefits at lower parcellation resolutions. We hope that our work enables computationally efficient integration of spatial smoothing into established structural connectome mapping pipelines.

HighlightsO_LIWe establish a network equivalent of image smoothing for structural connectomes.
C_LIO_LIConnectome Spatial Smoothing (CSS) improves connectome test-retest reliability, identifiability and sensitivity.
C_LIO_LICSS also facilitates reliable inference and improves power to detect statistical associations.
C_LIO_LIBoth high-resolution and atlas-based connectomes can benefit from CSS.
C_LI

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Upregulation of Somatostatin Receptor Type 2 in a Receptor-Deficient In Vivo Pancreatic Neuroendocrine Tumor Model Improves Tumor Response to Targeted 177Lu-DOTATATE PurposeThe goal of this study was to test whether histone deacetylase inhibitors (HDACis) restore somatostatin receptor type 2 (SSTR2) expression in models of high-grade pancreatic neuroendocrine tumors (PNETs), thereby facilitating effective treatment with 177Lu-DOTATATE therapy.

MethodsTo assess tumor grade correlation with SSTR2 expression, we assessed human SSTR2 promoter methylation and expression levels in 96 NIH patient samples and merged the GSE149395 and GSE117852 datasets. We used three NET cell lines (QGP-1, BON-1, GOT-1) characterized by variable SSTR2 expression profiles for functional in vitro studies using HDACis. Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to analyze the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies.

ResultsHuman PNET SSTR2 promoter methylation showed a significant positive correlation with higher tumor grades (P = 0.000014). We also found a significant negative correlation (P < 0.0001) between SSTR2 promoter methylation and SSTR2 expression in three NET cell lines. In vitro, SSTR2 expression increased significantly in BON-1 and QGP-1 cells at 48 and 72 hours in a dose-dependent fashion using two different HDACis, valproic acid and CI-994. In vivo studies demonstrated a significant increase in 177Lu-DOTATATE tumor uptake in QGP-1-engrafted mice after 10 days of CI-994 pretreatment (P = 0.0175). Treatment with 177Lu-DOTATATE reduced tumor size in mice pretreated with CI-994 compared to 177Lu-DOTATATE alone (at 15 days, P = 0.0028).

ConclusionHDACis increase SSTR2 surface expression in models of high-grade, SSTR2-deficient PNETs. This approach has the potential to improve tumor response to targeted therapy with 177 Lu-DOTATATE in patients with receptor-negative, metastatic PNETs.

Translational Relevance StatementPancreatic neuroendocrine tumors (PNETs) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. Unfortunately, high-grade PNETs lose SSTR2 surface expression and thus become ineligible for SSTR2-targeted 177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT). Restoring SSTR2 expression through the reversal of inhibitory epigenetic gene silencing mechanisms has the potential for improving tumor responsiveness to PRRT. We demonstrate that histone deacetylase inhibitors (HDACis) upregulate SSTR2 surface expression in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies validate a significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared to controls. Furthermore, we show that this higher tumor uptake results in significant anti-tumor response when compared to standard PRRT alone. Our preclinical results thus provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

sentences: Whole genome sequencing of three mesorhizobia isolated from northern Canada to identify genomic adaptations promoting nodulation in cold climates The N2-fixing symbiosis between rhizobia and legumes is negatively impacted by numerous stresses, including low temperatures. To identify genomic features and biochemical pathways of rhizobia that could foster improved symbiotic function under low temperatures, we isolated and characterized three Mesorhizobium strains from legume nodules collected at two distant northern Canadian sites. Whereas the classical determinants of nodulation and nitrogen fixation are located on the chromosome of most mesorhizobia, whole genome sequencing revealed that these genes are on a large symbiotic megaplasmid in all three of the newly isolated strains. A pangenome-wide association study identified 25 genes putatively associated with mesorhizobia isolated from arctic or subarctic environments, with the genomic location of many of these genes implying a relationship with legume symbiosis. Phylogenetic and sequence analyses of the common nodulation genes revealed alleles that are highly conserved amongst mesorhizobia isolated from northern climates but uncommon in mesorhizobia isolated from similar plant hosts in other climatic regions, suggesting potential functional adaptive differences and the horizontal transfer of these alleles between northern rhizobia. We speculate that nod sequence divergence was driven by climatic factors, and that the encoded proteins may be particularly stable and/or active at low temperatures.

sentences: Spc1 regulates substrate selection for signal peptidase Signal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1 that is dispensable for the catalytic activity of SPase, and its role remains elusive. In the present study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using secretory protein CPY variants with SSs modified in the n and h regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences become more susceptible to cleavage by SPase without Spc1. Further, SPase-mediated processing of transmembrane (TM) segments in model membrane proteins was reduced upon overexpression of Spc1. Spc1 was co-immunoprecipitated with membrane proteins carrying uncleaved signal-anchored or TM segments. These results suggest a role of Spc1 in shielding TM segments from SPase action, thereby contributing to accurate substrate selection for SPase.

sentences: Gestational Ethanol Exposure Induces Sex-specific Striatal Acetylcholine and Dopamine Deficits Fetal alcohol exposure has deleterious consequences on the cognitive abilities and motor skills of patients affected by Fetal Alcohol Spectrum Disorder (FASD) and in pre-clinical models of gestational ethanol exposure (GEE). Deficits in striatal cholinergic and dopamine function impair action learning and execution, yet the effects of GEE on striatal acetylcholine and dopamine release remain unexplored. Here, we report that ethanol (EtOH) exposure during the first ten postnatal days (GEEP0-P10), which mimics EtOH consumption during the last gestational trimester in humans, induces enduring sex-specific anatomical and motor learning deficits in female mice. These behavioral impairments were accompanied by increased evoked-dopamine levels in the dorsolateral striatum (DLS) of GEEP0-P10 female, but not male, mice. Further experiments revealed impaired striatal {beta}2-containing nicotinic acetylcholine receptor (nAChRs)-modulation of electrically evoked dopamine release. Using a genetically encoded acetylcholine sensor (GACh3.0), we found a reduced decay of acetylcholine transients in DLS of GEEP0-P10 females in the presence of an acetylcholinesterase inhibitor galantamine. Finally, we showed that this effect is associated with decreased excitability of striatal cholinergic interneurons (CINs), pointing to activity-dependent defects in acetylcholine release. Altogether, these data shed a new light on striatal deficits that might underlie cognitive and motor learning symptoms of patients affected by FASD.

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pUL21 regulation of pUs3 Kinase Activity Influences the Nature of Nuclear Envelope Deformation by the HSV-2 Nuclear Egress Complex It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 ({Delta}UL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in {Delta}UL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

Author SummaryDuring all herpesvirus infections, the nuclear envelope undergoes deformation in order to enable viral capsids assembled within the nucleus of the infected cell to gain access to the cytoplasm for further maturation and spread to neighbouring cells. These nuclear envelope deformations are orchestrated by the viral nuclear egress complex (NEC), which, in HSV, is composed of two viral proteins, pUL31 and pUL34. How the membrane-deformation activity of the NEC is controlled during infection is incompletely understood. The studies in this communication reveal that the phosphorylation status of pUL31 and pUL34 can determine the nature of nuclear envelope deformations and that the viral protein pUL21 can modulate the phosphorylation status of both NEC components. These findings provide an explanation for why HSV-2 strains lacking pUL21 are defective in nuclear egress. A thorough understanding of how NEC activity is controlled during infection may yield strategies to disrupt this fundamental step in the herpesvirus lifecycle, providing the basis for novel antiviral strategies.

sentences: Homology between the flagellar export apparatus and ATP synthetase: evidence from synteny predating the Last Universal Common Ancestor Evidence of homology between proteins in the ATP synthetase and the bacterial flagellar motor (BFM) has been accumulating since the 1980s. Specifically, the BFMs Type 3 Secretion System (T3SS) export apparatus FliH, FliI, and FliJ are considered homologous to FO-b + F1-{delta}, F1-/{beta}, and F1-{gamma}, and have similar structure and interactions. We review the discoveries that advanced the homology hypothesis and then conduct a further test by examining gene order in the two systems and their relatives. Conservation of gene order, or synteny, is often observed between closely related prokaryote species, but usually degrades with phylogenetic distance. As a result, observed conservation of synteny over vast phylogenetic distances can be evidence of shared ancestral coexpression, interaction, and function. We constructed a gene order dataset by examining the order of fliH, fliI, and fliJ genes across the phylogenetic breadth of flagellar and nonflagellar T3SS. We compared this to published surveys of gene order in the F1FO-ATP synthetase, its N-ATPase relatives, and the bacterial/archaeal V- and A-type ATPases. Strikingly, the fliHIJ gene order was deeply conserved, with the few exceptions appearing derived, and exactly matching the widely conserved F-ATPase gene order atpFHAG, coding for subunits b-{delta}--{gamma}. The V/A-type ATPases have a similar conserved gene order shared for homologous components. Our results further strengthen the argument for homology between these systems, and suggest a rare case of synteny conserved over billions of years, dating back to well before the Last Universal Common Ancestor (LUCA).

sentences: Free spermidine evokes superoxide radicals that manifest toxicity Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli {Delta}speG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

sentences: Exploitation of the genetic variability in the genotype of Egyptian Wheat by studding Molecular Markers, Molecular Docking and Nano Composites. Regulation of leaf and stem rust This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

sentences: Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis mutants. To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating large deletions at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either FastRed, FastGreen or FastCyan fluorescent seed selection cassette. Both, FastRed and FastGreen binary vectors, can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors at the same time. Our approach provides fast and flexible cloning, avoids very big constructs and enables screening different order mutants in the same generation. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the dark, non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) or ubiquitous promoter (PcUBi4-2). Available enzymes are "classical" Cas9, a recently reported, intron-optimized version or Cpf1 (Cas12a). Finally, we have taken care to introduce convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in the final T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.

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Faster short-read mapping with strobemer seeds in syncmer space Read alignment to genomes is a fundamental computational step used in many bioinformatic analyses, and often, it is the computational bottleneck. Therefore, it is desirable to perform the alignment step as fast as possible without compromising accuracy. Most alignment algorithms consider a seed-and-extend approach, where the time-consuming seeding step identifies and decides on candidate mapping locations. Recently, several advances have been made on seeding methods for fast sequence comparison.

We combine two such methods, syncmers and strobemers, in a novel seeding approach for constructing dynamic-sized fuzzy seeds and implement the method in a short-read aligner, strobealign. We benchmark strobealign to other traditional and recently proposed aligners on simulated and biological data and show that strobealign is an order of magnitude faster than traditional aligners such as BWA and Bowtie2 while being both faster and more accurate than more recently proposed aligners. Our aligner can free up substantial time and computing resources needed for read alignment in many pipelines.

Availabilityhttps://github.com/ksahlin/strobealign.

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Insights into bear evolution from a Pleistocene polar bear genome The polar bear (Ursus maritimus) has become a symbol of the threat to biodiversity from climate change. Understanding polar bear evolutionary history may provide insights into apex carnivore responses and prospects during periods of extreme environmental perturbations. In recent years, genomic studies have examined bear speciation and population history, including evidence for ancient admixture between polar bears and brown bears (Ursus arctos). Here, we extend our earlier studies of a 130,000-115,000-year-old polar bear from the Svalbard Archipelago using a 10X coverage genome sequence and ten new genomes of polar and brown bears from contemporary zones of overlap in northern Alaska. We demonstrate a dramatic decline in effective population size for this ancient polar bears lineage, followed by a modest increase just before its demise. A slightly higher genetic diversity in the ancient polar bear suggests a severe genetic erosion over a prolonged bottleneck in modern polar bears. Statistical fitting of data to alternative admixture graph scenarios favors at least one ancient introgression event from brown bears into the ancestor of polar bears, possibly dating back over 150,000 years. Gene flow was likely bidirectional, but allelic transfer from brown into polar bear is the strongest detected signal, which contrasts with other published works. These findings may have implications for our understanding of climate change impacts: polar bears, a specialist Arctic lineage, may not only have undergone severe genetic bottlenecks, but also been the recipient of generalist, boreal genetic variants from brown bear during critical phases of Northern Hemisphere glacial oscillations.

SignificanceInterspecific hybridization is a widespread phenomenon, but measuring its extent, directionality, and adaptive importance remains challenging. Ancient genomes, however, can help illuminate the history of modern organisms. Here, we present a genome retrieved from a 130,000-115,000-year-old polar bear and perform genome analyses of modern polar and brown bears throughout their geographic range. We find that the principal direction of ancient allele sharing was from brown bear into polar bear, although gene flow between them has likely been bidirectional. This inverts the current paradigm of unidirectional gene flow from polar into brown bear, and it suggests that polar bears were recipients of external genetic variation prior to their extensive population decline.

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Characteristics and data reporting of rare disease clinical trials: Getting better but still room for improvement BackgroundIt is estimated that there are more than 7,000 rare diseases (RDs) worldwide, impacting the lives of approximately 400 million people and only 5% have an approved therapy. Facing special challenges, including patient scarceness, incomplete knowledge of the natural history and only few specialized clinical sites, clinical trials (CT) are limited, making the data from trials critical for research and clinical care. Despite the introduction of the U.S. Food and Drug Administration Amendment Act (FDAAA) in 2007 requiring certain CTs to post results on the registry ClinicalTrials.gov within 12 months following completion, compliance has been reportedly poor. Here, we describe general characteristics of RD CTs, identify trends, and evaluate result reporting practices under the FDAAA aiming to draw awareness to the problem of non-compliance.

MethodsCTs conducted between 2008 and 2015 were extracted from the public U.S. trial registry ClinicalTrials.gov using the text mining software I2E (Linguamatics). Disease names were matched with rare disease names from the Orphanet Rare Disease Ontology (ORDO, v2.5, Orphanet). Statistical analyses and data visualization were performed using GraphPad Prism 7 and R (v3.5). The Students t-test was employed to calculate significance using p-value cut-offs of <0.05 or <0.001.

ResultsWe analyzed 1,056 RD CTs of which 55.7% were phase 2, 7.7% phase 2/3 and 36.7% phase 3 trials. The studies were mostly one- and two-armed experimental CTs with the majority (60.2%) being funded by industry. Cystic fibrosis and sickle cell disease represented the most frequently investigated diseases (25.0% and 16.5%). Industry-led phase 2 RD CTs were significantly (p<0.0001) shorter than their equivalent led by academia/non-profit (22 vs. 33 months). Screening CTs completed before the end of 2015, we found that of the 725 analyzed studies, 55.2% predominantly phase 2 CTs, did not report results. Taking their potential applicability to the FDAAA into account, 25.2% industry-funded and 28.0% academia/non-profit-funded trials failed to disclose results on ClinicalTrial.gov.

ConclusionRD CTs tend to be comparatively small, industry-funded studies focusing on genetic and neurologic conditions. Sponsor-related differences in study design, duration, and enrollment were observed. There are still substantial shortcomings when it comes to result publication.

sentences: Selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.

sentences: A fast, scalable, MinHash-based k-mer tool to assess Sequence Read Archive next generation sequence submissions. Sequence Read Archive submissions to the National Center for Biotechnology Information often lack useful metadata, which limits the utility of these submissions. We describe a scalable k-mer based tool for fast assessment of taxonomic diversity intrinsic to submissions, independent of metadata. We show that our MinHash-based k-mer tool is accurate and scalable, offering reliable criteria for efficient selection of data for further analysis by the scientific community, at once validating submissions while also augmenting sample metadata with reliable, searchable, taxonomic terms.

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Modeling Cell Biological Features of Meiotic Chromosome Pairing During meiosis, homologous chromosomes become associated side by side in a process known as homologous chromosome pairing. Pairing requires long range chromosome motion through a nucleus that is full of other chromosomes. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we use a coarse-grained molecular dynamics model to investigate how specific features of meiosis, including motor-driven telomere motion, nuclear envelope interactions, and increased nuclear size, affect the rate of pairing and the mitigation/resolution of interlocks. By creating in silico versions of three yeast strains and comparing the results of our model to experimental data, we find that a more distributed placement of pairing sites along the chromosome is necessary to replicate experimental findings. Active motion of the telomeric ends speeds up pairing only if binding sites are spread along the chromosome length. Adding a meiotic bouquet significantly speeds up pairing but does not significantly change the number of interlocks. An increase in nuclear size slows down pairing while greatly reducing the number of interlocks. Interestingly, active forces increase the number of interlocks, which raises the question: How do these interlocks resolve? Our model gives us detailed movies of interlock resolution events which we then analyze to build a step-by-step recipe for interlock resolution. In our model, interlocks must first translocate to the ends, where they are held in a quasi-stable state by a large number of paired sites on one side. To completely resolve an interlock, the telomeres of the involved chromosomes must come in close proximity so that the cooperativity of pairing coupled with random motion causes the telomeres to unwind. Together our results indicate that computational modeling of homolog pairing provides insight into the specific cell biological changes that occur during meiosis.

Author SummaryEarly in meiosis, homologous chromosomes must find each other within the crowded nuclear space and become aligned along their entire length in a process known as homologous chromosome pairing. It remains unclear how the cell manages to align each pair of chromosomes quickly while mitigating and resolving interlocks. Here, we study this process by using a computational model. Our model attempts to capture the large-scale cell biological picture of meiotic pairing including the random initial 3D search, active motion of the chromosome ends, and meiosis specific constraints such as telomere attachment to the nuclear envelope. We use our model to study how these different features of meiosis affect the rate of pairing and the mitigation/resolution of interlocks. Importantly, our model gives us detailed movies of interlock resolution events, which we then analyze to build a step-by-step recipe for interlock resolution. We believe computational modeling of homolog pairing provides valuable insight into this complex biological process.

sentences: Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation Local hypoxia (low oxygen) occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most of the prior research has focused on hypoxia-inducible genes in hypoxia as opposed to those which are decreased. Using ATACseq, we demonstrate that chromatin accessibility is decreased in an oxygen-dependent manner, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing and the R-loop interactome. As R-loops accumulate in hypoxic conditions we hypothesized that an underlying mechanism could be the repression of the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts and in patient samples with hypoxic tumors. In addition, we identified TRIM5 as a novel hypoxia inducible E3 ligase which targets DDX5 for proteasomal degradation independently of changes at mRNA levels. We demonstrate multiple mechanisms contributing to reduced DDX5 expression in hypoxic conditions. Most interestingly, we found that when DDX5 is rescued in hypoxia, R-loop levels accumulate further, therefore demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors, however, as shown for DDX5, their role is specific and distinct.

sentences: Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent content mixing, which is essential for single entity analysis. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer fabrication method for DE drop makers. This design is powerful as it allows robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets effectively maintain the compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.

sentences: Degenerate primed randomly amplified polymorphic DNA (DP-RAPD) fingerprinting of bacteriophages of Vibrio harveyi from shrimp hatcheries in Southern India Vibrio harveyi is a significant pathogen of shrimp. Seventy-six bacteriophages infecting luminescent V. harveyi were isolated from a total of 194 water samples drawn from various sources of shrimp hatcheries located in South East coast and Andaman island of India. Degenerate primed randomly amplified polymorphic DNA (DP- RAPD) fingerprinting of these bacteriophages was carried out to determine their genetic relatedness. Similarity matrix based on Dice coefficient followed by construction of dendrogram by unweighted pair group method with arithmetic averages (UPGMA) revealed 12 major clusters. One phage was randomly selected from each of these major clusters for transmission electron microscopic observations. Eleven of them had an icosahedral head (46-115 nm) with a long non-contractile tail (132-329 nm), belonging to the Siphoviridae family and two phages had a short tail (15-27 nm), belonging to the family Podoviridae. The phylogenetic analysis of the phages using DP-RAPD fingerprinting correlated to some extent to the phenotypic nature of the host specifically with regard to sucrose fermentation and source of isolation. However, phages specifically infecting V. harveyi and those belonging to different families did not cluster together in the DP-PCR cluster analysis. Hence, the genetic diversity of phages infecting same host with respect to phenotypic difference was revealed by the DP-RAPD applied in this study.

sentences: Pathological and metabolic underpinnings of energetic inefficiency in temporal lobe epilepsy The human brain consumes a disproportionate amount of energy to generate neural dynamics. Yet precisely how energetic processes are altered in neurological disorders remains far from understood. Here, we use network control theory to profile the brains energy landscape, describing the rich dynamical repertoire supported by the structural connectome. This approach allows us to estimate the energy required to activate a circuit, and determine which regions most support that activation. Focusing on temporal lobe epilepsy (TLE), we show that patients require more control energy to activate the limbic network than healthy volunteers, especially ipsilateral to the seizure focus. Further, greater energetic costs are largely localized to the ipsilateral temporo-limbic regions. Importantly, the energetic imbalance between ipsilateral and contralateral temporo-limbic regions is tracked by asymmetric metabolic patterns, which in turn are explained by asymmetric gray matter volume loss. In TLE, failure to meet the extra energy demands may lead to suboptimal brain dynamics and inadequate activation. Broadly, our investigation provides a theoretical framework unifying gray matter integrity, local metabolism, and energetic generation of neural dynamics.

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Defining marine bacterioplankton community assembly rules by contrasting the importance of environmental determinants and biotic interactions Bacterioplankton communities play major roles in governing marine productivity and biogeochemical cycling, yet what drives the relative influence of the types of deterministic ecological processes which result in diversity patterns remains unclear. Here we examine how differing deterministic processes (environmental factors and biotic interactions) drive temporal dynamics of bacterioplankton diversity at three different oceanographic time-series locations, spanning 15 degrees of latitude, which are each characterized by different environmental conditions and varying degrees of seasonality. Monthly surface samples, collected over a period of 5.5 years, were analyzed using 16S rRNA amplicon sequencing. The high and mid-latitude sites of Maria Island and Port Hacking were characterized by high and intermediate levels of environmental heterogeneity respectively, with both alpha (local) diversity (72 % and 24 % of total variation) and beta diversity (32 % and 30 %) patterns within bacterioplankton assemblages primarily explained by environmental determinants, including day length, ammonium, and mixed layer depth. In contrast, at North Stradbroke Island, a sub-tropical location where environmental conditions are less seasonally variable, interspecific interactions were of increased importance in structuring bacterioplankton diversity (alpha diversity: 33 %; beta diversity: 26 %) with environment only contributing 11 and 13 % to predicting diversity, respectively. Our results demonstrate that bacterioplankton diversity is the result of both deterministic environmental and biotic processes and that the importance of these different deterministic processes varies, potential in response to environmental heterogeneity.

ImportanceMarine bacterioplankton drives important biological processes, including the cycling of key nutrients or fixing atmospheric carbon. Therefore, to predict future climate scenarios its critical to model these communities accurately. Processes that drive bacterioplankton diversity patterns in the oceans however remain unresolved, with most studies focusing on deterministic environmental drivers, ie temperature or available inorganic nutrients. Biotic deterministic processes including interactions among individuals are also important for structuring diversity patterns, however, this is rarely included to predict bacterioplankton communities. We develop an approach for determining the relative contribution of environmental and potential biotic interactions that structure marine bacterioplankton at three series at different latitudes. Environmental factors best predicted temporal trends in bacterioplankton diversity at the two high latitude time series, while biotic influence was most apparent at the low latitude time series. Our results suggest environmental heterogeneity is an important attribute driving the contribution of varying deterministic influence of bacterioplankton diversity.

sentences: Fish as model systems to study epigenetic drivers in human self-domestication and neurodevelopmental cognitive disorders Modern humans exhibit phenotypic traits that are shared across independent domestication events, suggesting the human self-domestication hypothesis. Epigenetic changes may facilitate early self-domestication in humans, since they can be the first layer of response to a novel environment. Here, we argue that fish provide model systems to study epigenetic drivers in human self-domestication. To do this, we compare genes that carry epigenetic changes in early domesticates of European sea bass with 1) anatomically modern humans and 2) neurodevelopmental cognitive disorders with abnormal self-domestication traits, i.e., schizophrenia, Williams syndrome and autism spectrum disorders. We found that genes with epigenetic changes in fish and in modern vs ancient humans were shared and were involved in processes like limb morphogenesis and phenotypes like abnormal snout morphology and hypopigmentation. Moreover, early domestication in fish and neurodevelopmental cognitive impairment in humans affected paralogue genes involved in processes such as neural crest differentiation and ectoderm differentiation. We conclude that parallel epigenetic changes may occur at the initial steps of domestication in absence of deliberate selection in phylogenetically distant vertebrates. These findings pave the way for future studies using fish as models to investigate epigenetic changes as drivers of human-self domestication and as triggers of cognitive disorders.

sentences: Peripheral neuropathy linked mRNA export factor GANP reshapes gene regulation in human motor neurons Loss-of-function of the mRNA export protein GANP (MCM3AP gene) cause early-onset sensorimotor neuropathy, characterised by axonal degeneration in long peripheral nerves. GANP functions as a scaffold at nuclear pore complexes, contributing to selective nuclear export of mRNAs. Here, we aimed to identify motor neuron specific transcripts that are regulated by GANP and may be limiting for local protein synthesis in motor neuron axons. We compared motor neurons with a gene edited mutation in the Sac3 mRNA binding domain of GANP to isogenic controls. We also examined patient-derived motor neurons. RNA sequencing of motor neurons as well as nuclear and axonal subcompartments showed that mutant GANP had a profound effect on motor neuron transcriptomes, with alterations in nearly 40 percent of all expressed genes and broad changes in splicing. Expression changes in multiple genes critical for neuronal functions, combined with compensatory upregulation of protein synthesis and early-stage metabolic stress genes, indicated that RNA metabolism was abnormal in GANP-deficient motor neurons. Surprisingly, limited evidence was found for large-scale nuclear retention of mRNA. This first study of neuropathy-linked GANP defects in human motor neurons shows that GANP has a wide gene regulatory role in a disease-relevant cell type that requires long-distance mRNA transport.

sentences: Effects of phenotypic variation on consumer coexistence and prey community structure A popular idea in ecology is that trait variation among individuals from the same species may promote the coexistence of competing species. However, theoretical and empirical tests of this idea have yielded inconsistent findings. We manipulated intraspecific trait diversity in a ciliate competing with a nematode for bacterial prey in experimental microcosms. We found that intraspecific trait variation inverted the original competitive hierarchy to favor the consumer with variable traits, ultimately resulting in competitive exclusion. This competitive outcome was driven by foraging traits (size, speed, and directionality) that increased the ciliates fitness ratio and niche overlap with the nematode. The interplay between consumer trait variation and competition resulted in non-additive cascading effects - mediated through prey defense traits - on prey community assembly. Our results suggest that predicting consumer competitive population dynamics and the assembly of prey communities will require understanding the complexities of trait variation within consumer species.

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Chemical screen identifies a small molecule antagonizing JA-Ile perception and auxin responses. The phytohormone JA-Ile regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein COI1 (Coronatine Insensitive 1) and a JAZ (Jasmonate ZIM-domain) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signalling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1- JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signalling, J4 works as an antagonist of the closely-related auxin signalling pathway, preventing TIR1/Aux-IAA interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways.

One sentence summaryA chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and non-vascular plants.

sentences: Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted BMP4. Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (iTRAQ) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced osteoblasts was confirmed by immunohistochemistry of MDA-PCa118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through 5{beta}1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

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Inferring species compositions of complex fungal communities from long- and short-read sequence data BackgroundThe kingdom fungi is crucial for life on earth and is highly diverse. Yet fungi are challenging to characterize. They can be difficult to culture and may be morphologically indistinct in culture. They can have complex genomes of over 1 Gb in size and are still underrepresented in whole genome sequence databases. Overall their description and analysis lags far behind other microbes such as bacteria. At the same time, classification of species via high throughput sequencing without prior purification is increasingly becoming the norm for pathogen detection, microbiome studies, and environmental monitoring. However, standardized procedures for characterizing unknown fungi from complex sequencing data have not yet been established.

ResultsWe compared different metagenomics sequencing and analysis strategies for the identification of fungal species. Using two fungal mock communities of 44 phylogenetically diverse species, we compared species classification and community composition analysis pipelines using shotgun metagenomics and amplicon sequencing data generated from both short and long read sequencing technologies. We show that regardless of the sequencing methodology used, the highest accuracy of species identification was achieved by sequence alignment against a fungi-specific database. During the assessment of classification algorithms, we found that applying cut-offs to the query coverage of each read or contig significantly improved the classification accuracy and community composition analysis without significant data loss.

ConclusionOverall, our study expands the toolkit for identifying fungi by improving sequence-based fungal classification, and provides a practical guide for the design of metagenomics analyses.

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CaPTrends: A global database of Carnivoran Population Trends MotivationPopulation trend information is an  Essential Biodiversity Variable for monitoring change in biodiversity over time. Here, we present a global dataset of 1122 population trends describing changes in abundance over time in large mammals from the Order Carnivora - some of the worlds most charismatic and functionally important fauna.

Main types of variables includedKey data fields for each record: species, coordinates, trend timeframe, methods of data collection and analysis, and population timeseries or summarised trend value. Population trend values are reported using quantitative metrics in 75% of records that collectively represent more than 6500 population estimates. The remaining records qualitatively describe population change (e.g. increase).

Spatial location and grainRecords represent locations across the globe (latitude: -51.0 to 80.0; longitude: -166.0 to 166.0) with more trends found within the northern temperate zone.

Time period and grainRecords span from 1726 to 2017, with 92% of trends starting after 1950.

Major taxa and level of measurementWe conducted a semi-systematic search for population trend data in 87 species from four families in the order Carnivora: Canidae, Felidae, Hyaenidae and Ursidae. We compiled data for 50 of the 87 species.

Software format.csv

sentences: A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.

sentences: Principal component analysis of RNA-seq data unveils a novel prostate cancer-associated gene expression signature Prostate cancer (Pca) is a highly heterogeneous disease and the second more common tumor in males. Molecular and genetic profiles have been used to identify subtypes and guide therapeutic intervention. However, roughly 26% of primary Pca are driven by unknown molecular lesions. We use Principal Component Analysis (PCA) and custom RNAseq-data normalization to identify a gene expression signature which segregates primary PRAD from normal tissues. This Core-Expression Signature (PRAD-CES) includes 33 genes and accounts for 39% of data complexity along the PC1-cancer axis. The PRAD-CES is populated by protein-coding (AMACR, TP63, HPN) and RNA-genes (PCA3, ARLN1), validated/predicted biomarkers (HOXC6, TDRD1, DLX1), and/or cancer drivers (PCA3, ARLN1, PCAT-14). Of note, the PRAD-CES also comprises six over-expressed LncRNAs without previous Pca association, four of them potentially modulating drivers genes TMPRSS2, PRUNE2 and AMACR. Overall, our PCA capture 57% of data complexity within PC1-3. GO enrichment and correlation analysis comprising major clinical features (i.e., Gleason Score, AR Score, TMPRSS2-ERG fusion and Tumor Cellularity) suggest that PC2 and PC3 gene signatures may describe more aggressive and inflammation-prone transitional forms of PRAD. Of note, surfaced genes may entail novel prognostic biomarkers and molecular alterations to intervene. Particularly, our work uncovered RNA genes with appealing implications on Pca biology and progression.

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Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence.

One-sentence summaryPhospho-regulation mediates quiescence and antibiotic persistence in Staphylococcus aureus.

sentences: Hydrozoan sperm-specific H2B histone variants stabilize chromatin and block transcription without enhancing chromatin condensation Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.

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

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

sentences: A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids (IOs) are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

sentences: The SARS-CoV-2 reproduction number R0 in cats Domestic cats are susceptible to SARS-CoV-2 virus infection and given that they are in close contact with people, assessing the potential risk cats represent for the transmission and maintenance of SARS-CoV-2 is important. Assessing this risk implies quantifying transmission from humans-to-cats, from cats-to-cats and from cats-to-humans. Here we quantified the risk of cat-to-cat transmission by reviewing published literature describing transmission either experimentally or under natural conditions in infected households. Data from these studies were collated to quantify the SARS-CoV-2 reproduction number R0 among cats. The estimated R0 was significantly higher than 1, hence cats could play a role in the transmission and maintenance of SARS-CoV-2. Questions that remain to be addressed are the risk of transmission from humans-to-cats and cats-to-humans. Further data on household transmission and data on virus levels in both the environment around infected cats and their exhaled air could be a step towards assessing these risks.

sentences: Cerebellar connectivity maps embody individual adaptive behavior From planification to execution, cerebellar microcircuits encode different features of skilled movements. However, it is unknown whether cerebellar synaptic connectivity maps encode movement features in a motor context specific manner. Here we investigated the spatial organization of excitatory synaptic connectivity in mice cerebellar cortex in different locomotor contexts: during development and in normal, trained or altered locomotor conditions. We combined optical, electrophysiological and graph modelling approaches to describe synaptic connectivity between granule cells (GCs) and Purkinje cells (PCs). Synaptic map maturation during development revealed a critical period in juvenile animals before the establishment of a stereotyped functional organization in adults. However, different locomotor conditions lead to specific GC-PC connectivity maps in PCs. Ultimately, we demonstrated that the variability in connectivity maps directly accounts for individual specific behavioral features of mice locomotion, suggesting that GC-PC networks encode a general motor context as well as individual specific internal models underlying motor adaptation.

sentences: GABA administration limits viral replication and pneumonitis in a mouse model of COVID-19 Despite the availability of vaccines for COVID-19, serious illness and death induced by coronavirus infection will remain a global health burden because of vaccination hesitancy, possible virus mutations, and the appearance of novel coronaviruses. Accordingly, there is a need for new approaches to limit severe illness stemming from coronavirus infections. Cells of the immune system and lung epithelia express receptors for GABA (GABA-Rs), a widely used neurotransmitter within the CNS. GABA-R agonists have anti-inflammatory effects and can limit acute lung injury. We previously showed that GABA treatment effectively reduced disease severity and death rates in mice following infection with a coronavirus (MHV-1) which provides a potentially lethal model of COVID-19. Here, we report that GABA treatment also reduced viral load in the lungs, suggesting that GABA-Rs may provide a new druggable target to limit pulmonary coronavirus replication. Histopathological analysis revealed that GABA treatment reduced lung inflammatory infiltrates and damages. Since GABA is safe for human consumption, inexpensive, and available worldwide, it is a promising candidate to help treat COVID-19.

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Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms The evolution of bacteria is driven by random genetic mutations and horizontal gene transfer (HGT) of genetic material from other bacteria. HGT can occur via transformation, transduction, and conjugation. Here, we present a potential new mechanism of HGT which occurs in a syntrophic Clostridium coculture. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, the two organisms undergo heterologous cell fusion, which includes fusion of the peptidoglycan cell walls and membranes. Heterologous cell fusion facilitated a large-scale exchange of cytoplasmic protein and RNA between the two organisms, leading to the formation of hybrid bacterial cells containing cytoplasmic material of the two parent organisms. Here we present new evidence that cell fusion events also facilitate the exchange of plasmid DNA between the two organisms of the syntrophic coculture. Through the use of a selective subculturing process, we successfully isolated wild-type C. acetobutylicum clones which have acquired a portion of the plasmid DNA - containing the antibiotic resistance marker - from a recombinant strain of C. ljungdahlii. Fusion events led to formation of persistent aberrant hybrid cells with distinct morphogenetic characteristics. Furthermore, our data support the concept of a novel, interspecies, mechanism of acquiring antibiotic resistance. Since neither organism contains any known conjugation machinery or mechanism, these findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.

IMPORTANCEInvestigations of natural multispecies microbiomes and the field of synthetic and syntrophic microbial cocultures are attracting renewed interest based on their potential application in biotechnology, ecology, and medical fields. A variety of synthetic and natural cocultures have been examined in terms of their metabolic output, but relatively few systems have been interrogated at the cellular and molecular level. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms, and leads to the formation of hybrid bacterial cells. Continuing this line of investigation, we now show that heterologous cell fusion between the two Clostridium organisms can also facilitate the exchange of DNA between the two organisms. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Fusion led to persistent hybrid cells containing DNA from both parents but with distinct properties and morphologies. Moreover, we provide evidence for a novel mechanism of acquiring antibiotic resistance mediated by the syntrophic interactions of this system. This is a major finding that may shed light on a new mechanism of bacterias ability to acquire antibiotic resistance.

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Microbial communities in retail draft beers and the biofilms they produce In the beer brewing industry, microbial spoilage presents a consistent threat that must be monitored and controlled to ensure the palatability of a finished product. Many of the predominant beer spoilage microbes have been identified and characterized, but the mechanisms of contamination and persistence remain an open area of study. Post-production, many beers are distributed as kegs that are attached to draft delivery systems in retail settings where ample opportunities for microbial spoilage are present. As such, restaurants and bars can experience substantial costs and downtime for cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the retail side of the beer industry is often overlooked, yet this arena may represent one of the largest threats to the profitability of a beer if its flavor profile becomes substantially distorted. In this study, we sampled and cultured microbial communities found in beers dispensed from a retail draft system to identify the contaminating bacteria and yeasts. We also evaluated their capability to establish new biofilms in a controlled setting. Among four tested beer types, we identified over a hundred different contaminant bacteria and nearly twenty wild yeasts. The culturing experiments demonstrated that most of these microbes were viable and capable of joining new biofilm communities. From these data, we provide an important starting point for the efficient monitoring of beer spoilage in draft systems and provide suggestions for cleaning protocol improvements that can benefit the retail community.

ImportanceBeer production, packaging, and service are each vulnerable to contamination by microbes that metabolize beer chemicals and impart undesirable flavors, which can result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to reduce and monitor contamination during production and packaging. A commonly overlooked quality control stage of a beer supply chain is at the retail service end, where beer kegs supply draft lines in bars and restaurants under non-sterile conditions. We found that retail draft line contamination is rampant and that routine line cleaning methods are insufficient to efficiently suppress beer spoilage. Thus, many customers unknowingly experience spoiled versions of the beers they consume. This study identified the bacteria and yeast that were resident in draft beer samples and also assessed their abilities to colonize tubing material as members of stable biofilm communities.

sentences: 3D Reconstructions of Mouse Skeletal Muscle Reveal a Decrease in the MICOS Complex and Altered Mitochondrial Networks Background: Skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. Studies of bioenergetics and protein turnover show that mitochondria mediate this decline in function. Mitochondria cristae are essential for the production of ATP. While mitochondrial aging is associated with endoplasmic reticulum stress, fragmented mitochondria, and decreased mitochondrial capacity, the genes associated with morphological changes in mitochondria during aging still requires further elucidation Further, it is not completely understood how 3D mitochondrial networks and the specialization of mitochondria are altered during aging. Methods: We measured changes in mitochondrial morphology and mitochondrial connectivity during the aging of the mouse gastrocnemius muscle through serial block facing-scanning electron microscopy and 3D reconstruction. Nanotunnels were also measured through 3D reconstruction. CRISPR/Cas9 KO was performed to examine changes in mitochondria upon loss of the mitochondrial contact site and cristae organizing system (MICOS) complex and optic atrophy 1 (OPA-1). Metabolomics were used to find key metabolite pathways changed upon off of the MICOS complex. Results: We found changes in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, cristae organizing system (MICOS) dynamics and gene expression in skeletal muscle during aging. We also found an association of OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Further, the loss of the MICOS complex was linked with decreased oxidative capacity, and altered mitochondrial metabolism. Conclusions: To our knowledge, studies of mitochondrial changes in skeletal muscle throughout aging remains limited. 3D reconstructions of nanotunnels elucidated novel patterns of mitochondrial aging in skeletal muscle. MICOS proteins decreased with age and mitochondrial morphology was similar between aged skeletal muscle and that of young mice with MICOS protein loss. In tandem, our data suggest a relationship between the MICOS complex and aging, which could be potentially linked to disease states with additional 3D reconstruction.

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Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guyana, Peru, and Costa Rica, suggest a limited role of bats in Zika transmission. Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested that could play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for Flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried an experimental infection in wild-caught Artibeus bats and sampled several free-living neotropical bats over three countries of the region. Experimental ZIKV infection was made in free-ranging adult bats (4 females and 5 males). The most relevant gross findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggestive to be caused by ZIKV, however, since these are wild-caught bats, we can not exclude other agents. Excretion of ZIKV by qPCR was detected (low titles) in only two urine samples in two inoculated animals. All other animals and tissues tested negative. Finally, no virus-neutralizing Abs were found in any animal. To determine ZIKV infection in nature, a total of 2056 bats were blood sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%); Anoura sp. (14%), and Molossus sp. (13.7 %). No sample of any tested species resulted positive to ZIKV by qPCR.

These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

Author summaryIn previous works made in 2008-2009, we have found the presence of antibodies against Flaviviruses and viral RNA has been detected in Neotropical chiropterans of Mexico, which led us to support the hypothesis that these animals could be reservoirs of Flaviviruses. As controversial opinions have been exposed, and based on a previous (2019) experimental ZIKV infection made in Colorado State University using adult Artibeus males from a captive colony, in this work we also experimentally infected adult Artibeus males complementarily adding females and using free-living animals instead of laboratory bats. We also monitored a diverse range of natural bat populations in Latin America for the presence of viral RNA against ZIKV in blood. A plaque reduction seroneutralization test was used for the detection of antibodies against ZIKV. Similar to the previous work, we found histopathological alteration in male testicles but also in ovaries and oviducts of females, as well as gliosis and multifocal necrosis in pyramidal neurons and Purkinge cells of inoculated animals. Only two urine samples from inoculated animals showed viral RNA. Additionally, leukopenia and lymphoid follicular splenic hyperplasia were evidenced. Differing to what was reported, no neutralizing antibodies against ZIKV were detected in any sample. Viral RNA within the blood was not present in any of the 2056 bat samples collected in French Guyana, Peru and Costa Rica and proceeding from 33 bat genera. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and might not have an important role on ZIKV transmission dynamics.

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Common ALS/FTD risk variants in UNC13A exacerbate its cryptic splicing and loss upon TDP-43 mislocalization Variants within the UNC13A gene have long been known to increase risk of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43. Here, we show that TDP-43 depletion induces robust inclusion of a cryptic exon (CE) within UNC13A, a critical synaptic gene, resulting in nonsense-mediated decay and protein loss. Strikingly, two common polymorphisms strongly associated with ALS/FTD risk directly alter TDP-43 binding within the CE or downstream intron, increasing CE inclusion in cultured cells and in patient brains. Our findings, which are the first to demonstrate a genetic link specifically between loss of TDP-43 nuclear function and disease, reveal both the mechanism by which UNC13A variants exacerbate the effects of decreased nuclear TDP-43 function, and provide a promising therapeutic target for TDP-43 proteinopathies.

One-Sentence SummaryShared ALS/FTD risk variants increase the sensitivity of a cryptic exon in the synaptic gene UNC13A to TDP-43 depletion.

sentences: Exaggerated cytokine production in human peripheral blood mononuclear cells by recombinant SARS-CoV-2 spike glycoprotein S1 and its inhibition by dexamethasone An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNF, IL-6, IL-1{beta} and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-{kappa}B p65 and I{kappa}B, and I{kappa}B degradation. DNA binding of NF-{kappa}B p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 {micro}M) resulted in inhibition of spike glycoprotein S1-induced NF-{kappa}B activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNF, IL-6, IL-1{beta} and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1{beta} production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-{kappa}B, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 is possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.

sentences: Exploration of polyyne biosynthetic gene cluster diversity in bacteria leads to the discovery of the Pseudomonas polyyne protegencin Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources. In bacteria their biosynthesis has been defined, however, the distribution of polyyne biosynthetic gene clusters (BGCs), and their evolutionary relationship to alkyne biosynthesis, have not been addressed. We explored the distribution of alkyne biosynthesis gene cassettes throughout bacteria, revealing evidence of multiple horizontal gene transfer events. Investigating the evolutionary connection between alkyne and polyyne biosynthesis identified a monophyletic clade possessing a conserved seven-gene cassette for polyyne biosynthesis. Mapping the diversity of these conserved genes revealed a phylogenetic clade representing a polyyne BGC in Pseudomonas, pgn, and subsequent pathway mutagenesis and analytical chemistry characterised the associated metabolite, protegencin. In addition to unifying and expanding our knowledge of polyyne diversity, our results show that alkyne and polyyne biosynthetic gene clusters are promiscuous within bacteria. Systematic mapping of conserved biosynthetic genes across bacterial genomic diversity has proven to be a successful method for discovering natural products.

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Cotranslational folding and assembly of the dimeric E. coli inner membrane protein EmrE In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally, i.e., while at least one of the two chains is still attached to the ribosome. Whether this is possible also for integral membrane proteins is unknown, however. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion - to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the E. coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in TMH1 and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins can thus both start to fold and start to dimerize during the cotranslational membrane-insertion process.

Significance statementMany water-soluble proteins are known to fold and even dimerize cotranslationally, i.e., when still attached to the ribosome. Here, we show that an E. coli inner membrane protein can also start to fold and dimerize cotranslationally, establishing the generality of these cotranslational maturation processes.

sentences: Genetic effect modification of cis-acting C-reactive protein variants in cardiometabolic disease status Mendelian randomization (MR) studies carried out among patients with a particular health condition should establish the genetic instrument influences the exposure in that subgroup, however this is normally investigated in the general population. Here, we investigated whether the genetic associations of four cis-acting C-reactive protein (CRP) variants differed between participants with and without three cardiometabolic conditions: obesity, type 2 diabetes, and cardiovascular disease. Associations of cis-genetic variants with CRP differed between obese and non-obese individuals. A multivariable analysis suggested strong independent associations of the gene-by-body mass index (BMI) interaction on CRP (P<1.18x10-8 for the CRP variants). Applying MR, we observed strong causal effect of BMI on CRP (P=2.14x10-65). In summary, our study indicates that genetic associations with CRP differ across disease sub-groups, with evidence to suggest that BMI is an effect modifier. MR studies of disease progression should report on the genetic instrument-exposure association in the disease subgroup under investigation.

sentences: XPD protects CTCF-Cohesin binding sites from somatic mutagenesis Xeroderma pigmentosum group D (XPD) is a DNA helicase involved in transcription initiation and nucleotide excision repair. Missense mutations in XPD are putative drivers in bladder cancer (BLCA) and are associated with a specific single base substitution mutational signature. However, the impact of XPD on the genome-wide distribution of somatic mutations remains unexplored. We analysed somatic mutation distribution in whole-genome sequenced (WGS) BLCA samples with (XPD mutant) and without XPD mutations (WT). XPD genotype had a large impact on the distribution of somatic mutations. XPD mutant samples had increased mutation density at open chromatin, including striking mutation hotspots at CTCF-cohesin binding sites (CBS). We validate these findings in additional WGS cohorts and BLCA exomes. Analysis of XPD occupancy and CBS hotspot mutations in other cancer types suggest that XPD protects CBS from DNA damage. Our study implicates XPD in genomic integrity maintenance at topologically-associating domain boundaries marked by CTCF-cohesin binding.

sentences: Predictable fluctuations in excitatory synaptic strength due to natural variation in presynaptic firing rate Many controlled, in vitro studies have demonstrated how postsynaptic responses to presynaptic spikes are not constant but depend on short-term synaptic plasticity (STP) and the detailed timing of presynaptic spikes. However, the effects of short-term plasticity (depression and facilitation) are not limited to short, sub-second timescales. The effects of STP appear on long timescales as changes in presynaptic firing rates lead to changes in steady-state synaptic transmission. Here we examine the relationship between natural variations in the presynaptic firing rates and spike transmission in vivo. Using large-scale spike recordings in awake mice from the Allen Institute Neuropixels dataset, we first detect putative excitatory synaptic connections based on cross-correlations between the spike trains of millions of pairs of neurons. For the subset of pairs where a transient, excitatory effect was detected, we use a model-based approach to track fluctuations in synaptic efficacy and find that efficacy varies substantially on slow ([~]1 minute) timescales over the course of these recordings. For many connections, the efficacy fluctuations are correlated with fluctuations in the presynaptic firing rate. To understand the potential mechanisms underlying this relationship, we then model the detailed probability of postsynaptic spiking on a millisecond timescale, including both slow changes in postsynaptic excitability and monosynaptic inputs with short-term plasticity. The detailed model reproduces the slow efficacy fluctuations observed with many putative excitatory connections, suggesting that these fluctuations can be both directly predicted based on the time-varying presynaptic firing rate and, at least partly, explained by the cumulative effects of STP.

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Nitrous Oxide Enhances Focused Ultrasound Mediated Delivery of Viral Gene Therapy Transcranial Magnetic Resonance Guided Focused Ultrasound (MRgFUS) can be used in conjunction with intravenous microbubbles to open the blood brain barrier (BBB) in discrete brain regions. This method is limited by the microbubble dose, the amount of energy that can be safely delivered across an intact skull, and the inability to target areas of the brain outside of central brain regions. We find that N2O administration substantially decreases the required dose of microbubbles and/or focused ultrasound pressure while still reliably opening the blood brain barrier. Additionally, we observe that the use of nitrous oxide improves the delivery of adeno-associated virus (AAV9) vector when compared to medical air. Our findings may help overcome focused ultrasound limitations and improve targeted delivery of therapeutics in humans.

One Sentence SummaryNitrous Oxide potentiates Blood Brain Barrier opening with Focused Ultrasound and enhances viral vector delivery.

sentences: Targeting neuronal homeostasis to prevent seizures Manipulating neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, no anticonvulsant compounds have yet been reported that directly manipulate neuronal homeostasis. Here, we describe a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB), with a mode-of-action that includes increased expression of the homeostatic regulator Pumilio (Pum). In Drosophila and mouse we use a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy. The pyrazole analogue (RAB216) demonstrates best efficacy, protecting 50% of mice from PTZ-induced seizure. Knock-down of Pum, in Drosophila, blocks anticonvulsive effects, whilst analysis of validated Pum targets show significant reductions following exposure of mouse brain to 4-TBB. This study provides proof-of-principle that anticonvulsant effects can be achieved through regulation of neuronal homeostasis and identifies a chemical lead compound for future development.

sentences: Dual RNA modulation of protein mobility and stability within phase-separated condensates One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.

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Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations ObjectiveTo determine if mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis.

DesignThe ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis.

SettingThe vivarium of a research laboratory

SubjectsBalb/c mice

Interventions: Mice were challenged with an LD80 dose of either lipopolysaccharide (LPS / endotoxin), TNF, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis.

Measurement and Main ResultsEach group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs (1) were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time and dose dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animal if administered early and at a high dose.

ConclusionsOur conclusions are MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAbs efficacy is both time and dose dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens-of-thousands of lives annually and could result in improved antibiotic stewardship.

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AnchorWave: sensitive alignment of genomes with high diversity, structural polymorphism and whole-genome duplication variation Millions of species are currently being sequenced and their genomes are being compared. Many of them have more complex genomes than model systems and raised novel challenges for genome alignment. Widely used local alignment strategies often produce limited or incongruous results when applied to genomes with dispersed repeats, long indels, and highly diverse sequences. Moreover, alignment using many-to-many or reciprocal best hit approaches conflicts with well-studied patterns between species with different rounds of whole-genome duplication or polyploidy levels. Here we introduce AnchorWave, which performs whole-genome duplication informed collinear anchor identification between genomes and performs base-pair resolution global alignments for collinear blocks using the wavefront algorithm and a 2-piece affine gap cost strategy. This strategy enables AnchorWave to precisely identify multi-kilobase indels generated by transposable element (TE) presence/absence variants (PAVs). When aligning two maize genomes, AnchorWave successfully recalled 87% of previously reported TE PAVs between two maize lines. By contrast, other genome alignment tools showed almost zero power for TE PAV recall. AnchorWave precisely aligns up to three times more of the genome than the closest competitive approach, when comparing diverse genomes. Moreover, AnchorWave recalls transcription factor binding sites (TFBSs) at a rate of 1.05-74.85 fold higher than other tools, while with significantly lower false positive alignments. AnchorWave shows obvious improvement when applied to genomes with dispersed repeats, active transposable elements, high sequence diversity and whole-genome duplication variation.

Significance statementOne fundamental analysis needed to interpret genome assemblies is genome alignment. Yet, accurately aligning regulatory and transposon regions outside of genes remains challenging. We introduce AnchorWave, which implements a genome duplication informed longest path algorithm to identify collinear regions and performs base-pair resolved, end-to-end alignment for collinear blocks using an efficient 2-piece affine gap cost strategy. AnchorWave improves alignment of partially synthetic and real genomes under a number of scenarios: genomes with high similarity, large genomes with high TE activity, genomes with many inversions, and alignments between species with deeper evolutionary divergence and different whole-genome duplication histories. Potential use cases for the method include genome comparison for evolutionary analysis of non-genic sequences and population genetics of taxa with complex genomes.

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Landscaping the behavioural ecology of primate stone tool use Ecology is fundamental to the development, transmission, and perpetuity of primate technology. Previous studies on tool site selection have addressed the relevance of targeted resources and raw materials for tools, but few have considered the broader foraging landscape. In this first landscape-scale study of the ecological contexts of wild chimpanzee (Pan troglodytes verus) tool-use, we investigate the conditions required for nut-cracking to occur and persist over time at discrete locations in Bossou (Guinea). We examine this at three levels: selection, frequency of use, and inactivity. We find that, further to the presence of a nut tree and availability of raw materials, abundance of food-providing trees as well as proximity to nest sites were significant predictors of nut-cracking occurrence. This suggests that the spatial distribution of nut-cracking sites is mediated by the broader behavioural landscape and is influenced by non-extractive foraging of predictable resources, as well as non-foraging activities. Additionally, the number of functional tools was greater at sites with higher frequency of nut-cracking and was negatively correlated with site inactivity. Our findings indicate that the technological landscape of the Bossou chimpanzees shares affinities with the  favoured places model of hominin site formation and provides new insights for reconstructing ancient patterns of landscape use.

ResumeLecologie est fondamentale pour le developpement, la transmission et la perennite de la technologie des primates. Des etudes anterieures ont identifie la disponibilite des ressources cibles ainsi que les matieres premieres pour les outils comme des facteurs influents dans la selection des emplacements pour les activites technologiques. Cependant, il y a peu detudes qui abordent cette recherche a lechelle du paysage et du comportement fourrager. Dans cette premiere etude paysagere sur lutilisation doutils par le chimpanze sauvage (Pan troglodytes verus), nous recherchons les conditions ecologiques qui influencent la selection, lutilisation et linactivite des emplacements utilises pour le cassage des noix en Bossou, Guinee. Nos resultats montrent quen plus de la presence dun noyer et de la disponibilite des matieres premieres, labondance darbres nourriciers ainsi que la proximite des sites de nidification etaient des predicteurs significatifs de loccurrence du cassage des noix. Cela suggere que la distribution spatiale des sites de cassage de noix est influencee par le paysage comportemental et est influencee par le fourrage non-extractive de ressources previsibles, ainsi que par des activites non-fourragers. Nos resultats indiquent que le paysage technologique des chimpanzes de Bossou partage des affinites avec le modele des << lieux favoris >> de la formation des sites hominides et fournit de nouvelles perspectives pour reconstruire les modes dutilisation du paysage anciens.

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Dynamics of deep water and N uptake under varied N and water supply AimsEnhanced nitrogen (N) and water uptake from deep soil layers may increase resource use efficiency whilst maintaining yield under stressed conditions. Winter oilseed rape (Brassica napus L.) can develop deep roots and access deep-stored resources such as N and water, while this potential has large uncertainties in variable environments. In this study, we aimed to evaluate the effects of reduced N and water supply on deep N and water uptake.

MethodsOilseed rape plants grown in outdoor rhizotrons were supplied with 240 and 80 kg N ha-1 respectively in 2019 whereas a well-watered and a water-deficit treatment were established in 2020. To track deep water and N uptake, a mixture of 2H2O and Ca(15NO3)2 was injected into the soil column at 0.5 and 1.7 m depths. {delta}2H in transpiration water and {delta}15N in leaves were measured after injection. {delta}15N in biomass samples were also measured.

ResultsDifferences in N or water supply had little effect on root growth. The low N treatment reduced water uptake throughout the soil profile, but caused a non-significant increment in 15N uptake efficiency at both 0.5 and 1.7 m. Water deficit in the upper soil layers led to compensatory deep water, while N uptake was not altered by soil water status.

ConclusionOur findings demonstrate that for winter oilseed rape, high N application and water deficiency in shallow layers increases deep water uptake, and that the efficiency of deep N uptake is mainly sensitive to N supply rather than water supply.

sentences: Dissociable mechanisms of reward learning co-mature during human adolescence as predicted by macaque lesion models Reward-guided learning and decision-making is a fundamental adaptive ability and depends on a number of component processes. We investigate how such component processes mature during human adolescence. Our approach was guided by analyses of the effects of lateral orbitofrontal lesions in macaque monkeys, as this part of the brain shows clear developmental maturation in humans during adolescence. Using matched tasks and analyses in humans (n=388, 11-35yrs), we observe developmental changes in two key learning mechanisms as predicted from the monkey data. First, choice-reward credit assignment - the ability to link a specific outcome to a specific choice - is reduced in adolescents. Second, the effects of the global reward state - how good the environment is overall recently - exerts a distinctive pattern of influence on learning in humans compared to other primates and across adolescence this pattern becomes more pronounced. Both mechanisms were correlated across participants suggesting that associative learning of correct reward assignments and GRS based learning constitute two complementary mechanisms of reward-learning that co-mature during adolescence.

sentences: The 3D structure of lipidic fibrils of α-synuclein -synuclein (Syn) is abundant in neurons, but its misfolding and abnormal fibrillization are associated with severe neurodegenerative diseases. Although interactions between Syn and phospholipid membranes are relevant during Syn fibril assembly, insights into the interactions of Syn fibrils with phospholipids have remained elusive. Here, we present six novel polymorphic atomic structures of Syn fibrils aggregated in the presence of phospholipids. The structures reveal that phospholipids favor a novel protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities between the protofilaments. These findings provide a structural rationale for fibril-induced lipid extraction, a mechanism likely to be involved in the development of -synucleinopathies.

sentences: Fracture Mechanics of Human Blood Clots: Measurements of Toughness and Critical Length scales Blood coagulates to plug vascular damage and stop bleeding, and thus the function of blood clots in hemostasis depends on their resistance against rupture (toughness). Despite the significance, fracture mechanics of blood clots remains largely unexplored, particularly the measurements of toughness and critical length scales governing clot fracture. Here, we study the fracture behavior of human whole blood clots and platelet-poor plasma clots. The fracture energy of whole blood clots and platelet-poor plasma clots determined using modified lap-shear method is 5.90{+/-}1.18 J/m2 and 0.96{+/-}0.90 J/m2, respectively. We find that the measured toughness is independent of the specimen geometry and loading conditions. These results reveal a significant contribution of blood cells to the clot fracture, as well as the dissipative length scale and nonlinear elastic length scale governing clot fracture.

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

sentences: Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.

sentences: FAMOSS, a conserved 41-aa peptide involved in plant tip growth regulation Recent evidence shows that small open reading frame (smORF; <100 codons)-encoded peptides (SEPs) containing transmembrane domains are preadapted to be progenitors of novel functional genes. A dozen of such SEPs translated from long non-coding RNAs (lncRNAs) are already functionally characterised in animals. However, functional plant lncRNA-smORF-coded peptides are not yet described. Here, we report detailed functional characterization of a 41-aa peptide encoded by lncRNA-smORFs in the moss Physcomitrium patens, which was named "FAst-growing MOSS" (FAMOSS). We found that the FAMOSS interacts with the Rab-type small GTPase proteins and its overexpression leads to faster moss growth rate and more intensive vesicular transport in apical cells, while its knockout results in the opposite effect. The FAMOSS contains a predicted transmembrane domain and possible orthologs from streptophyta algae to flowering plants have a very conserved structure. Thus, the FAMOSS peptide is a previously unknown conserved player of Rab-mediated processes in plants. Our findings are in line with functional studies of transmembrane SEPs in animals and prove the principles of SEPs evolution. This study provides new insights into functions of plant lncRNA-smORFs.

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Metagenomic sequencing for rapid identification of Xylella fastidiosa from leaf samples Xylella fastidiosa (Xf) is a globally distributed plant pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to strain level in single and mixed infected plant samples at concentrations as low as one picogram of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (Cq > 35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. We hope this pipeline can be used for early detection of Xf and incorporated as a tool to inform disease management strategies.

IMPORTANCEXylella fastidiosa (Xf) destructive outbreaks in Europe highlight this pathogens capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than three days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

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

sentences: In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging 1.Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo-fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo-FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells.

sentences: Dual avatars of E. coli grxB encoded Glutaredoxin 2 perform ascorbate recycling and ion channel activities Glutaredoxins (Grxs) are single-domain redox enzymes of the thioredoxin superfamily, and primarily function as glutathione (GSH) dependent disulphide reductases. Whereas, the E. coli Glutaredoxin 2 (EcGrx2) encoded by grxB has two conserved GST-fold domains, it still lacks a classical Grx-like functions. In this study, we show for the first time, that EcGrx2 exists in both soluble and membrane integrated forms. The soluble form associates with a previously unidentified GSH dependent dehydroascrobate (DHA) reductase, and the membrane integrated form possesses ion channel activities. Using enzyme kinetic data and structural data we unequivocally demonstrate that EcGrx2 recycles ascorbate (AsA) from DHA. This ability to recycle AsA is inhibited by Zinc (Zn2+). We also show that both wildtype and the E. coli grxB deletion mutant can be rescued from H2O2-induced oxidative stress using ascorbate as an antioxidant, which otherwise is only known as a carbon source in bacteria. Moreover, the grxB- mutant is susceptible to intracellular killing by ROS producing macrophages. We further discovered that EcGrx2 integrates into the native E. coli membrane and show that the purified soluble protein readily inserts into artificial lipid bilayer membrane and conducts ions in vitro. Our data demonstrates a highly conserved functional similarity among EcGrx2-orthologs and highlights that the utilization and subsequent recycling of ascorbate as an antioxidant by grxB harbouring gram-negative bacteria, including human pathogens, may provide a survival advantage under hostile oxidative environments.

sentences: Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a Overexpression of Arabidopsis Dehydration Response Element Binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. While the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be colocalized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding when compared to non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a overexpressing lines, suggesting antagonism between heat and dehydration stress pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and rapid response towards stress tolerance as well as attenuation of the signal to prevent deleterious effects of the runoff response.

sentences: Encoding-linked pupil response is modulated by expected and unexpected novelty: Implications for memory formation and neurotransmission Whether a novel stimulus is expected or unexpected may have implications for the kind of ensuing encoding and the type of subsequent memory. Pupil response was used in the present study to explore the way expected and unexpected stimuli are encoded and whether encoding-linked pupil response is modulated by expectation. Participants first established a contingency relationship between a series of symbols and the type of stimulus (man-made or natural) that followed each one. At encoding, some of the target stimuli violated the previously established relationship (i.e., unexpected), while the majority conformed to this relationship (i.e., expected). Expectation at encoding had opposite effects on familiarity and recollection, the two types of memory that support recognition, and modulated differently the way pupil responses predicted subsequent memory. Encoding of unexpected novel stimuli was associated with increased pupil dilation as a predictor of subsequent memory type and strength. In contrast, encoding of expected novel stimuli was associated with decreased pupil response (constriction), which was predictive of subsequent memory type and strength. The findings support the close link between pupil response and memory formation, but critically indicate that this is modulated by the type of novelty as defined by expectation. These novel findings have important implications for the encoding mechanisms involved when different types of novelty are detected and is proposed to indicate the operation of different neurotransmitters in memory formation.

sentences: Category-specific item encoding in the medial temporal lobe and beyond: The role of reward. Forming new memories is a fundamental part of human life, and the medial temporal lobe (MTL) is central to memory formation. Recent research suggests that within MTL, the perirhinal and parahippocampal cortices (PRC, PHC) process object and scene memory, respectively, whereas the hippocampus (HC) is agnostic to stimulus category. It is unclear, however, whether MTL category specificity extends to item encoding. Furthermore, MTL does not act in isolation: Reward-related memories are formed in interplay with the dopaminergic midbrain (substantia nigra/ventral tegmental area, SNVTA) and amygdala (AMY), but it is unclear whether reward modulates neural item encoding in a category-specific way. To address these questions, we had 39 healthy volunteers (27 for all memory-based analyses) undergo functional magnetic resonance imaging while they solved an incidental encoding task, which paired objects or scenes with high or low reward, followed by a next-day surprise recognition test. Behaviourally, high reward preferably enhanced object memory. Importantly, neural activity in PRC and PHC reflected item encoding of objects and scenes, respectively. Moreover, AMY encoding effects were selective for high-reward objects, with a similar pattern in PRC. SNVTA and HC showed no clear evidence of item encoding. The behavioural and neural asymmetry of reward-related encoding effects may be conveyed through an anterior-temporal memory system, including AMY and PRC, potentially in interplay with the ventromedial prefrontal cortex (vmPFC).

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Caspases switch off m6A RNA modification pathway to reactivate a ubiquitous human tumor virus The methylation of RNA at the N6 position of adenosine (m6A) orchestrates multiple biological processes to control development, differentiation, and cell cycle, as well as various aspects of the virus life cycle. How the m6A RNA modification pathway is regulated to finely tune these processes remains poorly understood. Here, we discovered the m6A reader YTHDF2 as a caspase substrate via proteome-wide prediction, followed by in vitro and in vivo validations. We further demonstrated that cleavage-resistant YTHDF2 blocks, while cleavage-mimicking YTHDF2 fragments promote, the replication of a common human oncogenic virus, Epstein-Barr virus (EBV). Intriguingly, our study revealed a feedback regulation between YTHDF2 and caspase-8 via m6A modification of CASP8 mRNA and YTHDF2 cleavage during EBV replication. Further, we discovered that caspases cleave multiple components within the m6A RNA modification pathway to benefit EBV replication. Together, our study establishes that caspase disarming of the m6A RNA modification machinery fosters EBV reactivation.

Teaser

Cellular m6A RNA modification machinery is cleaved by caspases to foster the reproduction of a common human tumor virus

sentences: Stem girth changes in response to soil water potential in lowland dipterocarp forest in Borneo: a time-series analysis Time series data offer a way of investigating the causes driving ecological processes as phenomena. To test for possible differences in water relations between species of different forest structural guilds at Danum (Sabah, NE Borneo), daily stem girth increments (gthi), of 18 trees across six species were regressed individually on soil moisture potential (SMP) and temperature (TEMP), accounting for temporal autocorrelation (in GLS-arima models), and compared between a wet and a dry period. The best-fitting significant variables were SMP the day before and TEMP the same day. The first resulted in a mix of positive and negative coefficients, the second largely positive ones. An adjustment for dry-period showers was applied. Interactions were stronger in dry than wet period. Negative relationships for overstorey trees can be interpreted in a reversed causal sense: fast transporting stems depleted soil water and lowered SMP. Positive relationships for understorey trees meant they took up most water at high SMP. The unexpected negative relationships for these small trees may have been due to their roots accessing deeper water supplies (if SMP was inversely related to that of the surface layer), and this was influenced by competition with larger neighbour trees. A tree-soil flux dynamics manifold may have been operating. Patterns of mean diurnal girth variation were more consistent among species, and time-series coefficients were negatively related to their maxima. Expected differences in response to SMP in the wet and dry periods did not clearly support a previous hypothesis differentiating drought and non-drought tolerant understorey guilds. Trees within species showed highly individual responses when tree size was standardized. Data on individual root systems and SMP at several depths are needed to get closer to the mechanisms that underlie the tree-soil water phenomena in these tropical forests. Neighborhood stochasticity importantly creates varying local environments experienced by individual trees.

sentences: Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To better understand the underlying pathobiology, we used the nitrofen rat model of CDH and characterized the proteome of hypoplastic CDH lungs at the alveolar stage (E21). Amongst the 218 significantly altered proteins between CDH and control lungs were Tenascin C, CREBBP, LYN and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen and human fetal CDH lungs. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially reversed the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3 associated cytokines (IL-15, IL-9, IL-2) are increased in fetal tracheal aspirates of CDH survivors compared to non-survivors after fetoscopic tracheal occlusion. Using pathway analysis for significantly altered proteins in our proteomic analysis, we observed an enrichment in inflammatory response associated with Epstein Barr Virus and cytokine signaling in nitrofen CDH lungs. However, we were unable to detect EBV mRNA via in-situ Hybridization in human CDH lungs. With our unbiased proteomics approach, we show for the first time that inflammatory processes are likely underlying the pathogenesis of abnormal lung development in CDH.

sentences: Species ecology explains the various spatial components of genetic diversity in tropical reef fishes Intraspecific genetic diversity should be dependent on species ecology, but the influence of ecological traits on interspecific differences in genetic variation is yet to be explored. Generating sequenced data for 20 tropical reef fish species of the Western Indian Ocean, we investigate how species ecology influences genetic diversity patterns from local to regional scales. We distinguish between the , {beta} and{gamma} components of genetic diversity, which we subsequently link to six ecological traits. In contrast to what is expected by the neutral theory of molecular evolution, we find that the  and{gamma} components of genetic diversity are negatively associated with species abundance, which can be explained by larger variance in reproductive success in large populations and/or higher introgression in less frequent species. Pelagic larval duration, an important dispersal trait in marine fishes, is found to be negatively related to genetic {beta} diversity, as expected by theory. We conclude that the neutral theory of molecular evolution may not be sufficient to explain genetic diversity in tropical reef fishes and that additional processes influence those relationships.

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Multiple bouts of high-intensity interval exercise reverse age-related functional connectivity disruptions without affecting motor learning in older adults Exercise has emerged as an intervention that may mitigate age-related resting state functional connectivity and sensorimotor declines. Here, 42 healthy older adults rested or completed 3 sets of high-intensity interval exercise for a total of 23 minutes, then immediately practiced an implicit motor task with their non-dominant hand across five separate sessions. Participants completed resting stage functional MRI before the first and after the fifth day of practice; they also returned 24-hours and 35-days later to complete short- and long-term retention. Independent component analysis of resting state functional MRI revealed increased connectivity in the frontoparietal, the dorsal attentional, and cerebellar networks and group. seed-based analysis showed strengthened connectivity between the limbic system and right cerebellum, and between the right cerebellum and bilateral middle temporal gyri in the exercise group. There was no motor learning advantage for the exercise group. Our data suggest that exercise paired with an implicit motor learning task in older adults can augment resting state functional connectivity without enhancing behaviour beyond that stimulated by skilled motor practice.

Impact statementFive separate bouts of exercise paired with skilled motor practice strengthens resting state networks in brain regions that are susceptible to declines in older adults without affecting motor acquisition or learning.

sentences: Determining the stability of genome-wide factors in BMI between ages 40 to 69 years. Genome-wide association studies (GWAS) have successfully identified common variants associated with BMI. However, the stability of genetic variation influencing BMI from midlife and beyond is unknown. By analyzing BMI data collected from 165,717 men and 193,073 women from the UKBiobank, we performed BMI GWAS on six independent five-year age intervals between 40 and 73 years. We then applied genomic structural equation modeling (gSEM) to test competing hypotheses regarding the stability of genetic effects for BMI. LDSR genetic correlations between BMI assessed between ages 40 to 73 were all very high and ranged 0.89 to 1.00. Genomic structural equation modeling revealed that genetic variance in BMI at each age interval could not be explained by the accumulation of any age-specific genetic influences or autoregressive processes. Instead, a common set of stable genetic influences appears to underpin variation in BMI from middle to early old age in men and women alike.

sentences: Multi-Scale LM/EM Neuronal Imaging from Brain to Synapse with a Tissue Clearing Method, ScaleSF The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates 1) brain-wide macroscopic observation, 2) mesoscopic circuit mapping, 3) microscopic subcellular imaging, and 4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

sentences: Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors Angiogenesis is essential for sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Thus, understanding mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1 in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1 at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases (PHDs) to bind and hydroxylate HIF-1, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Overexpression of PIM1 is sufficient to stabilize HIF-1 in normoxia and stimulate angiogenesis in a HIF-1-dependent manner in vivo. CRISPR mutants of HIF-1 (Thr455D) showed increased tumor growth, proliferation and angiogenesis. Moreover, T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

sentences: Quantifying the potential for red blood cell β-adrenergic sodium-proton exchangers to protect oxygen transport in hypoxic and hypercapnic white seabass White seabass (Atractoscion nobilis) are increasingly experiencing periods of low oxygen (O2; hypoxia) and high carbon dioxide (CO2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O2 carrier in the blood and in many teleost fishes Hb-O2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically-stimulated sodium-proton-exchangers ({beta}-NHE). We hypothesized that RBC {beta}-NHEs in white seabass are an important mechanism that can protect the blood O2-carrying capacity during hypoxia and hypercapnia. We determined the O2-binding characteristics of white seabass blood, the response of RBCs to adrenergic stimulation, and quantified the protective effect of {beta}-NHE activity on Hb-O2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O2 affinity (PO2 at half-saturation; P50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient -0.92; Root effect 52%). The presence of RBC {beta}-NHEs was confirmed by functional, molecular and bioinformatic data and super-resolution imaging revealed, for the first time, the subcellular location of {beta}-NHE protein in vesicle-like structures and on the RBC membrane, and its translocation after adrenergic stimulation. The activation of RBC {beta}-NHEs increased Hb-O2 saturation by [~]8% in normoxia at 1 kPa PCO2, and by up to 20% in hypoxia. Our results confirm that RBC {beta}-NHE activity in white seabass has great potential to protect arterial O2 transport in environmentally relevant conditions of hypoxia and hypercapnia, but also reveal a potential vulnerability of fish to combinations of these stressors.

sentences: Sex differences in age- and episodic memory-related effects on task-based functional connectivity Aging is associated with episodic memory decline and changes in functional brain connectivity. Understanding whether and how biological sex influences age- and memory performance-related functional connectivity has important theoretical and clinical implications for our understanding of brain and cognitive aging. Yet, little is known about the effect of sex on neurocognitive aging. Here, we scanned 161 healthy adults between 19-76 yrs of age in an event-related functional magnetic resonance imaging (fMRI) study of face-location spatial context memory. Adults were scanned while performing easy and difficult versions of the task at both encoding and retrieval. We used multivariate whole-brain partial least squares (PLS) connectivity to test the hypothesis that there are sex differences in age- and episodic memory performance-related functional connectivity. We examined how individual differences in age and retrieval accuracy correlated with task-related connectivity. We then repeated this analysis after disaggregating the data by self-reported sex. We found that increased encoding and retrieval-related connectivity within the dorsal attention network (DAN), and between DAN and frontoparietal network (FPN) and visual networks, was positively correlated to retrieval accuracy and negatively correlated with age in both sexes. We also observed sex differences in age- and performance-related functional connectivity: i) greater between-network integration was apparent at both levels of task difficulty in women only, and ii) increased DAN-DMN connectivity with age was observed in men and was correlated with poorer memory performance. Therefore, the neural correlates of age-related episodic memory decline differ in women and men and has important theoretical and clinical implications for the cognitive neuroscience of memory, aging and dementia prevention.

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Antitumor Effects Of Phytochemicals Of Hot Three Plants On Gastric Adenocarcinoma BACKGROUNDThymoquinone (TQ) and beta-sitosterol (BS), the bioactive constituents derived from the medicinal plant Atragene sibirica L, Ranunculus repens L, Pulsatilla Bungeana have been used for anti-cancer treatment in Traditional Mongolian Medicine. Recent studies reported that TQ, BS exhibited anti-proliferative effects on several cancer cell lines. This study was performed to investigate the antitumor effects of phytochemicals on gastric adenocarcinoma in vitro.

METHODSTo this aim, we performed a cell apoptosis assay WST1 and several cancer-related gene expressions (Bcl, BIRC5, p53, BAX). NCI-N87, MKN74 cells were seeded at a density of 5x104 cells per well in 96-well culture plates. After overnight incubation, the fresh medium containing different concentrations of TQ (20, 40, 60, 80, and 100 mM/ml) and beta-sitosterol (10, 1, 0.1, 0.001, 0.001 mM/ml) were applied. Following a 24-h incubation, the cell viability was determined by WST-1 assay. We also quantified the expression levels of mRNAs of these genes through qRT-PCR experiments.

RESULTSOur results showed that TQ induced a higher percentage of cell apoptosis in the human gastric adenocarcinoma cell lines compared to that of control. We observed a significant 4.8-fold change in BAX/BCL2 ratio. These increases were highly correlated with a concentration-dependent manner (p=0.00178). Moreover, the expression of p53 and BIRC5 was downregulated in MKN74 cells after treatment with TQ.

CONCLUSIONSOur results demonstrate that TQ effectively inhibits cell proliferation through several gene expressions in vitro. Moreover, inhibition of the downstream molecule of these genes would explain the underlying mechanism of the antitumor activity in cancer cell lines.

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Intergenerational transfer and sex differences of DNA methylation patterns in the Pacific oyster (Crassostrea gigas) Apart from DNA-sequence-based inheritance, inheritance of epigenetic marks such as DNA methylation is controversial across the tree of life. In mammals, post-fertilization and primordial germ cell reprogramming processes erased most parental DNA methylation information. In nonmammalian vertebrates and insects, it has been proposed that DNA methylation is an essential hereditary carrier. However, how and to what extent general DNA methylation reprogramming affects intergenerational inheritance in molluscs remains unclear. Here, we investigated genome-wide DNA methylation in a mollusc model, the Pacific oyster (Crassostrea gigas), to test how epigenetic information transfers from parents to offspring. Analysis of global methylome revealed that the DNA methylation patterns are highly conserved within families. Almost half of the differentially methylated CpG dinucleotides (DMCs) between families in parents could transfer to offspring. These results provided the direct evidence for the hypothesis that the Pacific oyster DNA methylation patterns are inherited in generations. Moreover, distinct DNA methylation differences between male and female somatic tissues in C. gigas are revealed in this study. These sex-differential methylated genes significantly enriched in the regulation of Rho protein signal transduction process, which indicated that DNA methylation might have an essential role in the sexual differentiation of somatic tissues in C. gigas.

Author SummaryTransgenerational inheritance of DNA methylation marks varies across the tree of life. In mammals, post-fertilization and primordial germ cell reprogramming processes obstructed the DNA methylation transmission from parents to child, and only some CpG dinucleotides retain gamete-inherited methylation. However, the DNA methylation inheritance seems apparent in nonmammalian vertebrates and insects. As one of the essential mollusc models, the Pacific oyster Crassostrea gigas have received the most substantial epigenetic studies, mainly focusing on the DNA methylation profiles. While a previous study suggested the existence of paternal inheritance of DNA methylation patterns in C. gigas, more data are needed to confirm this hypothesis. In this study, genome-wide DNA methylation analysis was performed to investigate the epigenetic inheritance in C. gigas. Almost half of the DNA methylation differences between families in parents were found to be transferred to children, indicating the absence of global DNA methylation reprogramming in C. gigas. Besides, extensive hypomethylation in C. gigas females compared with males were also unveiled. These hypomethylated genes were significantly enriched in the regulation of Rho protein signal transduction process. For example, guanine nucleotide exchange factors, including KALRN, FGD1, and FGD6, were hypomethylated in C. gigas females, and the corresponding transcriptions were significantly upregulated. Our findings provided insights into the evolution of DNA methylation patterns, transgenerational epigenetic inheritance, and sexual differentiation in molluscs.

sentences: Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature Single-Cell Analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.

sentences: Cells function as a ternary logic gate to decide migration direction under integrated chemical and fluidic cues Cells sense various environmental cues and process intracellular signals to decide their migration direction in many physiological and pathological processes. Although several signaling molecules have been identified in these directed migrations, it still remains elusive how cells decipher multiple cues, specifically chemical and fluidic cues. Here, we investigated the cellular signal processing machinery by reverse-engineering directed cell migration under integrated chemical and fluidic cues. We exposed controlled chemical and fluidic cues to cells using a microfluidic platform and analyzed the extracellular coupling of the cues with respect to the cellular detection limit. Then, the cells migratory behavior was reverse-engineered to build the cells intrinsic signal processing system as a logic gate. Our primary finding is that the cellular signal processing machinery functions as a ternary logic gate to decipher integrated chemical and fluidic cues. The proposed framework of the ternary logic gate suggests a systematic approach to understand how cells decode multiple cues to make decisions in migration.

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Multilineage plasticity in prostate cancer through expansion of stem-like luminal epithelial cells with elevated inflammatory signaling Lineage plasticity is a well-established mechanism of resistance to targeted therapies in lung and prostate cancer, where tumors transition from adenocarcinoma to small-cell or neuroendocrine carcinoma. Through single-cell analysis of a cohort of heavily-treated castration-resistant human prostate cancers (CRPC), we report a greater degree of plasticity than previously appreciated, with multiple distinct neuroendocrine (NEPC), mesenchymal (EMT-like), and other subpopulations detected within single biopsies. To explore the steps leading to this plasticity, we turned to two genetically engineered mouse models of prostate cancer that recapitulate progression from adenocarcinoma to neuroendocrine disease. Time course studies reveal expansion of stem-like luminal epithelial cells (Sca1+, Psca+, called L2) that, based on trajectories, gave rise to at least 4 distinct subpopulations, NEPC (Ascl1+), POU2F3 (Pou2f3+), TFF3 (Tff3+) and EMT-like (Vim+, Ncam1+)--these populations are also seen in human prostate and small cell lung cancers. Transformed L2-like cells express stem-like and gastrointestinal endoderm-like transcriptional programs, indicative of reemerging developmental plasticity programs, as well as elevated Jak/Stat and interferon pathway signaling. In sum, while the magnitude of multilineage heterogeneity, both within and across patients, raises considerable treatment challenges, the identification of highly plastic luminal cells as the likely source of this heterogeneity provides a target for more focused therapeutic intervention.

One Sentence SummaryMultilineage plasticity results from expansion of stem-like luminal cells with JAK/STAT activation, serving as a therapeutic target.

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Direct cochlear recordings in humans show a theta rhythmic modulation of the auditory nerve by selective attention The architecture of the efferent auditory system enables prioritization of strongly overlapping spatiotemporal cochlear activation patterns elicited by relevant and irrelevant inputs. So far, attempts at finding such attentional modulations of cochlear activity delivered indirect insights in humans or required direct recordings in animals. The extent to which spiral ganglion cells forming the human auditory nerve are sensitive to selective attention remains largely unknown. We investigated this question by testing the effects of attending to either the auditory or visual modality in human cochlear implant (CI) users (3 female, 13 male). Auditory nerve activity was directly recorded with standard CIs during a silent (anticipatory) cue-target interval. When attending the upcoming auditory input, ongoing auditory nerve activity within the theta range (5-8 Hz) was enhanced. Crucially, using the broadband signal (4-25 Hz), a classifier was even able to decode the attended modality from single-trial data. Follow-up analysis showed that the effect was not driven by a narrow frequency in particular. Using direct cochlear recordings from deaf individuals, our findings suggest that cochlear spiral ganglion cells are sensitive to top-down attentional modulations. Given the putatively broad hair-cell degeneration of these individuals, the effects are likely mediated by alternative efferent pathways as compared to previous studies using otoacoustic emissions. Successful classification of single-trial data could additionally have a significant impact on future closed-loop CI developments that incorporate real-time optimization of CI parameters based on the current mental state of the user.

Significance StatementThe efferent auditory system in principle allows top-down modulation of auditory nerve activity, however evidence for this is lacking in humans. Using cochlear recordings in participants performing an audiovisual attention task, we show that ongoing auditory nerve activity in the silent cue-target period is directly modulated by selective attention. Specifically, ongoing auditory nerve activity is enhanced within the theta range when attending upcoming auditory input. Furthermore, over a broader frequency range, the attended modality can be decoded from single-trial data. Demonstrating this direct top-down influence on auditory nerve activity substantially extends previous works that focus on outer hair cell activity. Generally, our work could promote the use of standard cochlear implant electrodes to study cognitive neuroscientific questions.

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In vitro evolution of Remdesivir resistance reveals genome plasticity of SARS-CoV-2 Remdesivir (RDV) is used widely for COVID-19 patients despite varying results in recent clinical trials. Here, we show how serially passaging SARS-CoV-2 in vitro in the presence of RDV selected for drug-resistant viral populations. We determined that the E802D mutation in the RNA-dependent RNA polymerase was sufficient to confer decreased RDV sensitivity without affecting viral fitness. Analysis of more than 200,000 sequences of globally circulating SARS-CoV-2 variants show no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we also observed changes in the Spike (i.e., H69 E484, N501, H655) corresponding to mutations identified in emerging SARS-CoV-2 variants indicating that they can arise in vitro in the absence of immune selection. This study illustrates SARS-CoV-2 genome plasticity and offers new perspectives on surveillance of viral variants.

One Sentence SummarySARS-CoV-2 drug resistance & genome plasticity

sentences: Structural basis for late maturation steps of the human mitoribosomal large subunit Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2-6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.

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Characterization of retinal development in 13-lined ground squirrels PurposeThe cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human foveal region but retinal development in this useful rodent species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize the species as a practical alternative animal model for investigating cone-based vision in health and disease.

MethodsThe spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Changes in the postnatal gene expression were also assessed by qPCR.

ResultsThe 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell type-specific marker labeling was: at E18, ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E24-25.5 shortly before birth, photoreceptor progenitor (Otx2, Recoverin), horizontal and amacrine cells (Lhx1, Oc1); and at P15, bipolar cells (Vsx1, CaBP5) and Muller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin+ outer segments and PNA labeling of cone sheaths was completed at the time of eye opening, P21-24.

ConclusionsThe timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. This provides a baseline for future examinations of developmental, disease model, and stem cell approach studies employing this emerging rodent model of human vision.

sentences: Enhanced habit formation in Tourette syndrome : dopamine release and striatal disinhibition modulate shortcut connections in a hierarchical model of cortico-basal ganglia loops In the Gilles de la Tourette syndrome, tics are often considered as habitual responses towards unwanted premonitory urges. Support for the relationship between tics and habits comes from devaluation protocols, which reveal that unmedicated Tourette patients show an increased tendency towards responses to devalued outcomes. We use a neuro-computational model of hierarchically organized cortico-basal ganglia-thalamo-cortical loops to shed more light on enhanced habit formation of Tourette patients. In our model, habitual behavior emerges from cortico-thalamic shortcut connections, where enhanced habit formation can be linked to faster plasticity in the shortcut or to a stronger feedback from the shortcut to the basal ganglia. Irregular activity in such shortcut connections may have different pathophysiological origins. Based on our model, we explore decreased local striatal inhibition, which may correspond to a loss of inhibitory interneurons, and increased dopaminergic modulation of striatal medium spiny neurons as causes for irregular shortcut plasticity or activation. Both lead to higher rates of response towards devalued outcomes in our model, similar to what is observed in Tourette patients. Our results support the view of tics in Tourette syndrome as maladaptive habits. We suggest to reveal more shortcuts between cortico-basal ganglia-thalamo-cortical loops in the human brain and study their potential role in the development of the Tourette syndrome.

sentences: Transcranial photobiomodulation enhances visual working memory capacity in humans Transcranial photobiomodulation (tPBM) is a novel and noninvasive intervention, which has shown promise for improving cognitive performances. Whether tPBM can modulate brain activity and thereby enhance working memory (WM) capacity in humans remains unclear. In this study, we delivered double-blind and sham-control tPBM with different wavelengths to the prefrontal cortex (PFC) in 90 healthy participants and conducted four electroencephalography (EEG) experiments to investigate whether individual visual working memory capacity and related neural response could be modulated. We found that 1064 nm tPBM applied to the right PFC has both a substantial impact on visual working memory capacity and occipitoparietal contralateral delay activity (CDA), no matter orientation or color feature of the memorized objects. Importantly, the CDA set-size effect during the retention mediated the effect between 1064 nm tPBM and subsequent WM performance. However, these behavioral benefits and the corresponding changes of CDA set-size effect were absent with tPBM at 852 nm wavelength or with the stimulation on the left PFC. Our findings provide converging evidence that 1064 nm tPBM applied on the right PFC can improve visual working memory capacity, as well as explain the individuals electrophysiological changes about behavioral benefits.

sentences: Climatic niche conservatism in a clade of disease vectors (Diptera: Phlebotominae) Sandflies of the family Psychodidae show notable diversity in both disease vector status and climatic niche. Some species (in the subfamily Phlebotominae) transmit Leishmania parasites, responsible for the disease leishmaniasis. Other Psychodidae species do not. Psychodid species ranges can be solely tropical, confined to the temperate zones, or span both. Studying the relationship between the evolution of disease vector status and that of climatic niche affords an understanding not only of the climate conditions associated with the presence and species richness of Leishmania vectors, but also allows the study of the extent to which psychodid flies climatic niches are conserved, in a context with implications for global human health. We obtained observation site data, and associated climate data, for 223 psychodid species to understand which aspects of climate most closely predict distribution. Temperature and seasonality are strong determinants of species occurrence within the clade. We built a mitochondrial DNA phylogeny of Psychodidae, and found a positive relationship between pairwise genetic distance and climate niche differentiation, which indicates strong niche conservatism. This result is also supported by strong phylogenetic signals of metrics of climate differentiation. Finally, we used ancestral trait reconstruction to infer the tropicality (i.e., proportion of latitudinal range in the tropics minus the proportion of the latitudinal range in temperate areas) of ancestral species, and counted transitions to and from tropicality states, finding that tropical and temperate species respectively produced almost entirely tropical and temperate descendant species, a result consistent for vector and non-vector species. Taken together, these results imply that while vectors of Leishmania can survive in a variety of climates, their climate niches are strongly predicted by phylogeny.

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Quasi-Entropy Closure: A Fast and Reliable Approach to Close the Moment Equations of the Chemical Master Equation MotivationThe Chemical Master Equation is the most comprehensive stochastic approach to describe the evolution of a (bio-)chemical reaction system. Its solution is a time-dependent probability distribution on all possible configurations of the system. As the number of possible configurations is typically very large, the Master Equation is often practically unsolvable. The Method of Moments reduces the system to the evolution of a few moments of this distribution, which are described by a system of ordinary differential equations. Those equations are not closed, since lower order moments generally depend on higher order moments. Various closure schemes have been suggested to solve this problem, with different advantages and limitations. Two major problems with these approaches are first that they are open loop systems, which can diverge from the true solution, and second, some of them are computationally expensive.

ResultsHere we introduce Quasi-Entropy Closure, a moment closure scheme for the Method of Moments which estimates higher order moments by reconstructing the distribution that minimizes the distance to a uniform distribution subject to lower order moment constraints. Quasi-Entropy closure is similar to Zero-Information closure, which maximizes the information entropy. Results show that both approaches outperform truncation schemes. Moreover, Quasi-Entropy Closure is computationally much faster than Zero-Information Closure. Finally, our scheme includes a plausibility check for the existence of a distribution satisfying a given set of moments on the feasible set of configurations. Results are evaluated on different benchmark problems.

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sentences: Predicting Composition of Genetic Circuits with Resource Competition: Demand and Sensitivity The design of genetic circuits typically relies on characterization of constituent modules in isolation to predict the behavior of modules composition. However, it has been shown that the behavior of a genetic module changes when other modules are in the cell due to competition for shared resources. In order to engineer multi-module circuits that behave as intended, it is thus necessary to predict changes in the behavior of a genetic module when other modules load cellular resources. Here, we introduce two characteristics of circuit modules: the demand for cellular resources and the sensitivity to resource loading. When both are known for every genetic module in a circuit library, they can be used to predict any modules behavior upon addition of any other module to the cell. We develop an experimental approach to measure both characteristics for any circuit module using a resource sensor module. Using the measured resource demand and sensitivity for each module in a library, the outputs of the modules can be accurately predicted when they are inserted in the cell in arbitrary combinations. These resource competition characteristics may be used to inform the design of genetic circuits that perform as predicted despite resource competition.

sentences: A Machine Learning Framework to Predict Subcellular Morphology of Endothelial Cells for Digital Twin Generation Gaining insight into different cell behaviors is key to better understanding different pathologies. These behaviors may be explained in part through close observation of 3D cell morphology. Therefore, the objective of this research was to develop a machine learning (ML) framework that can predict 3D subcellular morphological variation of endothelial cells (ECs) to generate digital twins. ECs were cultured and their membrane, nucleus, and focal adhesion (FA) sites were stained and imaged with confocal microscopy. The multicellular confocal z stacks were segmented resulting in a total of 60 single-cell stacks. Fifty randomly picked cells were augmented 20-fold to train the ML framework, and the remaining 10 were used for an independent test of prediction accuracy. The ML framework was based on an open-source conditional generative adversarial network (cGAN), which was expanded to make 3D predictions using membrane only as input to predict nucleus and FA morphology. After training the framework, the results on the independent test showed an average prediction accuracy of [~]87% for nucleus and [~]70% for FA sites. The predictions were used to build a digital twin of each EC and compared to their respective ground truth, showing an average [~]79% global accuracy and [~]84% accuracy in FA-Nucleus distribution. The results presented show the effectiveness of the developed ML framework to generate digital twins of Ecs using limited amount of data. These digital twins can be used to couple EC morphology with different behaviors. The ML framework can be potentially expanded to predict morphology of other subcellular structures as well as to study other types of cells.

sentences: Explaining heterogeneity in medial entorhinal cortex with task-driven neural networks Medial entorhinal cortex (MEC) supports a wide range of navigational and memory related behaviors. Well-known experimental results have revealed specialized cell types in MEC -- e.g. grid, border, and head-direction cells -- whose highly stereotypical response profiles are suggestive of the role they might play in supporting MEC functionality. However, the majority of MEC neurons do not exhibit stereotypical firing patterns. How should the response profiles of these more "heterogeneous" cells be described, and how do they contribute to behavior? In this work, we took a computational approach to addressing these questions. We first performed a statistical analysis that shows that heterogeneous MEC cells are just as reliable in their response patterns as the more stereotypical cell types, suggesting that they have a coherent functional role. Next, we evaluated a spectrum of candidate models in terms of their ability to describe the response profiles of both stereotypical and heterogeneous MEC cells. We found that recently developed task-optimized neural network models are substantially better than traditional grid cell-centric models at matching most MEC neuronal response profiles -- including those of grid cells themselves -- despite not being explicitly trained for this purpose. Specific choices of network architecture (such as gated nonlinearities and an explicit intermediate place cell representation) have an important effect on the ability of the model to generalize to novel scenarios, with the best of these models closely approaching the noise ceiling of the data itself. We then performed in silico experiments on this model to address questions involving the relative functional relevance of various cell types, finding that heterogeneous cells are likely to be just as involved in downstream functional outcomes (such as path integration) as grid and border cells. Finally, inspired by recent data showing that, going beyond their spatial response selectivity, MEC cells are also responsive to non-spatial rewards, we introduce a new MEC model that performs reward-modulated path integration. We find that this unified model matches neural recordings across all variable-reward conditions. Taken together, our results point toward a conceptually principled goal-driven modeling approach for moving future experimental and computational efforts beyond overly-simplistic single-cell stereotypes.

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Metabotropic group II glutamate receptors in the basolateral amygdala mediate cue-triggered increases in incentive motivation RationaleReward-associated cues can trigger incentive motivation for reward and invigorate reward-seeking behaviour via Pavlovian-to-instrumental transfer (PIT). Glutamate signaling within the basolateral amygdala (BLA) modulates cue-triggered increases in incentive motivation. However, the role of BLA metabotropic group II glutamate (mGlu2/3) receptors is largely unknown.

ObjectivesIn Experiment 1, we characterized cue-triggered increases in incentive motivation for water reward using the PIT paradigm. In Experiment 2, we assessed the influence of intra-BLA microinjections of the mGlu2/3 receptor agonist LY379268 on this effect.

MethodsWater-restricted male Sprague-Dawley rats learned to press a lever for water. Separately, they learned to associate one of two auditory cues with free water. On test days, rats could lever press under extinction conditions (no water), with intermittent, non-contingent CS+ and CS- presentations. In Experiment 1, rats were tested under baseline conditions. In Experiment 2, rats received intra-BLA microinjections of LY379268 (0, 3 and 6 g/hemisphere) before testing.

ResultsAcross experiments, CS+, but not CS- presentations increased water-associated lever pressing during testing, even though responding was reinforced neither by water nor the CS+. Intra-BLA LY379268 abolished both CS+ potentiated pressing on the water-associated lever and CS+ evoked conditioned approach to the site of water delivery. LY379268 did not influence locomotion or instrumental and Pavlovian response rates during intervals between CS presentations or during the CS-, indicating no motor effects.

ConclusionsmGlu2/3 receptor activity in the BLA mediates CS-triggered potentiation of incentive motivation for reward, suppressing both CS-induced increases in instrumental pursuit of the reward and anticipatory approach behaviour.

sentences: Slow-cycling cells in glioblastoma: a specific population in the cellular mosaic of cancer stem cells AO_SCPLOWBSTRACTC_SCPLOWGlioblastoma (GBM) exhibits populations of cells that drive tumorigenesis, treatment resistance, and disease progression. Cells with such properties have been described to express specific surface and intracellular markers or exhibit specific functional features including being slow-cycling or quiescent with the ability to generate proliferative progenies. In GBM, each of these cellular fractions was shown to harbor cardinal features of cancer stem cells 1-7. In this study we focus on the comparison of these cells and present evidence of great phenotypic and functional heterogeneity in brain cancer cell populations with stemness properties, especially between slow-cycling cells and cells phenotypically defined based on the expression of markers commonly used to enrich for cancer stem cells (CSCs). Our data support the cancer stem cell mosaicism model with slow-cycling cells representing critical tesserae.

sentences: Evolution of Antibody Immunity to SARS-CoV-2 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with development of variable levels of antibodies with neutralizing activity that can protect against infection in animal models. Antibody levels decrease with time, but the nature and quality of the memory B cells that would be called upon to produce antibodies upon re-infection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection. We find that IgM, and IgG anti-SARS-CoV-2 spike protein receptor binding domain (RBD) antibody titers decrease significantly with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by five-fold in pseudotype virus assays. In contrast, the number of RBD-specific memory B cells is unchanged. Memory B cells display clonal turnover after 6.2 months, and the antibodies they express have greater somatic hypermutation, increased potency and resistance to RBD mutations, indicative of continued evolution of the humoral response. Analysis of intestinal biopsies obtained from asymptomatic individuals 4 months after coronavirus disease-2019 (COVID-19) onset, using immunofluorescence, or polymerase chain reaction, revealed persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 volunteers. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

sentences: Rapid screening of pest resistance genes in maize using a sugarcane mosaic virus vector Spodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production world-wide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although several additional transgenes have confirmed insecticidal activity in other plants, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of transgenes for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defense signaling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome-inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti-insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) spider and scorpion toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth on maize was reduced by transient SCMV-mediated overexpression of genes encoding these proteins. Additionally, experiments with some of the SCMV-expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors can be exploited as a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

sentences: Genetically engineered MRI-trackable extracellular vesicles as SARS-CoV-2 mimetics for mapping ACE2 binding in vivo The elucidation of viral-receptor interactions and an understanding of virus-spreading mechanisms are of great importance, particularly in the era of pandemic. Indeed, advances in computational chemistry, synthetic biology, and protein engineering have allowed precise prediction and characterization of such interactions. Nevertheless, the hazards of the infectiousness of viruses, their rapid mutagenesis, and the need to study viral-receptor interactions in a complex in vivo setup, call for further developments. Here, we show the development of biocompatible genetically engineered extracellular vesicles (EVs) that display the receptor binding domain (RBD) of SARS-CoV-2 on their surface as coronavirus mimetics (EVsRBD). Loading EVsRBD with iron oxide nanoparticles makes them MRI-visible, and thus, allows mapping of the binding of RBD to ACE2 receptors non-invasively in live subjects. Importantly, the proposed mimetics can be easily modified to display the RBD of SARS-CoV-2mutants, namely Delta and Omicron, allowing rapid screening of newly raised variants of the virus. The proposed platform thus shows relevance and cruciality in the examination of quickly evolving pathogenic viruses in an adjustable, fast, and safe manner.

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A genome-wide circular RNA transcriptome in Rat BackgroundCircular RNAs are a novel class of non-coding RNAs that backsplice from 5 donor site and 3 acceptor site to form a circular structure. A number of circRNAs have been discovered in model organisms including human, mouse, Drosophila, among other organisms. There are a few candidate-based studies on circular RNAs in rat, a well studied model organism. The availability of a recent dataset of transcriptomes encompassing 11 tissues, 4 developmental stages and 2 genders motivated us to explore the landscape of circular RNAs in the organism.

MethodologyIn order to understand the difference among different pipelines, we have used the same bodymap RNA sequencing dataset. A number of pipelines have been published to identify the backsplice junctions for the discovery of circRNAs but studies comparing these tools have suggested that a combination of tools would be a better approach to identify high-confidence circular RNAs. We employed 5 different combinations of tools including tophat_CIRCexplorer2, segemehl_CIRCexplorer2, star_CIRCexplorer, Bowtie2_findcirc and Bowtie2_findcirc (noHisat2) to identify circular RNAs from the dataset.

ResultsOur analysis identified a number of tissue-specific, developmental stage specific and gender specific circular RNAs. We further independently validated 16 circRNA junctions out of 24 selected candidates in 5 tissue samples. We additionally estimated the quantitative expression of 5 circRNA candidates using real-time PCR and our analysis suggests 3 candidates as tissue-enriched

ConclusionThis study is one of the most comprehensive studies that provides a circular RNA transcriptome as well as to understand the difference among different computational pipelines in Rat.

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Predicting genotype-specific gene regulatory networks Understanding how each persons unique genotype influences their individual patterns of gene regulation has the potential to improve our understanding of human health and development and to refine genotype-specific disease risk assessments and treatments. However, the effects of genetic variants are not typically considered when constructing gene regulatory networks, despite the fact that many disease-associated genetic variants are thought to have regulatory effects, including the disruption of transcription factor (TF) binding. We developed EGRET (Estimating the Genetic Regulatory Effect on TFs), which infers a genotype-specific gene regulatory network (GRN) for each individual in a study population. EGRET begins by constructing a genotype-informed TF-gene prior network derived using TF motif predictions, eQTL data, individual genotypes, and the predicted effects of genetic variants on TF binding. It then uses message passing to integrate this prior network with gene expression and TF protein-protein interaction data to produce a refined, genotype-specific regulatory network. We used EGRET to infer GRNs for two blood-derived cell lines and identified genotype-associated, cell-line specific regulatory differences that we subsequently validated using allele-specific expression, chromatin accessibility QTLs, and differential ChIP-seq TF binding. We also inferred EGRET GRNs for three cell types from each of 119 individuals and identified cell type-specific regulatory differences associated with diseases related to those cell types. EGRET is, to our knowledge, the first method that infers networks that reflect individual genetic variation in a way that provides insight into genetic regulatory associations that drive complex phenotypes.

EGRET is available through the Network Zoo R package (netZooR v0.9; netzoo.github.io).

sentences: A simple and viable approach to estimate population trends Changes in population abundances over time are of central concern for environmental conservation and the understanding of population dynamics. The standard slope estimator has restricting assumptions and lacks a constant range, which makes its interpretation less intuitive. Herein, more robust measures of trend. It is based on proportional difference and can be used for data on various scale types. If it is applied to ranks it yields the correlation coefficient. Related measures of association are described that assess the relationship between species, including a rank-order correlation that is sensitive to top ranks and a correlation for continuous data that is more robust than the correlation coefficient. All proposed measures have a range between -1 and +1. Furthermore, they can provide a common ground for evaluating trend strength and strength of association for populations undergoing very different dynamics.

sentences: Gene network module changes associated with the vertebrate fin to limb transition Evolutionary phenotypic transitions, such as the fin to limb transition in vertebrate evolution, result from changes in associated genes and their interactions, often in response to changing environment. Identifying the associated changes in gene networks is vital to achieve a better understanding of these transitions. Previous experimental studies have been typically limited to manipulating a small number of genes. To expand the number of analyzed genes and hence, biological knowledge, we computationally isolated and compared the gene modules for paired fins (pectoral fin, pelvic fin) of fishes (zebrafish) to those of the paired limbs (forelimb, hindlimb) of mammals (mouse) using quality-enhanced gene networks from zebrafish and mouse. We ranked module genes according to their weighted-degrees and identified the highest-ranking hub genes, which were important for the module stability. Further, we identified genes conserved during the fin to limb transition and investigated the fates of zebrafish-specific and mouse-specific module genes in relation to their involvements in newly emerged or lost anatomical structures during the aquatic to terrestrial vertebrate transition. This paper presents the results of our investigations and demonstrates a general network-based computational workflow to study evolutionary phenotypic transitions involving diverse model organisms and anatomical entities.

sentences: Multi-Pathway DNA Double-Strand Break Repair Reporters Reveal Extensive Cross-Talk Between End-Joining, Single Strand Annealing, and Homologous Recombination DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to extensive mutagenesis and genomic loss. Repair pathway cross-talk and compensation within the DSB-repair network is incompletely understood, despite its importance for genomic stability, oncogenesis, and the outcome of genome editing by CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple distinct pathways to repair of a DSB. These reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibition of the essential c-NHEJ factor DNA-PKcs not only increases repair by HR, but also results in a substantial increase in mutagenic repair by SSA. We show that SSA-mediated repair of Cas9-generated DSBs can occur between Alu elements at endogenous genomic loci, and is enhanced by inhibition of DNA-PKcs. Finally, we demonstrate that the short-range end-resection factors CtIP and Mre11 promote both SSA and HR, whereas the long-range end-resection factors DNA2 and Exo1 promote SSA, but reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the rapid and comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.

sentences: Genomic time-series data show that gene flow maintains high genetic diversity despite substantial genetic drift in a butterfly species Effective population size affects the efficacy of selection, rate of evolution by drift, and neutral diversity levels. When species are subdivided into multiple populations connected by gene flow, evolutionary processes can depend on global or local effective population sizes. Theory predicts that high levels of diversity might be maintained by gene flow, even very low levels of gene flow, consistent with species long-term effective population size, but tests of this idea are mostly lacking. Here, we show that Lycaeides butterfly populations maintain low contemporary (variance) effective population sizes (e.g., [~]200 individuals) and thus evolve rapidly by genetic drift. Contemporary effective sizes were consistent with local census populations sizes. In contrast, populations harbored high levels of genetic diversity consistent with an effective population size several orders of magnitude larger. We hypothesized that the differences in the magnitude and variability of contemporary versus long-term effective population sizes were caused by gene flow of sufficient magnitude to maintain diversity but only subtly affect evolution on generational time scales. Consistent with this hypothesis, we detected low but non-trivial gene flow among populations. Furthermore, using population-genomic time-series data, we documented patterns consistent with predictions from this hypothesis, including a weak but detectable excess of evolutionary change in the direction of the mean (migrant gene pool) allele frequencies across populations, and consistency in the direction of allele frequency change over time. The documented decoupling of diversity levels and short-term change by drift in Lycaeides has implications for our understanding of contemporary evolution and the maintenance of genetic variation in the wild.

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The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication.

IMPORTANCEEvidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.

sentences: The hippocampus shows an own-age bias during unfamiliar face viewing The present study investigated the neural correlates of the own-age bias for face recognition in a repetition suppression paradigm. Healthy young and older adults viewed upright and inverted unfamiliar faces. Some of the upright faces were repeated following one of two delays (lag 0 or lag 11). Repetition suppression effects were observed in bilateral fusiform cortex. However, there were no significant effects indicating an own-age bias in repetition suppression. The absence of these effects is arguably inconsistent with perceptual expertise accounts of own-age biases in face processing. By contrast, the right anterior hippocampus showed an own-age bias (greater activity for own-than other-age faces) when viewing an unfamiliar face for the first time. Given the importance of the hippocampus for episodic memory encoding, we conjecture that the increased hippocampal activity for own-age relative to other-age faces reflects differential engagement of neural processes supporting the episodic encoding of faces and might provide insight into the neural underpinnings of own-age biases in face recognition memory.

sentences: Differential susceptibility of neuronal neurotransmitter phenotypes to HHV6 infection Within the family Herpesviridae, sub-family {beta}-herpesvirinae, and genus Roseolovirus, there are only three human herpesviruses that have been discovered and described: HHV-6A, HHV-6B, and HHV-7. Initially, HHV-6A and HHV-6B were considered to be simply two variants of the same virus (i.e., HHV6). Despite high overall genetic sequence identity (~90%), HHV-6A and HHV-6B are now recognized as two distinct viruses of the genus. Limited sequence identity (e.g., <70%) in key coding regions as well as significant differences in physiological and biochemical profiles (e.g., preferential use of different receptors for viral entry into susceptible hosts) underscore the conclusion that HHV-6A and HHV-6B are distinct virus species. Likewise, each virus appears to differentially contribute as putative etiologic agents to a variety of neurological disorders, including: multiple sclerosis, epilepsy, and chronic fatigue syndrome. Despite being implicated as causative agents in nervous system dysfunction, mechanisms of action and relative contributions of each virus to neural disorders remain elusive. Unresolved questions regarding: cell receptor use and binding affinity (i.e., CD49 versus CD134); cell tropism; the role of HHV-7 superinfection; and, relative virulence between HHV-6A versus HHV-6B - prevent a complete characterization. Although it has been demonstrated that both HHV-6A and HHV-6B can infect glia and, more recently, cerebellar Purkinje cells, cell tropism of HHV-6A versus HHV-6B for different nerve cell types remains vague. In this study, we demonstrate that both HHV-6A and HHV-6B can infect different nerve cell types (i.e., glia versus neurons) and different neuronal neurotransmitter phenotypes derived from the differentiation of human neural stem cells. We further show that both HHV-6A and HHV-6B induce cytopathic effects (CPEs) in susceptible nerve cells. However, the time-course and severity of CPEs appear to differ between HHV-6A versus HHV-6B infections and are dependent upon multiplicity of infection (MOI). As demonstrated by immunofluorescence, although both the HHV-6A and HHV-6B viruses productively infected VGluT1-containing cells (i.e., glutamatergic neurons) and dopamine-containing cells (i.e., dopaminergic neurons), neither HHV-6A nor HHV-6B challenge resulted in the productive infection of GAD67-containing cells (i.e., GABAergic cells). The reason underlying the apparent resistance of GABAergic cells to HHV-6A and HHV-6B infection remains unclear. Morphometric and image analyses of neurite extension and retraction dynamics as well as the time-course of cell aggregation phenomena (e.g., syncytia formation) during infection also indicate that HHV-6A induces more severe CPEs than HHV-6B at the same time-point and MOI. These data suggest that HHV-6A is more virulent than HHV-6B on susceptible human neural stem cells (HNSCs) differentiated into neuronal phenotypes, while neither virus is able to infect GABAergic cells. If these in vitro data hold in vivo, the inhibitory interneuron dysfunction hypothesis for HHV6-driven seizures may be ruled out as a potential mechanism for HHV6-induced epileptogenesis.

sentences: Rapid prototyping of metabolites detection by bacterial biosensors in human fecal samples. Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LCMS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a protocol for feces processing and gut metabolites detection using bacterial biosensors (bactosensors), enabling rapid evaluation of their operational capacity in these samples. We show that a simple filtration step is enough to remove host microbes and reproducibly obtain a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measured how fecal samples affect the performance of biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and found that bactosensors are highly sensitive to fecal matrices. Sensitivity to the matrix is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for bactosensors operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bactosensor can be used for metabolites monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

sentences: An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.

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A subset of gut leukocytes have telomerase-dependent 'hyper-long' telomeres and require telomerase for function in zebrafish Telomerase, the enzyme capable of elongating telomeres, is usually restricted in human somatic cells, which contributes to progressive telomere shortening with cell-division and ageing. Using the zebrafish model, we show that subsets gut immune cells have telomerase-dependent "hyper-long" telomeres, which we identified as being predominantly macrophages and dendritics (mpeg1.1+ and cd45+mhcII+). Notably, mpeg+ macrophages have much longer telomeres in the gut than in their haematopoietic tissue of origin, suggesting that there is a gut-specific modulation of telomerase in these cells.

Moreover, we show that a subset of gut mpeg+ cells express telomerase (tert) in young WT zebrafish, but that the relative proportion of these cells decreases with ageing. Importantly, this is accompanied by telomere shortening and DNA damage responses with ageing and a telomerase-dependent decrease in expression of autophagy and immune activation markers. Finally, these telomerase-dependent molecular alterations are accompanied by impaired phagocytosis of E.coli and increased gut permeability in vivo. Together, our data show that limiting levels of telomerase lead to changes in gut immunity and gut permeability, which, together, are likely contributors to local and systemic tissue degeneration and increased susceptibility to infection with ageing.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/478480v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@141a6caorg.highwire.dtl.DTLVardef@12d24baorg.highwire.dtl.DTLVardef@c64267org.highwire.dtl.DTLVardef@319449_HPS_FORMAT_FIGEXP  M_FIG C_FIG Our data show that limiting levels of telomerase contribute to alterations in gut immunity, namely increased apoptosis, decreased autophagy and immune activation of mpeg+ macrophages in the gut. This is accompanied by a decreased ability to clear pathogens and increased gut permeability. Together, these are likely contributors to local and systemic tissue degeneration and susceptibility to infection with ageing.

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The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain.

Author SummaryIn the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.

sentences: Functional divergence of the bag of marbles gene in the Drosophila melanogaster species group In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and D. simulans, but not in the outgroup species D. ananassae. Here, we test the null hypothesis that bams differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in D. teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bams critical gametogenesis role being newly derived in D. melanogaster, D. simulans, D. yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.

sentences: A Genotype-to-Phenotype Modeling Framework to Predict Human Pathogenicity of Novel Coronaviruses Leveraging prior viral genome sequencing data to make predictions on whether an unknown, emergent virus harbors a  phenotype-of-concern has been a long-sought goal of genomic epidemiology. A predictive phenotype model built from nucleotide-level information alone has previously been considered un-tenable with respect to RNA viruses due to the ultra-high intra-sequence variance of their genomes, even within closely related clades. Building from our prior work developing a degenerate k-mer method to accommodate this high intra-sequence variation of RNA virus genomes for modeling frameworks, and leveraging a taxonomic  group-shuffle-split paradigm on complete coronavirus assemblies from prior to October 2018, we trained multiple regularized logistic regression classifiers at the nucleotide k-mer level capable of accurately predicting withheld SARS-CoV-2 genome sequences as human pathogens and accurately predicting withheld Swine Acute Diarrhea Syndrome coronavirus (SADS-CoV) genome sequences as non-human pathogens. LASSO feature selection identified several degenerate nucleotide predictor motifs with high model coefficients for the human pathogen class that were present across widely disparate classes of coronaviruses. However, these motifs differed in which genes they were present in, what specific codons were used to encode them, and what the translated amino acid motif was. This emphasizes the importance of a phenetic view of emerging pathogenic RNA viruses, as opposed to the canonical phylogenetic interpretations most-commonly used to track and manage viral zoonoses. Applying our model to more recent Orthocoronavirinae genomes deposited since October 2018 yields a novel contextual view of pathogen-potential across bat-related, canine-related, porcine-related, and rodent-related coronaviruses and critical adaptations which may have contributed to the emergence of the pandemic SARS-CoV-2 virus. Finally, we discuss the utility of these predictive models (and their associated predictor motifs) to novel biosurveillance protocols that substantially increase the  pound-for-pound information content of field-collected sequencing data and make a strong argument for the necessity of routine collection and sequencing of zoonotic viruses.

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To See, Not to See, or to See Poorly: Perceptual Quality and Guess Rate as a Function of Electroencephalography (EEG) Brain Activity in an Orientation Perception Task Detection of visual stimuli fluctuates over time, and these fluctuations have been shown to correlate with time-domain evoked activity and frequency-domain periodic activity. However, it is unclear if these fluctuations are related to a change in guess rate, perceptual quality, or both. Here we determined whether the quality of perception randomly varies across trials or is fixed so that the variability is the same. Then we estimated how perceptual quality and guess rate on an orientation perception task relates to EEG activity. Response errors were fitted to variable precision models and the standard mixture model to determine whether perceptual quality is from a varying or fixed distribution. Overall, the best fit was the standard mixture model that assumes response variability can be defined by a fixed distribution.

The power and phase of 2-7 Hz post-target activities were found to vary along with task performance in that more accurate trials had greater power, and the preferred phase differed significantly between accurate and guess trials. Guess rate and {sigma} were significantly lower on trials with high 2-3 Hz power than low and the difference started around 250 ms post-target.

These effects coincide with changes in the P3 ERP: there was a more positive deflection in the accurate trials vs guesses. These results suggest that the spread of errors (perceptual quality) can be characterized by a fixed range of values. Where the errors fall within that range is modulated by the post-target power in the lower frequency bands and their analogous ERPs.

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RNA-Seq analysis and transcriptome assembly of Salicornia neei reveals powerful system for ammonium detoxification BackgroundSalicornia neei is a halophyte plant that has been proposed for use in the phytoremediation of the saline wastewater generated by land-based aquaculture, which usually contains elevated concentrations of ammonium resulting from protein metabolism. To identify the molecular mechanisms related to ammonium response, we analyzed the transcriptome of S. neei in response to growth in saline water containing 3 mM ammonium and the Michaelis-Menten ammonium removal biokinetics.

ResultsThe RNA sequencing generated a total of 14,680,108 paired-end reads from the control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,020 transcripts and a reference transcriptome with an N50 of 683 bp.

A total of 45,327 genes were annotated, representing 51.2% of the contig predicted from de novo assembly. As regards DEGs, a total of 9,140 genes were differentially expressed in response to ammonium in saline water; of these, 7,396 could be annotated against functional databases. The upregulated genes were mainly involved in cell wall biosynthesis, transmembrane transport and antiporter activities, including biological KEGG pathways linked to the biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, and nitrogen metabolism. In addiction, a set of 72 genes was directly involved in ammonium metabolism, including glutamine synthetase 1 (GLN1), glutamate synthase 1 (GLT1), and ferredoxin-dependent glutamate synthase chloroplastic (Fd-GOGAT). Finally, we observed that the ammonium uptake rate increased with increasing ammonium concentrations, and tended toward saturation in the range of 3 to 4 mM.

ConclusionOur results support the hypothesis that an ammonium detoxification system mediated by glutamine and glutamate synthase was activated in S. neei when exposed to ammonium and saline water. The present transcriptome profiling method could be useful when investigating the response of halophyte plants to saline wastewater from land-based aquaculture.

sentences: A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

sentences: Validation of a small molecule inhibitor of PDE6D-RAS interaction with potent anti-leukemic effects RAS mutations prevalent in high-risk leukemia have been linked to relapse and chemotherapy resistance. Efforts to directly target RAS proteins have been largely unsuccessful. However, since RAS-mediated transformation is dependent on signaling through the RAS-related C3 botulinum toxin substrate (RAC) small GTPase, we hypothesized that targeting RAC may be an effective therapeutic approach in RAS mutated tumors. Here we describe multiple small molecules capable of inhibiting RAC activation in acute lymphoblastic leukemia cell lines. One of these, DW0254, also demonstrates promising anti-leukemic activity in RAS-mutated cells. Using chemical proteomics and biophysical methods, we identified the hydrophobic pocket of phosphodiester 6 subunit delta (PDE6D), a known RAS chaperone, as a target for this compound. Inhibition of RAS localization to the plasma membrane upon DW0254 treatment is associated with RAC inhibition through a phosphatidylinositol-3-kinase/AKT-dependent mechanism. Our findings provide new insights on the importance of PDE6D-mediated transport for RAS-dependent RAC activation and leukemic cell survival.

sentences: In situ polyadenylation enables spatial mapping of the total transcriptome Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We apply this Spatial Total RNA-Sequencing (STRS) approach to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration, and highlight host transcriptional responses associated with local viral RNA abundance. In situ polyadenylation requires the addition of only a single step to a widely used protocol for spatial RNA-sequencing, and thus could be broadly and quickly adopted. Spatial RNA-sequencing of the total transcriptome will enable new insights into spatial gene regulation and biology.

sentences: Astrocytes encode complex behaviorally relevant information Astrocytes, glial cells of the central nervous system, help to regulate neural circuit operation and adaptation. They exhibit complex forms of chemical excitation, most prominently calcium transients, evoked by neuromodulator and -transmitter receptor activation1-4. However, whether and how astrocytes contribute to cortical processing of complex behavior remains unknown1. One of the puzzling features of astrocyte calcium transients is the high degree of variability in their spatial and temporal patterns under behaving conditions. Here, we provide mechanistic links between astrocytes activity patterns, molecular signaling, and behavioral cognitive and motor activity variables by employing a visual detection task that allows for in vivo calcium imaging, robust statistical analyses, and machine learning approaches. We show that trial type and performance levels deterministically shape astrocytes spatial and temporal response properties. Astrocytes encode the animals decision, reward, and sensory properties. Our error analysis confirms that astrocytes carry behaviorally relevant information depending on and complementing neuronal coding. We also report that cell-intrinsic mechanisms curb astrocyte calcium activity. Additionally, we show that motor activity-related parameters strongly impact astrocyte responses and must be considered in sensorimotor study designs. Our data inform and constrain current models of astrocytes contribution to complex behavior and brain computation beyond their established homeostatic and metabolic roles.

sentences: Hypoxia is a dominant remodeler of the CD8+ T cell surface proteome relative to activation and regulatory T cell-mediated suppression Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the anti-tumor immune response. T cell surface receptors that influence interactions and function in the TME are already proven targets for cancer immunotherapy. However, surface proteome remodeling of primary human T cells in response to suppressive forces in the TME has never been characterized systematically. Using a reductionist cell culture approach with primary human T cells and SILAC-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly, the CD8+/Treg co-culture only modestly affected the CD8+ surfaceome, but did reverse several activation-induced surfaceomic changes. In contrast, hypoxia dramatically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia, revealing a novel hypoxia-induced surface receptor program. Our findings are consistent with the premise that hypoxic environments create a metabolic challenge for T cell activation, which may underlie the difficulty encountered in treating solid tumors with immunotherapies. Together, the data presented here provide insight into how suppressive TME factors remodel the T cell surfaceome and represent a valuable resource to inform future therapeutic efforts to enhance T cell function in the TME.

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Close-kin mark-recapture methods to estimate demographic parameters of mosquitoes Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when a total of 1,000 adult females are sampled biweekly-to-fortnightly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with a total of 4,000 larvae sampled biweekly over the same period. As the cost of genome sequencing declines, these methods hold great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

Author summaryClose-kin mark-recapture (CKMR) methods are a genetic analogue of traditional mark-recapture methods in which the frequency of marked individuals in a sample is used to infer demographic parameters such as census population size and mean dispersal distance. In CKMR, the mark is a close-kin relationship between individuals (parents and offspring, siblings, etc.). While CKMR methods have mostly been applied to aquatic species to date, opportunities exist to apply them to insects and other terrestrial species. Here, we explore the application of CKMR to mosquitoes, with Aedes aegypti, a primary vector of dengue, chikungunya and yellow fever, as a case study. By analyzing simulated Ae. aegypti populations, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration. Optimal sampling schemes are consistent with Ae. aegypti ecology and field studies, requiring only minor adjustments to current mosquito surveillance programs. This study represents the first theoretical exploration of the application of CKMR to an insect species, and demonstrates its potential for characterizing the demography of insects of epidemiological and agricultural importance.

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Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light). Presence of niche-dependent physiological time cues has been proposed, allowing local tissues flexibility of phase adjustment. However, to date, such stimuli have remained elusive. Here we show that cycles of mechanical loading and osmotic stimuli within physiological range drive rhythmic expression of clock genes and reset clock phase and amplitude in cartilage and intervertebral disc tissues. Hyperosmolarity (not hypo-osmolarity) resets clocks in young and ageing skeletal tissues through mTORC2-AKT-GSK3{beta} pathway, leading to genome-wide induction of rhythmic genes. These results advocate diurnal patterns of mechanical loading and consequent daily surges in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

One-Sentence SummaryCircadian clocks in aneural skeletal tissues sense the passage of time through rhythmic patterns of loading and osmolarity

sentences: A shared brain system forming confidence judgment across cognitive domains Confidence is typically defined as a subjective judgment about whether a decision is right. Decisions are based on sources of information that come from various cognitive domains and are processed in different brain systems. An unsettled question is whether the brain computes confidence in a similar manner whatever the domain or in a manner that would be idiosyncratic to each domain. To address this issue, human participants of both sexes performed two tasks probing confidence in decisions made about the same material (history and geography statements), but based on different cognitive processes: semantic memory for deciding whether the statement was true or false, and duration perception for deciding whether the statement display was long or short. At the behavioral level, we found that the same factors (difficulty, accuracy, response time and confidence in the preceding decision) predicted confidence judgments in both tasks. At the neural level, we observed using fMRI that confidence judgments in both tasks were associated to activity in the same brain regions: positively in the ventromedial prefrontal cortex and negatively in a prefronto-parietal network. Together, these findings suggest the existence of a shared brain system that generates confidence judgments in a similar manner across cognitive domains.

sentences: Effects of variability in manually contoured spinal cord masks on fMRI co-registration and interpretation Functional magnetic resonance imaging (fMRI) of the human spinal cord (SC) is an emerging method for understanding neurovascular responses to stimuli. Group-analysis of SC fMRI data involves co-registration of subject-level data to standard space, which requires manual masking of the cord and may result in bias of group-level SC fMRI results. To test this, we examined variability in SC masks drawn in fMRI data from 21 healthy participants from a completed study mapping responses to sensory stimuli of the C7 dermatome. Masks were drawn on temporal mean functional image by eight raters with varying levels of neuroimaging experience, and the rater from the original study acted as an expert reference. Spatial agreement between rater and expert masks was measured using the Dice Similarity Coefficient, and the influence of rater and dataset was examined using ANOVA. Each raters masks were used to register functional data to the PAM50 template. Gray matter-white matter signal contrast of registered functional data was used to evaluate the spatial normalization accuracy across raters. Subject- and group-level analyses of activation during left- and right-sided sensory stimuli were performed for each raters co-registered data. Agreement with the experts SC mask was associated with both rater experience level (F(7,140) = 32.12, P < 2x10-16,{eta} 2 = 0.29) and dataset (F(20,140) = 20.58, P < 2x10-16,{eta} 2 = 0.53). Dataset variations may reflect image quality metrics: the ratio between the signal intensity of spinal cord voxels and surrounding cerebrospinal fluid was correlated with DSC results (p<0.001). As predicted, variability in the manually-drawn masks influenced spatial normalization, and GM:WM contrast in the registered data showed significant effects of rater and dataset (rater: F(8,160) = 23.57, P < 2x10-16,{eta} 2 = 0.24; dataset: F(20,160) = 22.00, P < 2x10-16,{eta} 2 = 0.56). Registration differences propagated into subject-level activation maps which showed rater-dependent agreement with the expert. Although group-level activation maps differed between raters, no systematic bias was identified. Increasing consistency in manual contouring of spinal cord fMRI data improved co-registration and inter-rater agreement in activation mapping, however our results suggest that improvements in image acquisition and post-processing are also critical to address.

sentences: Integrin α6 upregulation in nucleus pulposus cell under high oxygen tension attenuates intervertebral disc degeneration The destruction of low oxygen microenvironment played critical roles in the pathogenesis of intervertebral disk degeneration (IVDD). In this study, high oxygen tension (HOT) treatment upregulated integrin 6(ITG 6) expression, which could be alleviated by blocking PI3K/AKT signaling pathway. And the levels of ITG 6 expression were increased in the NP tissue from IVDD patients and IVDD rat model with mild degeneration, which were reduced as degeneration degree increases. Further studies found that ITG 6 could protect NP cells against HOT-induced apoptosis and oxidative stress, and protect NP cells from HOT-inhibited ECM proteins synthesis. ITG 6 upregulation by HOT contributed to maintain a NP tissue homeostasis through the interaction with hypoxia inducible factor-1 (HIF-1). Furthermore, silencing of ITG 6 in vivo could obviously accelerate puncture-induced IVDD. Taken together, ITG 6 upregulation by HOT in NP cells might be a protective factor in IVDD as well as restore NP cell function.

sentences: Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate Retriever-mediated recycling on endosomes Cell-surface receptors control how cells respond to their environment. Many cell-surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH and CCC complexes. Here we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces PI3P, which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition causes a loss of Retriever and CCC from endosomes, and mutation of the lipid binding site on a CCC subunit impairs its endosomal localization and delays integrin recycling. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.

sentences: Sleep problems in preschoolers with autism spectrum disorders are associated with sensory sensitivities and thalamocortical overconnectivity Projections between the thalamus and sensory cortices are established early in development and play an important role in sleep regulation as well as in relaying sensory information to cortex. Atypical thalamocortical functional connectivity frequently observed in children with autism spectrum disorders (ASD) might therefore be linked to sensory and sleep problems common in ASD. Here we investigated the relationship between auditory-thalamic functional connectivity measured during natural sleep fMRI, sleep problems, and sound sensitivities in 70 toddlers and preschoolers (1.5 to 5-year-olds) with ASD compared to a matched group of 46 typically developing (TD) children. In children with ASD, sleep problems and sensory sensitivities were positively correlated, and increased sleep latency was associated with overconnectivity between the thalamus and auditory cortex in a subsample with high quality MRI data (n=29). Additionally, auditory cortex BOLD signal amplitude was elevated in children with ASD, potentially reflecting reduced sensory gating or a lack of auditory habituation during natural sleep. These findings indicate that atypical thalamocortical functional connectivity can be detected early in development and may play a crucial role in sleep problems and sensory sensitivities in ASD.

sentences: Effective Cell Membrane Tension is Independent of Substrate Stiffness Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs.

sentences: The Role of the Human Brain Neuron-Glia-Synaptic Composition in Forming Resting State Functional Connectivity Networks While significant progress has been achieved in studying resting state functional networks in a healthy human brain and in a wide range of clinical conditions, many questions related to their relationship to the brains cellular constituents remain open. Herein we use quantitative Gradient-Recalled-Echo (qGRE) MRI for mapping human brain cellular composition, and BOLD (blood oxygen level dependent) MRI to explore how the brain cellular constituents relate to resting state functional networks. Results show that the BOLD-signal-defined synchrony of connections between cellular circuits in network-defined individual functional units is mainly associated with the regional neuronal density, while the between-functional-units connectivity strength is also influenced by the glia and synaptic components of brain tissue cellular constituents. These mechanisms lead to a rather broad distribution of resting state functional networks properties. Visual networks with the highest neuronal density (but lowest density of glial cells and synapses) exhibit the strongest coherence of BOLD signal, as well as the strongest intra-network connectivity. The Default Mode Network (DMN) is positioned near the opposite part of the spectrum with relatively low coherence of the BOLD signal but a remarkably balanced cellular content enabling DMN prominent role in the overall organization of the brain and the hierarchy of functional networks.

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Closed-loop optimization of transcranial magnetic stimulation with electroencephalography feedback BackgroundTranscranial magnetic stimulation (TMS) is widely used in brain research and treatment of various brain dysfunctions. However, the optimal way to target stimulation and administer TMS therapies, for example, where and in which electric field direction the stimuli should be given, is yet to be determined.

ObjectiveTo develop an automated closed-loop system for adjusting TMS parameters (in this work, the stimulus orientation) online based on TMS-evoked brain activity measured with electroencephalography (EEG).

MethodsWe developed an automated closed-loop TMS-EEG set-up. In this set-up, the stimulus parameters are electronically adjusted with multi-locus TMS. As a proof of concept, we developed an algorithm that automatically optimizes the stimulation orientation based on single-trial EEG responses. We applied the algorithm to determine the electric field orientation that maximizes the amplitude of the TMS- EEG responses. The validation of the algorithm was performed with six healthy volunteers, repeating the search twenty times for each subject.

ResultsThe validation demonstrated that the closed-loop control worked as desired despite the large variation in the single-trial EEG responses. We were often able to get close to the orientation that maximizes the EEG amplitude with only a few tens of pulses.

ConclusionOptimizing stimulation with EEG feedback in a closed-loop manner is feasible and enables effective coupling to brain activity.

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Rcompadre and Rage - two R packages to facilitate the use of the COMPADRE and COMADRE databases and calculation of life history traits from matrix population models O_LIMatrix population models (MPMs) are an important tool for biologists seeking to understand the causes and consequences of variation in vital rates (e.g., survival, reproduction) across life cycles. Empirical MPMs describe the age- or stage-structured demography of organisms and usually represent the life history of a population during a particular time frame at a specific geographic location.
C_LIO_LIThe COMPADRE Plant Matrix Database and COMADRE Animal Matrix Database are the most extensive resources for MPM data, collectively containing >12,000 individual projection matrices for >1,100 species globally. Although these databases represent an unparalleled resource for researchers, land managers, and educators, the current computational tools available to answer questions with MPMs impose significant barriers to potential COM(P)ADRE database users by requiring advanced knowledge to handle diverse data structures and program custom analysis functions.
C_LIO_LITo close this knowledge gap, we present two interrelated R packages designed to (i) facilitate the use of these databases by providing functions to acquire, quality control, and manage both the MPM data contained in COMPADRE and COMADRE, and a users own MPM data (Rcompadre), and (ii) present a range of functions to calculate life history traits from MPMs in support of ecological and evolutionary analyses (Rage). We provide examples to illustrate the use of both.
C_LIO_LIRcompadre and Rage will facilitate demographic analyses using MPM data and contribute to the improved replicability of studies using these data. We hope that this new functionality will allow researchers, land managers, and educators to unlock the potential behind the thousands of MPMs and ancillary metadata stored in the COMPADRE and COMADRE matrix databases, and in their own MPM data.
C_LI

sentences: Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face and one to the cytosolic. Two are specific to VGLUT2 whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

sentences: Tuberculosis treatment failure associated with evolution of antibiotic resilience Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

sentences: cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

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Discovery, diversity and functional associations of crAss-like phages in human gut metagenomes from four Dutch cohorts The crAss-like phages are a diverse group of related viruses that includes one of the most abundant viruses of the human gut. To explore their diversity and functional role in human population and clinical cohorts, we analyzed gut metagenomic data collected from more than 2000 individuals from the Netherlands. We discovered 125 novel species-level and 32 novel genus-level clusters of crAss-like phages, all belonging to five previously recognized groups associated with the human gut. Analysis of their genomic features revealed that closely related crAss-like phages can possess strikingly divergent regions responsible for transcription, presumably acquired through recombination. Prediction of crAss-like phage hosts pointed primarily to bacteria of the phylum Bacteroidetes, consistent with previous reports. Finally, we explored the temporal stability of crAss-like phages over a 4-year period and identified associations between the abundance of crAss-like phages and several human phenotypes, including depletion of crAss-like phages in inflammatory bowel disease patients.

HighlightsO_LI125 tentative new species of crAss-like phages were discovered
C_LIO_LIClosely related crAss-like phages often possess highly divergent transcription gene modules possibly acquired via recombination
C_LIO_LICrAss-like fraction of the human gut virome remains relatively stable over the period of 4 years
C_LIO_LIPrevalence of crAss-like phages in the human gut is associated with several metabolic, dietary and health phenotypes
C_LIO_LIGut crAss-like phages are depleted in inflammatory bowel disease patients
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sentences: Slow fluctuations in ongoing brain activity decrease in amplitude with ageing yet their impact on task-related evoked responses is dissociable from behaviour In humans, ageing is characterized by decreased brain signal variability and increased behavioural variability. To understand how reduced brain variability segregates with increased behavioural variability, we investigated the association between reaction time variability, evoked brain responses and ongoing brain signal dynamics, in young (N = 36) and older adults (N = 39). We studied the electroencephalogram (EEG) and pupil size fluctuations to characterize the cortical and arousal responses elicited by a cued go/no-go task. Evoked responses were strongly modulated by slow (< 2 Hz) fluctuations of the ongoing signals, which presented reduced power in the older participants. Although variability of the evoked responses was lower in the older participants, once we adjusted for the effect of the ongoing signal fluctuations, evoked responses were equally variable in both groups. Moreover, the modulation of the evoked responses caused by the ongoing signal fluctuations had no impact on reaction time, thereby explaining why although ongoing brain signal variability is decreased in older individuals, behavioural variability is not. Finally, we showed that adjusting for the effect of the ongoing signal was critical to unmask the link between neural responses and behaviour.

sentences: The neuropeptides VIP and PACAP inhibit SARS-CoV-2 replication in monocytes and lung epithelial cells, decrease the production of proinflammatory cytokines, and VIP levels are associated with survival in severe COVID-19 patients. Infection by SARS-CoV-2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID-19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, VIP and PACAP, highly similar neuropeptides, decreased the SARS-CoV-2 genome replication in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS-CoV-2-induced activation of NF-kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID-19.

sentences: Altered brain criticality in Schizophrenia: New insights from MEG Schizophrenia has a complex etiology and symptomatology that is difficult to untangle. After decades of research, important advancements towards a central biomarker are still lacking. One of the missing pieces is a better understanding of how non-linear neural dynamics are altered in this patient population. In this study, the resting-state neuromagnetic signals of schizophrenia patients and healthy controls were analyzed in the framework of criticality. When biological systems like the brain are in a state of criticality, they are thought to be functioning at maximum efficiency (e.g., optimal communication and storage of information) and with maximum adaptability to incoming information. Here, we assessed the self-similarity and multifractality of resting-state brain signals recorded with magnetoencephalography in patients with schizophrenia patients and in matched controls. Our analysis showed a clear ascending, rostral to caudal gradient of self-similarity values in healthy controls, and an opposite gradient for multifractality (descending values, rostral to caudal). Schizophrenia patients had similar, although attenuated, gradients of self-similarity and multifractality values. Statistical tests showed that patients had higher values of self-similarity than controls in fronto-temporal regions, indicative of more regularity and memory in the signal. In contrast, patients had less multifractality than controls in the parietal and occipital regions, indicative of less diverse singularities and reduced variability in the signal. In addition, supervised machine-learning, based on logistic regression, successfully discriminated the two groups using measures of self-similarity and multifractality as features. Our results provide new insights into the baseline cognitive functioning of schizophrenia patients by identifying key alterations of criticality properties in their resting-state brain data.

sentences: CircHIPK3 dysregulation of the miR-30c/DLL4 axis is essential for KSHV lytic replication Non-coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposis sarcoma-associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA-mediated regulation of target protein-coding transcripts; however, we have identified a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV-mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR-30c, leading to increased levels of the miR-30c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.

sentences: POU2AF2/C11orf53 functions as a co-activator of POU2F3 by maintaining chromatin accessibility and enhancer activity Small cell lung cancer (SCLC), accounting for around 13% of all lung cancers, often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. The POU class 2 homeobox 3 (POU2F3) is a master regulator of tuft cell identity and defines a subtype of SCLC tumors (SCLC-P subtype) that lack the expression of neuroendocrine (NE) markers. Here, we have identified a previously uncharacterized protein, C11orf53, which is co-expressed with POU2F3 in both SCLC cell lines and patient samples, defining the same SCLC-P subtype. C11orf53 is an essential gene for the cell survivability of SCLC-P subtype, based on genome-wide CRISPR screening and genetic depletion of C11orf53 or POU2F3, inducing apoptosis in different SCLC-P subtype cell lines. Furthermore, our biochemical and genome-wide studies have demonstrated that C11orf53 is a novel chromatin-bound protein that occupies broad active enhancer domains and regulates gene expression at active enhancers. Mechanistically, C11orf53 directly interacts with POU2F3 at the POU domain, and is recruited to chromatin by POU2F3. Consequently, depletion of C11orf53 dramatically reduced enhancer H3K27ac levels and chromatin accessibility, resulting in a reduction of the expression of POU2F3-dependent tuft cell markers. Based on the expression pattern and molecular function of C11orf53, we have renamed it as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2). In summary, our study has identified a new co-activator of POU2 family transcription factors, and sheds light on the therapeutic potential of targeting POU2AF2/POU2F3 heterodimer in human SCLC.

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Neuron tracing and quantitative analyses of dendritic architecture reveal symmetrical three-way-junctions and phenotypes of git-1 in C. elegans Complex dendritic trees are a distinctive feature of neurons. Alterations to dendritic morphology are associated with developmental, behavioral and neurodegenerative changes. The highly-arborized PVD neuron of C. elegans serves as a model to study dendritic patterning; however, quantitative, objective and automated analyses of PVD morphology are missing. Here, we present a method for neuronal feature extraction, based on deep-learning and fitting algorithms. The extracted neuronal architecture is represented by a database of structural elements for abstracted analysis. We obtain excellent automatic tracing of PVD trees and uncover that dendritic junctions are unevenly distributed. Surprisingly, these junctions are three-way-symmetrical on average, while dendritic processes are arranged orthogonally. We quantify the effect of mutation in git-1, a regulator of dendritic spine formation, on PVD morphology and discover a localized reduction in junctions. Our findings shed new light on PVD architecture, demonstrating the effectiveness of our objective analyses of dendritic morphology and suggest molecular control mechanisms.

Author SummaryNerve cells (neurons) collect input signals via branched cellular projections called dendrites. A major aspect of the study of neurons, dating back over a century, involves the characterization of neuronal shapes and of their dendritic processes.

Here, we present an algorithmic approach for detection and classification of the tree-like dendrites of the PVD neuron in C. elegans worms. A key feature of our approach is to represent dendritic trees by a set of fundamental shapes, such as junctions and linear elements. By analyzing this dataset, we discovered several novel structural features. We have found that the junctions connecting branched dendrites have a three-way-symmetry, although the dendrites are arranged in a crosshatch pattern; and that the distribution of junctions varies across distinct sub-classes of the PVDs dendritic tree. We further quantified subtle morphological effects due to mutation in the git-1 gene, a known regulator of dendritic spines. Our findings suggest molecular mechanisms for dendritic shape regulation and may help direct new avenues of research.

sentences: Narrative Event Segmentation in the Cortical Reservoir During continuous perception of movies or stories, awake humans display cortical activity patterns that reveal hierarchical segmentation of event structure. Sensory areas like auditory cortex display high frequency segmentation related to the stimulus, while semantic areas like posterior middle cortex display a lower frequency segmentation related to transitions between events (Baldassano et al. 2017). These hierarchical levels of segmentation are associated with different time constants for processing. Chien and Honey (2020) observed that when two groups of participants heard the same sentence in a narrative, preceded by different contexts, neural responses for the groups were initially different and then gradually aligned. The time constant for alignment followed the segmentation hierarchy: sensory cortices aligned most quickly, followed by mid-level regions, while some higher-order cortical regions took more than 10 seconds to align. These hierarchical segmentation phenomena can be considered in the context of processing related to comprehension. Uchida et al. (2021) recently described a model of discourse comprehension where word meanings are modeled by a language model pre-trained on a billion word corpus (Yamada et al 2020). During discourse comprehension, word meanings are continuously integrated in a recurrent cortical network. The model demonstrates novel discourse and inference processing, in part because of two fundamental characteristics: real-world event semantics are represented in the word embeddings, and these are integrated in a reservoir network which has an inherent gradient of functional time constants due to the recurrent connections. Here we demonstrate how this model displays hierarchical narrative event segmentation properties. The reservoir produces activation patterns that are segmented by the HMM of Baldassano et al (2017) in a manner that is comparable to that of humans. Context construction displays a continuum of time constants across reservoir neuron subset, while context forgetting has a fixed time constant across these subsets. Virtual areas formed by subgroups of reservoir neurons with faster time constants segmented with shorter events, while those with longer time constants preferred longer events. This neurocomputational recurrent neural network simulates narrative event processing as revealed by the fMRI event segmentation algorithm of Baldassano et al (2017), and provides a novel explanation of the asymmetry in narrative forgetting and construction observed by Chien and Honey (2020). The model extends the characterization of online integration processes in discourse to more extended narrative, and demonstrates how reservoir computing provides a useful model of cortical processing of narrative structure.

sentences: Construction of a potent pan-vaccine based on the evolutionary tendency of SARS-CoV-2 spike protein. SARS-CoV-2 continued to spread globally along with different variants. Here, we systemically analyzed viral infectivity and immune-resistance of SARS-CoV-2 variants to explore the underlying rationale of viral mutagenesis. We found that the Beta variant harbors both high infectivity and strong immune resistance, while the Delta variant is the most infectious with only a mild immune-escape ability. Remarkably, the Omicron variant is even more immune-resistant than the Beta variant, but its infectivity increases only in Vero E6 cells implying a probable preference for the endocytic pathway. A comprehensive analysis revealed that SARS-CoV-2 spike protein evolved into distinct evolutionary paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance, resulting in a narrow spectrum of the current single-strain vaccine. In light of these findings and the phylogenetic analysis of 2674 SARS-CoV-2 S-protein sequences, we generated a consensus antigen (S6) taking the most frequent mutations as a pan-vaccine against heterogeneous variants. As compared to the ancestry SWT vaccine with significantly declined neutralizations to emerging variants, the S6 vaccine elicits broadly neutralizing antibodies and full protections to a wide range of variants. Our work highlights the importance and feasibility of a universal vaccine strategy to fight against antigen drift of SARS-CoV-2.

sentences: The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium, and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. The results of our structural and mutational studies support a model wherein IdeR uses an indirect readout mechanism, identifying its targets via the sequence-specific DNA backbone structure rather than through direct contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.

sentences: Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex, a first-in-class molecular glue. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809).

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The Global Register of Introduced and Invasive Species: Country Compendium The Country Compendium of the Global Register of Introduced and Invasive Species (GRIIS) is a collation of data across 196 individual country checklists of alienspecies, along with a designation of those species with evidence of impact at a country level. The Compendium provides a baseline for monitoring the distribution and invasion status of all major taxonomic groups, and can be used for the purpose of global analyses of Introduced (alien, non-native, exotic) and Invasive species (invasive alien species), including regional, single and multi-species taxon assessments and comparisons. It enables exploration of gaps and inferred absences of species for countries, and provides a means for short to medium term refinement of GRIIS Checklists. The Country Compendium is, for example, instrumental, along with data on first records of introduction, for assessing and reporting on invasive alien species targets, including for the Convention on Biological Diversity and Sustainable Development Goals. The GRIIS Country Compendium provides a baseline and mechanism for tracking the spread of introduced and invasive alien species across countries globally.

O_TBL View this table:
org.highwire.dtl.DTLVardef@eb30fdorg.highwire.dtl.DTLVardef@dd338aorg.highwire.dtl.DTLVardef@62b8daorg.highwire.dtl.DTLVardef@1561763org.highwire.dtl.DTLVardef@119988e_HPS_FORMAT_FIGEXP  M_TBL C_TBL

sentences: Barcode aware adaptive sampling for Oxford Nanopore sequencers. Adaptive sampling enables selection of individual DNA molecules from sequencing libraries, a unique property of nanopore sequencing. Here we develop our adaptive sampling tool readfish to become "barcode-aware" enabling selection of different targets within barcoded samples or filtering out individual barcodes. We show that multiple human genomes can be assessed for copy number and structural variation on a single sequencing flow cell using sample specific customised target panels on both GridION and PromethION devices.

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Linear-Nonlinear Cascades Capture Synaptic Dynamics Short-term synaptic dynamics differ markedly across connections and strongly regulate how action potentials communicate information. To model the range of synaptic dynamics observed in experiments, we have developed a flexible mathematical framework based on a linear-nonlinear operation. This model can capture various experimentally observed features of synaptic dynamics and different types of heteroskedasticity. Despite its conceptual simplicity, we show that it is more adaptable than previous models. Combined with a standard maximum likelihood approach, synaptic dynamics can be accurately and efficiently characterized using naturalistic stimulation patterns. These results make explicit that synaptic processing bears algorithmic similarities with information processing in convolutional neural networks.

Author summaryUnderstanding how information is transmitted relies heavily on knowledge of the underlying regulatory synaptic dynamics. Existing computational models for capturing such dynamics are often either very complex or too restrictive. As a result, effectively capturing the different types of dynamics observed experimentally remains a challenging problem. Here, we propose a mathematically flexible linear-nonlinear model that is capable of efficiently characterizing synaptic dynamics. We demonstrate the ability of this model to capture different features of experimentally observed data.

sentences: West Nile virus detection in horses in three Brazilian states. We report genetic evidence of WNV circulation from southern and northeastern Brazilian states isolated from equine red blood cells. In the northeastern state the tenth human case was also detected, presenting neuroinvasive disease compatible with WNV infection. Our analyses demonstrate that much is still unknown on the virus local epidemiology. We advocate for a shift to active surveillance, to ensure adequate control for future epidemics with spill-over potential to humans.

sentences: Competitive outcomes between native and invasive plants are linked to shifts in the bacterial rhizosphere microbiome Rhizosphere microbiomes have received growing attention in recent years for their role in plant health, stress tolerance, soil nutrition, and invasion. Still, relatively little is known about how these microbial communities are altered under plant competition, and even less about whether these shifts are tied to competitive outcomes between native and invasive plants. We investigated the structure and diversity of rhizosphere bacterial and fungal microbiomes of native annual forbs and invasive annual grasses individually and in competition using high-throughput amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS region. We assessed how differentially abundant microbial families correlate to plant biomass under competition We find that bacterial diversity and structure differ between native forbs and invasive grasses, but fungal diversity and structure do not. Further, bacterial community structures under competition are distinct from individual bacterial community structures. We also identified five bacterial families that varied in normalized abundance between treatments and that were correlated with plant biomass under competition. We speculate that invasive grass dominance over these natives may be partially due to effects on the rhizosphere community, with changes in specific bacterial families potentially benefiting invaders at the expense of natives.

sentences: Cochlear organoids reveal epigenetic and transcriptional programs of postnatal hair cell differentiation from supporting cells We explored the transcriptional and epigenetic programs underlying the differentiation of hair cells from postnatal progenitor cells in cochlear organoids. Heterogeneity in the cells including cells with the transcriptional signatures of mature hair cells allowed a full picture of possible cell fates. Construction of trajectories identified Lgr5+ cells as progenitors for hair cells and the genomic data revealed gene regulatory networks leading to hair cells. We validated these networks, demonstrating dynamic changes both in expression and predicted binding sites of these transcription factors during organoid differentiation. We identified known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and predicted novel regulatory factors, Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for HC regeneration which is limited in the adult.

sentences: A non-canonical lipid droplet metabolism regulates the conversion of alpha-Synuclein to proteolytic resistant forms in neurons of a Drosophila model of Parkinson disease Parkinsons disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (Syn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between Syn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human Syn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/Midway, fatty acid transport protein/dFatp) and catabolic (lipase Brummer) enzymes, indicating that non-canonical mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by several distinct LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of Syn, which was found at the surface of LDs both in photoreceptors neurons of Drosophila and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of Syn to proteolytic digestion, a phenomenon associated with Syn aggregation in human neurons. We thus propose that Syn cooperates with LD proteins to inhibit lipolysis and that binding of Syn to LDs contributes to the pathogenic misfolding and aggregation of Syn in neurons.

sentences: Interpretable Drug Target Predictions using Self-Expressiveness The identification of missing drug targets is critical for the development of treatments and for the molecular elucidation of drug side effects. Drug targets have been predicted by exploiting molecular, biological or pharmacological features of drugs and protein targets. Yet, developing integrative and interpretable machine learning models for predicting drug targets remains a challenging task. We present Inception, an integrative and interpretable matrix completion model for predicting drug targets. Inception is a self-expressive model that learns two similarity matrices: one for drugs and another for protein targets. These learned similarity matrices are key for our models interpretability: they can explain how a predicted drug-target interaction can be explain in terms of a linear combination of chemical, biological and pharmacological similarities. We develop a novel objective function with efficient closed-form solution. To demonstrate the ability of Inception at recovering missing drug-target interactions (DTIs), we perform cross-validation experiments with stringent controls of data imbalance, chemical similarities between drugs and sequence similarities between targets. We also assess the performance of our model using a simulated prospective approach. Having trained our model with DTIs from a snapshot 2011 of the DrugBank database, we test whether we could predict DTIs from a 2020 snapshot of DrugBank. Inception outperforms two state-of-the-art drug target prediction models in all the scenarios. This suggests that Inception could be useful for predicting missing drug target interactions while providing interpretable predictions.

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A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs (rRNA) and in eukaryotic genomes are typically present as one or more arrays of tandem repeats. Species have characteristic rDNA copy numbers, ranging from tens to thousands of copies, with the number thought to be redundant for rRNA production. However, the tandem rDNA repeats are prone to recombination-mediated changes in copy number, resulting in substantial intra-species copy number variation. There is growing evidence that these copy number differences can have phenotypic consequences. However, we lack a comprehensive understanding of what determines rDNA copy number, how it evolves, and what the consequences are, in part because of difficulties in quantifying copy number. Here, we developed a genomic sequence read approach that estimates rDNA copy number from the modal coverage of the rDNA and whole genome to help overcome limitations in quantifying copy number with existing mean coverage-based approaches. We validated our method using strains of the yeast Saccharomyces cerevisiae with previously-determined rDNA copy numbers, and then applied our pipeline to investigate rDNA copy number in a global sample of 788 yeast isolates. We found that wild yeast have a mean copy number of 92, consistent with what is reported for other fungi but much lower than in laboratory strains. We also show that different populations have different rDNA copy numbers. These differences can partially be explained by phylogeny, but other factors such as environment are also likely to contribute to population differences in copy number. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.

Author summaryThe ribosomal RNA gene repeats (rDNA) form large tandem repeat arrays in most eukaryote genomes. Their tandem arrangement makes the rDNA prone to copy number variation, and there is increasing evidence that this copy number variation has phenotypic consequences. However, difficulties in measuring rDNA copy number hamper investigation into rDNA copy number dynamics and their significance. Here we developed a novel bioinformatics method for measuring rDNA copy number from whole genome sequence data that is based on the modal sequence read coverage. We established parameters for optimal performance of the method and validated it using yeast strains of known rDNA copy numbers. We then applied the method to a dataset of almost 800 global yeast isolates and demonstrate that yeast populations have different rDNA copy numbers that partially correlate with phylogeny. Our work provides a simple and accurate method for determining rDNA copy number that leverages the growing number of whole genome datasets, and highlights the dynamic nature of rDNA copy number.

sentences: Modulating short-term auditory memory with focal transcranial direct current stimulation applied to the Supramarginal Gyrus Previous studies have shown that transcranial direct current stimulation (tDCS) can affect performance by decreasing regional excitability in a brain region that contributes to the task of interest. To our knowledge, no research to date has found both enhancing and diminishing effects on performance, depending upon which polarity of current is applied. The supramarginal gyrus (SMG) is an ideal brain region for testing tDCS effects because it is easy to identify using the 10-20 EEG coordinate system, and results of neuroimaging studies have implicated the left SMG in short-term memory for phonological and non-phonological sounds. In the present study, we found that applying tDCS to the left SMG affected pitch memory in a manner that depended upon the polarity of stimulation: cathodal tDCS had a negative impact on performance while anodal tDCS had a positive impact. These effects were significantly different from sham stimulation, which did not influence performance; they were also specific to the left hemisphere - no effect was found when applying cathodal stimulation to the right SMG - and were unique to pitch memory as opposed to memory for visual shapes. Our results provide further evidence that the left SMG is a nodal point for short-term auditory storage and demonstrate the potential of tDCS to influence cognitive performance, and to causally examine hypotheses derived from neuroimaging studies.

sentences: Humoral immune responses against seasonal coronaviruses predict efficiency of SARS-CoV-2 spike targeting, FcγR activation, and corresponding COVID-19 disease severity Despite SARS-CoV-2 being a "novel" coronavirus, several studies suggest that detection of anti-spike IgG early in infection may be attributable to the amplification of humoral memory responses against seasonal hCoVs in severe COVID-19 patients. In this study, we examined this concept by characterizing anti-spike IgG from a cohort of non-hospitalized convalescent individuals with a spectrum of COVID-19 severity. We observed that anti-spike IgG levels positively correlated with disease severity, higher IgG cross-reactivity against betacoronaviruses (SARS-CoV-1 and OC43), and higher levels of proinflammatory Fc gamma receptor 2a and 3a (Fc{gamma}R2a & Fc{gamma}R3a) activation. In examining the levels of IgG targeting betacoronavirus conserved and immunodominant epitopes versus disease severity, we observed a positive correlation with the levels of IgG targeting the conserved S2FP region, and an inverse correlation with two conserved epitopes around the heptad repeat (HR) 2 region. In comparing the levels of IgG targeting non-conserved epitopes, we observed that only one of three non-conserved immunodominant epitopes correlated with disease severity. Notably, the levels of IgG targeting the receptor binding domain (RBD) were inversely correlated with severity. Importantly, targeting of the RBD and HR2 regions have both been shown to mediate SARS-CoV-2 neutralization. These findings show that, aside from antibody (Ab) targeting of the RBD region, humoral memory responses against seasonal betacoronaviruses are potentially an important factor in dictating COVID-19 severity, with anti-HR2-dominant Ab profiles representing protective memory responses, while an anti-S2FP dominant Ab profiles indicate deleterious recall responses. Though these profiles are masked in whole antigen profiling, these analyses suggest that distinct Ab memory responses are detectable with epitope targeting analysis. These findings have important implications for predicting severity of SARS-CoV-2 infections (primary and reinfections), and may predict vaccine efficacy in subpopulations with different dominant antibody epitope profiles.

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Sponging of glutamate at the outer plasma membrane surface reveals roles for glutamate in development Plants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), number of lateral roots, vegetative growth and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.

SIGNIFICANCE STATEMENTGlutamate is an important signaling molecule in animals and plants. The affinity-dependent effects of surface-displayed glutamate sensors on plant development intimate that glutamate levels in the vicinity of the plasma membrane play important roles in signaling in plants.

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GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery Dynamin2 GTPase (Dyn2) is a crucial player in clathrin-mediated endocytosis. Dyn2 is tetrameric in cytoplasm and self-assembles into functional units upon membrane binding. How the curvature activities and functionality of Dyn2 emerge during self-assembly and are regulated by lipids remains unknown. Here we reconstituted the Dyn2 self-assembly process using membrane nanotubes (NT) and vesicles and characterized it using single- molecule fluorescence microscopy, optical tweezers force spectroscopy and cryo-electron microscopy. On NTs, Dyn2 first forms small subhelical oligomers, which are already curvature active and display pronounced curvature sensing properties. Conical lipids and GTP promote their further self-assembly into helical machinery mediating the NT scission. In the presence of large unilamellar vesicles (LUVs), an alternative self- assembly pathway emerges where the subhelical oligomers form membrane tethering complexes mediating LUV-NT binding. Reconstitution of tethering in the LUV system revealed that lipid mixing is controlled by conical lipid species, divalents, GTP, and SH3 binding partners of Dyn2. In membranes with a high content of lipids with negative intrinsic curvature, cryo-EM revealed putative membrane contact sites made by Dyn2 clusters. On such membranes, with GTP lowered to 0.2 mM, both membrane fission and tethering activities become possible, indicating functional promiscuity of Dyn2.

We conclude that GTP and lipids control both extent and topology of Dyn2 functional self-assembly. The function of Dyn2 oligomers evolves from curvature sensing, seen in subhelical Dyn2 oligomers, to curvature creation and fission, seen in Dyn2 helices. Under specific circumstances, such as downregulation of SH3 partners of Dyn2 and GTP depletion, membrane tethering activity can emerge in membrane systems enriched with conical lipids. Hence the Dyn2 functionality is actively adapted to lipidome, explaining its large habitat in the cells and tissues.

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Microtubule-based mitotic spindles contain a micron-sized mixed-nucleotide zone Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro. In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing "HURP-gap" which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone.

One Sentence SummaryWe reveal that the microtubules of the mitotic spindle contain a third, uncharacterized domain, a mixed nucleotide zone that resides between the GTP-cap and the GDP-tubulin lattice.

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Cuttlefish: Fast, parallel, and low-memory compaction of de Bruijn graphs from large-scale genome collections MotivationThe construction of the compacted de Bruijn graph from collections of reference genomes is a task of increasing interest in genomic analyses. These graphs are increasingly used as sequence indices for short and long read alignment. Also, as we sequence and assemble a greater diversity of genomes, the colored compacted de Bruijn graph is being used as the basis for efficient methods to perform comparative genomic analyses on these genomes. Therefore, designing time and memory efficient algorithms for the construction of this graph from reference sequences is an important problem.

ResultsWe introduce a new algorithm, implemented in the toolCuttlefish, to construct the (colored) compacted de Bruijn graph from a collection of one or more genome references. Cuttlefish introduces a novel approach of modeling de Bruijn graph vertices as finite-state automata; it constrains these automatas state-space to enable tracking their transitioning states with very low memory usage. Cuttlefish is fast and highly parallelizable. Experimental results demonstrate that it scales much better than existing approaches, especially as the number and the scale of the input references grow. On our test hardware, Cuttlefish constructed the graph for 100 human genomes in under 9 hours, using ~29 GB of memory while no other tested tool completed this task. On 11 diverse conifer genomes, the compacted graph was constructed by Cuttlefish in under 9 hours, using ~84 GB of memory, while the only other tested tool that completed this construction on our hardware took over 16 hours and ~289 GB of memory.

AvailabilityCuttlefish is written in C++14, and is available under an open source license at https://github.com/COMBINE-lab/cuttlefish.

Contactrob@cs.umd.edu

Supplementary informationSupplementary text are available at Bioinformatics online.

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ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants ARP2/3 is a heteroheptameric protein complex evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. We demonstrate here that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile dot-like structures in the cytoplasm that are associated with plant peroxisomes. ARP2/3 dot structure is found at the peroxisome periphery and contains assembled ARP2/3 complex and WAVE/SCAR complex subunit NAP1. This dot occasionally colocalizes with the autophagosome, and under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than WT, we link the ARP2/3 complex on peroxisomes to the process of peroxisome degradation by autophagy called pexophagy. Additionally, several other peroxisomal proteins colocalize with ARP2/3 dot on plant peroxisomes. Our results suggest a specific role of ARP2/3 and actin in the peroxisome periphery, presumably in membrane remodelling. We hypothesize that this role of ARP2/3 aids processes at the peroxisome periphery such as peroxisome degradation through autophagy or regulation of peroxisomal proteins localization or function.

Significance statementARP2/3 complex-positive dots associate exclusively with peroxisomes in plant cells, where it colocalizes with autophagosome marker ATG8f and several other proteins. Our experiments link ARP2/3 to pexophagy: colocalization between ARP2/3 dots and autophagosome increases when autophagy processes are induced or inhibited; ARP2/3 and ATG8f colocalize and co-immunoprecipitate, and finally, ARP2/3 mutants cells contain more peroxisomes than WT. Our results suggest a novel role of ARP2/3 in peroxisome structure and function regulation.

sentences: Spatial modeling of extinct Southeast Asian Hobbit Homo floresiensis habitat suitability based on its diet availability and climatic parameters as a proxy The discovery of a new prehistoric species of the genus Homo remains a significant matter of intense interest. One of significant discovery recently is the Homo floresiensis, representing a small-bodied and small-brained hominin, excavated, and found in Liang Bua Cave in Flores Island, East Nusa Tenggara, Indonesia. This species height was only about 106 cm (36") and the weight was 30-40 kg (66-86 lbs). H. floresiensis was known consumed extant murine rodents as its diets as this was evidence found in Liang Bua Cave. Then this study aims to model the H. floresiensis suitable habitat using maximum entropy method and 2 extant murine rodents, Rattus exulans and R. rattus as a proxy. The results show that the most suitable habitats for H. floresiensis indicated by suitable habitat values close to 1 were concentrated in the central of Flores Island that was overlapped with mountainous areas with elevation ranging from 1500 to 2000 m. These suitable habitats were also overlapped with dense vegetation covers, volcanic rock, and Kiro rock formation. Climatic parameters that limit the distributions of H. floresiensis were annual mean temperature, isothermality, minimum temperature of coldest period, and precipitation seasonality. Parts of Flores Island with the low temperature below 20 {degrees}C were favorable for H. floresiensis while an increase in isothermality limits the H. floresiensis distributions.

sentences: Neural correlates and determinants of approach-avoidance conflict in the prelimbic prefrontal cortex The recollection of environmental cues associated with threat or reward allows animals to select the most appropriate behavioral responses. Neurons in the prelimbic cortex (PL) respond to both threat- and reward-associated cues. However, it remains unknown whether PL regulates threat-avoidance vs. reward-approaching responses when an animals decision depends on previously associated memories. Using a conflict model in which male Long-Evans rats retrieve memories of shock- and food-paired cues, we observed two distinct phenotypes during conflict: i) rats that continued to press a lever for food (Pressers); and ii) rats that exhibited a complete suppression in food seeking (Non-Pressers). Single-unit recordings revealed that increased risk-taking behavior in Pressers is associated with persistent food-cue responses in PL, and reduced spontaneous activity in PL glutamatergic (PLGLUT) neurons during conflict. Activating PLGLUT neurons in Pressers attenuated food-seeking responses in a neutral context, whereas inhibiting PLGLUT neurons in Non-Pressers reduced defensive responses and increased food approaching during conflict. Our results establish a causal role for PLGLUT neurons in mediating individual variability in memory-based risky decision making by regulating threat-avoidance vs. reward-approach behaviors.

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GAMES: A dynamic model development workflow for rigorous characterization of synthetic genetic systems Mathematical modeling is invaluable for advancing understanding and design of synthetic biological systems. However, the model development process is complicated and often unintuitive, requiring iteration on various computational tasks and comparisons with experimental data. Ad hoc model development can pose a barrier to reproduction and critical analysis of the development process itself, reducing potential impact and inhibiting further model development and collaboration. To help practitioners manage these challenges, we introduce GAMES: a workflow for Generation and Analysis of Models for Exploring Synthetic systems that includes both automated and human-in-the-loop processes. We systematically consider the process of developing dynamic models, including model formulation, parameter estimation, parameter identifiability, experimental design, model reduction, model refinement, and model selection. We demonstrate the workflow with a case study on a chemically responsive transcription factor. The generalizable workflow presented in this tutorial can enable biologists to more readily build and analyze models for various applications.

Graphical Abstract

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sentences: Single-molecule, full-length transcript isoform sequencing reveals disease mutation-associated RNA isoforms in cardiomyocytes Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we established an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generated a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms (http://steinmetzlab.embl.de/iBrowser/). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identified 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by novel IMMT isoforms. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms as the functional unit, instead of genes or exons, providing more immediate biological interpretation and higher resolution transcriptome comparisons.

sentences: Fungal community turnover along steep environmental gradients from geothermal soils in Yellowstone National Park Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 {degrees}C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.

sentences: Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine intestine, including SELLhi{gamma}{delta} T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular CD4 {beta} T cells, and cycling T/ILCs were enriched in ileum with Peyers patches, while non-cycling{gamma}{delta} T, CD8 {beta} T, and group 1 ILCs were enriched in ileum without Peyers patches. scRNA-seq findings were leveraged to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral porcine ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.

sentences: Cardenolide Increase in Foxglove During Stresses Corresponds to Cholesterol and Phytosterol Biosynthesis Cardenolides are a group of steroidal metabolites found in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Here we show that cardenolides were increased upon methyl jasmonate (MJ), sorbitol, potassium (KCl) chloride, and salicylic acid analog (BTH: 2,1,3-benzothiadiazole) treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5{beta}-reductases (P5{beta}R and P5{beta}R2) did not correlate with cardenolide increase. The expression of 3{beta}-hydroxysteroid dehydrogenase (3{beta}HSD) correlated with cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: SSR1 (sterol sidechain reductase), SMO1, and SMO3 (sterol methyl oxidase). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that both cholesterol and phytosterol are involved in cardenolide biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and revealed a novel relationship between phytosterol and cardenolide biosynthesis.

sentences: High current production of Shewanella oneidensis with electrospun carbon nanofiber anodes is directly linked to biofilm formation 1We study the current production of Shewanella oneidensis MR-1 with electro-spun carbon fiber anode materials and analyze the effect of the anode morphology on the micro-, meso-and macroscale. The materials feature fiber diameters in the range between 108 nm and 623 nm, resulting in distinct macropore sizes and surface roughness factors. A maximum current density of (255{+/-} 71) {micro}A cm-2 was obtained with 286 nm fiber diameter material. Additionally, micro-and mesoporosity were introduced by CO2 and steam activation which did not improve the current production significantly. The current production is directly linked to the biofilm dry mass under anaerobic and micro-aerobic conditions. Our findings suggest that either the surface area or the pore size related to the fiber diameter determines the attractiveness of the anodes as habitat and there-fore biofilm formation and current production. Reanalysis of previous works supports these findings.

sentences: Genetic characteristics of Apodemus speciosus at Akiyoshidai Quasi-National Park in Yamaguchi Prefecture The large Japanese field mouse (Apodemus speciosus) is a small rodent endemic to Japan. The mice have a genetic characteristic in which the number of chromosomes differs between those from western Japan and those from eastern Japan. A. spesiosus, found throughout Japan, is used as a model animal for geogenetics and monitoring of radiation effects of wildlife. In this present study, to elucidate the genetic characteristics of the mice Akiyoshidai Quasi-National Park in Yamaguchi Prefecture, we investigated mitochondrial DNA and chromosome numbers. As a result, A. speciosus from Yamaguchi Prefecture were classified into the Honshu-Shikoku-Kyushu group and had a western Japan-type chromosome set of 2n=46; however, some Yamaguchi Prefecture mice formed a genetic cluster in Yamaguchi Prefecture, suggesting that continuous monitoring is needed to reveal the dynamics of genetic diversity.

sentences: Neural and cognitive correlates of performance in dynamic multi-modal settings The endeavour to understand human cognition has largely relied upon investigation of task-related brain activity. However, resting-state brain activity can also offer insights into individual information processing and performance capabilities. Previous research has identified electroencephalographic resting-state characteristics (most prominently: the individual alpha frequency; IAF) that predict cognitive function. However, it has largely overlooked a second component of electrophysiological signals: aperiodic 1/f activity. The current study examined how both oscillatory and aperiodic resting-state EEG measures, alongside traditional cognitive tests, can predict performance in a dynamic and complex, semi-naturalistic cognitive task. Participants resting-state EEG was recorded prior to engaging in a Target Motion Analysis (TMA) task in a simulated submarine control room environment (CRUSE), which required participants to integrate dynamically changing information over time. We demonstrated that the relationship between IAF and cognitive performance extends from simple cognitive tasks (e.g., digit span) to complex, dynamic measures of information processing. Further, our results showed that individual 1/f parameters (slope and intercept) differentially predicted performance across practice and testing sessions, whereby flatter slopes were associated with improved performance during learning, while higher intercepts were linked to better performance during testing. In addition to the EEG predictors, we demonstrate a link between cognitive skills most closely related to the TMA task (i.e., spatial imagery) and subsequent performance. Overall, the current study highlights (1) how resting-state metrics - both oscillatory and aperiodic - have the potential to index higher-order cognitive capacity, while (2) emphasising the importance of examining these electrophysiological components within more dynamic settings and over time.

sentences: Glomerular function and urinary biomarker changes between vancomycin and vancomycin plus piperacillin-tazobactam in a translational rat model. Clinical studies have reported additive nephrotoxicity associated with the combination of vancomycin (VAN) and piperacillin-tazobactam (TZP). This study assessed differences in glomerular filtration rate (GFR) and urinary biomarkers between rats receiving VAN and those receiving VAN+TZP. Male Sprague-Dawley rats (n=26) were randomized to receive 96 hours of intravenous VAN at 150mg/kg/day, intraperitoneal TZP at 1400 mg/kg/day, or VAN+TZP. Kidney function was evaluated using fluorescein-isothiocyanate sinistrin and a transdermal sensor to estimate real-time glomerular filtration rate (GFR). Kidney injury was evaluated via urinary biomarkers including kidney injury molecule-1 (KIM-1), clusterin, and osteopontin. Compared to a saline control, only rats in the VAN group showed significant declines in GFR by day 4 (-0.39 mL/min/100 g body weight, 95% CI: -0.68 to -0.10, p=0.008). When the VAN+TZP and VAN alone treatment groups were compared, significantly higher urinary KIM-1 was observed in the VAN alone group on day 1 (18.4 ng, 95% CI: 1.4 to 35.3, p=0.03), day 2 (27.4 ng, 95% CI: 10.4 to 44.3, p=0.002), day 3 (18.8 ng, 95% CI: 1.9 to 35.8, p=0.03), and day 4 (23.2 ng, 95% CI: 6.3 to 40.2, p=0.007). KIM-1 was the urinary biomarker that most correlated with decreasing GFR on day 3 (Spearmans rho: -0.45, p = 0.022) and day 4 (Spearmans rho: - 0.41, p = 0.036). Kidney function decline and increased KIM-1 were observed among rats that received VAN only, but not TZP or VAN+TZP. Addition of TZP to VAN does not worsen kidney function or injury in a validated translational rat model.

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Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

One Sentence SummaryOmicron, carrying [~]30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.

sentences: Characterization of extracellular vesicles and artificial nanoparticles with four orthogonal single-particle analysis platforms We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

sentences: Biogenesis of mature piRNAs in Caenorhabditis elegans is dependent on orchestration between multiple ribonucleases and PRG-1 Piwi-interacting RNAs (piRNAs) are an animal-specific class of germline-enriched small non-coding RNAs that shape transcriptome, as well as ensure genomic integrity and fertility by regulating transposons and other selfish genetic elements. In Caenorhabditis elegans mature piRNAs are 21-nucleotides long, begin with a monophosphorylated uridine, and they associate with PRG-1 to form piRISCs that scan the transcriptome for  non-self sequences. However, these piRNAs are born as longer 5-capped transcripts, where PARN-1, a 3-5 exoribonuclease, contributes to the formation of the mature 3-end. But, till date, the 5-processing events remain elusive. We demonstrate that the recently identified endoribonuclease activity of XRN-2 is involved in the processing of the 5-end of precursor piRNAs in worms. Depletion of XRN-2 results in reduced mature piRNA levels, with concomitant increase in levels of the 5-capped precursors. We also reveal that the piRNAs born as longer precursor molecules ([≥]60 nt), prior to 5-end processing, undergo ENDU-1-mediated endoribonucleolytic processing of their 3-ends. Our in vitro RNA-protein interaction studies unravel the mechanistic interactions between XRN-2 and PRG-1 towards the formation of mature 5-ends of piRNAs. In vivo experiments employing prg-1 mutant worms indicate that XRN-2 has the potential to perform clearance of precursors that are not bound and protected by PRG-1. Finally, we also demonstrate that XRN-2 is not only important for the generation of mature piRNAs and piRNA-dependent endo-siRNAs, but through yet unknown pathways, it also affects piRNA-independent endo-siRNAs that shape transcriptome, as well as contribute to genomic integrity via regulation of transposable elements.

sentences: The Effects of Prenatal Nicotine and THC E-Cigarette Exposure on Motor Development in Rats In the United States, nicotine and cannabis are the most common licit and illicit drugs used among pregnant women. Importantly, nicotine and cannabis are now being combined for consumption via e-cigarettes, an increasingly popular route of administration. Both nicotine and tetrahydrocannabinol (THC), the primary psychoactive component of cannabis, cross the placenta barrier. However, the consequences of prenatal cannabis use are not well understood, and less is known about potential combination effects when consumed with nicotine, especially via e-cigarettes. The present study used a rodent model to examine how prenatal e-cigarette exposure to nicotine, THC, and the combination impacts motor development among offspring. Pregnant Sprague-Dawley rats were exposed to nicotine (36 mg/mL), THC (100 mg/mL), the combination, or vehicle via e-cigarette inhalation from gestational days (GD) 5-20. One sex pair per litter was tested on an early sensorimotor development task (postnatal days [PD] 12-20) and a parallel bar motor coordination task (PD 30-32). Combined prenatal exposure to nicotine and THC delayed sensorimotor development, even though neither drug produced impairments on their own. In contrast, prenatal exposure to either nicotine or THC impaired motor coordination, whereas combined exposure exacerbated these effects, particularly among females. These data illustrate that prenatal exposure to either nicotine or THC may alter motor development, and that the combination may produce more severe effects. These findings have important implications for pregnant women as we better understand the teratogenic effects of these drugs consumed via e-cigarettes.

sentences: Vitronectin acts as a key regulator of adhesion and migration in human umbilical cord-derived MSCs under different stress conditions Mesenchymal stem cell (MSC)-based therapy gets compromised as adverse microenvironmental conditions like nutrient deprivation, ischemia, hypoxia at the target site affect migration, engraftment and viability, of MSCs post transplantation. To improve the treatment efficacy, it is critical to identify factors involved in regulating migration and adhesion of MSCs under such microenvironmental stress conditions. In our study, we observed that human Whartons jelly-derived MSCs (WJ-MSCs) exhibited increase in cell spread area and adhesion, with reduction in cellular migration under serum starvation stress. The changes in adhesion and migration characteristics were accompanied by formation of large number of super mature focal adhesions along with extensive stress fibres and altered ECM gene expression with notable induction in vitronectin (VTN) expression. NF-{kappa}{beta} was found to be a positive regulator of VTN expression while ERK pathway regulated it negatively. Molecular and phenotypic comparison studies with inhibition of these signalling pathways or knocking down of VTN under serum starvation established the correlation between increase in VTN expression and increased cellular adhesion with corresponding reduction in cell migration. VTN knockdown also resulted in reduction of super mature focal adhesions and extensive stress fibres, which were formed under serum starvation. Additionally, VTN induction was not detected in hypoxia-treated WJ-MSCs, and the MSCs showed no significant change in the adhesion or migration properties under hypoxia. However, when VTN expression was induced under hypoxia by ERK pathway inhibition, similar increase in cell spread area and adhesion were observed. Our study thus highlights VTN as a key player which is induced under serum starvation stress and possibly regulates the adhesion and migration properties of WJ-MSCs via focal adhesion signalling.

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Neural Flip-Flops II: Short-Term Memory and Electroencephalography By enabling many brain structures state changes, the explicit cascaded oscillators proposed here can generate the rhythmic neural activity found in EEGs. The function of such synchronization in information processing systems is timing error avoidance. The narrow requirement for the oscillator input pulse duration suggests a possible relationship to the abnormal electrical activity characteristic of epileptic seizures. Together, flip-flops and synchronization by oscillators suggest a resolution to the longstanding controversy of whether short-term memory depends on neurons firing persistently or in brief, coordinated bursts.

The proposed cascade of oscillators consists of a ring oscillator and four toggle flip-flops connected in sequence. The novel oscillator and toggle are composed of three and six neurons, respectively. Their operation depends only on minimal properties of excitatory and inhibitory inputs.

The hypothesis that cascaded oscillators produce EEG phenomena implies that the distribution of EEG frequencies is determined by just two parameters, the mean (d) and standard deviation ({sigma}d) of the delay times of neurons that make up the initial oscillators in the cascades. For example, if d and {sigma}d are measured in milliseconds, the boundary separating the alpha and beta frequency bands is O_FD O_INLINEFIG[Formula]C_INLINEFIGC_FD

With 4 and 1.5 ms being the best available estimates for d and {sigma}d, respectively, this predicted boundary value is 14.9 Hz, which is within the range of commonly cited estimates obtained empirically from EEGs. Four and 1.5 ms also accurately predict the peaks and other boundaries of the five major EEG frequency bands.

Significance statementThe neuronal model proposed here implies several major aspects of electroencephalography. The matched periods of neural activity found in EEGs, and their wide distribution across the brain and across the frequency spectrum, follow from selective pressure for a biologically useful function: timing error avoidance for diverse brain functions in the trade-off between speed and accuracy. This activity can be achieved with a simple organization of synaptic connections and minimal neuron capabilities of excitation and inhibition. The multimodal distribution of EEG frequencies is an explicit function of the mean and variance of neuron delay times. The model suggests a relationship to epileptic seizures and a resolution to a short-term memory controversy. Two EEG characteristics make other models implausible.

sentences: Antioxidant Response Activating nanoParticles (ARAPas) localize to atherosclerotic plaque and locally activate the Nrf2 pathway Atherosclerotic disease is the leading cause of death world-wide with few novel therapies available despite the ongoing health burden. Redox dysfunction is a well-established driver of atherosclerotic progression; however, the clinical translation of redox-based therapies is lacking. One of the challenges facing redox-based therapies is their targeted delivery to cellular domains of redox dysregulation. In the current study, we sought to develop Antioxidant Response Activating nanoParticles (ARAPas), encapsulating redox-based interventions, that exploit macrophage biology and the dysfunctional endothelium in order to selectively accumulate in atherosclerotic plaque. We employed flash nanoprecipitation (FNP) to synthesize bio-compatible polymeric nanoparticles encapsulating the hydrophobic Nrf2 activator drug, CDDO-Methyl (CDDOMe-ARAPas). Nuclear factor erythroid 2-related factor 2 (Nrf2)-activators are a promising class of redox-active drug molecules whereby activation of Nrf2 results in the expression of several antioxidant and cyto-protective enzymes that can be athero-protective. In this study, we characterize the physiochemical properties of CDDOMe-ARAPas as well as confirm their in vitro internalization by murine macrophages. Drug release of CDDOMe was determined by Nrf2-driven GFP fluorescence. Moreover, we show that these CDDOMe-ARAPas exert anti-inflammatory effects in classically activated macrophages. Finally, we show that CDDOMe-ARAPas selectively accumulate in atherosclerotic plaque of two widely-used murine models of atherosclerosis: ApoE-/- and LDLr-/- mice, and are capable of increasing gene expression of Nrf2-transcriptional targets in the atherosclerotic aortic arch. Future work will assess the therapeutic efficacy of intra-plaque Nrf2 activation with CDDOMe-ARAPas to inhibit atherosclerotic plaque progression. Overall, our present studies underline that targeting of atherosclerotic plaque is an effective means to enhance delivery of redox-based interventions.

sentences: Extending the gap and loading RecA: the presynaptic phase plays a pivotal role in modulating lesion tolerance pathways During replication, the presence of unrepaired lesions results in the formation of single stranded DNA (ssDNA) gaps that need to be repaired to preserve genome integrity and cell survival. All organisms have evolved two major lesion tolerance pathways to continue replication: Translesion Synthesis (TLS), potentially mutagenic, and Homology Directed Gap Repair (HDGR), that relies on homologous recombination. In Escherichia coli, the RecF pathway repairs such ssDNA gaps by processing them to produce a recombinogenic RecA nucleofilament during the presynaptic phase. In this study, we show that the presynaptic phase is crucial for modulating lesion tolerance pathways. Indeed, impairing either the extension of the ssDNA gap (mediated by the nuclease RecJ and the helicase RecQ) or the loading of RecA (mediated by the RecFOR complex) leads to a decrease in HDGR. We suggest a model where defects in the presynaptic phase delay the formation of the D-loop and increase the time window allowed for TLS. We indeed observe an increase in TLS independent of SOS induction. In addition, we revealed an unexpected synergistic interaction between recF and recJ genes, that results in a recA deficient-like phenotype in which HDGR is almost completely abolished.

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Potential Pharmacodynamic Mechanism of the Main ingredients in Licorice for Chronic Obstructive Pulmonary Disease PurposeThis study aimed to investigate the effect of essential ingredients of licorice on the chronic obstructive pulmonary disease (COPD).

MethodThe ingredients information were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/), related genes about COPD was collected from geneCards (http://www.genecards.org/). Network pharmacology was utilized in this study.

ResultThe intersection data set contains 20 molecular targets between COPD and liquorice. Protein-protein interaction network showed that there are a total of 58 nodes and 137 edges involved. The link number of AKT1 in PPI network was 39, which is the highest level of interaction. MAPK1 is an important target of Licorice on COPD.

ConclusionMAPK signaling pathway could be the important key target of main ingredients of licorice on COPD.

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biPACT: a method for three-dimensional visualization of mouse spinal cord circuits of long segments with high resolution BackgroundThe spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injuries. Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging.

New MethodWe present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord.

ResultsCorticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading,

Comparison with Existing MethodsThe present method does not require special equipment, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. It enables to visualize individual neural fiber of specific neural circuit in long spinal cord segments.

ConclusionsBy using simple neural staining, and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord.

HighlightsO_LINo special devices or genetic tracers are required for a new clearing method
C_LIO_LINeuronal fibers are individually depicted in long segments of mouse spinal cord.
C_LIO_LIRe-staining of neuronal fiber is possible.
C_LIO_LIStereotaxic observation is achieved by 3-D reconstruction with open-source software.
C_LI

sentences: Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50 of 0.086 g mL-1 (DL4) and 0.385 g mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 0.414 g mL-1 for Alpha, 1.060 g mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

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Characterizing the body morphology of the first metacarpal in the Homininae using 3D geometric morphometrics ObjectivesThe morphological characteristics of the thumb are of particular interest due to its fundamental role in enhanced manipulation. Despite its possible importance regarding this issue, the body of the first metacarcapal (MC1) has not been fully characterized using morphometrics. This could provide further insights into its anatomy, as well as its relationship with manipulative capabilities. Hence, this study quantifies the shape of the MC1s body in the extant Homininae and some fossil hominins to provide a better characterization of its morphology.

Materials and methodsThe sample includes MC1s of modern humans (n=42), gorillas (n=27) and chimpanzees (n=30), as well as Homo neanderthalensis, Homo naledi and Australopithecus sediba. 3D geometric morphometrics were used to quantify the shape of MC1s body.

ResultsThe results show a clear distinction among the three extant genera. H. neanderthalensis mostly falls within the modern human range of variation. H. naledi varies slightly from modern humans, although also showing some unique trait combination, whereas A. sediba varies to an even greater extent. When classified using a discriminant analysis, the three fossils are categorized within the Homo group. Conclusion: The modern human MC1 is characterized by a distinct suite of traits, not present to the same extent in the great apes, that are consistent with an ability to use forceful precision grip. This morphology was also found to align very closely with that of H. neanderthalensis. H. naledi shows a number of human-like adaptations, whilst A. sediba presents a mix of both derived and more primitive traits.

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Manganese toxicity disrupts indole acetic acid homeostasis and suppresses CO2 assimilation reaction in rice plants Despite the essentiality of Mn in terrestrial plants, its excessive accumulation in plant tissues causes growth defects, known as Mn toxicity. Mn toxicity can be divided into apoplastic and symplastic types depending on its onset. For growth defects, symplastic rather than apoplastic Mn toxicity is hypothesised to be more critical. However, details of the relationship between growth defects and symplastic Mn toxicity remains elusive. In this study, we aimed to elucidate the molecular mechanisms of symplastic Mn toxicity in rice plants. We found that under excess Mn conditions, CO2 assimilation was inhibited by stomatal closure, and both carbon anabolic and catabolic activities were decreased. In addition to stomatal dysfunction, stomatal and leaf anatomical development were also altered by excess Mn accumulation. Furthermore, the indole acetic acid (IAA) concentration was decreased, and auxin-responsive gene expression analyses showed IAA-deficient symptoms in leaves due to excess Mn accumulation. These results suggest that excessive Mn accumulation causes IAA deficiency, and low IAA concentrations suppress plant growth by suppressing stomatal opening and leaf anatomical development for efficient CO2 assimilation in leaves.

HIGHLIGHTIncreased Mn concentration lowers auxin concentrations in rice leaves, which suppresses photosynthesis by changing stomatal function and development.

sentences: Quality assessment and refinement of chromatin accessibility data using a sequence-based predictive model Chromatin accessibility assays are central to the genome-wide identification of gene regulatory elements associated with transcriptional regulation. However, the data have highly variable quality arising from several biological and technical factors. To surmount this problem, we use the predictability of open-chromatin peaks from DNA sequence-based machine-learning models to evaluate and refine chromatin accessibility data. Our framework, gapped k-mer SVM quality check (gkmQC), provides the quality metrics for a sample based on the prediction accuracy of the trained models. We tested 886 samples with DNase-seq from the ENCODE/Roadmap projects to demonstrate that gkmQC can effectively identify high-quality samples underperforming owing to marginal read depths. Peaks identified in high-quality samples by gkmQC are more accurately aligned at functional regulatory elements, show greater enrichment of regulatory elements harboring functional variants from genome-wide association studies (GWAS), and explain greater heritability of phenotypes from their relevant tissues. Moreover, gkmQC can optimize the peak-calling threshold to identify additional peaks, especially for single-cell chromatin accessibility data as well as bulk data. Here we provide a standalone open-source toolkit (https://github.com/Dongwon-Lee/gkmQC) for such analyses and share improved regulatory maps using gkmQC. These resources will contribute to the functional interpretation of disease-associated regulatory genetic variation.

sentences: Preserved motor memory in Parkinson's disease Patients with Parkinsons disease, who lose the dopaminergic projections to the striatum, are impaired in certain aspects of motor learning. Recent evidence suggests that, in addition to its role in motor performance, the striatum plays a key role in the memory of motor learning. Whether Parkinsons patients have impaired motor memory and whether motor memory is modulated by dopamine at the time of initial learning is unknown. To address these questions, we measured memory of a learned motor sequence in Parkinsons patients who were either On or Off their dopaminergic medications. We compared them to a group of older and younger controls. Contrary to our predictions, motor memory was not impaired in patients compared to older controls, and was not influenced by dopamine state at the time of initial learning. To probe post-learning consolidation processes, we also tested whether learning a new sequence shortly after learning the initial sequence would interfere with later memory. We found that, in contrast to younger adults, neither older adults nor patients were susceptible to this interference. These findings suggest that motor memory is preserved in Parkinsons patients and raise the possibility that motor memory in patients is supported by compensatory non-dopamine sensitive mechanisms. Furthermore, given the similar performance characteristics observed in the patients and older adults and the absence of an effect of dopamine, these results raise the possibility that aging and Parkinsons disease affect motor memory in similar ways.

sentences: On the Design of a PID Bio-controller with Set Point Weighting and Filtered Derivative Action Effective and robust regulation of biomolecular processes is crucial for designing reliable synthetic bio-devices functioning in uncertain and constantly changing biological environments. Proportional-Integral-Derivative (PID) controllers are undeniably the most common way of implementing feed-back control in modern technological applications. Here, we introduce a highly tunable PID bio-controller with set point weighting and filtered derivative action presented as a chemical reaction network with mass action kinetics. To demonstrate its effectiveness, we apply our PID scheme on a simple biological process of two mutually activated species, one of which is assumed to be the output of interest. To highlight its performance advantages we compare it to PI regulation using numerical simulations in both the deterministic and stochastic setting.

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Dual STDP processes at Purkinje cells contribute to distinct improvements in accuracy and vigor of saccadic eye movements. Saccadic eye-movements play a crucial role in visuo-motor control by allowing rapid foveation onto new targets. However, the neural processes governing saccades adaptation are not fully understood. Saccades, due to the short-time of execution (20-100 ms) and the absence of sensory information for online feedback control, must be controlled in a ballistic manner. Incomplete measurements of the movement trajectory, such as the visual end-point error, are supposedly used to form internal predictions about the movement kinematics resulting in predictive control. In order to characterize the synaptic and neural circuit mechanisms underlying predictive saccadic control, we have reconstructed the saccadic system in a digital controller embedding a spiking neural network of the cerebellum with spike timing-dependent plasticity (STDP) rules driving parallel fiber - Purkinje cell long-term potentiation and depression (LTP and LTD). This model implements a control policy based on a dual plasticity mechanism, resulting in the identification of the roles of LTP and LTD in optimizing saccade movement control: it turns out that LTD regulates the accuracy and LTP the speed (vigor) of the ballistic eye movement. The control policy also required cerebellar PCs to be divided into two subpopulations, characterized by burst or pause responses. To our knowledge, this is the first model that explains in mechanistic terms the accuracy and vigor regulation of ballistic eye movements in forward mode exploiting spike-timing to regulate firing in different populations of the neuronal network. This elementary model of saccades could be extended and applied to other more complex cases in which single jerks are concatenated to compose articulated and coordinated movements.

Author summaryIt has been suggested that the cerebellum plays a crucial role in oculomotor adaptation. Computationally, the cerebellum is described as a supervised learner whose activity can be adjusted by synaptic changes proportional to the amount of mismatch between expected and actual movements outcomes (sensorimotor-errors). However, the spike-timing-dependent-plasticity (STDP) underlying adaptation has been so far modeled in behaviors where the error is continuously available as tracking-error. Such models depend on detailed tracking-errors to improve movement quality. We asked if the cerebellum can maintain good motor control even if the error is not completely available? This is important considering that error-dependent-STDP is only a subset among a family of STDP processes in the cerebellum. Moreover, even the physiological signals that were generally thought to carry sensorimotor-errors (called complex-spikes) are shown to encode multiple types of information regarding the movement. In this context, we characterize the role of cerebellar STDPs in saccade control, where the error information is constrained because of sensory suppression. We show that even in the absence of detailed error, the cerebellum can leverage two of its STDPs to increase movement quality. Hence, we emphasize the need to go beyond error-centric view to understand how the cerebellum improves motor behavior.

sentences: Individual differences in frontoparietal plasticity in humans Neuroplasticity, defined as the brains potential to change in response to its environment, has been extensively studied at the cellular and molecular levels. Work in animal models suggests that stimulation to the ventral tegmental area (VTA) enhances plasticity, and that myelination constrains plasticity. Little is known, however, about whether proxy measures of these properties in the human brain are associated with learning. Here, we investigated the plasticity of the frontoparietal system by asking whether VTA resting-state functional connectivity and myelin map values (T1w/T2w ratios) predicted learning after short-term training on the adaptive n-back (n = 46, ages 18-25). We found that stronger baseline connectivity between VTA and lateral prefrontal cortex predicted greater improvements in accuracy. Lower myelin map values predicted improvements in response times, but not accuracy. Our findings suggest that proxy markers of neural plasticity can predict learning in humans.

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High Resolution Slide-seqV2 Spatial Transcriptomics Enables Discovery of Disease-Specific Cell Neighborhoods and Pathways High resolution spatial transcriptomics is a transformative technology that enables mapping of RNA expression directly from intact tissue sections; however, its utility for the elucidation of disease processes and therapeutically actionable pathways remain largely unexplored. Here we applied Slide-seqV2 to mouse and human kidneys, in healthy and in distinct disease paradigms. First, we established the feasibility of Slide-seqV2 in human kidney by analyzing tissue from 9 distinct donors, which revealed a cell neighborhood centered around a population of LYVE1+ macrophages. Second, in a mouse model of diabetic kidney disease, we detected changes in the cellular organization of the spatially-restricted kidney filter and blood flow regulating apparatus. Third, in a mouse model of a toxic proteinopathy, we identified previously unknown, disease-specific cell neighborhoods centered around macrophages. In a spatially-restricted subpopulation of epithelial cells, we also found perturbations in 77 genes associated with the unfolded protein response (UPR). Our studies illustrate and experimentally validate the utility of Slide-seqV2 for the discovery of disease-specific cell neighborhoods.

One-Sentence SummaryHigh resolution Slide-seqV2 spatial transcriptomics in human and mouse kidneys.

sentences: Standardizing workflows in imaging transcriptomics with the abagen toolbox Gene expression fundamentally shapes the structural and functional architecture of the human brain. Open-access transcriptomic datasets like the Allen Human Brain Atlas provide an unprecedented ability to examine these mechanisms in vivo; however, a lack of standardization across research groups has given rise to myriad processing pipelines for using these data. Here, we develop the abagen toolbox, an open-access software package for working with transcriptomic data, and use it to examine how methodological variability influences the outcomes of research using the Allen Human Brain Atlas. Applying three prototypical analyses to the outputs of 750,000 unique processing pipelines, we find that choice of pipeline has a large impact on research findings, with parameters commonly varied in the literature influencing correlations between derived gene expression and other imaging phenotypes by as much as{rho} [≥] 1.0. Our results further reveal an ordering of parameter importance, with processing steps that influence gene normalization yielding the greatest impact on downstream statistical inferences and conclusions. The presented work and the development of the abagen toolbox lay the foundation for more standardized and systematic research in imaging transcriptomics, and will help to advance future understanding of the influence of gene expression in the human brain.

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

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Delayed dynamics of migratory response to CTLA-4 blockade reveals a mechanistic view on potential T cells reinvigoration following immune checkpoint blockade Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) receptors, two clinically relevant targets for immunotherapy of cancer, are negative regulators of in immune cell activation and migration. However, optimizing therapeutic outcomes still requires fundamental research to reach a comprehensive insight into the coherent function of immune regulators.

Here, we investigated the statistical dynamics of T cells migration as a measure of the functional response to these pathways in an experimental setup of immune checkpoint blockade. For this purpose, we used a previously developed 3-dimensional organotypic culture of patient-derived tumor spheroids.

Experiment-based dynamical modeling remarked distinct characteristics of the receptors regulation followed through with the modification of their proportions in the immune modulation. We demonstrated that time-delayed kinetics of PD-1 activation just overrides its relatively more efficient cell-level function which potentially makes an operative contribution to the functional dominance of CTLA-4 in the tumor microenvironment. Simulation results showed good agreement with data for tumor cells reduction and active immune cells count observed in each experiment.

These analyses propose a new mechanistic view on relative immunogenicity of PD-1 and CTLA-4 inhibitors manifested in literature and point the possible inherent obstacles in checkpoint inhibition-based immunotherapy of cancer to address in the future.

SignificanceEx vivo monitoring of temporal response to PD-1 and CTLA-4 in the closure of T cell movement dynamics and elucidating their feasible commitment to the kinetic constraints at cell-level resolution. Delayed dynamics of migratory response to CTLA-4 inhibition revealed a mechanistic view on potential T cell reinvigoration following immune checkpoint blockade.

sentences: EMC10 reduction in human neurons and adult mouse brain rescues cellular and behavioral deficits linked to 22q11.2 deletion Up-regulation of Mirta22/Emc10 is a major transcriptional effect of the 22q11.2-associated microRNA dysregulation and underlies key cellular as well as behavioral deficits. EMC10 is a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. Here we show that EMC10 expression is elevated in hiPSC-derived neurons from 22q11.2 deletion carriers and that reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling. Moreover, using injection of Antisense Oligonucleotides, we demonstrate that normalization of Emc10 levels in adult mouse brain rescues social memory deficits. The observations that elevated EMC10 expression is deleterious in 22q11.2 deletion carriers and that sustained elevation of EMC10 throughout the adult life can interfere with neural processes point to manipulations of EMC10 levels and downstream targets as a specific venue to ameliorate or even prevent disease progression in 22q11.2 deletion syndrome.

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Swine H1N1 influenza virus variants with enhanced polymerase activity and HA stability promote airborne transmission in ferrets Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor HA stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits.

IMPORTANCEDiverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both -2,6- and -2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

sentences: Persistence of Cajal-Retzius cells in the postnatal hippocampus is required for development of dendritic spines of CA1 pyramidal cells Cajal-Retzius (CR) cells are a transient type of neuron that populate the postnatal hippocampus. The role of transient cell types and circuits have been vastly addressed in neocortical regions, but poorly studied in the hippocampus. To understand how CR cells persistence influences the maturation of hippocampal circuits, we specifically ablated CR cells from the postnatal hippocampus. Our results highlighted layer-specific effects on dendritic spines and synaptic-related genes and revealed a critical role of CR cells in the establishment of the hippocampal network.

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Single-cell profiling of lncRNAs in human germ cells and molecular analysis reveals transcriptional regulation of LNC1845 on LHX8 Non-coding RNAs exert diverse functions in many cell types. In addition to transcription factors from coding genes, non-coding RNAs may also play essential roles in shaping and directing the fate of germ cells. Here, we report the presence of many long non-coding RNAs (lncRNAs) which were specifically expressed in the germ cells during human gonadal development by single-cell profiling of the reported datasets, and the functional characterization of one divergent lncRNA, LNC1845. Comprehensive bioinformatic analysis of these lncRNAs indicates that divergent lncRNAs occupied the majority of female and male germ cells. Integrating lncRNA expression into the bioinformatic analysis also enhances the cell-type classification of female germ cells. Functional dissection using in vitro differentiation of human pluripotent stem cells to germ cells revealed the regulatory role of LNC1845 on a transcription factor essential for ovarian follicle development, LHX8, by modulating the levels of histone modifications, H3K4me3 and H3K27Ac. Hence, this study provides a comprehensive analysis of lncRNAs in developing germ cells and elucidates how a lncRNA function as a cis regulator during human germ cell development.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/486705v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@31290forg.highwire.dtl.DTLVardef@17ec505org.highwire.dtl.DTLVardef@1b8cd0dorg.highwire.dtl.DTLVardef@19e4de3_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIlncRNAs are expressed at significantly higher levels in both male and female germ cells than in somatic cells during human gonadal development.
C_LIO_LIDivergent lncRNAs are enriched in the female meiotically and male mitotically arrested germ cells.
C_LIO_LILNC1845 regulates the transcriptional expression of LHX8 by modulating H3K4me3 and H3K27Ac levels.
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Complete genomic and epigenetic maps of human centromeres Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics.

One-sentence summaryDeep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.

sentences: polishCLR: a Nextflow workflow for polishing PacBio CLR genome assemblies Long-read sequencing has revolutionized genome assembly, yielding highly contiguous, chromosome-level contigs. However, assemblies from some third generation long read technologies, such as Pacific Biosciences (PacBio) Continuous Long Reads (CLR), have a high error rate. Such errors can be corrected with short reads through a process called polishing. Although best practices for polishing non-model de novo genome assemblies were recently described by the Vertebrate Genome Project (VGP) Assembly community, there is a need for a publicly available, reproducible workflow that can be easily implemented and run on a conventional high performance computing environment. Here, we describe polishCLR (https://github.com/isugifNF/polishCLR), a reproducible Nextflow workflow that implements best practices for polishing assemblies made from CLR data. PolishCLR can be initiated from several input options that extend best practices to suboptimal cases. It also provides re-entry points throughout several key processes including identifying duplicate haplotypes in purge_dups, allowing a break for scaffolding if data are available, and throughout multiple rounds of polishing and evaluation with Arrow and FreeBayes. PolishCLR is containerized and publicly available for the greater assembly community as a tool to complete assemblies from existing, error-prone long-read data.

sentences: Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an "aromatic cage" at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

sentences: Vestibular and auditory hair cell regeneration following targeted ablation of hair cells with diphtheria toxin in zebrafish Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turn over during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 dpf zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

sentences: The incentive circuit: memory dynamics in the mushroom body of Drosophila melanogaster Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting and assimilation of memories. We propose that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short-term to long-term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control.

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GBRAP: a tool to retrieve, parse and analyze GenBank files of viral and bacterial species SummaryGenBank files contain genomic data of sequenced living organisms. Here, we present GBRAP (GenBank Retrieving, Analyzing and Parsing software), a tool written in Python 3 that can be used to easily download, parse and analyze viral and bacterial GenBank files, even when contain more than one genomic sequence for each species. GBRAP can analyze more files simultaneously through single command-line parameters that give as output a single table showing the genomic characteristics of each organism. It is also able to calculate Shannon, LZSS (Lempel-Ziv-Storer-Szymanski) and topological entropy for both the entire genome and its constitutive elements such as genes, rRNAs, tRNAs, tmRNAs and ncRNAs together with Chargaffs second parity rule scores obtained using different mathematical methods. Moreover, GBRAP can calculate, the number, the length and the nucleotides abundance of genomic components for each DNA strand and for the overlapping regions among the two complementary helixes. To our knowledge, this is the only software capable of providing this type of genomic analyses all together in a single tool, that, therefore can be used by the scientists interested in both genomics and evolutionary research.

Availability and implementationThe data underlying this article are available from the corresponding author on reasonable request.

sentences: Decoding cellular deformation from pseudo-simultaneously observed Rho GTPase activities The inability to simultaneously observe all of the important Rho GTPases (Cdc42, Rac1, and RhoA) has prevented us from obtaining evidence of their coordinated regulation during cell deformation. Here, we propose Motion-Triggered Average (MTA), an algorithm that converts individually observed GTPases into pseudo-simultaneous observations. Using the time series obtained by MTA and mathematical model, we succeeded for the first time in decoding the cell edge velocity from the three GTPase activities to provide clear numerical evidence for coordinated cell edge regulation by the three GTPases. We found that the characteristics of the obtained activities were consistent with those of previous studies, and that GTPase activities and their derivatives were involved in edge regulation. Our approach provides an effective strategy for using single-molecule observations to elucidate problems hampered by the lack of simultaneous observations.

sentences: Quantification of in-plane flexoelectricity in lipid bilayers Lipid bilayers behave as 2D dielectric materials that undergo polarization and deformation in the presence of an electric field. This effect has been previously modeled by continuum theories which assume a polarization field oriented normal to the membrane surface. However, the molecular architecture of the lipids reveals that the heqadgroup dipoles are primarily oriented tangential to the membrane surface. Here, we perform atomistic and coarse-grained molecular dynamics simulations to quantify the in-plane polarization undergone by a flat bilayer and a spherical vesicle in the presence of an applied electric field. We use these predictions to compute an effective in-plane flexoelectric coefficient for four different lipid types. Our findings provide the first molecular proof of the in-plane polarization undergone by lipid bilayers and furnish the material parameter required to quantify membrane-electric field interactions.

sentences: Is Melanopsin Activation Affecting Large Field Color Matching Functions? Color theory is based on the exclusive activation of cones. However, since the discovery of melanopsin expressing cells in the human retina, evidence of its intrusion in brightness and color vision is increasing. We aimed to assess if differences between peripheral or large field and foveal color matches can be accounted for melanopsin activation or rod intrusion. Photopic color matches by young observers showed that differences between extrafoveal and foveal results cannot be explained by rod intrusion. Furthermore, statistical analyses on existing color matching functions suggest a role of melanopsin activation, particularly, in Large Field S Fundamentals.

sentences: Mutation rate variation shapes genome-wide diversity in Drosophila melanogaster What shapes the distribution of nucleotide diversity along the genome? Attempts to answer this question have sparked debate about the roles of neutral stochastic processes and natural selection in molecular evolution. However, the mechanisms of evolution do not act in isolation, and integrative models that simultaneously consider the influence of multiple factors on diversity are lacking; without them, confounding factors lurk in the estimates. Here we present a new statistical method that jointly infers the genomic landscapes of genealogies, recombination rates and mutation rates. In doing so, our model captures the effects of genetic drift, linked selection and local mutation rates on patterns of genomic variation. Guided by our causal model, we use linear regression to estimate the individual contributions of these micro-evolutionary forces to levels of nucleotide diversity. Our analyses reveal the signature of selection in Drosophila melanogaster, but we estimate that the mutation landscape is the major driver of the distribution of diversity in this species. Furthermore, our simulation study suggests that in many evolutionary scenarios the mutation landscape will be a crucial force shaping diversity, depending notably on the genomic window size used in the analysis. We argue that incorporating mutation rate variation into the null model of molecular evolution will lead to more realistic inference in population genomics.

sentences: Differential Regulation of Single Microtubules and Bundles by a Three-Protein Module A remarkable feature of the microtubule cytoskeleton is co-existence of sub-populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (i) Strong rescue activity of CLASP1, which overcomes weaker effects of Kif4A on single microtubules, (ii) Lower microtubule and PRC1-binding affinity of CLASP1, which permit dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule lengths, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.

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TAD-like single-cell domain structures exist on both active and inactive X chromosomes and persist under epigenetic perturbations BackgroundTopologically associating domains (TADs) are important building blocks of three-dimensional genome architectures. The formation of TADs was shown to depend on cohesin in a loop-extrusion mechanism. Recently, advances in an image-based spatial genomics technique known as chromatin tracing led to the discovery of cohesin-independent TAD-like structures, also known as single-cell domains -- highly variant self-interacting chromatin domains with boundaries that occasionally overlap with TAD boundaries but tend to differ among single cells and among single chromosome copies. Recent computational modeling studies suggest that epigenetic interactions may underlie the formation of the single-cell domains.

ResultsHere we use chromatin tracing to visualize in female human cells the fine-scale chromatin folding of inactive and active X chromosomes, which are known to have distinct global epigenetic landscapes and distinct population-averaged TAD profiles, with inactive X chromosomes largely devoid of TADs and cohesin. We show that both inactive and active X chromosomes possess highly variant single-cell domains across the same genomic region despite the fact that only active X chromosomes show clear TAD structures at the population level. These X chromosome single-cell domains exist in distinct cell lines. Perturbations of major epigenetic components did not significantly affect the frequency or strength of the single-cell domains. Increased chromatin compaction of inactive X chromosomes occurs at a length scale above that of the single-cell domains.

ConclusionsIn sum, this study suggests that single-cell domains are genome architecture building blocks independent of the tested major epigenetic components.

sentences: TNF-mediated neuroinflammation is linked to neuronal necroptosis in Alzheimer's disease hippocampus The pathogenetic mechanisms underlying neuronal death and dysfunction in Alzheimers disease (AD) remain unclear. However, chronic neuroinflammation has been implicated in stimulating or exacerbating neuronal damage. The tumor necrosis factor (TNF) superfamily of cytokines are involved in many systemic chronic inflammatory and degenerative conditions and are amongst the key mediators of neuroinflammation. TNF binds to the TNFR1 and TNFR2 receptors to activate diverse cellular responses that can be either neuroprotective or neurodegenerative. In particular, TNF can induce programmed necrosis or necroptosis in an inflammatory environment. Although activation of necroptosis has recently been demonstrated in the AD brain, its significance in AD neuron loss and the role of TNF signaling is unclear. We demonstrate an increase in expression of multiple proteins in the TNF/TNF receptor-1-mediated necroptosis pathway in the AD post-mortem brain, as indicated by the phosphorylation of RIPK3 and MLKL, predominantly observed in the CA1 pyramidal neurons. The density of phosphoRIPK3+ and phosphoMLKL+ neurons correlated inversely with total neuron density and showed significant sexual dimorphism within the AD cohort. In addition, apoptotic signaling was not significantly activated in the AD brain compared to the control brain. Exposure of human iPSC-derived glutamatergic neurons to TNF increased necroptotic cell death when apoptosis was inhibited, which was significantly reversed by small molecule inhibitors of RIPK1, RIPK3, and MLKL. In the post-mortem AD brain and in human iPSC neurons to TNF, we show evidence of altered expression of proteins of the ESCRT III complex, which has been recently suggested as an antagonist of necroptosis and a possible mechanism by which cells can survive after necroptosis has been triggered. Taken together, our results suggest that neuronal loss in AD is due to TNF-mediated necroptosis rather than apoptosis, which is amenable to therapeutic intervention at several points in the signaling pathway.

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An interpretable connectivity-based decoding model for classification of chronic marijuana use BackgroundPsychiatric neuroimaging typically proceeds with one of two approaches: encoding models, which aim to model neural mechanisms, or decoding models, which aim to predict behavioral or clinical characteristics from brain imaging data. In this study, we seek to combine these aims by developing interpretable decoding models that offer both accurate prediction and novel neural insights. We demonstrate the effectiveness of this combined approach in a case study of chronic marijuana use.

MethodsChronic marijuana (MJ) users (n=195) and non-using healthy controls (n=128) completed a cue-elicited craving task during functional magnetic resonance imaging. Linear machine learning methods were used to classify individuals into chronic MJ users and non-users based on task-evoked, whole-brain functional connectivity. We then used graph theoretic analyses to identify  predictive functional connectivities among brain regions that contributed most substantially to the classification of chronic marijuana use.

ResultsWe obtained high (~80% out-of-sample) accuracy across four different classification models, demonstrating that task-evoked, whole-brain functional connectivity can successfully differentiate chronic marijuana users from non-users. Subsequent network analyses revealed key predictive regions (e.g., anterior cingulate cortex, dorsolateral prefrontal cortex, and precuneus) that are often implicated in neuroimaging studies of substance use disorders, as well as some key exceptions. We also identified a core set of networks of brain regions that contributed to successful classification, comprised of many of the same predictive regions.

ConclusionsOur dual aims of accurate prediction and interpretability were successful, producing a predictive model that also provides interpretability at the neural level. This novel approach may complement other predictive-exploratory approaches for a more complete understanding of neural mechanisms in drug use and other neuropsychiatric disorders.

sentences: A cryptic pocket in Ebola VP35 allosterically controls RNA binding Protein-protein and protein-nucleic acid interactions are often considered difficult drug targets because the surfaces involved lack obvious druggable pockets. Cryptic pockets could present opportunities for targeting these interactions, but identifying and exploiting these pockets remains challenging. Here, we apply a general pipeline for identifying cryptic pockets to the interferon inhibitory domain (IID) of Ebola viral protein 35 (VP35). VP35 plays multiple essential roles in Ebolas replication cycle but lacks pockets that present obvious utility for drug design. Using adaptive sampling simulations and machine learning algorithms, we predict VP35 harbors a cryptic pocket that is allosterically coupled to a key dsRNA-binding interface. Thiol labeling experiments corroborate the predicted pocket and mutating the predicted allosteric network supports our model of allostery. Finally, covalent modifications that mimic drug binding allosterically disrupt dsRNA binding that is essential for immune evasion. Based on these successes, we expect our pipeline is equally applicable to other proteins.

sentences: Hydrodynamic and Polyelectrolyte Properties of Actin Filaments: Theory and Experiments Actin filaments polyelectrolyte and hydrodynamic properties, their interactions with the biological environment, and external force fields play an essential role in their biological activities in eukaryotic cellular processes. In this article, we introduce a unique approach that combines dynamics and electrophoresis light scattering experiments, an extended semiflexible worm-like chain model, and an asymmetric polymer length distribution theory to characterize the polyelectrolyte and hydrodynamic properties of actin filaments in aqueous electrolyte solutions. A fitting approach was used to optimize the theories and filament models for hydrodynamic conditions. We used the same sample and experimental conditions and considered several g-actin and polymerization buffers to elucidate the impact of their chemical composition, reducing agents, pH values, and ionic strengths on the filament translational diffusion coefficient, electrophoretic mobility, structure factor, asymmetric length distribution, effective filament diameter, electric charge, zeta potential, and semiflexibility. Compared to those values obtained from molecular structure models, our results revealed a lower value of the effective G-actin charge and a more significant value of the effective filament diameter due to the formation of the double layer of the electrolyte surrounding the filaments. Contrary to the data usually reported from electron micrographs, the lower values of our results for the persistence length and average contour filament length agree with the significant difference in the association rates at the filament ends that shift to sub-micro lengths, the maximum of the length distribution.

sentences: Optimisation of unsteady flight in learned avian perching manoeuvres Perching at speed is amongst the most challenging flight behaviours that birds perform1,2, and beyond the capability of current autonomous vehicles. Smaller birds may touchdown by hovering3-8, but larger birds typically swoop upward to perch1,2 - presumably because the adverse scaling of their power margin prohibits slow flapping flight9, and because swooping transfers excess kinetic to potential energy1,2,10. Perching is risky in larger birds6,11, demanding precise control of velocity and pose12-15, but it is unknown how they optimize this challenging manoeuvre. More generally, whereas cruising flight behaviours such as migration and commuting are adapted to minimize cost-of-transport or time-of-flight16, the optimization of unsteady flight manoeuvres remains largely unexplored7,17. Here we show that swooping minimizes neither the time nor energy required to perch safely in Harris hawks Parabuteo unicinctus, but instead minimizes the distance flown under hazardous post-stall conditions. By combining motion capture data from 1,563 flights with flight dynamics modelling, we found that the birds choice of where to transition from powered dive to unpowered climb minimizes the distance from the landing perch over which very high lift coefficients are required. Time and energy are therefore invested to maintain the control authority needed to execute a safe landing, rather than being minimized continuously as in technical applications of autonomous perching under nonlinear feedback control13 and deep reinforcement learning18,19. Naive birds learn this behaviour on-the-fly, so our findings suggest an alternative reward function for reinforcement learning of autonomous perching in air vehicles.

sentences: Developmental emergence of sleep rhythms enables long-term memory capabilities in Drosophila In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day (1-4). The mechanism through which sleep rhythms emerge, and the consequent advantage conferred to a juvenile animal, are unknown. In 2nd instar Drosophila larvae (L2), like human infants, sleep is not under circadian control (5). Here, we identify the precise developmental timepoint when the circadian clock begins to regulate sleep in Drosophila, leading to the emergence of sleep rhythms at the early 3rd instar stage (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousalpromoting Dh44 neurons, bringing arousal under clock control to drive the emergence of circadian sleep. Finally, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of an aversive cue, and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

sentences: Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

sentences: Omicron BA.1 and BA.2 Variants Increase the Interactions of SARS-CoV-2 Spike Glycoprotein with ACE2 SARS-CoV-2 infection is initiated by binding of the receptor-binding domain (RBD) of its spike glycoprotein to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptors in host cells. Recently detected Omicron variant of SARS-CoV-2 (B.1.1.529) is heavily mutated on RBD. Currently, the most common Omicron variants are the original BA.1 Omicron strain and the BA.2 variant, which became more prevalent since it first appeared. To investigate how these mutations affect RBD-PD interactions, we performed all-atom molecular dynamics simulations of the BA.1 and BA.2 RBD-PD in the presence of full-length glycans, explicit water and ions. Simulations revealed that RBDs of BA.1 and BA.2 variants exhibit a more dispersed interaction network and make an increased number of salt bridges and hydrophobic interactions with PD compared to wild-type RBD. Although BA.1 and BA.2 differ in two residues at the RBD-ACE2 interface, no major difference in RBD-PD interactions and binding strengths were observed between these variants. Using the conformations sampled in each trajectory, the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method estimated ~34% and ~51% stronger binding free energies for BA.1 and BA.2 RBD, respectively, than wild-type RBD, which may result in higher binding efficiency of the Omicron variant to infect host cells.

sentences: Modeling Allelic Diversity of Multi-parent Mapping Populations Affects Detection of Quantitative Trait Loci The search for quantitative trait loci (QTL) that explain complex traits such as yield and flowering time has been ongoing in all crops. Methods such as bi-parental QTL mapping and genome-wide association studies (GWAS) each have their own advantages and limitations. Multi-parent advanced generation intercross (MAGIC) populations contain more recombination events and genetic diversity than bi-parental mapping populations and reduce the confounding effect of population structure that is an issue in association mapping populations. Here we discuss the results of using a MAGIC population of doubled haploid (DH) maize lines created from 16 diverse founders to perform QTL mapping. We compare three models that assume bi-allelic, founder, and ancestral haplotype allelic states for QTL. The three methods have different power to detect QTL for a variety of agronomic traits. Although the founder approach finds the most QTL, there are also QTL unique to each method, suggesting that each model has advantages for traits with different genetic architectures. A closer look at a well-characterized flowering time QTL, qDTA8, which contains vgt1, suggests a potential epistatic interaction and highlights the strengths and weaknesses of each method. Overall, our results reinforce the importance of considering different approaches to analyzing genotypic datasets, and show the limitations of binary SNP data for identifying multi-allelic QTL.9

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DAVI: a tool for clustering and visualising protein domain architectures The characterization of protein functions is one of the main challenges in bioinformatics. Proteins are often composed of individual units termed domains, motifs that can evolve independently. The domain architecture of a given protein is the particular order and the content of its numerous domains. Some computational approaches predict the most likely domain architecture for a set of proteins. However, a few numbers of visualization tools exist, and most of them are unavailable. Here we present DAVI, an efficient and user-friendly web server for protein domain architecture clustering and visualization. DAVI accepts the output of most used domain architecture prediction tools and also produces domain architectures for a set of protein sequences. It provides a rich visualization for comparing, analyzing, and visualizing domain architectures.

Availabilityhttp://genome.lcqb.upmc.fr/Domain-Architecture-Viewer

sentences: Regulation of lateral root development by shoot-sensed far-red light via HY5 is nitrate-dependent and involves the NRT2.1 nitrate transporter. Plants are very effective in responding to environmental changes during competition for light and nutrients. Low Red:Far-Red (low R:FR)-mediated neighbor detection allows plants to compete successfully with other plants for available light. This above-ground signal can also reduce lateral root growth by inhibiting lateral root emergence, a process that might help the plant invest resources in shoot growth. Nitrate is an essential nutrient for plant growth and Arabidopsis thaliana responds to low nitrate conditions by enhancing nutrient uptake and reducing lateral and main root growth. There are indications that low R:FR signaling and low nitrate signaling can affect each other. It is unknown which response is prioritized when low R:FR light- and low nitrate signaling co-occur. We investigated the effect of low nitrate conditions on the low R:FR response of the A. thaliana root system in agar plate media, combined with the application of supplemental Far-Red (FR) light to the shoot. We observed that under low nitrate conditions main and lateral root growth was reduced, but more importantly, that the response of the root system to low R:FR was suppressed. Consistently, a loss-of-function mutant of a nitrate transporter gene NRT2.1 lacked low R:FR-induced lateral root reduction and its root growth was hypersensitive to low nitrate. ELONGATED HYPOCOTYL5 (HY5) plays an important role in the root response to low R:FR and we found that it was less sensitive to low nitrate conditions with regards to lateral root growth. In addition, we found that low R:FR increases NRT2.1 expression and that low nitrate enhances HY5 expression. HY5 also affects NRT2.1 expression, however, it depended on the presence of ammonium in which direction this effect was. Replacing part of the nitrogen source with ammonium also removed the effect of low R:FR on the root system, showing that changes in nitrogen sources can be crucial for root plasticity. Together our results show that nitrate signaling can repress low R:FR responses and that this involves signaling via HY5 and NRT2.1.

sentences: A selective bottleneck during host entry drives the evolution of new legume symbionts During the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle-shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

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Differential timing for glucose assimilation in Prochlorococcus and coexistent microbial populations at the North Pacific Subtropical Gyre The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here we investigated whole microbial community and group-specific primary production and glucose assimilation, using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis.

Our results showed a circadian rhythm for glucose assimilation in Prochlorococcus, with maximum assimilation during the midday and minimum at midnight, which was different compared with that of the total microbial community. This suggests that rhythms in glucose assimilation have been adapted in Prochlorococcus to couple the active transport to photosynthetic light reactions producing energy, and possibly to avoid competition from the rest of the microbial community. High-light Prochlorococcus strains showed most transcriptional changes upon glucose enrichment. Pathways involved in glucose metabolism as the pentose phosphate, the Entner-Dudoroff, glycolysis, respiration and glucose transport showed an increase in the transcript level. A few genes of the low-light strains showed opposite changes, suggesting that glucose assimilation has been subjected to diversification along the Prochlorococcus evolution.

sentences: Bacillus thuringiensis Cry1A toxins divert progenitor cell fate toward enteroendocrine lineage by diminishing cell adhesion with intestinal stem cells Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, though not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and enteroendocrine functions.

sentences: Full-likelihood genomic analysis clarifies a complex history of species divergence and introgression: the example of the erato-sara group of Heliconius butterflies AO_SCPLOWBSTRACTC_SCPLOWIntrogressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species makes estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood analysis methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ full-likelihood methods under the multispecies coalescent (MSC) model with and without gene flow to analyze the genomic data from six members of the erato-sara clade of Heliconius butterflies and infer the species phylogeny and cross-species introgression events. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of H. telesiphe, a likely hybrid speciation origin for H. hecalesia, and gene flow between sister species H. erato and H. himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the multispecies coalescent makes it possible to extract rich historical information of species divergence and gene flow from genomic data.

sentences: Simultaneous association of grip effort with snack food items does not change preferences Effort is typically considered aversive such that rewards requiring less effort are preferred over identical value rewards that require greater effort, commonly referred to as "effort discounting". Although effort has been repeatedly shown to be weighed as a cost, there are indications to suggest that under some conditions it may increase preferences. An example for how effort affects preferences is the  contrast effect were items that follow effort production gain value. In the current study we examined whether the association of grip effort with snack food items would change preferences. In four experiments, we first identified each participants individual preferences of snack food items and subjective maximal grip force. We then associated different effort levels with items of similar preference. Finally, we tested for changes in preferences following the effort association. Across the four studies, effort association had no effect on preferences. Using Bayesian analyses, we conclude that simultaneous association of effort does not change preferences in our studies and call for a replication effort of previous findings.

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Reproducible data integration and visualization of biological networks in R MotivationCollaborative workflows in network biology not only require the documentation of the performed analysis steps but also of the network data on which the decisions were based. However, replication of the entire workflow or tracking of the intermediate networks used for a particular visualization remains an intricate task. Also, the amount and heterogeneity of the integrated data requires instruments to explore and thus comprehend the results.

ResultsHere we demonstrate a collection of software tools and libraries for network data integration, exploration, and visualization to document the different stages of the workflow. The integrative steps are performed in R, and the entire process is accompanied by an interchangeable toolset for data exploration and network visualization.

AvailabilityThe source code of the performed workflow is available as R markdown scripts at https://github.com/frankkramer-lab/reproducible-network-visualization. A compiled HTML version is also hosted on Github pages at https://frankkramer-lab.github.io/reproducible-network-visualization.

Contactflorian.auer@informatik.uni-augsburg.de

sentences: Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here we have characterized the structure and dynamics of DeCLIC through cryo-electron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD-simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

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Intracellular force comparison of pathogenic KIF1A, KIF5, and dynein by fluctuation analysis In mammalian cells, there exist approximately 40 types of microtubule motor proteins that are assigned to specific cargo deliveries. For example, the kinesin-1 family motor KIF5 is the major motor responsible for anterograde mitochondrial transport, whereas the kinesin-3 family motor KIF1A is responsible for synaptic vesicle precursor transport. In contrast, cytoplasmic dynein is responsible for retrograde transport of nearly all cargos. The force and velocity of these microtubule motors have been investigated in in-vitro single-molecule experiments. In the present study, we compared the intracellular force and velocity of various types of motors in the mammalian neuronal axon obtained by non-invasive force measurement (fluctuation analysis) and extreme value analysis with those obtained by previous single-molecule experiments. As we found a high correlation between our results and the previous results, we next investigated synaptic vesicle precursor transport by hereditary spastic paraplegia-associated KIF1A variants (V8M, R350G, and A255V). KIF1A-V8M and KIF1A-A255V exhibited force and velocity impairment in mammalian neuronal axons, whereas the physical property of KIF1A-R350G was similar to that of the wild type. We believe that the development of new analytical techniques for investigating intracellular cargo transports is helpful to elucidate the molecular mechanism of KIF1A-associated neurological disorders.

Statement of SignificanceRecent in-vitro single-molecule experiments have clearly revealed that microtubule motors only fully exert their functions when fully equipped with the proteins associated with cargo vesicle transport. This emphasizes the significance of intracellular physical measurements, in which the motors should fully function. In addition to motor force and velocity, the number of motors transporting a single cargo together is an important physical quantity to characterize cargo transport, but is difficult to estimate using in-vitro single-molecule experiments. In this study, we aimed to extract physical information on microtubule motors in the intracellular environment.

sentences: Restoration of DNA Integrity and Cell Cycle by Electric Stimulation in Planarian Tissues Damaged by Ionizing Radiation Exposure to high levels of ionizing {gamma}-radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity to reestablish stem cell activity in tissues damaged by ionizing radiation. We show that sub-threshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally {gamma}-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic [Ca2+] through the activation of L-type Cav channels and intracellular Ca2+ stores leading to the activation of immediate early genes and ectopic expression of stem cell markers in postmitotic cells. Overall, we show the potential of electric current stimulation to reverse damaging effects of high dose {gamma}-radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

sentences: Image-based phenotypic profiling of a chemogenomic screening library identifies novel druggable targets in the EGFR pathway The gene encoding epidermal growth factor receptor (EGFR) is a major driver gene in cancer. Many drugs targeting EGFR-associated molecules have been developed, yet many have failed in clinical trials due to a lack of efficacy and/or unexpected side effects. In this study, I used image-based phenotypic profiling to screen a pharmacologically active compound library with the aim of identifying new druggable targets in the EGFR pathway. As anticipated, the phenotypic screen identified compounds that produce phenotypes resulting from targeting a known specific molecule or pathway. The assay also showed that compounds with diverse known mechanisms of action produced similar, EGFR-related cellular phenotypes. Biochemical assays revealed that those compounds share a previously unappreciated common target/pathway, showing that the image-based assay can identify new target molecules that are independent of the compounds known target. Further experiments showed that ROCK1 and PSMD2 are novel druggable targets within the EGFR pathway.

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Immunokinetic Model for COVID-19 Patients In this paper, we develop a fractional-order differential model for the dynamics of immune responses to SARS-CoV-2 viral load in one host. In the model, a fractional-order derivative is incorporated to represent the effects of temporal long-run memory on immune cells and tissues for any age group of patients. The population of cytotoxic T-cells (CD8+), natural killer (NK) cells and infected viruses are unknown in this model. Some interesting sufficient conditions that ensure the asymptotic stability of the steady states are obtained.

This model indicates some complex phenomena in COVID-19 such as "immune exhaustion" and "Long COVID". Sensitivity analysis is also investigated for model parameters to determine the parameters that are effective in determining of the long COVID duration, disease control and future treatment as well as vaccine design. The model is verified with clinical and experimental data of 5 patients with COVID-19.

sentences: Multielectrode Cortical Stimulation Selectively Induces Unidirectional Wave Propagation Cortical stimulation is emerging as an experimental tool in basic research and a promising therapy for a range of neuropsychiatric conditions. As multielectrode arrays enter clinical practice, the possibility of using spatiotemporal patterns of electrical stimulation to induce desired physiological patterns has become theoretically possible, but in practice can only be implemented by trial-and-error because of a lack of predictive models. Experimental evidence increasingly establishes travelling waves as fundamental to cortical information-processing, but we lack understanding how to control wave properties despite rapidly improving technologies. This study uses a hybrid biophysical-anatomical and neural-computational model to predict and understand how a simple pattern of cortical surface stimulation could induce directional traveling waves via asymmetric activation of inhibitory interneurons. It reveals local circuit mechanisms to control spatiotemporal cortical dynamics and predicts interventions that can be developed to treat a broad range of cognitive disorders.

sentences: Placental structure, function and mitochondrial phenotype relate to fetal size and sex in mice Fetal growth depends on placental function, which requires energy from mitochondria. Here we investigated whether mitochondrial function in the placenta relates to growth of the lightest and heaviest fetuses of each sex within the litter of mice. Placentas from the lightest and heaviest fetuses were taken to evaluate placenta morphology (stereology), mitochondrial energetics (high-resolution respirometry), and mitochondrial regulators, nutrient transporters, hormone handling and signalling pathways (qPCR and western blotting). We found that mitochondrial complex I and II oxygen consumption rate was greater for placentas supporting the lightest female fetuses, although placental complex I abundance of the lightest females and complexes III and V of the lightest males were decreased compared to their heaviest counterparts. Expression of mitochondrial biogenesis (Nrf1) and fission (Drp1 and Fis1) genes was lower in the placenta from the lightest females, whilst biogenesis-related gene Tfam was greater in the placenta of the lightest male fetuses. Additionally, placental morphology and steroidogenic gene (Cyp17a1 and Cyp11a1) expression was aberrant for the lightest females, but glucose transporter (Glut1) expression was lower in only the lightest males versus their heaviest counterparts. Differences in intra-litter placental phenotype were related to sex-dependent changes in the expression of hormone responsive (androgen receptor) and metabolic signalling (AMPK, AKT, PPAR{gamma}) pathways. Thus, in normal mouse pregnancy, placental structure, function and mitochondrial phenotype are differentially responsive to growth of the female and the male fetus. This study may inform the design of sex- specific therapies for placental insufficiency and fetal growth abnormalities with life-long benefits for the offspring.

sentences: Towards rainy Arctic winters: experimental icing impacts tundra plant productivity and reproduction The Arctic is warming rapidly, and the rate of warming is twice as fast in winter as in summer. More frequent extreme warm spells and associated rain-on-snow events in winter can dramatically alter snowpack conditions, ultimately encapsulating the tundra vegetation in  basal ice for several months. As most tundra vegetation studies have focused on summer warming, potential icing effects on phenology, productivity, and reproduction remain largely unexplored. We addressed this gap by performing a pioneer field experiment in high Arctic Svalbard, simulating extreme rain-on-snow and resultant basal ice ( icing) in five consecutive winters and assessing vascular plant responses throughout each subsequent growing season. We also tested whether icing responses were modified by experimentally increased summer temperatures. Icing alone delayed early vegetative and reproductive phenology of the dominant dwarf shrub, Salix polaris, but with clear evidence for a  catch-up in terms of improved community-level primary production later in the growing season. This compensatory growth occurred at the expense of flower production and delayed seed maturation. Both the phenological delay and allocation trade-offs were associated with icing-induced lags in thawing out and subsequent warming up of the soil, crucial in regulating plant nutrient availability and acquisition. However, experimental summer warming modified icing effects by advancing plant phenology, thus increasing primary productivity already early in the growing season, and partially offsetting negative icing effects on reproduction. Consequently, both winter and summer warming must be considered simultaneously in order to predict climate change responses of Arctic tundra vegetation. Our findings demonstrate that more frequent extreme warm spells in winter have the potential to shape key aspects of high Arctic plant communities. As we are heading towards rain-dominated Arctic winters, this calls for similar experiments in coordinated circumpolar monitoring programs across tundra plant communities.

sentences: Ionic strength modulates HU protein-induced DNA supercoiling The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

sentences: Half-life of biodegradable plastics in the marine environment depends on material, habitat, and climate zone The performance of the biodegradable plastic materials polyhydroxyalkanoate (PHA), polybutylene sebacate (PBSe) and polybutylene sebacate co-terephthalate (PBSeT), and of polyethylene (LDPE) was assessed under marine environmental conditions in a three-tier approach. Biodegradation lab tests (20 {degrees}C) were complemented by mesocosm tests (20 {degrees}C) with natural sand and seawater and by field tests in the warm-temperate Mediterranean Sea (12 - 30 {degrees}C) and in tropical Southeast Asia (29 {degrees}C) in three typical coastal scenarios. Plastic film samples were exposed in the eulittoral beach, the pelagic open water and the benthic seafloor and their disintegration monitored over time. We used statistical modelling to predict the half-life for each of the materials under the different environmental conditions to render the experimental results numerically comparable across all experimental conditions applied. The biodegradation performance of the materials differed by orders of magnitude depending on climate, habitat and material and revealed the inaccuracy to generically term a material  marine biodegradable. The half-life t0.5 of a film of PHA with 85 m thickness ranged from 54 d on the seafloor in SE Asia to 1247 d in mesocosm pelagic tests. t0.5 for PBSe (25 m) ranged from 99 d in benthic SE Asia to 2614 d in mesocosm benthic tests, and for PBSeT t0.5 ranged from 147 d in the mesocosm eulittoral to 797 d in Mediterranean benthic field tests. For LDPE no biodegradation could be observed. These data can now be used to estimate the persistence of plastic objects should they end up in the marine environments considered here and will help to inform the life cycle (impact) assessment of plastics in the open environment.

sentences: Glucocorticoids Mediate Transcriptome-wide Alternative Polyadenylation: Mechanisms and Functional Implications Alternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3' ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome-wide the impact of glucocorticoids on APA in 30 human B-lymphoblastoid cell lines. We found that glucocorticoids could regulate APA of a subset of genes, possibly by changing the expression of 142 RNA-binding proteins, some with known APA-regulating properties. Interestingly, genes with glucocorticoid-mediated APA were enriched in viral translation-related pathways, while genes with glucocorticoid-mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid-mediated APA might result in functional consequences distinct from gene expression. Glucocorticoid-mediated APA was also cell-type-specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype-dependent glucocorticoid-mediated APA, providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of novel observations regarding the impact of glucocorticoids on APA, suggesting novel avenues for mechanistic studies of hormonal biology.

sentences: On the fitness of informative cues in complex environments To be able to deal with uncertainty is of primary importance to most living organisms. When cues provide information about the state of the environment, organisms can use them to respond flexibly. Life forms have evolved complex adaptations and sensory mechanisms to use these environmental cues and extract valuable information about the environment. Previous work has shown a theoretical limit to the amount of fitness benefit possible to be extracted from the cues. We show that the previously used information theoretical approaches can be generalised to scenarios involving any potential relationship between the number of possible phenotypes and environmental states. Such cases are relevant when physiological constraints or complex ecological scenarios lead to the number of environmental states exceeding potential phenotypes. We illustrate cases in which these scenarios can emerge: along environmental gradients, such as geographical transects or complex environments, where organisms adopt different bet-hedging strategies, switching stochastically between phenotypes or developing intermediate ones. In conclusion, we develop an information-theoretic extensible approach for investigating and quantifying fitness in ecological studies.

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GRAFIMO: variant and haplotype aware motif scanning on pangenome graphs Transcription factors (TFs) are proteins that promote or reduce the expression of genes by binding short genomic DNA sequences known as transcription factor binding sites (TFBS). While several tools have been developed to scan for potential occurrences of TFBS in linear DNA sequences or reference genomes, no tool exists to find them in pangenome variation graphs (VGs). VGs are sequence-labelled graphs that can efficiently encode collections of genomes and their variants in a single, compact data structure. Because VGs can losslessly compress large pangenomes, TFBS scanning in VGs can efficiently capture how genomic variation affects the potential binding landscape of TFs in a population of individuals. Here we present GRAFIMO (GRAph-based Finding of Individual Motif Occurrences), a command-line tool for the scanning of known TF DNA motifs represented as Position Weight Matrices (PWMs) in VGs. GRAFIMO extends the standard PWM scanning procedure by considering variations and alternative haplotypes encoded in a VG. Using GRAFIMO on a VG based on individuals from the 1000 Genomes project we recover several potential binding sites that are enhanced, weakened or missed when scanning only the reference genome, and which could constitute individual-specific binding events. GRAFIMO is available as an open-source tool, under the MIT license, at https://github.com/pinellolab/GRAFIMO and https://github.com/InfOmics/GRAFIMO.

Author summaryTranscription factors (TFs) are key regulatory proteins and mutations occurring in their binding sites can alter the normal transcriptional landscape of a cell and lead to disease states. Pangenome variation graphs (VGs) efficiently encode genomes from a population of individuals and their genetic variations. GRAFIMO is an open-source tool that extends the traditional PWM scanning procedure to VGs. By scanning for potential TBFS in VGs, GRAFIMO can simultaneously search thousands of genomes while accounting for SNPs, indels, and structural variants. GRAFIMO reports motif occurrences, their statistical significance, frequency, and location within the reference or alternative haplotypes in a given VG. GRAFIMO makes it possible to study how genetic variation affects the binding landscape of known TFs within a population of individuals.

sentences: A somatosensory computation that unifies limbs and tools It is often claimed that tools are embodied by the user, but whether the brain actually repurposes its body-based computations to perform similar tasks with tools is not known. A fundamental computation for localizing touch on the body is trilateration. Here, the location of touch on a limb is computed by integrating estimates of the distance between sensory input and its boundaries (e.g., elbow and wrist of the forearm). As evidence of this computational mechanism, tactile localization on a limb is most precise near its boundaries and lowest in the middle. We show that the brain repurposes trilateration to localize touch on a tool. In a large sample of participants, we found that localizing touch on a tool produced the signature of trilateration, with highest precision close to the base and tip of the tool. A computational model of trilateration provided a good fit to the observed localization behavior. To further demonstrate the computational plausibility of repurposing trilateration, we implemented it in a three-layer neural network that was based on principles of probabilistic population coding. This network determined hit location in tool-centered coordinates by using a tools unique pattern of vibrations when contacting an object. Simulations demonstrated the expected signature of trilateration, in line with the behavioral patterns. Our results have important implications for how trilateration may be implemented by somatosensory neural populations. We conclude that trilateration is a fundamental spatial computation that unifies limbs and tools.

sentences: Resistance training rejuvenates the mitochondrial methylome in aged human skeletal muscle Resistance training (RT) alters skeletal muscle nuclear DNA methylation patterns (or the methylome). However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older untrained males (65{+/-}7 years old) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 hours following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Various biochemical assays were also performed, and older male data were compared to younger trained males (22{+/-}2 years old, n=7). RT increased whole-body lean tissue mass (p=0.017), VL thickness (p=0.012), and knee extensor torque (p=0.029) in older males. RT also profoundly affected the mtDNA methylome in older males, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p<0.05). Notably, several mtDNA sites presented a more "youthful" signature after RT in older males when comparisons were made to younger males. The 1.12 kilobase D-loop/control region on mtDNA, which regulates mtDNA replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and increases in mitochondrial complex III and IV protein levels were also observed (p<0.05). This is the first study to show RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT remarkably evokes mitochondrial methylome profiles to mimic a more youthful signature in older males.

sentences: Improved fluorescent proteins for dual-color post-embedding CLEM Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScharlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScharlet-H, dual-color post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.

sentences: Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Forster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

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Spectral fiber-photometry derives hemoglobin-absorption changes for accurate measurement of fluorescent sensors Fiber-photometry is an emerging technique for recording fluorescent sensor activity in the brain. However, significant hemoglobin-absorption artifacts in fiber-photometry data may be misinterpreted as sensor activity changes. Because hemoglobin exists in nearly every location in the brain and its concentration varies over time, such artifacts could impede the accuracy of many photometry recording results. Here we present a novel use of spectral photometry technique and propose computational methods to quantify photon absorption effects by using activity-independent fluorescence signals, which can be used to derive oxy- and deoxy-hemoglobin concentration changes. Following time-locked neuronal activation in vivo, we observed that a 20% increase in CBV contributes to about a 4% decrease in green fluorescence signal and a 2% decrease in red fluorescence signal. While these hemoglobin concentration changes are often temporally delayed than the fast-responding fluorescence spikes, we found that erroneous interpretation may occur when examining pharmacology-induced sustained activity changes, and in some cases, hemoglobin-absorption could flip the GCaMP signal polarity. We provided hemoglobin-based correction methods to restore fluorescence signals across spectra and compare our results against the commonly used regression approach. We also demonstrated the utility of spectral fiber-photometry for delineating brain regional differences in hemodynamic response functions.

HighlightsO_LIHemoglobin-absorption compromises fiber-photometry recording in vivo
C_LIO_LISpectral photometry allows quantification of hemoglobin concentration changes for correction
C_LIO_LIThe proposed platform allows measuring regional differences in neurovascular transfer function
C_LI

sentences: Complexity-stability relationship in empirical microbial ecosystems Mays stability theory [1, 2], which holds that large ecosystems can be stable up to a critical level of complexity, a product of the number of resident species and the intensity of their interactions, has been a central paradigm in theoretical ecology [3-7]. So far, however, empirically demonstrating this theory in real ecological systems has been a long-standing challenge, with inconsistent results [8]. Especially, it is unknown whether this theory is pertinent in the rich and complex communities of natural microbiomes, mainly due to the challenge of reliably reconstructing such large ecological interaction networks [9-11]. Here, we introduce a novel computational framework for estimating an ecosystems complexity without relying on a priori knowledge of its underlying interaction network. By applying this method to human-associated microbial communities from different body sites [12] and sponge-associated microbial communities from different geographical locations [13], we found that in both cases the communities display a pronounced trade-off between the number of species and their effective connectance. These results suggest that natural microbiomes are shaped by stability constraints, which limit their complexity.

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Cognitive Deficits and Altered Cholinergic Innervation in Young Adult Mice Carrying a Parkinson's Disease LRRK2-G2019S Knockin Mutation Impaired executive function is a common and debilitating non-motor symptom of idiopathic and hereditary Parkinsons disease (PD), but there is little understanding of the underlying pathophysiological mechanisms and circuits. The G2019S mutation in the kinase domain of leucine-rich repeat kinase 2 (LRRK2) greatly increases risk for late-onset PD, and non-manifesting LRRK2-G2019S carriers also exhibit early and significant cognitive impairment. Here, we subjected young adult mice carrying a Lrrk2-G2019S knockin mutation to touchscreen-based operant tasks that measure attention, goal-directed learning and cognitive flexibility, all of which rely on prefrontal-striatal connectivity and are strongly modulated by cholinergic innervation. In a visuospatial attention task, mutant mice exhibited significantly more omissions and longer response latencies than controls that could not be attributed to deficits in motivation, visual sensory perception per se or locomotion, thereby suggesting impairment in divided attention and slower information processing speed. Pretreating mice with the acetylcholinesterase inhibitor donepezil normalized both higher omission rates and longer reward latencies in the mutants, but did not affect any performance metric in controls. Strikingly, cholinergic fiber density in mPFC and dorsomedial striatum was significantly sparser in mutants than in controls, while further behavioral interrogation of the mutants revealed significant impairments in action-outcome associations but preserved cognitive flexibility. These data suggest that the G2019S mutation impacts cholinergic innervation and impairs corticostriatal network function in young adulthood that may contribute to early PD-associated cognitive deficits.

STATEMENT OF SIGNIFICANCEThe LRRK2-G2019S mutation causes hereditary Parkinsons disease and is found in some idiopathic cases. Early cognitive impairment is a common symptom of hereditary and idiopathic PD, yet there is little mechanistic understanding of such impairment. Here, we tested young adult Lrrk2-G2019S knockin mice in a series of touchscreen-based visuospatial tasks. We found that mutants exhibited significant deficits in attention and goal-directed learning, and had significantly slower information processing speed. Treatment with an acetylcholinesterase inhibitor reversed some of these behavioral deficits, while anatomical analyses showed significantly sparser cholinergic innervation of brain structures important for executive function. These findings suggest the G2019S mutation alters cholinergic signaling in young adulthood, and thus may contribute to early PD-associated impairment in several cognitive domains.

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Novel canine high-quality metagenome-assembled genomes, prophages, and host-associated plasmids by long-read metagenomics together with Hi-C proximity ligation Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors.

We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range.

In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

sentences: Structural basis of Ty1 integrase tethering on RNA polymerase III for targeted DNA integration The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. Here, we report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Unexpectedly, IN1 binding associates with insertion of subunit C11 C-terminal Zn-ribbon into the Pol III funnel, which provides atomic evidence for a two-metal mechanism during RNA cleavage. Moreover, unstructured regions of subunits C53 and C37 reorganize close to C11, likely explaining the connection between the C37/C53 heterodimer and C11 during transcription reinitiation. Our results suggest that IN1 binding induces a Pol III configuration that favors chromatin residence, thus improving the likelihood of Ty1 integration.

sentences: Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan Occurrence of extended-spectrum {beta}-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units (TPU) ranging in size from 2,855 bp to 11,093 bp; the exception, blaCTX-M-2, was associated with ISCR1. In many cases, the TPU were directly inserted into chromosomes. However, in one case, the TPU was part of a 36-kbp multiresistance region bracketed by IS26, and the whole multiresistance region was inserted into the chromosome. We also found a case where a chromosomally-located TPU and an adjacent region had transposed into another chromosomal location, leading to duplication of blaCTX-M. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different STs (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some environmental E. coli can stably maintain blaCTX-M genes in the absence of antibiotic selection pressure, though further studies are needed to confirm this.

sentences: Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.

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Actin-dependent recruitment of Ago2 to the zonula adherens Adherens junctions are cadherin-based structures critical for cellular architecture. E-cadherin junctions in mature epithelial cell monolayers tether to an apical actomyosin ring to form the zonula adherens (ZA). We have previously shown that the adherens junction protein PLEKHA7 associates with and regulates the function of the core RNA interference (RNAi) component Ago2, specifically at the ZA. However, the mechanism mediating Ago2 recruitment to the ZA remained unexplored. Here, we reveal that this ZA-specific recruitment of Ago2 depends on both the structural and tensile integrity of the actomyosin cytoskeleton. We found that depletion of not only PLEKHA7, but also either of three PLEKHA7-interacting, LIM-domain family proteins, namely LMO7, LIMCH1, and PDLIM1, results in disruption of actomyosin organization and tension, as well as loss of Ago2 junctional localization. These results demonstrate that recruitment of Ago2 to the ZA is sensitive to actomyosin perturbations, introducing the idea of a mechanosensitive RNAi machinery, with potential implications in tissue remodeling and in disease.

SummaryE-cadherin junctions at the zonula adherens recruit the key RNAi component Ago2 through a still unexplored mechanism. The present work shows that junctional recruitment of Ago2 depends on the integrity and tension of the actin cytoskeleton, revealing a mechanosensitive aspect of RNAi.

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Exploration of 1 BAY 11-7082 as a novel antibiotic Exposure of the Gram-negative pathogen Pseudomonas aeruginosa to sub-inhibitory concentrations of antibiotics increases formation of biofilms. We exploited this phenotype to identify molecules with potential antimicrobial activity in a biofilm-based high-throughput screen. The anti-inflammatory compound BAY 11-7082 induced dose-dependent biofilm stimulation, indicative of antibacterial activity. We confirmed that BAY 11-7082 inhibits growth of P. aeruginosa and other priority pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). We synthesized 27 structural analogues, including a series based on the related scaffold 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC), 10 of which displayed increased anti-Staphylococcal activity. Because the parent molecule inhibits the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome, we measured the ability of select analogues to reduce IL-1{beta} production in mammalian macrophages, identifying minor differences in the structure-activity relationship for the anti-inflammatory and antibacterial properties of this scaffold. Although we could evolve stably resistant MRSA mutants with cross resistance to BAY 11-7082 and PSPC, their lack of shared mutations suggested that the two molecules could have multiple targets. Finally, we showed that BAY 11-7082 and its analogues potentiate the activity of penicillin G against MRSA, suggesting that this scaffold may serve as an interesting starting point for the development of antibiotic adjuvants.

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A new behavioral and neural marker of social vision Research on face perception has revealed highly specialized visual mechanisms such as configural processing, and provided markers of interindividual differences -including disease risks and alterations- in visuoperceptual abilities that traffic in social cognition. Is face perception unique in degree or kind of mechanisms, and in its relevance for social cognition? Combining functional MRI and behavioral methods, we address the processing of an uncharted class of socially relevant stimuli: minimal social scenes involving configurations of two bodies spatially close and face-to-face as if interacting (hereafter, facing dyads). We report category-specific activity for facing (vs. non-facing) two-body configurations in selective areas of the visual cortex. That activity shows face-like signatures of configural processing -i.e., stronger response, and greater susceptibility to stimulus inversion for facing (vs. non-facing) dyads-, and is predicted by performance-based measures of body-dyad perception (i.e., accuracy in a fast visual categorization task). Moreover, individual performance in body-dyad perception is reliable, stable-over-time and correlated with the individual social sensitivity, coarsely captured by the Autism-Spectrum Quotient. Further analyses clarify the relationship between single-body and body-dyad perception. We propose that facing dyads are processed through highly specialized mechanisms (and brain areas), analogously to other biologically/socially relevant stimuli such as faces. Like face perception, facing-dyad perception can reveal basic visual processes that lay the foundations for understanding others, their relationships and interactions.

Significance statementWith its specialization to faces and biological motion, vision houses the foundations of human social ability. Using a multimodal approach (meta-analysis, fMRI, visual-perception tasks and self-administered survey), we introduce a new class of visual stimuli -minimal social scenes with two face-to-face bodies-, whose processing highlights new behavioral and neural markers of visuoperceptual abilities that traffic in social cognition. Behavioral and neural effects of body-dyad perception reveal the recruitment of specialized configural processing, previously described for face perception. Furthermore, individual performance in body-dyad perception is stable over time, and predicts an individuals social sensitivity, measured in terms of autism-spectrum traits. Thus, body-dyad perception reveals uncharted aspects of visual functioning and specialization, which may critically contribute to human social life.

sentences: Identification of INSR protein structural differences in Diabetes Mellitus type 2 subjects from protein sequences In this paper, we constructed several models of the Insulin Receptor protein(abbreviated INSR) from protein sequences from pathogenic and benign variants of type 2 Diabetes Mellitus patients. Through modeling these variants, we were able to determine which sections of the tertiary structure of the INSR protein were linked to type 2 Diabetes Mellitus. Ultimately, we created a map of the INSR protein and indicated which parts of the protein structure had significant effects on type 2 Diabetes Mellitus. We concluded differences in the charged/polar amino acids on the lower right section of the structure(amino acid numbers 487 and 750) were associated with pathogenic variants, while charged/polar differences on the lower left of the structure(amino acid numbers 669 and 784) had no impact on pathogenicity. We observed several differences in the amino acid chain structure, however they were not unique to the pathogenic variants; they were also seen in the benign variants.

sentences: Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facilitys Extremely Brilliant Source: the worlds first high-energy 4th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organs structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organs individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

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A metabolically stable PET tracer for imaging synaptic vesicle protein 2A: Synthesis and preclinical characterization of SDM-16 PurposeTo investigate the synaptic vesicle glycoprotein 2A (SV2A) expression in the whole central nervous system and peripheral tissues, a metabolically stable SV2A radiotracer is desirable to minimize a potential confounding effect of radiometabolites. The aim of this study was to develop and evaluate a metabolically stable SV2A radiotracer, [18F]SDM-16, in nonhuman primate brains.

MethodsThe racemic SDM-16 (4-(3,5-difluorophenyl)-1-((2-methyl-1H-imidazol-1-yl)methyl)pyrrolidin-2-one) was synthesized and assayed for in vitro SV2A binding affinity. We synthesized the enantiopure [18F]SDM-16 using the corresponding arylstannane precursor. Nonhuman primate brain PET was performed on a FOCUS 220 system. Arterial blood was drawn for metabolite analysis and construction of plasma input function. Regional time-activity curves (TACs) were evaluated with the one-tissue compartment (1TC) model to obtain the volume of distribution (VT). Binding potential (BPND) was calculated using either the nondisplaceable volume of distribution (VND) or the centrum semiovale (CS) as the reference region.

ResultsRacemic SDM-16 was synthesized in 3 steps with 44% overall yield and has high affinity (Ki = 3.7 nM) to human SV2A. [18F]SDM-16 was prepared in greater than 99% radiochemical and enantiomeric purity. This radiotracer displayed high specific binding in brain and was metabolically more stable than other SV2A PET tracers. The plasma free fraction (fP) of [18F]SDM-16 was 69%, which was higher than those of [11C]UCB-J (46%), [18F]SynVesT-1 (43%), [18F]SynVesT-2 (41%), and [18F]UCB-H (43%). The TACs were well described with the 1TC. The averaged test-retest variability (TRV) was -9{+/-}8%, and averaged absolute TRV (aTRV) was 10{+/-}7% for all analyzed brain regions.

ConclusionWe have successfully synthesized a metabolically stable and high affinity SV2A PET tracer, [18F]SDM-16, which showed high specific and reversible binding in the NHP brain. [18F]SDM-16 may have potential application in the visualization and quantification of SV2A beyond the brain.

sentences: Passive Exposure Sparsifies Neural Activity in the Primary Visual Cortex A notable feature of neural activity is sparseness - namely, that only a small fraction of neurons in a local circuit have high activity at any moment. Not only is sparse neural activity observed experimentally in most areas of the brain, but sparseness has been proposed as an optimization or design principle for neural circuits. Sparseness can increase the energy efficiency of the neural code as well as allow for beneficial computations to be carried out. But how does the brain achieve sparse-ness? Here, we found that when neurons in the primary visual cortex were passively exposed to a set of images over several days, neural responses became more sparse. Sparsification was driven by a decrease in the response of neurons with low or moderate activity, while highly active neurons retained similar responses. We also observed a net decorrelation of neural activity. These changes sculpt neural activity for greater coding efficiency.

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Regulation of translation by lysine acetylation in Escherichia coli N{varepsilon}-lysine acetylation is a common post-translational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this post-translational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of the acetyl donors, acetyl phosphate or acetyl-Coenzyme A, inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but rather increases the proportion of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunit than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits most pronounced during late exponential and early stationary phase growth - the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell.

IMPORTANCENumerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from the central metabolites acetyl coenzyme A or acetyl phosphate to a lysine residue in a protein. This post-translational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt-bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering a new target of regulation, protein synthesis, and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

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FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation.

125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

sentences: Genetically independent phenotype analysis identifies LPA and VCAM1 as drug targets for human ageing The length and quality of life is important to us all, yet identification of promising drug targets for human ageing using genetics has had limited success. Here, we combine six large European-ancestry genome-wide association studies (GWAS) of human ageing traits--healthspan, father and mother lifespan, exceptional longevity, frailty index, and self-rated health--in a principal component framework that maximises their shared genetic architecture. The first principal component (GIP1) is more heritable than the original studies and shows strong genetic correlations with length of life as well as multiple indices of mental and physical wellbeing. We identify 27 genomic regions associated with GIP1, and provide additional, independent evidence for an effect on human ageing for loci near HTT and MAML3 using a study of Finnish and Japanese subject survival. Across the genome, GIP1 associations are enriched in genes involved in haem metabolism and pathways related to transcription, neurogenesis, homeostasis, proteolysis, intracellular signalling, immunity, and the muscle system. Finally, using proteome-wide two-sample Mendelian randomisation and colocalisation, we provide robust evidence for a detrimental effect of blood levels of apolipoprotein(a) (LPA) and vascular cell adhesion molecule 1 (VCAM1) on GIP1. Together, our results demonstrate that combining multiple ageing traits using genetic principal components enhances power to detect biological targets for human ageing.

sentences: Long-read sequencing of SARS-CoV-2 reveals novel transcripts and a diverse complex transcriptome landscape. Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2 (COVID-19), is a positive single-stranded RNA virus with a 30 kb genome that is responsible for the current pandemic. To date, the genomes of global COVID-19 variants have been primarily characterized via short-read sequencing methods. Here, we devised a long-read RNA (IsoSeq) sequencing approach to characterize the COVID-19 transcript landscape and expression of its [~]27 coding regions. Our analysis identified novel COVID-19 transcripts including a) a short [~]65-70 nt 5-UTR fused to various downstream ORFs encoding accessory proteins such as the envelope, ORF 8, and ORF 9 (nucleocapsid) proteins, that are relatively highly expressed, b) novel SNVs that are differentially expressed, whereby a subset are suggestive of partial RNA editing events, and c) SNVs at functional sites, whereby at least one is associated with a differentially expressed spike protein isoform. These previously uncharacterized COVID-19 isoforms, expressed genes, and gene variants were corroborated using ddPCR. Understanding this transcriptional complexity may help provide insight into the biology and pathogenicity of SARS-CoV-2 compared to other coronaviruses.

sentences: siRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insights into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have established techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced to two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for medusa-type cnidarians, or jellyfish, are quite limited, and reliable techniques are required to interrogate function of specific genes. Here, we present a method to knockdown target genes by small interfering RNA (siRNA) delivery into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knockdown gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNAs electroporation, which circumvents the technical difficulty of microinjecting small eggs. Unexpectedly, Wnt3 knockdown in Cladonema does not induce morphological defects in planula, suggesting a distinct mechanism that operates during embryogenesis. Our method for electroporation of siRNAs targeting specific genes is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.

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Cannabidiol (CBD) repurposing as antibacterial: promising therapy of CBD plus polymyxin B against superbugs Abstract

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria are a major worldwide public health problem. In the last decades, resistance to last-resort antibiotics such as polymyxin B (PB) have been increasingly observed among these superbugs, compromising the effectiveness of antimicrobial therapy. The present study aimed (i) to assess the ultrapure Cannabidiol (CBD) antibacterial activity against a broad diversity of Gram-negative (GN) and Gram-positive (GP) bacteria (44 different species, 95 strains), comprising standard strains and clinical isolates, and (ii) to investigate the antibacterial activity of the combination CBD + PB against GN bacteria, including chromosomal- and plasmid-acquired PB-resistant and intrinsically PB-resistant GNB. We evaluated CBD in vitro antibacterial activity using the standard broth microdilution method, and the antibacterial activity of the combination CBD + PB was screened using the standard broth microdilution and confirmed by checkerboard assay. CBD exhibited antibacterial activity against different GP bacterial species, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, and Moraxella catarrhalis), and Mycobacterium tuberculosis. The combination CBD + PB exhibited antibacterial activity against PB-resistant GNB (e.g., Klebsiella pneumoniae) as well as additive and/or synergistic effect against LOS-expressing GND. The antibacterial activity of the combination CBD + PB against Pseudomonas aeruginosa and plasmid-mediated colistin-resistant (MCR-1) E. coli strains could be only demonstrated in the presence of phenylalanine-arginine-{beta}-naphthylamide (PA{beta}N). In conclusion, our results show promising translational potential of the combination CBD + PB against MDR and XDR GNB, including PB-resistant K. pneumoniae, highlighting its potential as a rescue treatment for life-threatening infections caused by these superbugs.

One Sentence SummaryPromising combination of cannabidiol (CBD) + polymyxin B (PB) against superbugs (e.g., PB-resistant Gram-negative bacilli): Repurposing CBD

sentences: Scientific modelling can be accessible, interoperable and user friendly: An example for pasture and livestock modelling This article describes the adaptation of a non-spatial model of pastureland dynamics, including vegetation life cycle, livestock management and nitrogen cycle, for use in a spatially explicit and modular modelling platform (k.LAB) dedicated to make data and models more interoperable. The aim is to deliver an existing, locally successful monolithic model, into a more modular, transparent and accessible approach to potential end users, regional managers, farmers and other stakeholders. This allows better usability and adaptability of the model beyond its originally intended geographical scope (the Cantabrian Region in the North of Spain). The original model, named Puerto, is developed in the R language and includes 1,491 lines of code divided into 13 script files and linked to 19 input tables. The spatiotemporal rewrite is structured around a set of 10 namespaces called PaL (Pasture and Livestock), which includes 198 interoperable but independent models. The end user chooses the spatial and temporal context of the analysis through an intuitive web-based user interface called k.Explorer. Each model can be called individually or in conjunction with the others, by querying any PaL-related concepts in a search bar. A scientific workflow is built as a response, which is run to produce result datasets and a report with information on the data sources and modelling processes used, delivering results with full transparency. We argue that this work demonstrates key steps needed to create more Findable, Accessible, Interoperable and Reusable (FAIR) models. This is particularly essential in environments as complex as agricultural systems, where multidisciplinary knowledge needs to be integrated across diverse spatial and temporal scales in order to understand complex and changing problems.

sentences: Coursing hyenas and stalking lions: the potential for inter- and intraspecific interactions Resource partitioning promotes coexistence among guild members, and carnivores reduce interference competition through behavioural mechanisms that promote spatio-temporal separation. We analyzed sympatric lion and spotted hyena movements and activity patterns to ascertain the mechanisms facilitating their coexistence within semi-arid and wetland ecosystems. We identified recurrent high-use (revisitation) and extended stay (duration) areas within home ranges, and correlated environmental variables with movement-derived measures of inter- and intraspecific interactions. Spatial overlaps among lions and hyenas occurred at edges of home ranges, around water-points, along pathways between patches of high-use areas, and expanded during the wet season. Lions shared more of their home ranges with spotted hyenas in arid ecosystems, but shared more of their ranges with conspecifics in mesic environments. Despite shared space use, we found evidence for subtle temporal differences in the nocturnal movement and activity patterns between the two predators, suggesting a fine localized-scale avoidance strategy. Revisitation frequency and duration within home ranges were influenced by interspecific interactions, subsequent to land cover categories and diel cycles. Intraspecific interactions were also important for lions and, for hyenas, moon illumination and ungulates attracted to former anthrax carcass sites in Etosha, with distance to water in Chobe/Linyanti. Recursion and duration according to locales of competitor probabilities were similar among female lions and both sexes of hyenas, but different for male lions. Our results suggest that lions and spotted hyenas mediate the potential for interference competition through subtle differences in temporal activity, fine-scale habitat use differentiation, and localized reactive-avoidance behaviours. These findings enhance our understanding of the potential effects of interspecific interactions among large carnivore space-use patterns within an apex predator system, and show adaptability across heterogeneous and homogeneous environments. Future conservation plans should emphasize the importance of inter- and intraspecific competition within large carnivore communities, particularly moderating such effects within increasingly fragmented landscapes.

sentences: Extracellular matrix guidance determines proteolytic and non-proteolytic cancer cell patterning Metastatic tumor cell invasion into interstitial tissue is a mechanochemical process that responds to tissue cues and further involves proteolytic remodeling of the tumor stroma. How matrix density, tissue guidance and the ability of proteolytic tissue remodeling cooperate and determine decision-making of invading tumor cells in complex-structured three-dimensional (3D) tissue remains unclear. We here developed a collagen-based invasion assay containing a guiding interface of low collagen density adjacent to randomly organized 3D fibrillar lattice and examined the invasion of melanoma cells from multicellular spheroids in response to matrix density, guidance cues and collagenolysis. After 48 hours of culture, two invasion niches developed, (i) sheet-like collective migration along the interface and (ii) single cell- and strand-like invasion into randomly organized 3D matrix. High collagen density impeded migration into the random matrix, whereas migration along a high-density collagen interface was increased compared to the low-density matrix assay. In silico analysis predicted that facilitated interface migration in high-density matrix depended on physical guidance without collagen degradation, whereas migration into randomly organized matrix was strongly dependent on collagenolysis. When tested in 3D culture, inhibition of matrix metalloprotease (MMP)-mediated collagen degradation compromised migration into random matrix in dependence of density, whereas interface-guided migration remained effective. In conclusion, with increasing tissue density, matrix cues bordered by dense matrix, but not randomly organized matrix, support effective MMP-independent migration. This identifies the topology of interstitial tissue a primary determinant of switch behaviors between MMP-dependent and MMP-independent cancer cell invasion.

sentences: Dual pathway architecture underlying vocal learning in songbirds Song acquisition and production in songbirds is governed by a dedicated neural circuitry that involves two parallel pathways: a motor pathway for the production and a basal ganglia (BG) pathway for the acquisition. Juveniles learn by imitating adult vocalizations and proceed by trial and error, errors being conveyed by a dopaminergic signal. The complex nature of the relationship between neural control and syrinx musculature makes song learning a complicated problem to solve. Reinforcement learning (RL) has been widely hypothesized to underlie such sensorimotor learning even though this can lead to sub-optimal solutions under uneven contours in continuous action spaces. In this article, we propose to re-interpret the role of a dual pathway architecture, underlying avian vocal learning, that helps overcome these limitations. We posit that the BG pathway conducts exploration by inducing large daily shifts in the vocal production while the motor pathway gradually consolidates this exploration. This process can be understood as a modified form of a simulated annealing process. Simulations on Gaussian performance landscapes and a syrinx-based performance landscape are demonstrated and compared with standard approaches. Taking behavioral constraints into account (60 days of learning, 1000 trials per day), the model allows to reach the global optimum in complex landscapes and thus provides a sound insight into the role of the dual pathway architecture underlying vocal learning.

sentences: Normative Decision Rules in Changing Environments Models based on normative principles have played a major role in our understanding of how the brain forms decisions. However, these models have typically been derived for simple, stable environments, and their relevance to decisions under more naturalistic, dynamic conditions is unclear. We previously derived a normative decision model in which evidence accumulation is adapted to environmental dynamics (Glaze et al., 2015), but the evolution of commitment rules (e.g., thresholds on the accumulated evidence) under such dynamic conditions is not fully understood. Here we derive a normative model for decisions based on changing evidence or reward. In these cases, performance (reward rate) is maximized using adaptive decision thresholds that best account for diverse environmental changes, in contrast to predictions of many previous decision models. These adaptive thresholds exhibit several distinct temporal motifs that depend on the specific, predicted and experienced changes in task conditions. These adaptive decision strategies perform robustly even when implemented imperfectly (noisily) and can account for observed response times on a task with time-varying evidence better than commonly used constant-threshold or urgency-gating models. These results further link normative and neural decision-making while expanding our view of both as dynamic, adaptive processes that update and use expectations to govern both deliberation and commitment.

sentences: Fish diversity in a doubly landlocked country - a description of the fish fauna of Uzbekistan using DNA barcoding Uzbekistan is one of two doubly landlocked countries in the world, where all rivers are endorheic basins. Although fish diversity is relatively poor in Uzbekistan compared to other regions, the fish fauna of the region has not yet been fully studied. The aim of this study was to establish a reliable barcoding reference database for fish in Uzbekistan. A total of 666 specimens, belonging to 59 species within 39 genera, 16 families, and 9 orders, were subjected to polymerase chain reaction amplification in the barcode region and sequenced. The length of the 666 barcodes was 682 bp. The average K2P distances within species, genera, and families were 0.22%, 6.33%, and 16.46%, respectively. The average interspecific distance was approximately 28.8 times higher than the mean intraspecific distance. The Barcode Index Number (BIN) discordance report showed that 666 specimens represented 55 BINs, of which five were singletons, 45 were taxonomically concordant, and five were taxonomically discordant. The barcode gap analysis demonstrated that 89.3% of the fish species examined could be discriminated by DNA barcoding. These results provide new insights into fish diversity in the inland waters of Uzbekistan and can provide a basis for the development of further studies on fish fauna.

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Evolution and regulation of microbial secondary metabolism Microbes have disproportionate impacts on the macroscopic world. This is in part due to their ability to grow to large groups and cooperatively secrete massive amounts of secondary metabolites that impact their environment. Yet, the conditions enabling secondary metabolism without compromising primary needs remain unclear. Here we investigated the biosynthesis of rhamnolipids, a secondary metabolite that Pseudomonas aeruginosa makes to decrease the surface tension of surrounding liquid. Using a combination of genomics, metabolomics, transcriptomics, and mathematical modeling we show that biosynthesis of rhamnolipids from glycerol varies inconsistently across the phylogenetic tree; instead, non-producer lineages are also those worse at reducing the oxidative stress of primary glycerol metabolism. The link to oxidative stress explains the inconsistent distribution across the P. aeruginosa tree, adding a new layer to the regulation of rhamnolipids--a microbial secondary metabolite important for fitness in natural and clinical settings.

SignificanceThe bacterium Pseudomonas aeruginosa is a major source of hospital-acquired infections. This pathogens knack for virulence relies on its ability to multiply and secrete massive amounts of substances that overwhelm microbial competitors and weaken host defenses. It remains unclear how the bacteria conciliate their need to grow and multiply--a need at the individual-level-- with their ability to secrete products--a need of the population. Here we combined genomics, metabolomics and mathematical modeling to study the biosynthesis of rhamnolipids, a surfactant that P. aeruginosa makes to reduce the surface tension of surrounding liquids. Our study reveals a new link between oxidative stress and rhamnolipid synthesis, which helps explain how this important bacterial product has evolved and how it persists in many lineages of pathogens.

sentences: Harnessing the Intrinsic Photochemistry of Isoxazoles for the Development of Chemoproteomic Crosslinking Methods Photo-crosslinking is a powerful technique for identifying both coarse- and fine-grained information on protein binding by small molecules. However, the scope of useful functional groups remains limited, with most studies focusing on diazirine, aryl azide, or benzophenone-containing molecules. Here, we report a unique method for photo-crosslinking, employing the intrinsic photochemistry of the isoxazole, a common heterocycle in medicinal chemistry, to offer an alternative to existing strategies using more perturbing, extrinsic crosslinkers. In this initial report, this technique is applied both in vitro and ex vivo, used in a variety of common chemoproteomic workflows, and validated across multiple proteins, demonstrating the utility of isoxazole photo-crosslinking in a wide range of biologically relevant experiments.

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Integration of Diverse Transcriptomics Datasets using Random Forest to Predict Universal Functional Pathways in Tfr Cells MotivationT follicular regulatory (Tfr) cells are a specialized cell subset that controls humoral immunity. Despite a number of individual transcriptomic studies on these cells, core functional pathways have been difficult to uncover due to the substantial transcriptional overlap of these cells with other effector cell types, as well as transcriptional changes occurring due to disease settings. Developing a core transcriptional module for Tfr cells that integrates multiple cell type comparisons as well as diverse disease settings will allow a more accurate prediction of functional pathways. Researchers studying allergic reactions, immune responses to vaccines, autoimmunity and cancer could use this gene set to better understand the roles of Tfr cells in controlling disease progression.

Additional cell types beyond Tfr cells that have similar features of transcriptomic complexity within diverse disease settings may also be studied using similar approaches. High-throughput sequencing technologies allow the generation of large datasets that require specific tools to best interpret the data. The development of a core transcriptional module for Tfr cells will allow investigators to determine if Tfr cells may have functional roles within their biological systems with little knowledge of Tfr biology. With this work, we have addressed the need of core gene modules to define specific subsets of immune cells.

ResultsWe introduce an integrated "core Tfr cell gene module" that can be incorporated into GSEA analysis using various input sizes. The integrated core Tfr gene module was built using transcriptomic studies in Tfr cells from several different tissues, disease settings, and cell type comparisons. Random forest was used to integrate the transcriptomic studies to generate the core gene module. A GSEA gene set was formulated from the integrated core Tfr gene module for incorporation into end-user friendly GSEA. The gene sets are presented along with random genes taken from the GTEX data set and are presented as GMT files. The user can upload the gene set to the GSEA website or any gene set tool which takes GMT files. We also present the full results of the model including p-values calculated by random forest. This provides users with more flexibility in choosing a p-value cutoff that is most appropriate for the experimental setting.

AvailabilityThe core Tfr gene sets are freely available at: https://github.com/alosdiallo/TFR_Model. We have also included all of the code and data used in developing these gene sets. The code and results are released under an MIT license.

Supplementary informationSupplementary data are available at Bioinformatics online.

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Maize inbred line B96 is the source of large-effect loci for resistance to generalist but not specialist spider mites Maize (Zea mays subsp. mays) yield loss from arthropod herbivory is substantial. While the basis of resistance to major insect herbivores has been comparatively well-studied in maize, less is known about resistance to spider mite herbivores, which are distantly related to insects and feed by a different mechanism. Two spider mites, the generalist Tetranychus urticae, and the grass-specialist Oligonychus pratensis, are notable pests of maize, especially during drought conditions. We assessed the resistance to both mite species of 38 highly diverse maize lines, including several previously reported to be resistant to one or the other mite species. We found that line B96, as well as its derivatives B49 and B75, were highly resistant to T. urticae. In contrast, neither these three lines, nor any others included in our study, were notably resistant to O. pratensis. Quantitative trait locus (QTL) mapping with F2 populations from crosses of B49, B75, and B96 to susceptible B73 identified a large-effect QTL on chromosome 6 as underlying T. urticae resistance in each line, with an additional QTL on chromosome 1 in B96. Genome sequencing and haplotype analyses identified B96 as the apparent sole source of resistance haplotypes. Our study identifies loci for use in maize breeding programs for T. urticae resistance, as well as to assess if the molecular-genetic basis of spider mite resistance is shared with insect pests of maize, as B96 is also among the most resistant known maize lines to several insects, including the European corn borer, Ostrinia nubilalis.

Key message Maize(Zea mays subsp. mays) inbred lines B49, B75, and B96 harbor large-effect loci for resistance to the generalist spider mite Tetranychus urticae, but not the specialist Oligonychus pratensis.

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Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon BackgroundMonitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors.

MethodsAnopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile.

ResultsThe study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.02%) and oocyst (14.41%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.94%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.44%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations.

ConclusionThe emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

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Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation a critical role for alternative oxidase (AOX) regulation that concerns also microbiota effectiveness Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of plants inner and outer environment. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at systems levels. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as, germination, root and shoot growth, formation of adventitious, clonal and reproductive organs, fruits and seeds. Fermentative paths can help in acclimation and, to our view the role of alternative oxidase (AOX) in coordinating complex metabolic and physiologic adjustments is underestimated.

Cellular level of sucrose is an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 hours after imbibition (HAI) was beneficial to germination. However, parallel enhanced AOX expression was essential to control negative effects by prolonged sucrose treatment. Early down-regulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose, but suppressed early germination in its presence. Our results also suggest that seeds-inoculated arbuscular mycorrhizal fungi can buffer sucrose stress during germination to restore normal respiration more efficiently.

Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early selection on disease tolerance, predicting plant holobiont behavior and improving germination. Furthermore, our research strengthens the view that AOX can serve as powerful functional marker source for seed hologenomes.

sentences: FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons Neurons are believed to rely on dendritic localization and translation of mRNAs in order to generate activity-dependent changes in the synaptic plasticity. Here, we develop a strategy combining compartment-specific CLIP and TRAP in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. This revealed transcripts that have differentially localized alternative 3UTR and splicing isoforms. FMRP targets are overrepresented among dendritic mRNAs, and compartment-specific FMRP-CLIP defined 383 dendritic FMRP targets, and also allowed for segregation of whole-cell FMRP targets into functional modules that are locally regulated by FMRP. In the absence of FMRP, dendritic FMRP targets show increased ribosome association, consistent with reported roles for FMRP in translational repression. Together, the data support a model in which distinct patterns of FMRP localization allow it to differentially regulate the expression of nuclear proteins and synaptic proteins within different compartments of a single neuronal cell type.

sentences: Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

sentences: Neurocomputational evidence that conflicting prosocial motives guide distributive justice In the history of humanity, most conflicts within and between societies have originated from perceived inequality in resource distribution. How humans achieve and maintain distributive justice has therefore been an intensely studied issue. However, most research on the corresponding psychological processes has focused on inequality aversion and has been largely agnostic of other motives that may either align or oppose this behavioral tendency. Here we provide behavioral, computational, and neuroimaging evidence that distribution decisions are guided by three distinct motives - inequality aversion, harm aversion, and rank reversal aversion - that interact and in fact can also deter individuals from pursuing equality. At the neural level, we show that these three motives are encoded by separate neural systems, compete for representation in various brain areas processing equality and harm signals, and are integrated in the striatum, which functions as a crucial hub for translating the motives to behavior. Our findings provide a comprehensive framework for understanding the cognitive and biological processes by which multiple prosocial motives are coordinated in the brain to guide redistribution behaviors. This framework enhances our understanding of the brain mechanisms underlying equality-related behavior, suggests possible neural origins of individual differences in social preferences, and provides a new pathway to understand the cognitive and neural basis of clinical disorders with impaired social functions.

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Abortion occurs during double fertilisation and ovule development in Paeonia ludlowii Paeonia ludlowii (Stern & G.Taylor) D.Y.Hong, a rare and endangered species, is indigenous to Tibet, China and propagated only by seed. Its natural reproduction is constrained by low fecundity. Excess seed abortion is a key factor restricting its natural reproduction, cultivation, introduction, and protection. In this study, we examined the characteristics of aborted ovules, developmental differences after flowering of normal and aborted ovules, and their ratios at different positions in P. ludlowii ovary. During pollination, fertilisation, and seed development, ovule abortion was frequent, with a random abortion position. There were four types of abortion, namely, abnormal pistil, sterile ovules, abnormal embryo sac, embryo and endosperm abortions. Of these, embryo and endosperm abortions could be divided into early abortion, middle abortion, and late abortion. The early aborted ovules stopped growing on day 12, the endoblast and endosperm in the embryo sac aborted gradually. Furthermore, the shape of the embryo sac cavity changed. The volume of aborted ovules was significantly different from that of fertile ovules. At ripening, the external morphology of different types of aborted seeds was significantly different.

Summary statementElucidation of the origin and characteristics of ovule abortion in an endangered Chinese plant species, Paeonia ludlowii.

sentences: Template-based design of peptides to inhibit SARS-CoV-2 RNA-dependent RNA polymerase complexation. The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between the subunits of the RdRp complex are highly conserved, facilitating the design of inhibitors with high affinity for the interaction hotspots of the complex. Here, we report development and application of a structural bioinformatics protocol to design peptides that can inhibit RdRp complex formation by targeting the interface of its core subunit nonstructural protein (nsp) 12 with accesory factor nsp7. We adopt a top-down approach for protein design by using interaction hotspots of the nsp7-nsp12 complex obtained from a long molecular dynamics trajectory as template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in silico to determine peptide sequences with highest shape and interaction complementarity for the nsp7-nsp12 interface. Two lead designed peptide are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation and anti-viral activity. Their binding affinity to nsp7 (target), as determined from surface plasmon resonance (SPR) assay, is found to be comparable to that of the nsp7-nsp12 complex. Further, one of the designed peptides gives 46 % inhibition of nsp7-nsp12 complex at 10:1 peptide:nsp7 molar concentration (from ELISA assay). Further optimization of cell penetrability and target affinity of these designed peptides is expected to provide lead candidates with high anti-viral activity against SARS-CoV-2.

sentences: The beta component of gamma-band auditory steady-state responses in patients with schizophrenia The mechanisms underlying circuit dysfunctions in schizophrenia (SCZ) remain poorly understood. Auditory steady-state responses (ASSRs), especially in the gamma and beta band, have been suggested as a potential biomarker for SCZ. While the reduction of 40Hz power for 40Hz drive has been well established and replicated in SCZ patients, studies are inconclusive when it comes to an increase in 20Hz power during 40Hz drive. There might be several factors explaining the inconsistencies, including differences in the sensitivity of the recording modality (EEG vs MEG), differences in stimuli (click-trains vs amplitude-modulated tones) and large differences in the amplitude of the stimuli. Here, we used a computational model of ASSR deficits in SCZ and explored the effect of three SCZ-associated microcircuit alterations: reduced GABA activity, increased GABA decay times and NMDA receptor hypofunction. We investigated the effect of input strength on gamma (40 Hz) and beta (20 Hz) band power during gamma ASSR stimulation and saw that the pronounced increase in beta power during gamma stimulation seen experimentally could only be reproduced in the model when GABA decay times were increased and only for a specific range of input strengths. More specifically, when the input was in this specific range, the rhythmic drive at 40Hz produced a strong 40Hz rhythm in the control network; however, in the  SCZ-like network, the prolonged inhibition led to a so-called  beat-skipping, where the network would only strongly respond to every other input. This mechanism was responsible for the emergence of the pronounced 20Hz beta peak in the power spectrum. The other two microcircuit alterations were not able to produce a substantial 20 Hz component but they further narrowed the input strength range for which the network produced a beta component when combined with increased GABAergic decay times. Our finding that the beta component only existed for a specific range of input strengths might explain the seemingly inconsistent reporting in experimental studies and suggests that future ASSR studies should systematically explore different amplitudes of their stimuli. Furthermore, we provide a mechanistic link between a microcircuit alterations and an electrophysiological marker in schizophrenia and argue that more complex ASSR stimuli are needed to disentangle the nonlinear interactions of microcircuit alterations. The computational modelling approach put forward here is ideally suited to facilitate the development of such stimuli in a theory-based fashion.

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CINmetrics: An R package for chromosomal instability analysis Genomic instability has been an important hallmark in cancer and more recently in neurodegenrative diseases. Chromosomal instability, as a measure of genomic instability, has been used to characterize clinical and biological phenotypes associated with these diseases by measuring structural and numerical chromosomal alterations. There have been multiple Chromosomal Instability Scores developed across many studies in the literature; however, these scores have not been compared because of a lack of single tool available to calculate these various metrics. Here, we provide an R package CINmetrics, that calculates six different chromosomal instability scores and allows direct comparison between them. We also show how these scores differ by applying CINmetrics to breast cancer data from The Cancer Genome Atlas (TCGA).

AvailabilityThe package is available on CRAN at https://cran.r-project.org/package=CINmetrics and on github at https://github.com/lasseignelab/CINmetrics

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Axisymmetric diffusion kurtosis imaging with Rician bias correction: A simulation study PurposeTo compare the estimation accuracy of axisymmetric diffusion kurtosis imaging (DKI) and standard DKI in combination with Rician bias correction (RBC) under the influence of noise.

MethodsAxisymmetric DKI is more robust against noise-induced variation in the measured signal than standard DKI because of its reduced parameter space. However, its susceptibility to Rician noise bias at low signal-to-noise ratios (SNRs) is unknown. Here, we investigate two main questions: first, does Rician bias correction improve estimation accuracy of axisymmetric DKI?; second, is the estimation accuracy of axisymmetric DKI increased compared to standard DKI? Estimation accuracy was investigated on the five axisymmetric DKI tensor metrics (AxTM): the parallel and perpendicular diffusivity and kurtosis and the mean kurtosis, using a simulation study based on synthetic and in-vivo data.

ResultsWe found that RBC was most effective for increasing accuracy of the parallel AxTM in highly to moderately aligned white matter. For the perpendicular AxTM, axisymmetric DKI without RBC performed slightly better than with RBC. However, the combination of axisymmetric DKI with RBC was the overall best performing algorithm across all five AxTM and the axisymmetric DKI framework itself substantially improved accuracy in tissues with low fiber alignment.

ConclusionThe combination of axisymmetric DKI with RBC facilitates accurate DKI parameter estimation at unprecedented low SNRs ({approx} 15), possibly making it a valuable tool for neuroscience and clinical research studies where scan time is a limited resource. The tools used in this paper are publicly available in the open-source ACID toolbox for SPM.

sentences: CRISPR-Cas Controls Cryptic Prophages The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12 and thought to be inactive against prophages. Instead, we demonstrate here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., 5 cryptic) prophages. Specifically, compared to the wild-type strain, reducing interfering RNAs (crRNA) from the spacer region due to a cas2 polar mutation, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA and subsequent cleavage of the double-stranded RNA, rather than through cleavage of cryptic prophage DNA; i.e., self-targeting. Consistently, seven of the 13 E. coli spacers contain partial matches to the cryptic prophages, and inactivation of CRISPR-Cas increases the level of cryptic prophage CP4-57 and DLP-12 lytic gene transcripts for lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Also, lysis is clearly seen via transmission electron microscopy when the CRISPR-Cas array is deleted. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.

sentences: How plants minimize somatic evolution A remarkable property of plants is their ability to accumulate mutations at a very slow pace despite their potentially long lifespans, during which they continually form buds, each with the potential to become a new branch. Because replication errors in cell division represent an unavoidable source of mutations, minimizing mutation accumulation requires the minimization of cell divisions. Here we show that there exists a well defined theoretical minimum for the branching cost, defined as the number of cell divisions necessary for the creation of each branch. Most importantly, we also show that this theoretical minimum can be closely approached by a simple pattern of cell divisions in the meristematic tissue of apical buds during the generation of novel buds. Both the optimal pattern of cell divisions and the associated branching cost are consistent with recent experimental data, suggesting that plant evolution has led to the discovery of this mechanism.

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Quantitative 3D microscopy reveals a genetic network predicting the local activity of anti-Aβ compounds Genetic and biochemical evidence implicates amyloid-{beta} (A{beta}) in Alzheimers disease, yet many anti-A{beta} treatments are clinically ineffective. Regional heterogeneity of efficacy may contribute to these disappointing results. Here we mapped the regiospecificity of various anti-A{beta} treatments by high-resolution light-sheet imaging of amyloid plaques in electrophoretically clarified brains of Thy1-APP mice overexpressing A{beta}. We found that A{beta} plaques in whole brains progressed from 1.2*106 to 2.5*106 (standard deviation {+/-} 1.6*105 and {+/-} 4.3*105 respectively) over a 9-month period. We then assessed the regiospecific plaque clearance in mice subjected to {beta}-secretase inhibition, amyloid intercalation by polythiophenes, or anti-A{beta} antibodies. Each treatment showed unique spatiotemporal A{beta} clearance signatures, with polythiophenes emerging as potent anti-A{beta} compounds with promising pharmacokinetic properties. We then interrogated genes matching regiospecific A{beta} clearance by aligning voxels that showed drug effectiveness to spatial-transcriptomics atlases. Bace1 and Thy1 expression matched the regiospecific efficacy of BACE inhibition, confirming the validity of our analyses. Voxels cleared by polythiophenes correlated with transcripts encoding synaptic proteins, whereas voxels cleared by BACE inhibition correlated with oligodendrocyte-specific genes. The restricted regional susceptibility of A{beta} plaques to specific treatments may contribute to the clinical failure of anti-A{beta} therapies. The striking regiospecificity of the treatments studied suggests that combinatorial regimens may improve functional outcomes.

One sentence summary3D microscopy of A{beta} plaques reveals that Alzheimers disease therapeutics have regiospecific effects that can be mapped to distinct genetic networks.

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Novel and Reported Compensatory Mutations in rpoABC Associate Specifically with Predominant Mycobacterium tuberculosis Rifampicin Resistance Marker rpoB:S450L BackgroundRifampicin (RIF) is a key first-line drug used to treat tuberculosis, a pulmonary disease caused by Mycobacterium tuberculosis. However antibiotic resistance to RIF is prevalent despite an apparent fitness cost. RIF resistance is primarily caused by mutations in the RIF resistance determining region in the rpoB gene, at the cost of slower growth in rich media. Compensatory mutations in the genes rpoA and rpoC have been shown to alleviate this fitness cost. These compensatory mutations may explain how RIF resistant strains have spread so rapidly. However, the effect of compensation on transmission is still unclear, partly because of uncertainty over which rpoABC mutations compensate for which RIF resistance markers.

ObjectivesWe performed an association study on a globally representative set of 4309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations.

Results and ConclusionsOnly 20.0% (216/1079) of RIF resistant isolates carried previously reported high-probability compensatory mutations, suggesting existence of other compensatory mutations. Using a strict phylogenetic approach, we identified 18 novel putative compensatory mutations in rpoC, rpoB, and rpoA. Novel and previously reported compensatory mutations were strongly associated with the RIFR marker rpoB:S450L, suggesting compensation may be specific to particular RIFR markers. These findings will aid identification of RIF-resistant M. tuberculosis strains with restored fitness. Such strains pose a greater risk of causing resistant outbreaks.

sentences: The geometry of domain-general performance monitoring representations in the human medial frontal cortex Controlling behavior to flexibly achieve desired goals depends on the ability to monitor ones own performance. It is unknown how performance monitoring can be both flexible to support different tasks and specialized to perform well on each. We recorded single neurons in the human medial frontal cortex while subjects performed two tasks that involve three types of cognitive conflict. Neurons encoding predicted conflict, conflict, and error in one or both tasks were intermixed, forming a representational geometry that simultaneously allowed task specialization and generalization. Neurons encoding conflict retrospectively served to update internal estimates of control demand. Population representations of conflict were compositional. These findings reveal how representations of evaluative signals can be both abstract and task-specific and suggest a neuronal mechanism for estimating control demand.

sentences: Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.

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Extracellular vesicle microRNA cargo engineering reveals critical mechanisms underlying therapeutic efficacy BackgroundExtracellular vesicles (EVs) are key mediators of intercellular communication and function to transfer biological cargo, including microRNA (miR), from donor to recipient cells. EVs isolated from cardiosphere-derived cells (CDCs) have demonstrated therapeutic efficacy in pre-clinical models of ischemic heart disease, highlighting them as promising vectors for the treatment of CVD. Importantly, it has not yet been established whether miR cargo is necessary for the observed therapeutic benefit of CDC-EVs following acute MI (AMI).

MethodsCDCs were transfected with siRNA against Drosha, the initial endonuclease in the miRNA biogenesis pathway, to generate miR depleted DROSHA-EVs. EVs were characterized by size, morphology, and protein/miR expression. The role of EV miRNA on cardiac target cell apoptosis, proliferation and angiogenesis was examined using a series of in vitro assays. Mice with acute MI underwent delivery of human CDC EVs, DROSHA-EVs and placebo in a double-blind study. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of histological endpoints. In vitro sufficiency assays were performed using a combinatorial approach with individual candidate miRs to identify clusters exhibiting synergistic efficacy.

ResultsDROSHA-EVs exhibited global downregulation of miRNA cargo but were otherwise indistinguishable from wild-type CDC-EVs. miR cargo was responsible for mediating the beneficial effects of human CDC-EV treatment on cardiomyocyte apoptosis, fibroblast proliferation and angiogenesis in vitro. DROSHA-EVs were unable to promote recovery following AMI on a functional or histological level, highlighting the critical role of EV miRNAs in cardioprotection following ischemic injury. A potentially therapeutic miR cluster, miR-146a-370-126a, was identified which acted synergistically to reduce cardiomyocyte apoptosis and was sufficient to render inert EVs into therapeutic vectors.

ConclusionsThese results demonstrate for the first time that miRNAs are required for the regenerative potential of CDC-EVs following AMI and identify a novel miR cluster with therapeutic implications.

sentences: Fetal-like reversion in the regenerating intestine is regulated by mesenchymal Asporin Epithelial tissues undergo fetal-like cellular reprogramming to regenerate after damage1,2. Although the mesenchyme and the extracellular matrix (ECM) play critical roles in tissue homeostasis and regeneration2-5, their role in repurposing developmental programs in epithelium is unknown. To model epithelial regeneration, we culture intestinal epithelium on decellularized small intestinal scaffold (iECM), and identify Asporin (Aspn), an ECM bound proteoglycan, as a critical mediator of cellular reprogramming. Aspn is produced by the mesenchyme, and we show that its effect on epithelial Tgf{beta}-signalling via CD44 is critical for fetal-like conversion. Furthermore, we demonstrate that Aspn is transiently increased upon chemotherapy-induced damage and pivotal for a timely induction of the fetal-like state and tissue regeneration. In summary, we establish a platform for modelling epithelial injury responses ex vivo, and show that the mesenchymal Aspn-producing niche controls tissue repair by regulating epithelial fetal-like reprogramming.

sentences: A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19 The development of effective countermeasures against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal model of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.

sentences: A Guide for Quantifying and Optimizing Measurement Reliability for the Study of Individual Differences Characterizing individual variations is central to interpreting individual differences in neuroscience and clinical studies. While the field has examined multifaceted individual differences in brain functional organization, it is only in recent years that neuroimaging researchers have begun to place a priority on its quantification and optimization. Here, we highlight a potential analytic pitfall that can lead to contaminated estimates of inter-individual differences. We define a two-dimensional individual variation field map to decipher sources of individual variation and their relation to fingerprinting and measures of reliability. We illustrate theoretical gradient flow that represents the most effective direction for optimization when measuring individual differences. We propose to use this general framework for dissecting within- and between-individual variation and provide a supporting online tool for the purposes of guiding optimization efforts in biomarker discovery.

sentences: Carbon catabolite repression in pectin digestion by phytopathogen Dickeya dadantii The catabolism of pectin from the plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce a massive pectinolytic activity during the maceration phase. While previous studies identified the role of a positive feedback loop specific to the pectin degradation pathway, here we show that the pel expression pattern is controlled by a metabolic switch between glucose and pectin. We develop a dynamical and quantitative regulatory model of this process integrating the two main regulators CRP and KdgR related to these two sources of carbon, and reproducing the concentration profiles of the associated metabolites, cAMP and KDG respectively, quantified using a new HPLC method. The model involves only 5 adjustable parameters, and recapitulates the dynamics of these metabolic pathways during bacterial growth together with the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights their activity as an instance of carbon catabolite repression occurring at the transcriptional regulatory level, and directly related to the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitative different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, explaining their evolutionary conservation as separate genes in this species.

sentences: A critical period of translational control during brain development at codon resolution Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

sentences: Molecular characterization of inversion breakpoints in the Drosophila nasuta species group Chromosomal inversions are fundamental drivers of genome evolution. In the Drosophila genus, inversions have been widely characterized cytologically, and play an important role in local adaptation. Here, we characterize chromosomal inversions in the Drosophila nasuta species group using chromosome-level, reference-quality assemblies of seven species and subspecies in this clade. Reconstruction of ancestral karyotypes allowed us to infer the order in which the 22 identified inversions occurred along the phylogeny. We found a higher rate of inversions on the X chromosome, and heterogeneity in the rate of accumulation across the phylogeny. We molecularly characterize the breakpoints of six autosomal inversions, and found that repeated sequences are associated with inversion breakpoints in four of these inversions, suggesting that ectopic recombination is an important mechanism in generating inversion. Characterization of inversions in this species group provides a foundation for future population genetic and functional studies in this recently diverged species group.

sentences: Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this "counterion pathway". The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

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Point-of-care analyte quantification and digital readout via lysate-based cell-free biosensors interfaced with personal glucose monitors Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate production of the reporter enzyme {beta}-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.

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sentences: Invertebrate-induced priority effects and secondary metabolites control fungal community composition in dead wood During decomposition of organic matter, microbial communities may follow different successional trajectories depending on the initial environment and colonizers. The timing and order of the assembly history can lead to divergent communities through priority effects. We explored how assembly history and resource quality affected fungal dead wood communities and decomposition, 1.5 and 4.5 years after tree felling. Additionally, we investigated the effect of invertebrate exclusion during the first two summers. For aspen (Populus tremula) logs, we measured initial resource quality of bark and wood, and surveyed the fungal communities by DNA metabarcoding at different time points during succession. We found that a gradient in fungal community composition was related to resource quality and discuss how this may reflect tolerance-dominance trade-offs in fungal life history strategies. As with previous studies, the initial amount of bark tannins was negatively correlated with wood decomposition rate over 4.5 years. The initial fungal community explained variation in community composition after 1.5, but not 4.5 years, of succession. Although the assembly history of initial colonizers may cause alternate trajectories in successional communities, our results indicate that the communities may easily converge with the arrival of secondary colonizers. We also identified a strong invertebrate-induced priority effect of fungal communities, even after 4.5 years of succession, thereby adding crucial knowledge to the importance of invertebrates in affecting fungal community development. By measuring and manipulating aspects of assembly history and resource quality that have rarely been studied, we expand our understanding of the complexity of fungal community dynamics.

sentences: Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet specific transition mechanisms ensuring ECM proteostasis and their regulatory impact are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of mouse Adamts6 and Adamts10 mutant embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics identification of ADAMTS6-cleaved sites, demonstrates a transcriptionally adapted system for fibrillin-2 proteolysis that contributes to postnatal fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development resulting from a drastic reduction of aggrecan, cartilage link protein and impaired BMP, but not TGF{beta} signaling in cartilage. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of these defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

sentences: Transcriptomic changes highly similar to Alzheimer's disease are observed in a subpopulation of individuals during normal brain aging Aging is a major risk factor for late-onset Alzheimers disease (LOAD). How aging contributes to the development of LOAD remains elusive. In this study, we examined multiple large-scale transcriptomic data from both normal aging and LOAD brains to understand the molecular interconnection between aging and LOAD. We found that shared gene expression changes between aging and LOAD are mostly seen in the hippocampal and several cortical regions. In the hippocampus, the expression of phosphoprotein, alternative splicing and cytoskeleton genes are commonly changed in both aging and AD, while synapse, ion transport, and synaptic vesicle genes are commonly down-regulated. Aging-specific changes are associated with acetylation and methylation, while LOAD-specific changes are more related to glycoprotein (both up- and down-regulations), inflammatory response (up-regulation), myelin sheath and lipoprotein (down-regulation). We also found that normal aging brain transcriptomes from relatively young donors (45-70 years old) clustered into several subgroups and some subgroups showed gene expression changes highly similar to those seen in LOAD brains. Using brain transcriptomic data from another cohort of older individuals (> 70 years), we found that samples from cognitively normal older individuals clustered with the "healthy aging" subgroup while AD samples mainly clustered with the "AD similar" subgroups. This may imply that individuals in the healthy aging subgroup will likely remain cognitively normal when they become older and vice versa. In summary, our results suggest that on the transcriptome level, aging and LOAD have strong interconnections in some brain regions in a subpopulation of cognitively normal aging individuals. This supports the theory that the initiation of LOAD occurs decades earlier than the manifestation of clinical phenotype and it may be essential to closely study the "normal brain aging" to identify the very early molecular events that may lead to LOAD development.

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Deep learning identifies pathological abnormalities predictive of graft loss in kidney transplant biopsies BackgroundInterstitial fibrosis, tubular atrophy, and inflammation are major contributors to renal allograft failure. Here we seek an objective, quantitative pathological assessment of these lesions to improve predictive utility.

MethodsWe constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte (MNL) infiltrates from Periodic acid-Schiff (PAS) stained slides of transplant biopsies (training: n=60, testing: n=33) that quantified pathological lesions specific for interstitium, tubules and MNL infiltration. The pipeline was applied to 789 whole slide images (WSI) from baseline (n=478, pre-implantation) and 12-month post-transplant (n=311) protocol biopsies in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss.

ResultsOur model accurately recognized kidney tissue compartments and MNLs. The digital features significantly correlated with Banff scores, but were more sensitive to subtle pathological changes below the thresholds in Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of 1-year graft loss (p=2.8e-05), while a Composite Damage Score (CDS) in 12-month post-transplant protocol biopsies predicted later graft loss (p=7.3e-05). ITAS and CDS outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITAS or CDS also demonstrated significantly higher incidence of eGFR decline and subsequent graft damage.

ConclusionsThis deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies, and demonstrated superior ability for prediction of posttransplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

sentences: Percolation transition determines protein size limit for passive transport through the nuclear pore complex Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex"s central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

sentences: Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends Growing microtubule ends provide platforms for the accumulation of plus-end tracking proteins that organize into comets of mixed protein composition. Using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions appear to facilitate the non-stoichiometric accumulation of cargo Tip1. We dissected the contribution of two disordered protein regions in Mal3 and found that both are required for the ability to form droplets and Tip1 accumulation, while autonomous Mal3 comet formation only requires one of them. Using theoretical modeling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends.

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Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogens evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide single nucleotide polymorphisms (SNPs) among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over one year throughout the Ngorongoro Conservation Area, Tanzania, an area hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes within the study area. However, phylogeographic structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis, with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence as well as co-infection.

ImportancePathogens transmitted between animals and people affect the health and livelihoods of farmers, particularly in developing countries dependent on livestock. Understanding over what distances these pathogens are transmitted and how they evolve is important to inform control strategies towards reducing disease impacts. Information on the circulation of Bacillus anthracis, which causes the often-lethal disease anthrax, is lacking for settings where the disease is commonplace. Consequently, we examined its genetic variability in an area in Tanzania where anthrax is widespread. We found no clear link between how closely cases were sampled and their genetic similarity. We suspect this lack of congruence is primarily driven by large-scale livestock movements, which control efforts should take into consideration. Another significant finding was the co-occurrence of multiple B. anthracis types within individual hosts, suggesting animals are commonly infected with a mixture of variants. This needs to be accounted for when investigating possible connections between cases.

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Connecting conformational stiffness of the protein with energy landscape by a single experiment Proteins are versatile biopolymers whose functions are determined by their structures. Understanding the structural dynamicity, with respect to energy landscape, is essential to describe their biological functions. The ability to study the dynamical properties of a single protein molecule is thus crucial, but ensuring that multiple physical properties can be simultaneously extracted within a single experiment on the exact same protein molecule in real-time has hitherto been infeasible.

Here, we present magnetic tweezers technology that surmounts this limitation, providing real-time dynamic information about changes in several molecular properties ({Delta}G0, conformation, and mean first passage time of unfolding and refolding) from a single experiment, by remeasuring the very same protein molecule in varying chemical condition. We illustrate the versatility of the method by studying electrolyte-dependent conformational flexibility and the energy landscape of substrate protein L under force. Changing salt concentrations reshapes the energy landscape by two specific ways: it speeds-up refolding kinetics while slowing down unfolding kinetics. From the same trajectory, we calculate the stiffness of the protein polymer, a quantity that varies with salt concentration. The data is described within the framework of a modified  electrolyte FJC model that we propose and study here. The observed correlation between{Delta} G0, kinetics and polymer elasticity connect protein chain physics and the energy landscape, while the experimental methodology we describe of getting energy landscape from a single experiment could have wide-ranging applications.

sentences: RUV-III-NB: Normalization of single cell RNA-seq Data Despite numerous methodological advances, the normalization of single cell RNA-seq (scRNA-seq) data remains a challenging task and the performance of different methods can vary greatly across datasets. Part of the reason for this is the different kinds of unwanted variation, including library size, batch and cell cycle effects, and the association of these with the biology embodied in the cells. A normalization method that does not explicitly take into account cell biology risks removing some of the signal of interest. Furthermore, most normalization methods remove the effects of unwanted variation for the cell embedding used for clustering-based analysis but not from gene-level data typically used for differential expression (DE) analysis to identify marker genes. Here we propose RUV-III-NB, a statistical method that can be used to remove unwanted variation from both the cell embedding and gene-level counts. RUV-III-NB explicitly takes into account its potential association with biology when removing unwanted variation via the use of pseudo-replicates. The method can be used for both UMI or sequence read counts and returns adjusted counts that can be used for downstream analyses such as clustering, DE and pseudotime analyses. Using five publicly available datasets that encompass different technological platforms, kinds of biology and levels of association between biology and unwanted variation, we show that RUV-III-NB manages to remove library size and batch effects, strengthen biological signals, improve differential expression analyses, and lead to results exhibiting greater concordance with independent datasets of the same kind. The performance of RUV-III-NB is consistent across the five datasets and is not sensitive to the number of factors assumed to contribute to the unwanted variation. It also shows promise for removing other kinds of unwanted variation such as platform effects. The method is implemented as a publicly available R package available from https://github.com/limfuxing/ruvIIInb.

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Formal meta-analysis of hypoxic gene expression profiles reveals a universal gene signature and cell type-specific effects BackgroundIntegrating transcriptional profiles results in the identification of gene expression signatures that are more robust than those obtained for individual datasets. However, direct comparison of datasets derived from heterogeneous experimental conditions is not possible and their integration requires the application of specific meta-analysis techniques. The transcriptional response to hypoxia has been the focus of intense research due to its central role in tissue homeostasis and in prevalent diseases. Accordingly, a large number of studies have determined the gene expression profile of hypoxic cells. Yet, in spite of this wealth of information, little effort have been done to integrate these dataset to produce a robust hypoxic signature.

ResultsWe applied a formal meta-analysis procedure to a dataset comprising 425 RNAseq samples derived from 42 individual studies including 33 different cell types, to derive a pooled estimate of the effect of hypoxia on gene expression. This approach revealed that a large proportion of the transcriptome (8556 genes out of 20888) is significantly regulated by hypoxia. However, only a small fraction of the differentially expressed genes (1265 genes, 15%) show an effect size that, according to comparisons to gene pathways known to be regulated by hypoxia, is likely to be biologically relevant. By focusing on genes ubiquitously expressed we identified a signature of 291 genes robustly and consistently regulated by hypoxia. Finally, by a applying a moderator analysis we found that endothelial cells show a characteristic gene expression pattern that is significantly different from other cell types.

ConclusionBy the application of a formal meta-analysis to hypoxic gene profiles, we have developed a robust gene signature that characterizes the transcriptomic response to low oxygen. In addition to identifying a universal set of hypoxia-responsive genes, we found a set of genes whose regulation is cell-type specific and suggest a unique metabolic response of endothelial cells to reduced oxygen tension.

sentences: An elite broadly neutralizing antibody protects SARS-CoV-2 Omicron variant challenge The strikingly high transmissibility and antibody evasion of SARS-CoV-2 Omicron variant have posted great challenges on the efficacy of current vaccines and antibody immunotherapy.Here, we screened 34 BNT162b2-vaccinees and cloned a public broadly neutralizing antibody (bNAb) ZCB11 from an elite vaccinee. ZCB11 neutralized all authentic SARS-CoV-2 variants of concern (VOCs), including Omicron and OmicronR346K with potent IC50 concentrations of 36.8 and 11.7 ng/mL, respectively. Functional analysis demonstrated that ZCB11 targeted viral receptor-binding domain (RBD) and competed strongly with ZB8, a known RBD-specific class II NAb. Pseudovirus-based mapping of 57 naturally occurred single mutations or deletions revealed that only S371L resulted in 11-fold neutralization resistance, but this phenotype was not observed in the Omicron variant. Furthermore,prophylactic ZCB11 administration protected lung infection against both the circulating pandemic Delta and Omicron variants in golden Syrian hamsters. These results demonstrated that vaccine-induced ZCB11 is a promising bNAb for immunotherapy against pandemic SARS-CoV-2 VOCs.

sentences: Mitophagy antagonism by Zika virus reveals Ajuba as a regulator of PINK1-Parkin signaling, PKR-dependent inflammation, and viral invasion of tissues. Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens, Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV NS5 antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplified the production of pro-inflammatory chemokines through PKR sensing of mitochondrial RNA. In Ajuba-/- mice, ZIKV induced early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy, and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

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Unbiased population heterozygosity estimates from genome-wide sequence data O_LIHeterozygosity is a metric of genetic variability frequently used to inform the management of threatened taxa. Estimating observed and expected heterozygosities from genome-wide sequence data has become increasingly common, and these estimates are often derived directly from genotypes at single nucleotide polymorphism (SNP) markers. While many SNP markers can provide precise estimates of genetic processes, the results of  downstream analysis with these markers may depend heavily on  upstream filtering decisions.
C_LIO_LIHere we explore the downstream consequences of sample size, rare allele filtering, missing data thresholds and known population structure on estimates of observed and expected heterozygosity using two reduced-representation sequencing datasets, one from the mosquito Aedes aegypti (ddRADseq) and the other from a threatened grasshopper, Keyacris scurra (DArTseq).
C_LIO_LIWe show that estimates based on polymorphic markers only (i.e. SNP heterozygosity) are always biased by global sample size (N), with smaller N producing larger estimates. By contrast, results are unbiased by sample size when calculations consider monomorphic as well as polymorphic sequence information (i.e. genome-wide or autosomal heterozygosity). SNP heterozygosity is also biased when differentiated populations are analysed together, while autosomal heterozygosity remains unbiased. We also show that when nucleotide sites with missing genotypes are included, observed and expected heterozygosity estimates diverge in proportion to the amount of missing data permitted at each site.
C_LIO_LIWe make three recommendations for estimating genome-wide heterozygosity: (i) autosomal heterozygosity should be reported instead of (or in addition to) SNP heterozygosity; (ii) sites with any missing data should be omitted; (iii) populations should be analysed in independent runs. This should facilitate comparisons within and across studies and between observed and expected measures of heterozygosity.
C_LI

sentences: Coordinate regulation of liver ferroportin degradation and de novo synthesis determines serum iron levels in mice The iron hormone hepcidin is transcriptionally activated by iron or inflammation via distinct, partially overlapping pathways. We addressed how iron affects inflammatory hepcidin levels and the ensuing hypoferremic response. Dietary iron overload did not mitigate hepcidin induction in LPS-treated wt mice but prevented effective inflammatory hypoferremia. Likewise, LPS modestly decreased serum iron in hepcidin-deficient Hjv-/- mice, model of hemochromatosis. Synthetic hepcidin triggered hypoferremia only in control but not iron-loaded wt animals. Furthermore, it dramatically decreased hepatic and splenic ferroportin in Hjv-/- mice on standard or iron-deficient diet, but only triggered hypoferremia in the latter. Mechanistically, iron induced liver ferroportin mRNA translation, thereby antagonizing hepcidin-mediated hypoferremia. Conversely, iron depletion suppressed de novo ferroportin synthesis in Hjv-/- livers, allowing exogenous hepcidin to cause hypoferremia. Consequently, prolonged LPS treatment eliminating ferroportin mRNA permitted hepcidin-mediated hypoferremia in iron-loaded mice. Thus, liver ferroportin mRNA translation is critical determinant of serum iron and finetunes hepcidin-dependent functional outcomes. Our data indicate a crosstalk between hepcidin/ferroportin and IRE/IRP systems. Moreover, they suggest that hepcidin supplementation therapy is more efficient combined with iron depletion.

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A species-specific signature residue in the PB2 subunit of the bat influenza virus polymerase restricts viral RNA synthesis. Bat influenza A viruses (H17N10 and H18N11) are genetically distant from conventional influenza A viruses and replicates poorly in non-bat hosts species. However, the reason behind the lower replication fitness of these viruses are yet to be elucidated. In this work, we have identified species-specific signature residues, present in viral PB2 protein, which is a major determinant of polymerase fitness in human, avian and bat cell lines. Through extensive sequence and structural comparison between the bat and non-bat influenza virus RNA polymerases, we have identified a previously uncharacterized PB2-282 residue, which is serine in bat virus PB2 protein but harbours highly conserved glutamic acid in conventional influenza A viruses. Introduction of these bat specific signatures in the polymerase of a human adapted strain of influenza A/H1N1 virus drastically reduces its polymerase activity and replication efficiency in cell lines of human, bat and canine origin. In contrast, introduction of the human specific signatures in bat virus PB2 (H17N10), significantly enhances its function in the context of a chimeric RNA polymerase. Interestingly, the PB2-282 resides within an evolutionary conserved "S-E-S" motif present across different genera of influenza viruses but is replaced with a "S-S-T" motif in bat influenza viruses, indicating that this E to S transition may serve as a species-specific adaptation signature that modulates the activity of bat virus polymerase in other host species.

ImportanceRecent isolation of influenza A like viruses (H17N10 and H18N11) in bats raised concerns about their potential of zoonotic transmission in human. Here we present species-specific signature residues present in the bat influenza virus polymerase, which may act as critical modulators of bat virus propagation in non-bat host species. We utilize bioinformatics based comparative analysis followed by functional screening in order to identify the PB2-282nd position, which harbors a highly conserved glutamic acid in conventional influenza A viruses, but contains an unusual serine in case of bat influenza viruses. Human adapted polymerase, harboring bat specific signature (PB2-S282) performs poorly, while bat PB2 protein harboring human specific signature (PB2-E282) shows increased fitness in human cells. Together, our data identifies novel species-specific signatures present within the influenza virus polymerase that may serve as a key factor in the adaptation of influenza viruses from bat to non-bat host species and vice versa.

sentences: Digital twin predicting diet response before and after long-term fasting Today, there is great interest in diets proposing new combinations of macronutrient compositions and fasting schedules. Unfortunately, there is little consensus regarding the impact of these different diets, since available studies measure different sets of variables in different populations, thus only providing partial, non-connected insights. We lack an approach for integrating all such partial insights into a useful and interconnected big picture. Herein, we present such an integrating tool. The tool uses a novel mathematical model that describes mechanisms regulating diet-response and fasting metabolic fluxes, both for organ-organ crosstalk, and inside the liver. The tool can mechanistically explain and integrate data from several clinical studies, and correctly predict new independent data, including data from a new clinical study. Using this model, we can predict non-measured variables, e.g. hepatic glycogen and gluconeogenesis, and we can quantify personalized expected differences in outcome for any diet. This constitutes a new digital twin technology.

sentences: Quantifying Intratumor Heterogeneity by Key Genes Selected Using Concrete Autoencoder The tumor cell population in a cancer tissue has distinct molecular characteristics and exhibits different phenotypes, thus, resulting in different subpopulations. This phenomenon is known as Intratumor Heterogeneity (ITH), which is a major contributor in drug resistance, poor prognosis, etc. Therefore, quantifying the levels of ITH in cancer patients is essential and there are many algorithms which do so in different ways, using different types of omics data. DEPTH (Deviating gene Expression Profiling Tumor Heterogeneity) is the latest algorithm that uses transcriptomic data to evaluate the ITH score. It shows promising performance, has strong similarity with six other algorithms, and has advantage over two algorithms that uses same type of data (tITH, sITH). However, it has a major drawback that it uses expression values of all the genes ([~]20K genes) in quantifying ITH levels. We hypothesize that a subset of key genes is sufficient to quantify the ITH level for a tumor. To prove our hypothesis, we developed a deep learning-based computational framework using unsupervised Concrete Autoencoder (CAE) to select a set of cancer-specific key genes that can be used to evaluate the ITH score. For experiment, we used gene expression profile data of tumor cohorts of breast, kidney and lung cancer from TCGA repository. We selected three sets of key genes, each set related to breast, kidney, and lung tumor cohorts, using multi-run CAE. For the three cancers stated and three molecular subtypes of lung cancer, we calculated ITH level using all genes and key genes selected by CAE and performed a side-by-side comparison. It was found that similar conclusions can be reached for survival and prognostic outcomes based on ITH scores derived from all genes and the sets of key genes. Additionally, for subtypes of lung cancer, the comparative distribution of ITH scores derived from all genes and key genes remains similar. Based on these observations, it can be stated that, a subset of key genes, instead of all genes, is sufficient for ITH quantification. Our results also showed that many of the key genes are prognostically significant, which can be used as possible therapeutic targets.

sentences: ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by Molecular Dynamics simulations which also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

sentences: Utilizing proteomics and phosphoproteomics to predict ex vivo drug sensitivity across genetically diverse AML patients Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemia. This genetic heterogeneity is difficult to treat using conventional therapies that are generally based on the detection of a single driving mutation. Thus, the use of molecular signatures, consisting of multiple functionally related transcripts or proteins, in making treatment decisions may overcome this hurdle and provide a more effective way to inform drug treatment protocols. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify signatures that could predict patient-specific drug responses. The Clinical Proteomic Tumor Analysis Consortium is in the process of extending this cohort to collect proteomic and phosphoproteomic measurements from a subset of these patient samples to evaluate the hypothesis that proteomic signatures can robustly predict drug response in AML patients. We sought to examine this hypothesis on a sub-cohort of 38 patient samples from Beat AML with proteomic and drug response data and evaluate our ability to identify proteomic signatures that predict drug response with high accuracy. For this initial analysis we built predictive models of patient drug responses across 26 drugs of interest using the proteomics and phosphproteomics data. We found that proteomics-derived signatures provide an accurate and robust signature of drug response in the AML ex vivo samples, as well as related cell lines, with better performance than those signatures derived from mutations or mRNA expression. Furthermore, we found that in specific drug-resistant cell lines, the proteins in our prognostic signatures represented dysregulated signaling pathways compared to parental cell lines, confirming the role of the proteins in the signatures in drug resistance. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to predict drug sensitivity in AML.

sentences: Rnf149-related is an FGF/MAPK-independent regulator of pharyngeal muscle fate specification During embryonic development, cell fate specification gives rise to dedicated lineages that underlie tissue formation. In olfactores, which comprise tunicates and vertebrates, the cardiopharyngeal field is formed by multipotent progenitors to both cardiac and branchiomeric muscles. The ascidian Ciona is a powerful model to study the cardiopharyngeal fate specification with cellular resolution, as only 2 pairs of cardiopharyngeal multipotent progenitors give rise to the heart and to pharyngeal muscles (aka atrial siphon muscles, ASM). These progenitors are multilineage primed, in as much as they express a combination of early ASM- and heart-specific transcripts that become restricted to their corresponding precursors, following oriented asymmetric divisions. Here, we identify the primed gene Rnf149-related (Rnf149-r), which becomes restricted to the heart progenitors, but appears to regulate pharyngeal muscle fate specification in the cardiopharyngeal lineage. CRISPR/Cas9-mediated loss knock-out of Rnf149-r function impairs atrial siphon muscle morphogenesis, and down-regulates Tbx1/10 and Ebf, two key determinants of the pharyngeal muscle fate, while upregulating heart-specific gene expression. These phenotypes are reminiscent of loss of FGF-MAPK signaling in the cardiopharyngeal lineage, and integrated analysis of lineage-specific bulk RNA-seq profiling of loss-of-function perturbations identified a significant overlap between FGF-MAPK and Rnf149-r targets. However, functional interaction assays suggested the Rnf149-r does not directly modulate the activity of the FGF-MAPK-Ets1/2 pathway. Instead, we propose that Rnf149-r acts both in parallel to the FGF-MAPK signaling on shared targets, as well as on FGF-MAPK-independent targets through (a) separate pathway(s).

sentences: Simultaneous inference of parental admixture proportions and admixture times from unphased local ancestry calls Population genetic analyses of local ancestry tracts routinely assume that the ancestral admixture process is identical for both parents of an individual, an assumption that may be invalid when considering recent admixture. Here we present Parental Admixture Proportion Inference (PAPI), a Bayesian tool for inferring the admixture proportions and admixture times for each parent of a single admixed individual. PAPI analyzes unphased local ancestry tracts and has two components models: a binomial model that exploits the informativeness of homozygous ancestry regions to infer parental admixture proportions, and a hidden Markov model (HMM) that infers admixture times from tract lengths. Crucially, the HMM employs an approximation to the pedigree crossover dynamics that accounts for unobserved within-ancestry recombination, enabling inference of parental admixture times. We compared the accuracy of PAPIs admixture proportion estimates with those of ANCESTOR in simulated admixed individuals and found that PAPI outperforms ANCESTOR by an average of 46% in a representative set of simulation scenarios, with PAPIs estimates deviating from the ground truth by 0.047 on average. Moreover, PAPIs admixture time estimates were strongly correlated with the ground truth in these simulations (R = 0.76), but have an average downward bias of 1.01 generations that is partly attributable to inaccuracies in local ancestry inference. As an illustration of its utility, we ran PAPI on real African Americans from the PAGE study (N = 5, 786) and found strong evidence of assortative mating by ancestry proportion: couples ancestry proportions are closer to each other than expected by chance (P < 10-6), and are highly correlated (R = 0.87). We anticipate that PAPI will be useful in studying the population dynamics of admixture and will also be of interest to individuals seeking to learn about their personal genealogies.

sentences: DNA double-strand break end synapsis by DNA loop extrusion DNA double-strand breaks (DSBs) occur every cell cycle and must be efficiently repaired. Non-homologous end joining (NHEJ) is the dominant pathway for DSB repair in G1-phase. The first step of NHEJ is to bring the two DSB ends back into proximity (synapsis). However, although synapsis is generally assumed to occur through passive diffusion, we show here that passive diffusion is unlikely to be consistent with the speed and efficiency of NHEJ observed in cells. Instead, we hypothesize that DNA loop extrusion facilitates synapsis. By combining experimentally constrained simulations and theory, we show that the simplest loop extrusion model only modestly facilitates synapsis. Instead, a loop extrusion model with targeted loading of loop extruding factors (LEFs), a small portion of long-lived LEFs as well as LEF stabilization by boundary elements and DSB ends achieves fast synapsis with near 100% efficiency. We propose that loop extrusion plays an underappreciated role in DSB repair.

sentences: A genome assembly of the Atlantic chub mackerel (Scomber colias): a valuable teleost fishing resource The Atlantic chub mackerel, Scomber colias Gmelin, 1789, is a medium-size pelagic fish with substantial importance in the fisheries of the Atlantic Ocean and the Mediterranean Sea. Over the past decade, this species has gained special relevance being one of the main targets of pelagic fisheries in the NE Atlantic. Here, we sequenced and annotated the first high-quality draft genome assembly of S. colias, produced with Pacbio HiFi long reads and Illumina Paired-End short reads. The estimated genome size is 814 Mb distributed into 2,028 scaffolds and 2,093 contigs with an N50 length of 4,19 and 3,34 Mb, respectively. We annotated 27,675 protein-coding genes and the BUSCO analyses indicated high completeness, with 97.3 % of the single-copy orthologs in the Actinopterygii library profile. The present genome assembly represents a valuable resource to address the biology and management of this relevant fishery. Finally, this is the fourth high-quality genome assembly within the Order Scombriformes and the first in the genus Scomber.

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UbE3-APA: A Bioinformatic Strategy to Elucidate Ubiquitin E3 Ligase Activities in Quantitative Proteomics Study MotivationUbiquitination is widely involved in protein homeostasis and cell signaling. Ubiquitin E3 ligases are critical regulators of ubiquitination that recognize and recruit specific ubiquitination targets for the final rate-limiting step of ubiquitin transfer reactions. Understanding the ubiquitin E3 ligase ac-tivities will provide knowledge in the upstream regulator of the ubiquitination pathway and reveal po-tential mechanisms in biological processes and disease progression. Recent advances in mass spec-trometry-based proteomics have enabled deep profiling of ubiquitylome in a quantitative manner. Yet, functional analysis of ubiquitylome dynamics and pathway activity remains challenging.

ResultsHere, we developed a UbE3-APA, a computational algorithm and stand-alone python-based software for Ub E3 ligase Activity Profiling Analysis. Combining an integrated annotation database with statistical analysis, UbE3-APA identifies significantly activated or suppressed E3 ligases based on quantitative ubiquitylome proteomics datasets. Benchmarking the software with published quantitative ubiquitylome analysis confirms the genetic manipulation of SPOP enzyme activity through overexpres-sion and mutation. Application of the algorithm in the re-analysis of a large cohort of ubiquitination proteomics study revealed the activation of PARKIN and the co-activation of other E3 ligases in mito-chondria depolarization-induced mitophagy process. We further demonstrated the application of the algorithm in the DIA-based quantitative ubiquitylome analysis.

AvailabilitySource code and binaries are freely available for download at URL: https://github.com/Chenlab-UMN/Ub-E3-ligase-Activity-Profiling-Analysis, implemented in python and supported on Linux and MS Windows

Contactyuechen@umn.edu

Supplementary informationSupplementary data are available.

sentences: Chronic sodium bromide treatment relieves autistic-like behavioral deficits in three mouse models of autism Autism Spectrum Disorders (ASD) are neurodevelopmental disorders whose diagnosis relies on deficient social interaction and communication together with repetitive behavior. To date, no pharmacological treatment has been approved that ameliorates social behavior in patients with ASD. Based on the excitation/inhibition imbalance theory of autism, we hypothesized that bromide ions, long used as an antiepileptic medication, could relieve core symptoms of ASD. We evaluated the effects of chronic sodium bromide (NaBr) administration on autistic-like symptoms in three genetic mouse models of autism: Oprm1-/- , Fmr1-/- and Shank3{Delta}ex13-16-/- mice. We showed that chronic NaBr treatment relieved autistic-like behaviors in these three models. In Oprm1-/- mice, these beneficial effects were superior to those of chronic bumetanide administration. At transcriptional level, chronic NaBr in Oprm1 null mice was associated with increased expression of genes coding for chloride ions transporters, GABAA receptor subunits, oxytocin and mGlu4 receptor. Lastly, we uncovered synergistic alleviating effects of chronic NaBr and a positive allosteric modulator (PAM) of mGlu4 receptor on autistic-like behavior in Oprm1-/- mice. We evidenced in heterologous cells that bromide ions behave as PAMs of mGlu4, providing a molecular mechanism for such synergy. Our data reveal the therapeutic potential of bromide ions, alone or in combination with a PAM of mGlu4 receptor, for the treatment of ASDs.

sentences: The "Alu-ome" shapes the epigenetic environment of regulatory elements controlling cellular defense Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of this intervals greatly varies in-between tissues, being longer in stem-and shorter in immune-cells. The very shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with RNA polymerase II transcription through the first Alu and accumulation of H3K4me1 signal on this first Alu. Finally, DNA methylation at first-Alus was found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes.

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Identification of candidate genes for self-compatibility in perennial ryegrass (Lolium perenne L.) Self-incompatibility (SI) is a genetic mechanism preventing self-pollination in approximately 40% of plant species. Two multiallelic loci, called S and Z, control the gametophytic SI system of the grass family (Poaceae), which contains all major forage grasses. Loci independent from S and Z have been reported to disrupt SI and lead to self-compatibility (SC). A locus causing SC in perennial ryegrass (Lolium perenne L.) was previously mapped on linkage group (LG) 5 in a F2 population segregating for SC. Using a subset of the same population (n=68), we first performed low-resolution quantitative trait locus (QTL) mapping to exclude the presence of additional, previously undetected contributors to SC. The previously reported QTL on LG 5 explained 38.4% of the phenotypic variation, and no significant contribution from other genomic regions was found. This was verified by the presence of significantly distorted markers in the region overlapping with the QTL. Second, we fine mapped the QTL to 0.26 cM using additional 2,056 plants and 23 novel sequence-based markers. Using an Italian ryegrass (Lolium multiflorum Lam.) genome assembly as reference, the markers flanking SC were estimated to span a ~3 Mb region encoding for 57 predicted genes. Among these, seven genes were proposed as relevant candidate genes based on their annotation and function described in previous studies. Our work is a step forward to identify SC genes in forage grasses and provides diagnostic markers for marker-assisted introgression of SC into elite germplasm.

Key messageA previously reported QTL conferring self-compatibility to perennial ryegrass was fine mapped to a 0.26 cM locus, corresponding to an estimated ~ 3 Mb physical region containing 57 predicted protein-encoding genes.

sentences: A QuantCrit investigation of society's educational debts due to racism, sexism, and classism in biology student learning We investigated the intersectional relationships between racism, sexism, and classism in inequities in student conceptual knowledge in introductory biology courses using a quantitative critical framework. Using Bayesian hierarchical linear models, we examined students conceptual knowledge as measured by the Introductory Molecular and Cell Biology Assessment. The data came from the LASSO database and included 6,547 students from 87 introductory courses at 11 institutions. The model indicated that students with marginalized identities by race, gender, and class tended to start with lower scores than continuing-generation, White men. We conceptualized these differences as educational debts society owed these students due to racism, sexism, and classism. Instruction added to these educational debts for most marginalized groups, with the largest increases for students with multiple marginalized identities. After instruction, society owed Black and Hispanic, first-generation women an educational debt equal to 60-80% of the average learning in the courses. These courses almost all (85/87) used collaborative learning and half (45/87) supported instruction with learning assistants. While research shows collaborative learning better serves students than lecture-based instruction, these results indicate it does not repay educational debts due to racism, sexism, and classism.

sentences: ID1 and CEBPA Coordinate Epidermal Progenitor Cell Differentiation The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here we report that the transcriptional regulator ID1 is enriched in basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and established TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1 and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.

sentences: A versatile active learning workflow for optimization of genetic and metabolic networks The study, engineering and application of biological networks require practical and efficient approaches. Current optimization efforts of these systems are often limited by wet lab labor and cost, as well as the lack of convenient, easily adoptable computational tools. Aimed at democratization and standardization, we describe METIS, a modular and versatile active machine learning workflow with a simple online interface for the optimization of biological target functions with minimal experimental datasets. We demonstrate our workflow for various applications, from simple to complex gene circuits and metabolic networks, including several cell-free transcription and translation systems, a LacI-based multi-level controller and a 27-variable synthetic CO2-fixation cycle (CETCH cycle). Using METIS, we could improve above systems between one and two orders of magnitude compared to their original setup with minimal experimental efforts. For the CETCH cycle, we explored the combinatorial space of [~]1025 conditions with only 1,000 experiments to yield the most efficient CO2-fixation cascade described to date. Beyond optimization, our workflow also quantifies the relative importance of individual factors to the performance of a system. This allows to identify so far unknown interactions and bottlenecks in complex systems, which paves the way for their hypothesis-driven improvement, which we demonstrate for the LacI multi-level controller that we were able to improve by 34-fold after having identified resource competition as limiting factor. Overall, our workflow opens the way for convenient optimization and prototyping of genetic and metabolic networks with customizable adjustments according to user experience, experimental setup, and laboratory facilities.

sentences: Downstream Effects: Impact of Antibiotic Pollution on Aquatic Host-Parasite Interactions The global increase in antibiotic use has led to contamination of freshwater environments occupied by parasites and their hosts. Despite the identified impacts of antibiotics on humans and wildlife, the effect of antibiotics on host-parasite life cycles is relatively unexplored. We utilize the trematode parasite Schistosoma mansoni, and its snail intermediate host Biomphalaria glabrata to investigate the influence of an ecologically relevant antibiotic concentration on the life history characteristics of both parasite and host. Our results demonstrate that antibiotics not only accelerate parasite development time, but also increase host reproduction and delay parasite-induced host castration. We propose that antibiotic exposure alters host microbiome composition, leading to increased host susceptibility and higher parasite production. Using a mathematical model, we suggest that life history alterations associated with antibiotics are likely to increase parasite transmission and disease burden. Our study suggests that antibiotic pollution could impact freshwater ecosystems by influencing host-parasite dynamics and potentially increase the burden of schistosomiasis in endemic regions.

sentences: Territory-level temperature influences settlement decisions, breeding phenology and productivity in three forest passerine birds Temperature plays an important role in determining breeding phenology of temperate birds, with higher spring temperatures associated with earlier breeding. However, the effect of localised territory-scale temperature variations is poorly understood, with relationships between temperature and breeding phenology mostly studied using coarse-grained climatic indices. Here, we interpolate spring temperatures recorded at 150 m2 grid intersections encompassing 417 ha of forest to examine the influence of territory-scale temperature, and its interaction with mean annual temperature, on territory selection, breeding phenology, clutch size and fledgling success for three co-occurring single-brooded passerine birds using data from 672 nests over four years. All species exhibited significant trends in reproductive traits associated with territory-scale temperature. Pied flycatchers Ficedula hypoleuca settled in cooler territories first, where they raised more fledglings. Blue tits Cyanistes caeruleus laid larger clutches in warmer territories in warm years and always laid earlier at warmer territories irrespective of annual temperature variation. Contrastingly, pied flycatcher and wood warbler Phylloscopus sibilatrix breeding phenology was earlier at warmer territories in cool years and cooler territories in warm years, with wood warbler clutch size responding similarly to this interaction. Greater previous breeding experience and increased higher rates of historical territory occupancy (territory quality) also predicted earlier breeding phenology and higher fledging success for pied flycatchers. We suggest that the migratory pied flycatcher and wood warbler are best synchronised with their prey availability in cooler years at a local population level while resident blue tits match local phenology across all years, which is potentially advantageous under warmer predicted climate change scenarios. We conclude that temperature at the territory scale can be an important driver of settlement and breeding phenology and influence reproductive traits.

sentences: A catalogue of omics biological ageing clocks reveals substantial commonality and associations with disease risk Biological age (BA), a measure of functional capacity and prognostic of health outcomes that discriminates between individuals of the same chronological age (chronAge), has been estimated using a variety of biomarkers. Previous comparative studies have mainly used epigenetic models (clocks), we use ~1000 participants to create eleven omics ageing clocks, with correlations of 0.45-0.97 with chronAge, even with substantial sub-setting of biomarkers. These clocks track common aspects of ageing with 94% of the variance in chronAge being shared among clocks. The difference between BA and chronAge - omics clock age acceleration (OCAA) - often associates with health measures. One years OCAA typically has the same effect on risk factors/10-year disease incidence as 0.46/0.45 years of chronAge. Epigenetic and IgG glycomics clocks appeared to track generalised ageing while others capture specific risks. We conclude BA is measurable and prognostic and that future work should prioritise health outcomes over chronAge.

sentences: Activated MST2 kinase is free of kinetic regulation Canonically, MST1/2 functions as a core kinase of the Hippo pathway and non-canonically is both activated during apoptotic signaling and acts in concert with RASSFs in T-cells. Faithful signal transduction relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made understanding the molecular mechanisms regulating activation of MST1/2 and identifying downstream signaling events. Here we present a kinetic analysis analyzing how the ability of MST1/2 to phosphorylate substrates is regulated. Using a steady state kinetic system, we parse the contribution of different factors including the domains of MST2, phosphorylation, caspase cleavage, and complex formation to MST2 activity. In the unphosphorylated state, we find the SARAH domain stabilizes substrate binding. Phosphorylation, we also determine, drives activation of MST2 and that once activated the kinase domain is free of regulation. The binding partners SAV1, MOB1A, and RASSF5 do not alter the kinetics of phosphorylated MST2. We also show that the caspase cleaved MST2 fragment is as active as full-length suggesting that the linker region of MST2 does not inhibit the catalytic activity of the kinase domain but instead regulates MST2 activity through non-catalytic mechanisms. This kinetic analysis helps establish a framework for interpreting how signaling events, mutations, and post-translational modifications contribute to signaling of MST2 in vivo.

sentences: Introduction of a condensed, reverse tricarboxylic acid cycle for additional CO2 fixation in plants Plants employ the Calvin-Benson cycle (CBC) to fix atmospheric CO2 for the production of biomass. The flux of carbon through the CBC is limited by the activity and selectivity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Alternative pathways that do not use RuBisCO to fix CO2 exist but occur only in anaerobic microorganisms. Rather than modifying existing routes of carbon metabolism in plants, we have developed a synthetic carbon fixation cycle that does not exist in nature, but is inspired by metabolisms of bacterial autotrophs. This synthetic cycle uses endogenous plant metabolites to fix CO2 and yield glyoxylate as a product. In this work, we build and characterize a condensed, reverse tricarboxylic acid (crTCA) cycle in vitro and in planta. We demonstrate that a simple, synthetic cycle can be used to fix carbon in vitro under aerobic and mesophilic conditions and that these enzymes retain activity when expressed transiently in planta. We then evaluate stable transgenic lines of Camelina sativa that have both phenotypic and physiologic changes. Transgenic C. sativa are shorter than controls with increased rates of photosynthetic CO2 assimilation and changes in photorespiratory metabolism. This first iteration of a build-test-learn phase of the crTCA cycle provides promising evidence that this pathway can be used to increase photosynthetic capacity in plants.

sentences: Overlapping influences shape motor activity during hasty sensorimotor decisions Humans and other animals are able to adjust their speed-accuracy tradeoff (SAT) at will depending on the urge to act, favoring either cautious or hasty decision policies in different contexts. An emerging view is that SAT regulation relies on influences exerting broad changes on the motor system, tuning its activity up globally when hastiness is at premium. The present study aimed to test this hypothesis. Fifty subjects performed a task involving choices between left and right index fingers, in which incorrect choices led either to a high or to a low penalty in two contexts, inciting them to emphasize either cautious or hasty policies. We applied transcranial magnetic stimulation on multiple motor representations, eliciting motor evoked potentials (MEP) in nine finger and leg muscles. MEP amplitudes allowed us to probe activity changes in the corresponding finger and leg representations, while subjects were deliberating about which index to choose. Our data indicate that hastiness entails a broad amplification of motor activity, though this amplification was limited to the chosen side. On top of this effect, we identified a local suppression of motor activity, surrounding the chosen index representation. Hence, a decision policy favoring speed over accuracy appears to rely on overlapping processes producing a broad (but not global) amplification and a surround suppression of motor activity. The latter effect may help to increase the signal-to-noise ratio of the chosen representation, as supported by single-trial correlation analyses indicating a stronger differentiation of activity changes in finger representations in the hasty context.

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Evaluation of BOLD effects in the rat cortex PurposeThis study aimed to characterize Blood oxygen level-dependent (BOLD) effects in 1H-MR spectra obtained during optogenetic activation of the rat forelimb cortex for the correction and estimation of accurate metabolite concentration changes.

MethodsT2*-induced effects were characterized by linewidth changes and amplitude changes of water, NAA and tCr spectral peaks during the stimulation paradigm. Spectral linewidth-matching procedures were used to correct for the line-narrowing effect induced by BOLD. For an increased understanding of spectroscopic BOLD effects and the optimized way to correct them, a 1 Hz line-narrowing effect was also simulated on mouseproton MR spectrum

 1H-fMRS data acquired using STEAM acquisitions at 9.4T in rats (n=8) upon optogenetic stimulation of the primary somatosensory cortex were used. Data were analyzed with MATLAB routines and LCModel. Uncorrected and corrected 1H-MR spectra of simulated and in-vivo data were quantified and compared. BOLD-corrected difference spectra were also calculated and analyzed.

ResultsSignificant mean increases in water and NAA peak heights (+ 1.1% and +4.5%, respectively) were found accompanied by decreased linewidths (-0.5 Hz and -2.8%) upon optogenetic stimulation. These estimates were used for further definition of an accurate line-broadening factor (lb). Usage of an erroneous lb introduced false-positive errors in metabolite concentration change estimates thereby altering the specificity of findings. Using different water scalings within LCModel, the water and metabolite BOLD contributions were separated.

ConclusionThe linewidth-matching procedure using a precise lb factor remains the most performant approach for the accurate quantification of small ({+/-}0.3 mol/g) metabolic changes in 1H-fMRS studies. A simple and preliminary compartmentation of BOLD effects was proposed, which will require validation.

sentences: Topological morphogenesis of neuroepithelial organoids Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function. However, how topology emerges during organ morphogenesis remains elusive. Here, we combine tissue reconstitution and quantitative microscopy to show that trans and cis epithelial fusion govern tissue topology and shape. These two modes of topological transitions can be regulated in neuroepithelial organoids, leading to divergent topologies. The morphological space can be captured by a single control parameter which is analogous to the reduced Gaussian rigidity of an epithelial surface. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of trans and cis fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues.

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Neural activity in the anterior cingulate cortex is required for effort-based decision making Adaptive decision making requires the evaluation of cost-benefit tradeoffs to guide action selection. Effort-based decision making involves weighing predicted gains against effort costs and is disrupted in several neuropsychiatric disorders. The ACC is postulated to control effort-base choice via its role in encoding the value of overcoming effort costs in rodent effort-based decision making assays. However, temporally precise methods of manipulating neural activity have rarely been applied to effort-based decision making. We developed and validated a mouse version of the barrier T-maze, and used optogenetics to inhibit ACC excitatory neurons at specific times during this task. Bilateral inhibition of ACC rapidly and reversibly impaired preference to exert greater effort for a larger reward when a less rewarded, low effort alternative was available. Equalizing effort for potential choice options led mice to choose the high reward arm of the maze regardless of whether the ACC was inhibited or not. The mechanics of choice behavior were altered during trials where the ACC was inhibited, but there were no effects on overall mobility or tendency to exert effort in an unrelated assay. These findings establish causality between ACC neural activity during a choice trial and effortful action selection during cost-benefit decision making.

SIGNIFICANCE STATEMENTDisturbances in evaluating effort-based costs during decision making occur in depression, schizophrenia, addiction and Parkinsons disease. Precisely resolving the function of prefrontal brain regions in mediating these processes will reveal key loci of dysfunction and potential therapeutic intervention in these disorders.

sentences: Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell senses the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP- dependent mechanism by which the stem/progenitor cells recognize the ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP- induced stem cell expansion strategy.

sentences: Optogenetic Manipulation of Cell Migration with High Spatiotemporal Resolution Using Lattice Lightsheet Microscopy Lattice lightsheet microscopy (LLSM) is modified with the aim of manipulating cellular behavior with subcellular resolution through three-dimensional (3D) optogenetic activation. In this study, we report a straightforward implementation of the activation source in LLSM in which the stimulating light can be generated by changing the spatial light modulator (SLM) patterns and the annual masks. As a result, a Bessel beam as a stimulation source is integrated into the LLSM without changing the optical configuration, achieving better spatiotemporal control of photoactivation. We show that the energy power required for optogenetic reactions is lower than 1 nW (or 24 mW/cm2), and membrane ruffling can be activated at different locations within a cell with subcellular resolution. We also demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable damage to cells.

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Genomic patterns of divergence in the early and late steps of speciation of the deep-sea vent thermophilic worms of the genus Alvinella. BackgroundThe transient and fragmented nature of the deep-sea hydrothermal environment made of ridge subduction, plate collision and the emergence of new rifts is currently acting to the separation of vent populations, local adaptation and is likely to promote bursts of speciation and species specialization. The tube-dwelling worms Alvinella pompejana called the Pompeii worm and its sister species A. caudata live syntopically on the hottest part of deep-sea hydrothermal chimneys along the East Pacific Rise. They are exposed to extreme thermal and chemical gradients, which vary greatly in space and time, and thus represent ideal candidates for understanding the evolutionary mechanisms at play in the vent fauna evolution.

ResultsIn the present study, we explored genomic patterns of divergence in the early and late steps of speciation of these emblematic worms using transcriptome assemblies and the first draft genome to better understand the relative role of geographic isolation and habitat preference in their genome evolution. Analyses were conducted on allopatric populations of Alvinella pompejana (early stage of separation) and between A. pompejana and its syntopic species Alvinella caudata (late stage of speciation). We first identified divergent genomic regions and targets of selection as well as their position in the genome over collections of orthologous genes and, then, described the speciation dynamics by documenting the annotation of the most divergent and/or positively selected genes involved in the isolation process. Gene mapping clearly indicated that divergent genes associated with the early stage of speciation, although accounting for nearly 30% of genes, are highly scattered in the genome without any island of divergence and not involved in gamete recognition or mito-nuclear incompatibilities. By contrast, genomes of A. pompejana and A. caudata are clearly separated with nearly all genes (96%) exhibiting high divergence. This congealing effect however seems to be linked to habitat specialization and still allows positive selection on genes involved in gamete recognition, as a possible long-duration process of species reinforcement.

ConclusionOur analyses pointed out the non-negligible role of natural selection on both the early and late stages of speciation in the emblematic thermophilic worms living on the walls of deep-sea hydrothermal chimneys. They shed ligth on the evolution of gene divergence during the process of speciation and species specialization over a very long period of time.

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Rapid identification of mosquito species, sex and age by mass spectrometric analysis BackgroundA rapid, accurate method to identify and to age grade mosquito populations would be a major advance in predicting the risk of pathogen transmission and evaluating the public health impact of vector control interventions. Whilst other chemometric methods show promise, current approaches rely on challenging morphological techniques or simple binary classifications that cannot identify the subset of the population old enough to be infectious. In this study, the ability of Rapid Evaporative Ionisation Mass Spectrometry (REIMS) to identify the species, sex and age of mosquitoes reared in the laboratory and derived from the wild was investigated.

ResultsThe accuracy of REIMS in identifying morphologically identical species of the Anopheles gambiae complex exceeded 97 % using principal component/linear discriminant analysis (PC-LDA) and 84 % based on random forest analysis. Age separation into 3 different age categories (1 d, 5-6 d, 14-15 d.) was achieved with 99 % (PC-LDA) and 94 % (random forest) accuracy, after including three species and storing samples at room temperature for up to 11 days. When tested on wild mosquitoes from the UK, REIMS data could determine the species and age of the specimens with accuracies of 91 and 90 % respectively.

ConclusionsThe accuracy of REIMS to resolve the species and age of Anopheles mosquitoes is comparable to that achieved by chemometric approaches. The processing time and ease of use represent significant advantages over current, dissection-based methods. Importantly the accuracy was maintained when using wild mosquitoes reared under differing environmental conditions, and when mosquitoes were stored frozen or desiccated. This high throughput approach thus has potential to conduct rapid, real- time monitoring of vector populations, providing entomological evidence of the impact of alternative interventions.

sentences: Genome size drives morphological evolution in organ-specific ways Morphogenesis is an emergent property of biochemical and cellular interactions during development. Genome size and the correlated trait of cell size can influence these interactions through its effects on developmental rate and tissue geometry, ultimately driving the evolution of morphology. We tested the effects of genome size and body size evolution on heart and liver morphology using nine species of the salamander genus Plethodon (genome sizes 29.3-67 Gb). Our results show that whole organ size is determined by body size, whereas tissue structure changes dramatically with evolutionary increases in genome size. In the heart, increased genome size is correlated with a reduction of myocardia in the ventricle, yielding proportionally less force-producing mass and more empty space. In the liver, increased genome size is correlated with fewer and larger vascular structures, positioning hepatocytes farther from the circulatory vessels that transport key metabolites. Although these structural changes should have obvious impacts on organ function, their effects on organismal performance and fitness are likely negligible because low metabolic rates in salamanders relax selective pressure on key metabolic organ performance. Overall, this study reveals the effects of large genome and cell size on the developmental systems producing the heart and liver.

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In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras Generation of bone marrow (BM) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce all BM hematopoietic cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into hematopoietic progenitor cells and multiple hematopoietic cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and resulted in long-term contribution of donor cells to hematopoietic cell lineages. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as "bioreactors".

KEY POINTS- We used blastocyst complementation to simultaneously produce all bone marrow hematopoietic cell lineages from mouse ESCs in a rat.

- ESC-derived cells from mouse-rat chimeras were fully functional and exhibited normal gene expression signatures and cell surface markers.

sentences: Depression-like state induced by low-frequency repetitive transcranial magnetic stimulation to ventral medial frontal cortex in monkeys The medial frontal cortex (MFC), especially its ventral part, has long been of great interest with respect to the pathology of mood disorders. A number of human brain imaging studies have demonstrated the abnormalities of this brain region in patients with mood disorders, however, whether it is critically involved in the pathogenesis of such disorders remains to be fully elucidated. In this study, we conducted a causal study to investigate how the suppression of neural activity in the ventral region of the MFC (vMFC) affects the behavioral and physiological states of monkeys by using repetitive transcranial magnetic stimulation (rTMS). By using low-frequency rTMS (LF-rTMS) as an inhibitory intervention, we found that LF-rTMS targeting the vMFC induced a depression-like state in monkeys, which was characterized by a reduced spontaneous behavioral activity, increased plasma cortisol level, impaired sociability, and decreased motivation level. On the other hand, no such significant changes in behavioral and physiological states were observed when targeting the other MFC regions, dorsal or posterior. We further found that the administration of an antidepressant agent, ketamine, ameliorated the abnormal behavioral and physiological states induced by the LF-rTMS intervention. These findings indicate the causal involvement of the vMFC in the regulation of mood and affect and the validity of the LF-rTMS-induced dysfunction of the vMFC as a nonhuman primate model of the depression-like state.

sentences: Temporal-spectral signaling of sensory information and expectations in the cerebral processing of pain The perception of pain is shaped by somatosensory information about threat. However, pain is also influenced by an individuals expectations. Such expectations can result in clinically relevant modulations and abnormalities of pain. In the brain, sensory information, expectations (predictions), and discrepancies thereof (prediction errors) are signaled by an extended network of brain areas. These brain areas generate evoked potentials and oscillatory responses at different latencies and frequencies. Recent evidence has provided first insights into how oscillatory responses at different frequencies signal predictions and prediction errors. However, a comprehensive picture of how evoked and oscillatory brain responses signal sensory information, predictions, and prediction errors in the processing of pain is lacking so far. We therefore built upon a neuroimaging study which investigated the spatial signalling of sensory information, predictions and predictions errors in the processing of pain (Geuter et al., 2017). To complement and extend this study, we applied brief painful stimuli to 48 healthy human participants and independently modulated sensory information (stimulus intensity) and expectations of pain intensity while measuring brain activity using electroencephalography (EEG). Pain ratings confirmed that pain intensity was shaped by both sensory information and expectations. In contrast, Bayesian analyses revealed that stimulus-induced EEG responses at different latencies (the N1, N2, and P2 components) and frequencies (alpha, beta, and gamma oscillations) were shaped by sensory information but not by expectations. Expectations, however, shaped alpha and beta oscillations before the painful stimuli. These findings indicate that commonly analyzed EEG responses to painful stimuli are more involved in signaling sensory information than in signaling expectations or mismatches of sensory information and expectations. Moreover, they indicate that the effects of expectations on pain are served by brain mechanisms which differ from those conveying effects of sensory information on pain.

sentences: Genome-wide screen identifies curli amyloid fibril as a bacterial component promoting host neurodegeneration Growing evidence indicate that gut microbiota play a critical role in regulating the progression of neurodegenerative diseases, such as Parkinsons disease (PD). The molecular mechanism underlying such microbe-host interaction is unclear. In this study, by feeding C. elegans expressing human -syn with E. coli knockout mutants, we conducted a genome-wide screen to identify bacterial genes that promote host neurodegeneration. The screen yielded 38 genes that fall into several genetic pathways, including curli formation, lipopolysaccharide assembly, adenosylcobalamin biosynthesis among others. We then focused on the curli amyloid fibril and found that genetically deleting or pharmacologically inhibiting the curli major subunit CsgA in E. coli reduced -syn-induced neuronal death, restored mitochondrial health, and improved neuronal functions. CsgA secreted by the bacteria colocalized with -syn inside neurons and promoted -syn aggregation through cross-seeding. Similarly, curli also promoted neurodegeneration in C. elegans models of AD, ALS, and HD and in human neuroblastoma cells.

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DeepHBV: A deep learning model to predict hepatitis B virus (HBV) integration sites. Hepatitis B virus (HBV) is one of the main causes for viral hepatitis and liver cancer. Previous studies showed HBV can integrate into host genome and further promote malignant transformation. In this study, we developed an attention-based deep learning model DeepHBV to predict HBV integration sites by learning local genomic features automatically. We trained and tested DeepHBV using the HBV integration sites data from dsVIS database. Initially, DeepHBV showed AUROC of 0.6363 and AUPR of 0.5471 on the dataset. Adding repeat peaks and TCGA Pan Cancer peaks can significantly improve the model performance, with an AUROC of 0.8378 and 0.9430 and an AUPR of 0.7535 and 0.9310, respectively. On independent validation dataset of HBV integration sites from VISDB, DeepHBV with HBV integration sequences plus TCGA Pan Cancer (AUROC of 0.7603 and AUPR of 0.6189) performed better than HBV integration sequences plus repeat peaks (AUROC of 0.6657 and AUPR of 0.5737). Next, we found the transcriptional factor binding sites (TFBS) were significantly enriched near genomic positions that were paid attention to by convolution neural network. The binding sites of AR-halfsite, Arnt, Atf1, bHLHE40, bHLHE41, BMAL1, CLOCK, c-Myc, COUP-TFII, E2A, EBF1, Erra and Foxo3 were highlighted by DeepHBV attention mechanism in both dsVIS dataset and VISDB dataset, revealing the HBV integration preference. In summary, DeepHBV is a robust and explainable deep learning model not only for the prediction of HBV integration sites but also for further mechanism study of HBV induced cancer.

Author summaryHepatitis B virus (HBV) is one of the main causes for viral hepatitis and liver cancer. Previous studies showed HBV can integrate into host genome and further promote malignant transformation. In this study, we developed an attention-based deep learning model DeepHBV to predict HBV integration sites by learning local genomic features automatically. The performance of DeepHBV model significantly improves after adding genomic features, with an AUROC of 0.9430 and an AUPR of 0.9310. Furthermore, we enriched the transcriptional factor binding sites of proteins by convolution neural network. In summary, DeepHBV is a robust and explainable deep learning model not only for the prediction of HBV integration sites but also for the further study of HBV integration mechanism.

sentences: Met-signaling controls Dendritic Cell migration by regulating podosome formation and function Signaling by the HGF receptor/Met in skin-resident Langerhans cells (LC) and dermal dendritic cells (dDC) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. Here we addressed the role of Met-signaling in distinct steps of LC/dDC emigration from skin by employing a conditional Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DC and concomitantly decreased proteolytic degradation of gelatin. Accordingly, Met-deficient LC failed to efficiently cross the extracellular matrix (ECM) rich basement membrane between epidermis and dermis. We further observed that Met-signaling by HGF reduced adhesion of bone marrow-derived LC to various ECM factors and enhanced the motility of Met-signaling competent DC in a 3D collagen matrix, which was not the case for Met-deficient LC/DC. We found no impact of Met-signaling on the integrin-independent amoeboid migration of DC in response to the CCR7 chemokine CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

sentences: Macrophage-derived VEGF-C decreases hematogenous metastatic dissemination by normalizing the tumor vasculature AO_SCPLOWBSTRACTC_SCPLOWExpression levels of the pro-lymphangiogenic vascular endothelial growth factor (VEGF)-C in the primary tumor correlates to the occurrence of proximal lymph node metastasis for most solid cancers including those of the breast. However, the role of VEGF-C in regulating tumor cell dissemination to distant organs such as the lungs, is currently unclear. By using an experimental tumor model of breast cancer (BC), we show that perivascular tumor-associated macrophages (TAMs), key regulators of hematogenous spreading, expressing VEGF-C, decrease tumor cell dissemination to the lungs. The mechanism whereby TAM-derived VEGF-C hampered pulmonary mechanism was ascribed to a phenotypic change of TAMs that augmented VEGFR3 expression on tumor vessels facilitating tumor vessel normalization and functionality. In contrast, tumor cell-derived VEGF-C increases vessel abnormalization and pulmonary metastases corresponding to lack of VEGFR3 expression on tumor vessels. Further evidence that TAM-derived VEGF-C may play a role in hematogenous spreading was obtained from human breast cancer specimens. In line with our experimental data, perivascular TAM-derived VEGF-C is inversely correlated with malignant grade and the occurrence of three-cell complexes composed of a perivascular macrophage, a tumor cell and an endothelial cell (collectively defined as tumor microenvironment of metastasis, or TMEM), which are sites of breast cancer intravasation and dissemination. Instead, cancer cell-derived VEGF-C correlated with malignant grade and TMEM complexes. Thus, our study reveals novel and apparent paradoxical roles of TAM-derived VEGF-C in regulating hematogenous tumor spreading in both experimental models and clinical samples.

sentences: Unilateral acoustic degradation delays attentional separation of competing speech Hearing loss is often asymmetric, such that hearing thresholds differ substantially between the two ears. The extreme case of such asymmetric hearing is single-sided deafness. A unilateral cochlear implant (CI) on the more severely impaired ear is an effective treatment to restore hearing. The interactive effects of unilateral acoustic degradation and spatial attention to one sound source in multi-talker situations are at present unclear. Here, we simulated some features of listening with a unilateral CI in young, normal-hearing listeners (N = 22) who were presented with 8-band noise-vocoded speech to one ear and intact speech to the other ear. Neural responses were recorded in the electroencephalogram (EEG) to obtain the spectro-temporal response function (sTRF) to speech. Listeners made more mistakes when answering questions about vocoded (versus intact) attended speech. At the neural level, we asked how unilateral acoustic degradation would impact the attention-induced amplification of tracking target versus distracting speech. Interestingly, unilateral degradation did not per se reduce the attention-induced amplification but instead delayed it in time: Speech encoding accuracy, modelled on the basis of the sTRF, was significantly enhanced for attended versus ignored intact speech at earlier neural response latencies (<~250 ms). This attentional enhancement was not absent but delayed for vocoded speech. These findings suggest that attentional selection of unilateral, degraded speech is feasible, but induces delayed neural separation of competing speech, which might explain listening challenges experienced by unilateral CI users.

sentences: Gut IgA Enhances Systemic IgG Responses to Pneumococcal Vaccines Through the Commensal Microbiota The gut microbiota enhances systemic immunoglobulin G (IgG) responses to vaccines. However, it is unknown whether this effect involves IgA, a mucosal antibody that coats intestinal microbes. Here we found that gut IgA increased peripheral IgG responses to pneumococcal vaccines, as these responses were profoundly impaired in mice with global or mucosa-restricted IgA deficiency. The positive effect of IgA on vaccine-induced IgG production implicated gut bacteria. Indeed, IgG responses to pneumococcal vaccines were also defective in ex-germ free mice recolonized with gut microbes from mouse or human IgA-deficient donors. IgA exerted this IgG-enhancing effect by constraining the systemic translocation of intestinal commensal antigens, which caused chronic immune activation, including T cell overexpression of programmed death-1. This immune inhibitory receptor hindered vaccine-specific IgG production by eliciting functional B cell unresponsiveness, which was reverted by anti-programmed death-1 treatment. Thus, gut IgA is functionally interconnected with systemic IgG via intestinal microbes.

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Geology and discovery record of the Trinil Pithecanthropus erectus site, Java The scientific utility of Eugene Dubois Pithecanthropus erectus (P.e.) Skullcap (Trinil 1), Femur I (Trinil 3) and associated paleontological specimens has been impaired for over a century by questions about their provenience. Firsthand accounts and contemporaneous field photographs, presented here, extensively document the site geology and discovery history.

The P.e. specimens and numerous-other fossils were unearthed in 1891-1893 from small excavations dug into a flat-lying bonebed exposed near the seasonal low-water level of the Solo River along its incised left embankment. Dubois on-site supervisors specified that the two P.e. fossils came from a [~]0.2-m-thick bonebed subunit traced at a single elevation for [~]12m from the 1891 Skullcap pit ([~]30m2) to the 1892 Femur-discovery excavation and across an enlarged 1892-1893 trench ([~]170m2). The depositional co-occurrence of the finds is supported by key documentation: the supervisors letters to Dubois about Femur I; his initial reporting to the Indies government; 1892-1893 accounts about expanding excavation of the Femur I stratum; Dubois 1891-1893 government submissions and 1894-1896 publications; confirmation by the Selenka Expedition in 1907-1908; Dubois annotations on unpublished site photographs; and a letter he wrote the year he died. Field studies in the 1930s to 1970s confirmed the essential aspects of the site geology.

The bonebed of 1891-1893 contained fossils referable to the extinct Trinil fauna species Axis lydekkeri, Duboisia santeng and Stegodon trigonocephalus. The Selenka Expedition excavations had a similar assemblage in the same stratigraphic position which they named the Hauptknochenschicht. The bonebed was thin bioclast-rich gravelly volcaniclastic sandstone with taphonomic and sedimentary features indicating an unusual origin. Bioclasts range from proboscidean craniums and logs to rat teeth, freshwater mollusc shells and leaves. The terrestrial-vertebrate skeletal elements are overwhelmingly disarticulated and frequently broken. Their surfaces are little-abraded by fluvial transport. The bone fossilization is quite uniform. More than one-hundred ungulate individuals perished. No evidence has been found of hominin- or terrestrial-carnivore involvement. The bioclasts varied in density from place-to-place and vertically, and were matrix supported in the bonebed. No substantial internal depositional hiatus was reported. In combination with Trinils paleogeographic context, these features implicate a catastrophic mortality of ungulates in a population aggregation along the floodplain of a perennial paleo-river, followed by lahar-flood transport and deposition of gravel-size lithic- and biotic-materials.

Trinil provides evidence favoring a broad archaic-hominin presence in southern Sundaland. The Trinil fauna is a lynch-pin in a long-lasting paleobiogeographic association between H. erectus and certain lineages of large bovids, cervids, proboscideans, rhinoceros, suids and tiger. The bonebeds paleogeographic setting exemplifies the stratovolcanic drainages that H. erectus occupied for >0.8 million years in Java, including the watershed of a marine delta [~]150km east of Trinil, a volcanic island [~]100km north of Trinil, and areas to its west for 500km (where species associated with Trinil H. erectus occur). In the Java Sea (Sunda Shelf), seismic data image immense Pleistocene river- and coastal-terranes which archaic hominins and other large-mammals, like those at Trinil, might have inhabited.

sentences: Autism in a dish: ES cell models of autism with copy number variations reveal cell-type-specific vulnerability Human genetics has identified numerous single nucleotide variations (SNVs) and copy number variations (CNVs) associated with autism spectrum disorders (ASD) and other psychiatric disorders. However, the lack of standardized biological resources impedes understanding of the common pathophysiology of ASD. Here, using next-generation chromosome engineering based on the CRISPR/Cas9 system, we established a biological resource including 65 genetically modified mouse embryonic stem cell (mESC) lines as genetic models of human SNVs and CNVs. To illustrate cell-type and CNV specific molecular features of ASD, we performed single-cell RNA sequencing (37,397 cells in total), morphological, and physiological analyses using 12 representative cell lines with CNVs highly associated with ASD. These results uncover gene ontology (GO) terms, canonical pathways, upstream regulators, and related neuropsychiatric disorders in a cell-type and CNV specific manner.

sentences: Cell type and subcellular compartment specific APEX2 proximity labeling proteomics in the mouse brain The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell type-specific neuroproteome is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteome in the mouse brain. Using adeno- associated viral transduction, we express subcellular-localized APEX2 to map the proteome of the nucleus, cytosol, and cell membrane of Drd1 receptor-positive striatal neurons. We show that each APEX2 construct can differentially and rapidly biotinylate proteins in situ across various subcellular compartments, confirmed by imaging, electron microscopy, and mass spectrometry. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome in the mouse brain.

sentences: Facultative variation across a shallow to deep torpor spectrum in hummingbirds Many small endotherms use torpor, saving energy by a controlled reduction of their body temperature and metabolic rate. Some species (e.g. arctic ground squirrels, hummingbirds) enter deep torpor, dropping their body temperatures by 23-37 {degrees}C, while others can only enter shallow torpor (e.g., pigeons, 3-10 {degrees}C reductions). However, deep torpor in mammals can increase predation risk (unless animals are in burrows or caves), inhibit immune function, and result in sleep deprivation, so even for species that can enter deep torpor, facultative shallow torpor might help balance energy savings with these potential costs. Deep torpor occurs in three avian orders. Although the literature hints that some bird species can use both shallow and deep torpor, little empirical evidence of such an avian torpor spectrum exists. We infrared imaged three hummingbird species that are known to use deep torpor, under natural temperature and light cycles, to test if they were also capable of shallow torpor. All three species used both deep and shallow torpor, often on the same night. Depending on the species, they used shallow torpor for 5-35% of the night. The presence of a bird torpor spectrum indicates a capacity for fine-scale physiological and genetic regulation of avian torpid metabolism.

sentences: PALB2 maintains redox and mitochondrial homeostasis in the brain and cooperates with ATG7 to suppress p53 dependent neurodegeneration The PALB2 tumor suppressor plays key roles in DNA repair and has been implicated in redox homeostasis. Autophagy maintains mitochondrial quality, mitigates oxidative stress and suppresses neurodegeneration. Here we show that Palb2 deletion in the mouse brain leads to motor deficits and that co-deletion of Palb2 with the essential autophagy gene Atg7 accelerates and exacerbates neurodegeneration induced by ATG7 loss. Palb2 deletion leads to elevated DNA damage, oxidative stress and mitochondrial markers, especially in Purkinje cells, and co-deletion of Palb2 and Atg7 results in accelerated Purkinje cell loss. Further analyses suggest that the accelerated Purkinje cell loss and severe neurodegeneration in the double deletion mice are due to oxidative stress and mitochondrial dysfunction, rather than DNA damage, and partially dependent on p53 activity. Our studies uncover a role of PALB2 in mitochondrial regulation and a cooperation between PALB2 and ATG7/autophagy in maintaining redox and mitochondrial homeostasis essential for neuronal survival.

sentences: Riemannian geometry of functional connectivity matrices for multi-site attention-deficit/hyperactivity disorder data harmonization The use of multi-site datasets in neuroimaging provides neuroscientists with more statistical power to perform their analyses. However, it has been shown that imaging-site introduces a variability in the data that cannot be attributed to biological sources. In this work, we show that functional connectivity matrices derived from resting-state multi-site data contain a significant imaging-site bias. To this aim, we exploited the fact that functional connectivity matrices belong to the manifold of symmetric positive-definite matrices, making possible to operate on them with Riemannian geometry. We hereby propose a geometry-aware harmonization approach, Rigid Log-Euclidean Translation, that accounts for this site bias. Moreover, we adapted other Riemannian-geometric methods designed for other domain adaptation tasks, and compared them to our proposal. Based on our results, Rigid Log-Euclidean Translation of multi-site functional connectivity matrices seems to be among the studied methods the most suitable one in a clinical setting. This represents an advance with respect to previous functional connectivity data harmonization approaches, which do not respect the geometric constraints imposed by the underlying structure of the manifold. In particular, when applying our proposed method on data from the ADHD-200 dataset, a multi-site dataset built for the study of attention-deficit/hyperactivity disorder, we obtained results that display a remarkable correlation with established pathophysiological findings and, therefore, represent a substantial improvement when compared to the non-harmonization analysis. Thus, we present evidence supporting that harmonization should be extended to other functional neuroimaging datasets, and provide a simple geometric method to address it.

sentences: A learned embedding for efficient joint analysis of millions of mass spectra Computational methods that aim to exploit publicly available mass spectrometry repositories primarily rely on unsupervised clustering of spectra. Here, we propose to train a deep neural network in a supervised fashion based on previous assignments of peptides to spectra. The network, called "GLEAMS," learns to embed spectra into a low-dimensional space in which spectra generated by the same peptide are close to one another. We use GLEAMS as the basis for a large-scale spectrum clustering, detecting groups of unidentified, proximal spectra representing the same peptide, and we show how to use these clusters to explore the dark proteome of repeatedly observed yet consistently unidentified mass spectra. We provide a software implementation of our approach, along with a tool to quickly embed additional spectra using a pre-trained model, to facilitate large-scale analyses.

sentences: Microfluidic bioprinting for the in vitro generation of novel biomimetic human testicular tissues Advances in cancer treatments have greatly improved pediatric cancer survival rates, leading to quality of life considerations and in particular fertility restoration. Accordingly, pre-pubertal patients have the option to cryopreserve testicular tissue for experimental restorative therapies, including in vitro spermatogenesis, wherein testicular tissue is engineered in vitro and spermatozoa are collected for in vitro fertilization (IVF). Current in vitro systems have been unable to reliably support the generation of spermatozoa from human testicular tissues, likely due to the inability for the dissociated testicular cells to recreate the native architecture of testicular tissue found in vivo. Recent advances in 3-D bioprinting can place cells into geometries at fine resolutions comparable to microarchitectures found in native tissues, and therefore hold promise as a tool for the development of a biomimetic in vitro system for human spermatogenesis. This study assessed the utility of bioprinting technology to recreate the precise architecture of testicular tissue and corresponding spermatogenesis for the first time. We printed testicular cell-laden hollow microtubules at similar resolutions to seminiferous tubules, and compared the results to testicular organoids. We show that the human testicular cells retain their viability and functionality post-printing, and illustrate an intrinsic ability to reorganize into their native cytoarchitecture. This study provides a proof of concept for the use of 3-D bioprinting technology as a tool to create biomimetic human testicular tissues.

sentences: Comprehensive Benchmarking of CITE-seq versus DOGMA-seq Single Cell Multimodal Omics The recently developed transcription, epitopes, and chromatin accessibility by sequencing (TEA-seq) and similar DOGMA-seq single-cell trimodal omics assays provide unprecedented opportunities for understanding cell biology, but independent optimization, benchmarking and evaluation are lacking. We explored the utility, pros and cons of DOGMA-seq compared to the bimodal cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) assay in activated and stimulated human peripheral blood T cells. We identified an optimal incubation time and concentration of digitonin (DIG) for cell permeabilization and found that single-cell trimodal omics measurements after DIG permeabilization were generally better than after an alternative "low-loss lysis" (LLL) permeabilization condition. Next, we found that DOGMA-seq with optimized DIG permeabilization and its ATAC library provides more information, even though its mRNA and cell surface protein antibody-derived tag (ADT) libraries have slightly inferior quality, compared to CITE-seq. Finally, we recognized the additional value of DOGMA-seq for studying lineage-specific T helper cells.

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Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5 OBJECTIVEExposure to air pollutants leads to the development of pulmonary and cardiovascular diseases, and thus air pollution is one of the major global threats to human health. Air pollutant particulate matter 2.5 (PM2.5)-induced cellular dysfunction impairs tissue homeostasis and causes vascular and cardiopulmonary damage. To test a hypothesis that elevated plasminogen activator inhibitor-1 (PAI-1) levels play a pivotal role in air pollutant-induced cardiopulmonary pathologies, we examined the efficacy of a drug-like novel inhibitor of PAI-1, TM5614, in treating PM2.5-induced vascular and cardiopulmonary pathologies.

APPROACH AND RESULTSResults from biochemical, histological, and immunohistochemical studies revealed that PM2.5 increases the circulating levels of PAI-1 and thrombin and that TM5614 treatment completely abrogates these effects in plasma. PM2.5 significantly augments levels of pro-inflammatory cytokine IL-6 in bronchoalveolar lavage fluid, and this also can be reversed by TM5614, indicating its efficacy in amelioration of PM2.5-induced increases in inflammatory and pro-thrombotic factors. TM5614 reduces PM2.5-induced increased levels of inflammatory markers Mac3 and pSTAT3, adhesion molecule VCAM1, and apoptotic marker cleaved caspase 3. Longer exposure to PM2.5 induces pulmonary and cardiac thrombosis, but TM5614 significantly ameliorates PM2.5-induced vascular thrombosis. TM5614 also reduces PM2.5-induced increased blood pressure and heart weight. In vitro cell culture studies revealed that PM2.5 induces the levels of PAI-1, type I collagen, fibronectin, and SREBP-1/2, a transcription factor that mediates profibrogenic signaling, in cardiac fibroblasts. TM5614 abrogated that stimulation, indicating that it may block PM2.5-induced PAI-1 and profibrogenic signaling through suppression of SREBP-1. Furthermore, TM5614 blocked PM2.5-mediated suppression of Nrf2, a major antioxidant regulator in cardiac fibroblasts.

CONCLUSIONSPharmacological inhibition of PAI-1 with TM5614 is a promising therapeutic approach to control air pollutant PM2.5-induced cardiopulmonary and vascular pathologies.

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sentences: Multicellular immune hubs and their organization in MMRd and MMRp colorectal cancer Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd patients. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across patient tumors and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines. By identifying interacting cellular programs, we thus reveal the logic underlying spatially organized immune-malignant cell networks.

sentences: A structural dynamic explanation for observed escape of SARS-CoV-2 BA.2 variant mutation S371L/F The SARS-CoV-2 Omicron sub-variants BA.1 and BA.2 have become the dominant variants worldwide due to enhanced transmissibility and immune evasion. In response to the rise of BA.1 and BA.2, two recent studies by Liu et al. and Iketani et al. provide a detailed analysis of loss of therapeutic antibody potency through evaluation of escape by pseudotyped viruses harboring BA.1 and BA.2 receptor binding domain (RBD) point mutations. Surprisingly, Liu et al. and Iketani et al. observed a profoundly broad escape effect for the individual mutations S371L and S371F. This result cannot be explained by known escape mechanisms of the SARS-CoV-2 RBD, and conflicts with existing computational and experimental escape measurements for S371 mutations performed on monomeric RBD. Through an examination of these conflicting datasets and a structural analysis of the antibodies assayed by Liu et al. and Iketani et al., we propose a mechanism to explain S371L/F escape according to a perturbation of spike trimer conformational dynamics that has not yet been described for any SARS-CoV-2 escape mutation. The proposed mechanism is relevant to Omicron and future variant surveillance as well as therapeutic antibody design.

sentences: A dissemination-prone morphotype enhances extrapulmonary organ entry by the fungus Cryptococcus neoformans Environmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (<10 m diameter) enlarge (>30 m diameter), then form a heterogeneous population. The brain contains uniformly small cells ([~]7 m). We demonstrate that formation of a small C. neoformans morphotype - called "seed" cells due to their disseminating ability - is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

sentences: A series of terribly unfortunate events: How environment and infection synergized to cause the Kihansi spray toad extinction Outbreaks of emerging infectious diseases are trained by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Extinction of the Tanzanian Kihansi spray toad (Nectophrynoides asperginis) in 2004 was contemporaneous with the construction of a dam, implicating habitat modification in the loss of this species. However, high burdens of a globally emerging infection, Batrachochytrium dendrobatidis (Bd) were synchronously observed implicating infectious disease in this toads extinction. Here, by shotgun sequencing skin DNA from archived toad mortalities and assembling chytrid mitogenomes, we prove this outbreak was caused by the BdCAPE lineage and not the panzootic lineage BdGPL that is widely associated with global amphibian extinctions. Molecular dating showed an invasion of BdCAPE across Southern Africa overlapping with the timing of the extinction event. However, post-outbreak surveillance of conspecific species inhabiting this mountainous region showed widespread infection by BdCAPE yet no signs of amphibian ill-health or species decline. Our findings show that despite efforts to mitigate the environmental impact caused by dams construction, invasion of the pathogen ultimately led to the loss of the Kihansi spray toad; a synergism between emerging infectious disease and environmental change that likely heralds wider negative impacts on biodiversity in the Anthropocene.

sentences: The extinction time under mutational meltdown AO_SCPLOWBSTRACTC_SCPLOWMutational meltdown describes an eco-evolutionary process in which the accumulation of deleterious mutations causes a fitness decline that eventually leads to the extinction of a population. Possible applications of this concept include medical treatment of RNA virus infections based on mutagenic drugs that increase the mutation rate of the pathogen. To determine the usefulness and expected success of such an antiviral treatment, estimates of the expected time to mutational meltdown are necessary. Here, we compute the extinction time of a population under high mutation rates, using both analytical approaches and stochastic simulations. Extinction is the result of three consecutive processes: (1) initial accumulation of deleterious mutations due to the increased mutation pressure; (2) consecutive loss of the fittest haplotype due to Mullers ratchet; (3) rapid population decline towards extinction. We find accurate analytical results for the mean extinction time, which show that the deleterious mutation rate has the strongest effect on the extinction time. We confirm that intermediatesized deleterious selection coefficients minimize the extinction time. Finally, our simulations show that the variation in extinction time, given a set of parameters, is surprisingly small.

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Cytostatic hypothermia and its impact on glioblastoma and survival Novel therapeutic approaches are needed for patients with glioblastoma (GBM) who otherwise have limited options. Here we studied and deployed non-freezing  cytostatic hypothermia to stunt GBM growth. This contrasts with ablative, cryogenic hypothermia: a double-edged sword against tumors infiltrating otherwise healthy tissue. We investigated three grades of hypothermia in vitro and identified a cytostatic window of 20-25{degrees}C. For some glioma lines, 18 h/d of cytostatic hypothermia was sufficient to halt division in vitro. Cytostatic hypothermia induced cell cycle arrest, reduced metabolite production and consumption, and reduced inflammatory cytokine synthesis. Next, we fabricated an experimental device to test local cytostatic hypothermia in vivo in two rodent models of GBM: utilizing the rat F98 and the human U-87 MG lines. Hypothermia more than doubled the median survival of F98 bearing rats from 3.9 weeks to 9.7 weeks and two rats survived through 12 weeks. All U-87 MG bearing rats that successfully received cytostatic hypothermia survived their study period. Thus, this approach lengthened survival without chemical interventions. Unlike targeted therapeutics that are successful in preclinical models but fail in clinical trials, cytostatic hypothermia affects multiple cellular processes simultaneously. This, alongside reduced cellular division, suggests that opportunities for tumor evolution are reduced and the likelihood of translation to larger species may be more likely. In addition, based on our work, designs, and the literature, engineering a patient-centric device is tangible. Taken together, cytostatic hypothermia could be a novel approach to cancer therapy and eventually serve a valuable role to patients with GBM.

One Sentence SummaryHypothermia influences multiple cellular pathways, can be a safe and effective approach to halt glioblastoma growth, and holds translational promise.

sentences: Whale shark rhodopsin adapted to its vertically wide-ranging lifestyle Spectral tuning of visual pigments often facilitates adaptation to new environments, and it is intriguing to study the visual ecology of pelagic sharks with expanded habitats. The whale shark, which dives into the deep sea of nearly 2,000 meters besides near-surface filter-feeding, was previously shown to possesses the  blue-shifted rhodopsin (RHO). In this study, our spectroscopy of recombinant whale shark RHO mutants revealed the dominant effect of the novel spectral tuning amino acid site 94, which is implicated in congenital stationary night blindness of humans, accounting for the blue shift. Thermal decay profiling revealed the reduction of the thermal stability of whale shark RHO, as typically observed for cone opsins, which was experimentally shown to be achieved by the site 178, as well as 94. The results suggest that these two sites cooperatively enhance the visual capacity in both the deep sea and the sea surface, enabling exceptionally wide vertical migration of this species.

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Evolution of a cis-acting SNP that controls Type VI Secretion in Vibrio cholerae Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes and other organisms to new niches. Comparative genomics can be used to infer rewiring of regulatory architecture based on large effect mutations like loss or acquisition of transcription factors but may be insufficient to identify small changes in non-coding, intergenic DNA sequence of regulatory elements that drive phenotypic divergence. In human-derived Vibrio cholerae, the response to distinct chemical cues triggers production of multiple transcription factors that can regulate the Type VI Secretion System (T6), a broadly distributed weapon for interbacterial competition. However, to date, the signaling network remains poorly understood because no regulatory element has been identified for the major T6 locus. Here we identify a conserved cis-acting single nucleotide polymorphism (SNP) controlling T6 transcription and activity. Sequence alignment of the T6 regulatory region from diverse V. cholerae strains revealed conservation of the SNP that we rewired to interconvert V. cholerae T6 activity between chitin-inducible and constitutive states. This study supports a model of pathogen evolution through a non-coding cis-regulatory mutation and preexisting, active transcription factors that confers a different fitness advantage to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches.

ImportanceOrganisms sense external cues with regulatory circuits that trigger the production of transcription factors, which bind specific DNA sequences at promoters ("cis" regulatory elements) to activate target genes. Mutations of transcription factors or their regulatory elements create phenotypic diversity, allowing exploitation of new niches. Waterborne pathogen Vibrio cholerae encodes the Type VI Secretion System "nanoweapon" to kill competitor cells when activated. Despite identification of several transcription factors, no regulatory element has been identified in the promoter of the major Type VI locus, to date. Combining phenotypic, genetic, and genomic analysis of diverse V. cholerae strains, we discovered a single nucleotide polymorphism in the Type VI promoter that switches its killing activity between a constitutive state beneficial outside hosts and an inducible state for constraint in a host. Our results support a role for non-coding DNA in adaptation of this pathogen.

sentences: Deep Bayesian networks for uncertainty estimation and adversarial resistance of white matter hyperintensity segmentation White matter hyperintensities (WMH) are frequently observed on structural neuroimaging of elderly populations and are associated with cognitive decline and increased risk of dementia. Many existing WMH segmentation algorithms produce suboptimal results in populations with vascular lesions or brain atrophy, or require parameter tuning and are computationally expensive. Additionally, most algorithms do not generate a confidence estimate of segmentation quality, limiting their interpretation. MRI-based segmentation methods are often sensitive to acquisition protocols, scanners, noise-level, and image contrast, failing to generalize to other populations and out-of-distribution datasets. Given these concerns, we propose a novel Bayesian 3D Convolutional Neural Network (CNN) with a U-Net architecture that automatically segments WMH, provides uncertainty estimates of the segmentation output for quality control and is robust to changes in acquisition protocols. We also provide a second model to differentiate deep and periventricular WMH. 432 subjects were recruited to train the CNNs from four multi-site imaging studies. A separate test set of 158 subjects was used for evaluation, including an unseen multi-site study. We compared our model to two established state-of-the-art techniques (BIANCA and DeepMedic), highlighting its accuracy and efficiency. Our Bayesian 3D U-Net achieved the highest Dice similarity coefficient of 0.89 {+/-} 0.08 and the lowest modified Hausdorff distance of 2.98 {+/-} 4.40 mm. We further validated our models highlighting their robustness on  clinical adversarial cases simulating data with low signal-to-noise ratio, low resolution, and different contrast (stemming from MRI sequences with different parameters). Our pipeline and models are available at: https://hypermapp3r.readthedocs.io

sentences: Memory shapes microbial populations Correct decision making is fundamental for all living organisms to thrive under environmental changes. The patterns of environmental variation and the quality of available information define the most favourable strategy among multiple options, from randomly adopting a phenotypic state to sensing and reacting to environmental cues. Memory - a phenomenon often associated with, but not restricted to, higher multicellular organisms - can help when temporal correlations exist. How does memory manifest itself in unicellular organisms? Through a combination of deterministic modelling and stochastic simulations, we describe the population-wide fitness consequences of phenotypic memory in microbial populations. Moving beyond binary switching models, our work highlights the need to consider a broader range of switching behaviours when describing microbial adaptive strategies. We show that multiple cellular states capture the empirical observations of lag time distributions, overshoots, and ultimately the phenomenon of phenotypic heterogeneity. We emphasise the implications of our work in understanding antibiotic tolerance, and, in general, survival under fluctuating environments.

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Movement-Dependent Electrical Stimulation for Volitional Strengthening of Cortical Connections in Behaving Monkeys Correlated activity of neurons can lead to long-term strengthening or weakening of the connections between them. In addition, the behavioral context, imparted by execution of physical movements or the presence of a reward, can modulate the plasticity induced by Hebbian mechanisms. In the present study, we have combined behavior and induced neuronal correlations to strengthen connections in the motor cortex of adult behaving monkeys. Correlated activity was induced using an electrical-conditioning protocol in which stimuli gated by voluntary movements were used to produce co-activation of neurons at motor-cortical sites involved in those movements. Delivery of movement-dependent stimulation resulted in small increases in the strength of associated cortical connections immediately after conditioning. Remarkably, when paired with further repetition of the movements that gated the conditioning stimuli, there were substantially larger gains in the strength of cortical connections, that occurred in a use-dependent manner, without delivery of additional conditioning stimulation. In the absence of such movements, little change was observed in the strength of motor-cortical connections. Performance of the motor behavior in the absence of conditioning also did not produce any changes in connectivity. Our results show that combining movement-gated stimulation with further natural use of the "conditioned" pathways after stimulation ends can produce use-dependent strengthening of connections in adult primates, highlighting an important role for behavior in cortical plasticity. Our data also provide strong support for combining movement-gated stimulation with use-dependent physical rehabilitation for strengthening connections weakened by a stroke or spinal-cord injury.

Significance StatementWe describe an electrical-conditioning protocol in adult behaving monkeys in which stimuli gated by voluntary movements were used to strengthen connections between motor-cortical neurons involved in those movements. Movement-gated stimulation created a plastic landscape in which repetition of the movements that gated conditioning stimuli produced strengthening of cortical connections, in a use-dependent manner, long after stimulation had ended, a finding that is both novel and unique. In the absence of such behavior, little change was observed in the strength of connections. Similarly, movements alone did not produce any changes in connectivity. Our data highlight a critical role for behavior in plasticity and provide strong support for combining movement-gated stimulation with use-dependent rehabilitation for strengthening connections weakened by injury or disease.

sentences: Accurate and robust inference of genetic ancestry from cancer-derived molecular data across genomic platforms Genetic ancestry-oriented cancer research requires the ability to perform accurate and robust ancestry inference from existing cancer-derived data, including whole exomes, transcriptomes and targeted gene panels, very often in the absence of matching cancer-free genomic data. In order to optimize and assess the performance of the ancestry inference for any given input cancer-derived molecular profile, we develop a data synthesis framework. In its core procedure, the ancestral background of the profiled patient is replaced with one of any number of individuals with known ancestry. Data synthesis is applicable to multiple profiling platforms and makes it possible to assess the performance of inference separately for each continental-level ancestry. This ability extends to all ancestries, including those without statistically sufficient representation in the existing cancer data. We further show that our inference procedure is accurate and robust in a wide range of sequencing depths. Testing our approach for three representative cancer types, and across three molecular profiling modalities, we demonstrate that global, continental-level ancestry of the patient can be inferred with high accuracy, as quantified by its agreement with the golden standard of the ancestry derived from matching cancer-free molecular data. Our study demonstrates that vast amounts of existing cancer-derived molecular data potentially are amenable to ancestry-oriented studies of the disease, without recourse to matching cancer-free genomes or patients self-identification by ancestry.

sentences: Monitoring the 5`UTR landscape reveals 5`terminal oligopyrimidine (TOP) motif switches to drive translational efficiencies Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here we use long-read sequencing to document the 5and 3untranslated region (UTR) isoform landscape of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. These 5UTR isoforms with identical coding sequences either include or exclude 5terminal oligopyrimidine (TOP) motifs and result in vastly altered translational efficiencies of the mRNA. Our findings suggest that switching between TOP and non-TOP motif-containing 5UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5UTR sequence.

sentences: FLIP: Benchmark tasks in fitness landscape inference for proteins Machine learning could enable an unprecedented level of control in protein engineering for therapeutic and industrial applications. Critical to its use in designing proteins with desired properties, machine learning models must capture the protein sequence-function relationship, often termed fitness landscape. Existing bench-marks like CASP or CAFA assess structure and function predictions of proteins, respectively, yet they do not target metrics relevant for protein engineering. In this work, we introduce Fitness Landscape Inference for Proteins (FLIP), a benchmark for function prediction to encourage rapid scoring of representation learning for protein engineering. Our curated tasks, baselines, and metrics probe model generalization in settings relevant for protein engineering, e.g. low-resource and extrapolative. Currently, FLIP encompasses experimental data across adeno-associated virus stability for gene therapy, protein domain B1 stability and immunoglobulin binding, and thermostability from multiple protein families. In order to enable ease of use and future expansion to new tasks, all data are presented in a standard format. FLIP scripts and data are freely accessible at https://benchmark.protein.properties.

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Murine blastocysts generated by in vitro fertilization show increased Warburg metabolism and altered lactate production In vitro fertilization (IVF) has resulted in the birth of over 8 million children. Although most of IVF-conceived children are healthy, several studies suggest an increased risk of altered growth rate, cardiovascular dysfunction, and glucose intolerance in this population compared to naturally conceived children. However, a clear understanding of how embryonic metabolism is affected by culture condition and how embryos reprogram their metabolism is unknown.

Here, we studied oxidative stress and metabolic alteration in blastocysts conceived by natural mating or by IVF and culture in physiologic (5%) or atmospheric (20%) oxygen. We found that IVF-generated blastocyst manifest increased reactive oxygen species, oxidative damage to DNA/lipid/proteins, and reduction in glutathione. Metabolic analysis revealed IVF-generated blastocysts display decreased mitochondria respiration and increased glycolytic activity suggestive of enhanced Warburg metabolism. These findings were corroborated by altered intracellular and extracellular pH and increased intracellular lactate levels in IVF-generated embryos. Comprehensive proteomic analysis and targeted immunofluorescence showed reduction of LDH-B and MCT1, enzymes involved in lactate metabolism. Importantly, these enzymes remained downregulated in tissues of adult IVF-conceived mice, suggesting that metabolic alterations in IVF-generated embryos may result in alteration in lactate metabolism. These findings suggest that alterations in lactate metabolism is a likely mechanism involved in genomic reprogramming and could be involved in the developmental origin of health and disease.

sentences: The structure of the Physcomitrium Patens Photosystem I Reveals a Unique Lhca2 Paralogue replacing Lhca4 The moss Physcomitrium patens diverged from green algae shortly after the colonization of land by ancient plants. This colonization posed new environmental challenges which drove evolutionary processes. The photosynthetic machinery of modern flowering plants is adapted to the high light conditions on land. Red shifted Lhca4 antennae are present in the photosystem I light harvesting complex of many green lineage plants but absent from P. patens. The Cryo-EM structure of the P. patens photosystem I light harvesting complex I supercomplex (PSI-LHCI) at 2.8 [A] reveals that Lhca4 is replaced by a unique Lhca2 paralogue in moss. This PSI-LHCI supercomplex also retains the PsaM subunit, present in cyanobacteria and several algal species but lost in higher plants, and the PsaO subunit responsible for binding light harvesting complex II. The blue shifted Lhca2 paralogue and chlorophyll b enrichment relative to higher plants make the P. patens PSI-LHCI spectroscopically unique among other green lineage supercomplexes. Overall, the structure represents an evolutionary intermediate PSI with the crescent shaped LHCI common in higher plants and contains a unique Lhca2 paralogue which facilitates the mosses adaptation to low light niches.

sentences: A modular, dynamic, DNA-based platform for regulating cargo distribution and transport between lipid domains Cell membranes regulate the distribution of biological machinery between phase-separated lipid domains to facilitate key processes including signalling and transport, which are among the life-like functionalities that bottom-up synthetic biology aims to replicate in artificial-cellular systems. Here, we introduce a modular approach to program partitioning of amphiphilic DNA nanostructures in co-existing lipid domains. Exploiting the tendency of different hydrophobic "anchors" to enrich different phases, we modulate the lateral distribution of our devices by rationally combining hydrophobes, and by changing nanostructure size and its topology. We demonstrate the functionality of our strategy with a bio-inspired DNA architecture, which dynamically undergoes ligand-induced reconfiguration to mediate cargo transport between domains via lateral re-distribution. Our findings pave the way to next-generation biomimetic platforms for sensing, transduction, and communication in synthetic cellular systems.

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Intramolecular carbon isotope signals reflect metabolite allocation in plants Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at treering glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesising leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference of leaf-cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis.

HighlightIntramolecular 13C/12C analysis resolves information about carbon uptake and allocation (and associated environmental controls) enabling more comprehensive assessments of carbon metabolism, plant-environment interactions, and environmental variability than whole-molecule 13C/12C analysis.

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Extending the application of the SCA/Sectors method for the identification of domain boundaries and subtype specific residues in multi-domain biosynthetic proteins: Application to Polyketide Synthases Polypeptides with multiple enzyme domains, such as type I polyketide synthases, produce chemically complex compounds that are difficult to produce via conventional chemical synthesis and are often pharmaceutically or otherwise commercially valuable. Engineering polyketide synthases, via domain swapping and/or site directed mutagenesis, in order to generate novel polyketides, has tended to produce either low yields of product or no product at all. The success of such experiments may be limited by our inability to predict the key functional residues and boundaries of protein domains. Computational tools to identify the boundaries and the residues determining the substrate specificity of domains could reduce the trial and error involved in engineering multi-domain proteins. In this study we use statistical coupling analysis to identify networks of co-evolving residues in type I polyketide synthases, thereby predicting domain boundaries. We extend the method to predicting key residues for enzyme substrate specificity. We introduce bootstrapping calculations to test the relationship between sequence length and the number of sequences needed for a robust analysis. Our results show no simple predictor of the number of sequences needed for an analysis, which can be as few as a hundred and as many as a few thousand. We find that polyketide synthases contain multiple networks of co-substituting residues: some are intradomain but most multiple domains. Some networks of coupled residues correlate with specific functions such as the substrate specificity of the acyl transferase domain, the stereo chemistry of the ketoreductase domain, or domain boundaries that are consistent with experimental data. Our extension of the method provides a ranking of the likely importance of these residues to enzyme substrate specificity, allowing us to propose residues for further mutagenesis work. We conclude that analysis of co-evolving networks of residues is likely to be an important tool for re-engineering multi-domain proteins.

Author summaryMany important compounds such as antibiotics or food flavourings are produced naturally by molecular factories within plant, fungal and bacterial cells. These molecular factories typically comprise a complex of multiple interacting enzymes, each enzyme being a stage in a molecular production line. Often the enzymes are connected together as subsections of the same amino acid chain, i.e. protein, with the amino acid chain folding into the separate functional enzymatic domains that comprise the production line. Polyketide synthases are such multi-domain proteins, and their products often have antibacterial, antifungal and antitumoric effects. Engineering polyketide synthases thus has the potential to produce novel drug candidates. We applied and developed statistical approaches to detect where in an amino acid sequence the boundaries are between different domains, potentially allowing these regions to be swapped around for the synthesis of novel compounds. We used the same approaches to identify parts of the amino acid chain important for the function of different types of domain, pointing to how they might be modified to make novel compounds. These analyses agree with published experimental data and allow us to make novel predictions, which we expect to help experimentalists produce novel compounds of commercial and pharmaceutical interest.

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Effects of perfluoroalkyl and polyfluoroalkyl substances on soil structure and function Soils are impacted at a global scale by several anthropogenic factors, including chemical pollutants. Among those, perfluoroalkyl and polyfluoroalkyl substances (PFAS) are of concern due to their high environmental persistence, and as they might affect soil health and functions. However, data on impacts of PFASs on soil structure and microbially-driven processes are currently lacking. This study explored the effects of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorobutanesulfonic acid (PFBS) at environmental-relevant nominal concentrations (1 ~ 1000 ng g-1) on soil functions, using a 6-week microcosm experiment. We measured soil respiration, litter decomposition, enzyme and microbial activities, soil aggregates, and bacterial abundance. PFAS (even at 1 ng g-1 for PFBS) significantly increased litter decomposition, associated with positive effects on bacterial abundance, and {beta}-glucosidase activities. This effect increased with PFAS concentrations. Soil respiration was significantly inhibited by PFAS in the 3rd week, while this effect was more variable in week 6. Water-stable aggregates were negatively affected by PFOS and PFOA, possibly related to microbial shifts. The general microbial activities and {beta}-D-cellobiosidase and phosphatase activities were barely affected by PFAS treatments. Our work highlights the potential effects of PFAS on soil health, and we argue that this substance class could be a factor of environmental change of potentially broad relevance in terrestrial ecosystem functioning.

SynopsisPFAS are likely to affect soil health.

Abstract Art

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Self-organizing Single-Rosette Brain Organoids from Human Pluripotent Stem Cells The field of brain organoid research is complicated by morphological variability with multiple neural rosette structures per organoid. We have developed a new human brain organoid technique that generates self-organizing, single-rosette spheroids (SOSRS) with reproducible size, cortical-like lamination, and cell diversity. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells (hPSCs) that is patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D transition from neural plate to neural tube that occurs during neurodevelopment. The vast majority of monolayer fragments form spheres with a single central lumen and consistent growth rates. Over time, the SOSRS develop appropriately ordered lamination consistent with six cortical layers by immunocytochemistry and single cell RNA-sequencing. The reproducibility of this method has allowed us to demonstrate robust structural phenotypes arising from chemical teratogen exposure or when modeling a genetic neurodevelopmental epileptic disorder. This platform should advance studies of human cortical development, brain disorder mechanisms, and precision therapies.

SUMMARY STATEMENTSimple procedure for generating reproducible single rosette cortical brain organoids used to identify robust structural phenotypes with neuroteratogen exposure and in a genetic neurodevelopmental disease model.

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Intrusive Experiences In Post-Traumatic Stress Disorder: Treatment Response Induces Changes In The Effective Connectivity Of The Anterior Insula BackgroundOne of the core features of posttraumatic stress disorder (PTSD) is reexperiencing the trauma. The anterior insula (AI) was proposed to play a crucial role in these intrusive experiences. However, the dynamic function of the AI in reexperiencing trauma, as well as its putative modulation by effective therapy, still need to be specified.

MethodsThirty PTSD patients were enrolled and exposed to traumatic memory reactivation therapy. Resting-state fMRI scans were acquired before and after treatment. To explore AI directed influences over the rest of the brain, we referred to a mixed-model using pre/post Granger causality analysis seeded on the AI as a within-subject factor and treatment response as a between-subject factor. To further identify correlates of reexperiencing trauma, we investigated how intrusive severity affected: (i) causality maps and (ii) the spatial stability of other intrinsic brain networks.

ResultsWe observed dynamic changes in AI effective connectivity in PTSD patients. Many within- and between-network causal paths were found to be less influenced by the AI after effective therapy. Insular influences were found positively correlated with flashback severity, while reexperiencing was linked with a stronger default mode network (DMN) and more unstable central executive network (CEN) connectivity.

ConclusionWe showed that directed changes in AI signaling to the DMN and CEN at rest may underlie the degree of intrusive symptoms in PTSD. A positive response to treatment further induced changes in network-to-network anticorrelated patterns. Such findings may guide targeted neuromodulation strategies in PTSD patients not suitably improved by conventional treatment.

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Systematic monitoring of photosynthetically-derived oxidative signals by a peroxiredoxin-based biosensor Transmission of reductive cues from the photosynthetic electron transport chain to redox-regulated proteins plays a crucial role in activating chloroplast metabolism. However, deciphering the role of their counterbalanced oxidative signals is challenging due to monitoring difficulties. Here, we demonstrate the light-depended redox modification of chloroplast-targeted 2-Cys peroxiredoxins and introduce peroxiredoxin-based biosensors to monitor photosynthetically-derived oxidative signals. By employing a set of genetically encoded biosensors, we show the induction of oxidative signals under habitual light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast metabolism. A faster increase in carbon assimilation rates during photosynthesis induction phase was measured in plants deficient in 2-Cys peroxiredoxins compared to wild-type, suggesting the involvement of oxidative signals in attenuating photosynthesis under variable light environments. We suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system.

One-Sentence SummaryA genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

sentences: MDH2 is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. Our work demonstrates the crucial role played by MDH2 in orchestrating the electron fuelling of the RC. On one hand, MDH2 mediated oxaloacetate (OAA) production allows mitochondria to metabolize pyruvate and glutamate and to feed complex I with NADH, whereas on the other hand, OAA inhibits complex II. This regulatory mechanism synergistically increases RCs NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA favouring the oxidation of malate produced by the MAS.

sentences: Partial loss of succinate dehydrogenase reduces high red cell distribution width and promotes healthy survival in chronically hypoxic mice Increased red cell distribution width (RDW), which measures erythrocyte size variability (anisocytosis), has been linked to early mortality in many diseases and normal aged population through unknown mechanisms. Hypoxia has been proposed to increase both RDW and mortality. However, experimental evidence, especially in animal models, is lacking. Here, we show that chronic hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce high RDW and immature reticulocyte fraction, and increase healthy lifespan in chronic hypoxia. Hemoglobin and erythrocyte numbers in hypoxia do not show statistically significant differences between Sdh chKO and WT mice. These results identify a mitochondrial mechanism regulating both RDW and organismal adaptation to chronic hypoxia, and suggest SDH as a potential therapeutic target to reduce high RDW-associated clinical mortality.

sentences: Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor of Toxoplasma gondii Infection Toxoplasma gondii is an important human pathogen infecting an estimated 1 in 3 people worldwide. The cytokine interferon gamma (IFN{gamma}) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction interferon stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFN{gamma}, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFN{gamma} induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFN{gamma} induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFN{gamma} mediated immunity to T. gondii infection is a complex, multifactorial process.

sentences: Migration without interbreeding: evolutionary history of a highly selfing Mediterranean grass inferred from whole genomes Wild plant populations show extensive genetic subdivision and are far from the ideal of panmixia which permeates population genetic theory. Understanding the spatial and temporal scale of population structure is therefore fundamental for empirical population genetics - and of interest in itself, as it yields insights into the history and biology of a species. In this study we extend the genomic resources for the wild Mediterranean grass Brachypodium distachyon to investigate the scale of population structure and its underlying history at whole-genome resolution. 86 accessions were sampled at local and regional scales in Italy and France, which closes a conspicuous gap in the collection for this model organism. The analysis of 196 accessions, spanning the Mediterranean from Spain to Iraq, suggests that the interplay of high selfing and seed dispersal rates has shaped genetic structure in B. distachyon. At the continental scale, the evolution in B. distachyon is characterized by the independent expansion of three lineages during the Upper Pleistocene. Today, these lineages may occur on the same meadow yet do not interbreed. At the regional scale, dispersal and selfing interact and maintain high genotypic diversity, thus challenging the textbook notion that selfing in finite populations implies reduced diversity. Our study extends the population genomic resources for B. distachyon and suggests that an important use of this wild plant model is to investigate how selfing and dispersal, two processes typically studied separately, interact in colonizing plant species.

sentences: Messages From the Small Intestine Carried by Extracellular Vesicles in Prediabetes: a Proteomic Portrait Extracellular vesicles (EVs) mediate communication in physiological and pathological conditions. In the pathogenesis of type 2 diabetes, inter-organ communication plays an important role in its progress and metabolic surgery leads to its remission. Moreover, gut dysbiosis is emerging as a diabetogenic factor. However, it remains unclear how gut senses metabolic alterations and whether this is transmitted to other tissues via EVs. Using a diet induced-prediabetic mouse model, we observed that protein packaging in gut derived EVs (GDE), specifically small intestine, is increased in prediabetes. Proteins related to lipid metabolism and to oxidative stress management were more abundant in prediabetic GDE compared to healthy controls. On the other hand, proteins related to glycolytic activity, as well as those responsible for the degradation of polyubiquitinated composites, were depleted in prediabetic GDE. Together, our findings show that protein packaging in GDE is markedly modified during prediabetes pathogenesis. Thus, suggesting that prediabetic alterations in small intestine are translated into modified GDE proteome, which are dispersed into the circulation where they can interact with and influence the metabolic status of other tissues. This study highlights the importance of the small intestine as a tissue that propagates prediabetic metabolic dysfunction throughout the body and the importance of GDE as the messenger.

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From Learning to Remembering: How Memory Consolidation Differs in Term and Preterm Born Children from Young Adults Human capacity to remember experienced episodes over a long period of time has its roots in childhood and develops throughout the lifespan. However, the neural regions supporting memory consolidation in the developing brain remain to be ascertained. The present study examined system-level memory consolidation of object-location associations after one night of sleep (short delay) and after two weeks (long delay), and its relation to structural brain measures in normally developing term born and preterm born 6-year-old children, as well as in young adults as a reference group of mature consolidation systems. We showed that final learning performance was reduced in preterm in comparison to term born children, who in turn were outperformed by young adults. There were no differences in short- and long-delay memory consolidation between term and preterm born children. Despite comparable short-delay memory consolidation in all groups, both term and preterm born children showed less efficient long-delay memory consolidation in comparison to young adults. Moreover, long-delay memory consolidation was positively associated with larger hippocampal volume in children, while a thinner medial orbitofrontal cortex was associated with better overall memory retention rates in all age groups. Thinner medial orbitofrontal cortex was furthermore associated with higher final learning performance in children. Taken together, the results suggest that temporal dynamics of memory consolidation and its association with structural brain measures in 6-year-old term born and preterm born children are comparable but differ from young adults.

RESEARCH HIGHLIGHTSO_LIShort-delay memory consolidation of object-location associations does not differ between 6-year-old preterm born and term born children, as well as between children and young adults.
C_LIO_LILong-delay memory consolidation in 6-year-old children is less efficient than in young adults.
C_LIO_LILarger HC volume is associated with higher long-delay retention rates in 6-year-old children, while thinner medial OFC predicted higher memory retention rates in all groups.
C_LIO_LIThinner medial OFC is associated with higher final learning performance in 6-year-old children and adults.
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sentences: Evidence of learning walks related to scorpion home burrow navigation The Navigation by Chemotextural Familiarity Hypothesis (NCFH) suggests that scorpions use their midventral pectines to gather chemical and textural information near their burrows and use this information as they subsequently return home. For NCFH to be viable, animals must somehow acquire home-directed "tastes" of the substrate, such as through path integration (PI) and/or learning walks. We conducted laboratory behavioral trials using desert grassland scorpions (Paruroctonus utahensis). Animals reliably formed burrows in small mounds of sand we provided in the middle of circular, sandlined behavioral arenas. We processed overnight infrared video recordings with a MATLAB script that tracked animal movements at 1-2 s intervals. In all, we analyzed the movements of 23 animals, representing nearly 1500 hours of video recording. We found that once animals established their home burrows, they immediately made one to several short, looping excursions away from and back to their burrows before walking greater distances. We also observed similar excursions when animals made burrows in level sand in the middle of the arena (i.e., no mound provided). These putative learning walks, together with recently reported PI in scorpions, may provide the crucial home-directed information requisite for NCFH.

sentences: An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.

sentences: Botrytis cinerea BcPTP1 is a late infection phase, cysteine rich protein cytotoxic effector Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious crops diseases. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcPTP1) that can also induce immune resistance in plants. BcPTP1 is a small (90 aa), cysteine rich protein without any known domains. Transiently expression of BcPTP1 in leaves caused chlorosis that intensifies with time and eventually lead to cell death. Point mutations in eight of the 10 cysteine residues of BcPTP1 abolished the toxic effect, however residual toxic activity remained after heating the peptide, suggesting contribution of unknown epitopes to protein phytotoxic effect. The transcript level of the bcptp1 gene was low during the first 36 h after inoculation and increased sharply upon transition to the late infection stage, suggesting a role of BcPTP1 in lesion spreading. While statistically insignificant, deletion of the bcptp1 gene led to slightly smaller lesions on bean leaves. Further analyses indicated that BcPTP1 is internalized into plant cells after secreting into the apoplast and its phytotoxic effect is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcPTP1 is a virulence factor that toxifies the host cells and facilitates lesion spreading during the late infection stage.

sentences: Brain gene co-expression networks link complement signaling with convergent synaptic pathology in schizophrenia The most significant common variant association for schizophrenia (SCZ) reflects increased expression of the complement component 4A (C4A). Yet, it remains unclear how C4A interacts with other SCZ risk genes and whether the complement system is more broadly implicated in SCZ pathogenesis. Here, we integrate several existing, large-scale genetic and transcriptomic datasets to interrogate the functional role of the complement system and C4A in the human brain. Surprisingly, we find no significant genetic enrichment among known complement system genes for SCZ. Conversely, brain co-expression network analyses using C4A as a seed gene revealed that genes down-regulated when C4A expression increased exhibit strong and specific genetic enrichment for SCZ risk. This convergent genomic signal reflected neuronal, synaptic processes and was sexually dimorphic and most prominent in frontal cortical brain regions. Overall, these results indicate that synaptic pathways--rather than the complement system--are the driving force conferring SCZ risk.

sentences: Single-Cell Spatial Proteomics Analyses of Head and Neck Squamous Cell Carcinoma Reveal Tumor Heterogeneity and Immune Architectures Associated with Clinical Outcome There is increasing evidence that the spatial organization of cells within the tumor-immune microenvironment (TiME) of solid tumors influences survival and response to therapy in numerous cancer types. Here, we report results and demonstrate the applicability of quantitative single-cell spatial proteomics analyses in the TiME of primary and recurrent human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors. Single-cell compositions of a nine patient, primary and recurrent (n=18), HNSCC cohort is presented, followed by deeper investigation into the spatial architecture of the TiME and its relationship with clinical variables and progression free survival (PFS). Multiple spatial algorithms were used to quantify the spatial landscapes of immune cells within TiMEs and demonstrate that neoplastic tumor-immune cell spatial compartmentalization, rather than mixing, is associated with longer PFS. Mesenchymal (SMA+) cellular neighborhoods describe distinct immune landscapes associated with neoplastic tumor-immune compartmentalization and improved patient outcomes. Results from this investigation are concordant with studies in other tumor types, suggesting that trends in TiME cellular heterogeneity and spatial organization may be shared across cancers and may provide prognostic value in multiple cancer types.

sentences: Detection of ancestry-derived variants of modern Japanese revealed the formation process of regional gradations of the current Japanese archipelago population Modern Japanese have two major ancestral populations: the indigenous Jomon people and continental immigrants. To figure out the formation process of current Japanese population, we developed a reference-free detection method of variants derived from ancestral populations using a summary statistic, the ancestry-marker index (AMI), and identified 208,648 SNPs that appear to have been derived from the Jomon people (Jomon-derived variants). The analysis of Jomon-derived variants in 10,842 modern Japanese individuals recruited from all over Japan revealed that the admixture proportions of the Jomon people varied between prefectures. The estimated allele frequencies of genome-wide SNPs in the ancestral populations of modern Japanese suggested their phenotypic characteristics possibly for adaptation to their respective livelihoods. According to our findings, we propose a formation model of modern Japanese population; regional variations in admixture proportions of the Jomon people and continental East Asians formed genotypic and phenotypic gradations of current Japanese archipelago populations.

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The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Ongoing work has identified several transcriptional regulators of nitrate signaling, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN 7 (NLP7), but additional regulators likely remain to be identified. Here, we characterized Arabidopsis NLP2 as a master upstream transcriptional regulator of early nitrate responses that interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, a DNA binding motif, and some target genes with NLP7. Additional genetic, genomic and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multi-tiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

One-sentence summaryNLP2 and NLP7 orchestrate plant responses to nitrate supply and control nitrate- dependent regulation of carbon and energy metabolism.

sentences: Rapid genomic evolution in Brassica rapa with bumblebee selection in experimental evolution Insect pollinators shape rapid phenotypic evolution of traits related to floral attractiveness and plant reproductive success. However, the underlying genomic changes and their impact on standing genetic variation remain largely unknown despite their importance in predicting adaptive responses in nature or in crops artificial selection. Here, based on a previous, nine generation experimental evolution study with fast cycling Brassica rapa plants adapting to bumblebees, we document genomic evolution associated to the adaptive process. We performed a genomic scan of the allele frequency changes along the genome and estimated the nucleotide diversity and genomic variance changes. We detected signature of selection associated with rapid changes in allelic frequencies on multiple loci. During experimental evolution, we detected an increase in overall genomic variance, whereas for loci under selection, a reduced variance was apparent in both replicates suggesting a parallel evolution. Our study highlights the polygenic nature of short-term pollinator adaptation and the importance of a such genetic architecture in the maintenance of genomic variance during strong natural selection by biotic factors.

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Insularity forcing on plant persistence strategies in edaphic island systems AimTrait-based approaches are increasingly implemented in island biogeography, providing key insights into the eco-evolutionary dynamics of insular systems. However, what determines persistence of plant species once they have arrived and established in an island remains largely unexplored. Here, we examined links between non-acquisitive persistence strategies and insularity across three terrestrial edaphic island systems, hypothesising that insularity promotes strategies for local persistence.

LocationEurope: Western Carpathians, Moravia, and Cantabrian Range.

Time periodPresent.

Major taxa studiedVascular plants.

MethodsFor each system, we used linear models at the island scale to test whether persistence-related plant trait patterns (average trait values and diversity) depend on three insularity metrics (island size, isolation and target effect). We focused on patterns of edaphic island specialists because, in contrast to matrix-derived species, their presence is confined to the edaphic islands.

ResultsWe found that insularity metrics explained large proportions in the variation of the average and diversity of persistence-related traits of edaphic island specialists. Insularity was associated with a decline in the proportion of island specialists that have clonal abilities, yet it affected trait values of specialists towards enhanced abilities to persist locally (e.g. more extensive lateral spread) while reducing trait variability. Higher degrees of insularity within the systems were translated to stronger effects on functional trait patterns.

Main conclusionsInsularity affects plant species diversity, distribution and forms in terrestrial island-like systems, similarly as it is assumed for true islands. Insularity - measured using a single (island size, isolation) or combined (target effect) predictors - may operate selecting for enhanced and less diverse persistence strategies. Ultimately, this process, which we call insularity forcing, operates as a selective process to promote species ability to avoid local extinction and to persist on terrestrial islands.

sentences: Noradrenergic Regulation of Two-Armed Bandit Performance Reversal learning depends on cognitive flexibility. Many reversal learning studies assess cognitive flexibility based on the number of reversals that occur over a test session. Reversals occur when an option is repeatedly chosen, e.g. eight times in a row. This design feature encourages win-stay behavior and thus makes it difficult to understand how win-stay decisions influence reversal performance. We used an alternative design, reversals over blocks of trials independent of performance, to study how perturbations of the medial orbital cortex and the noradrenergic system influence reversal learning. We found that choice accuracy varies independently of win-stay behavior and the noradrenergic system controls sensitivity to positive feedback during reversal learning.

sentences: Attentional brain rhythms during prolonged cognitive activity As routine and lower demand cognitive tasks are taken over by automated assistive systems, human operators are increasingly required to sustain cognitive demand over long periods of time. This has been reported to have long term adverse effects on cardiovascular and mental health. However, it remains unclear whether prolonged cognitive activity results in a monotonic decrease in the efficiency of the recruited brain processes, or whether the brain is able to sustain functions over time spans of one hour and more. Here, we show that during working sessions of one hour or more, contrary to the prediction of a monotonic decline, behavioral performance in both humans and non-human primates consistently fluctuates between periods of optimal and suboptimal performance at a very slow rhythm of circa 5 cycles per hour. These fluctuations are observed in both high attentional (in non-human primates) and low attentional (in humans) demand conditions. They coincide with fluctuations in pupil diameter, indicating underlying changes in arousal and information-processing load. Accordingly, we show that these rhythmic behavioral fluctuations correlate, at the neurophysiological level, with fluctuations in the informational attention orientation and perception processing capacity of prefrontal neuronal populations. We further identify specific markers of these fluctuations in LFP power, LFP coherence and spike-field coherence, pointing towards long-range rhythmic modulatory inputs to the prefrontal cortex rather than a local prefrontal origin. These results shed light on the resilience of brain mechanisms to sustained effort and have direct implications on how to optimize high cognitive demand working and learning environments.

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Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. The regulation of 5 DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and more recently, it was shown to be important for DNA end-bridging and inhibiting 5 resection mediated by Dna2-Sgs1. Nej1 interacts with Sae2 and this impacts DSB repair in three ways. First, Nej1 inhibits MRX-Sae2 interactions and Sae2 localization to a DSB. Second, Nej1 inhibits Sae2-dependent recruitment of Dna2 in the absence of Sgs1. Third, NEJ1 and SAE2 showed an epistatic relationship for DNA end-bridging, an event that restrains the broken ends and reduces the frequency of genomic deletions from developing at the DSB. Deletion of NEJ1 suppressed the synthetic lethality of sae2{Delta} sgs1{Delta} and was dependent on the nuclease activity of Dna2. These Nej1 functions promote end-joining DSB repair, but could also be relevant for controlling resection initiation during HR repair.

HighlightsO_LINej1 physically interacts with Sae2 and inhibits end-resection at a DSB.
C_LIO_LINej1 inhibits Sae2 interactions with the MRX complex.
C_LIO_LINej1 inhibits Sae2-dependent recruitment of Dna2 to a DSB.
C_LIO_LINEJ1 and SAE2 are epistatic for DNA end-bridging.
C_LIO_LIDeletion of NEJ1 suppresses the synthetic lethality of sae2{Delta} sgs1{Delta}, which is dependent on Dna2 nuclease activity.
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sentences: A fractal polymer with loops recapitulates key features of chromosome organization Chromosomes are exceedingly long polymers compacted in a cell nucleus. While it has long been suggested that mammalian chromosomes during interphase are folded into loops, experimental detection of such loops has remained a daunting task. The most comprehensive experimental information about chromosome spatial organization is provided by Hi-C experiments that measure the frequency of contacts between all chromosomal positions. The lack of a tractable physical model of a polymer folded into loops limits our ability to interpret experimental data. It thus remains unknown how to obtain accurate and quantitative information about the nature of chromosomal looping from Hi-C. Here, we introduce a model of a polymer with random loops, solve it analytically and extend it by simulations for real chains. Remarkably, our model faithfully reproduces complex shapes of experimental contact probability curves universal among mammalian cells. Furthermore, our model allows one to estimate sizes of randomly positioned chromosomal loops from experimental data. We also show that excluded volume in real chains can induce osmotic and topological repulsion between loops, stiffening interphase chromosomes. Thus, our new framework allows interpretation of experimental data and suggests that interphase chromosomes are crumpled polymers further folded into a sequence of randomly positioned loops.

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Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes Vector competence influences the ability of Anopheles mosquitoes to transmit Plasmodium parasites. The aim of this study was to determine the competence of male and female Anopheles mosquitoes to support the development of Plasmodium parasites. Male and female A. stephensi (STE2 strain) were infected with in vitro-cultured P. berghei (ANKA strain) ookinetes. We found that the number of oocysts produced was higher in males than in females. RNA-seq analysis of male and female mosquitoes injected with P. berghei ookinetes showed that predominantly genes of unknown function changed in expression levels in response to ookinete infection; however, further studies are required to elucidate their functions. Moreover, male mosquitoes were injected with in vitro-cultured P. falciparum (3D7 strain) gametocytes or zygotes. The development of P. falciparum in males was detected using nested polymerase chain reaction. We found the DNA content was higher on day 15 than on day 0, indicating that P. falciparum developed in the mosquito hemocoel. This study revealed promising new mechanisms underlying the interactions between Plasmodium and mosquitoes.

Author SummaryAnopheles mosquitoes transmit Plasmodium parasites that cause malaria disease. Vector competence is the ability of vectors to support pathogen development. The aim of this study was to determine the competence of male and female mosquitoes for Plasmodium parasites. Oocysts were formed in both male and female mosquitoes injected with ookinetes from in vitro-cultured Plasmodium berghei, which causes malaria in mice. The number of oocysts was higher in males, indicating that the competence in males was higher than that in females. RNA-seq analysis showed that the expression of genes of unknown function was highly variable in males and females. The genes defining the competence factors need further study, but the results indicate novel genes may be discovered. Furthermore, the development of Plasmodium falciparum, which causes the most severe malaria in humans, in male mosquitoes injected with in vitro-cultured gametocytes or zygotes was detected. As male mosquitoes do not suck blood, this method may allow us to experiment with Plasmodium more safely.

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Cytokinin oxidase/dehydrogenase family genes play important roles in the growth and development of rice Cytokinins play key roles in plant growth and development; hence, cytokinin biosynthesis and degradation have been extensively studied. Cytokinin oxidase/dehydrogenases (CKXs) are a group of enzymes that regulate oxidative cleavage to maintain cytokinin homeostasis. In rice, 11 OsCKX genes have been identified to date; however, most of their functions remain unknown. Here, we comprehensively analyzed the expression patterns and functions of OsCKX genes. Using CRISPR/Cas9 technology, we constructed mutants of all OsCKX genes to determine the functions of OsCKXs in rice development. The results revealed that the single osckx and higher-order osckx4 osckx9 mutant lines showed functional overlap and subfunctionalization. Notably, the osckx1 osckx2 and osckx4 osckx9 double mutants displayed contrasting phenotypic changes in tiller number and panicle size compared to the wild type. Moreover, we identified several genes with significantly altered expression in osckx4 and osckx9 single and double mutant plants. Many differentially expressed genes were found to be associated with auxin and cytokinin pathways. Additionally, the cytokinins in osckx4 osckx9 mutants were increased compared to the wild type. Overall, our findings provide new insights into the functions of OsCKX genes in rice growth and may be used as a foundation for future studies aimed at improving rice yield. Key words: Cytokinin, expression pattern, OsCKX, panicle, phenotype, rice, tiller

HighlightThe osckx4 osckx9 double mutant had a significantly greater number of tillers, whereas the osckx1 osckx2 double mutant showed the opposite phenotypic change, compared to the wild type

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Robust effects of working memory demand during naturalistic language comprehension in language-selective cortex A standard view of human language processing is that comprehenders build richly structured mental representations of natural language utterances, word by word, using computationally costly memory operations supported by domain-general working memory resources. However, three core claims of this view have been questioned, with some prior work arguing that (1) rich word-by-word structure building is not a core function of the language comprehension system, (2) apparent working memory costs are underlyingly driven by word predictability (surprisal), and/or (3) language comprehension relies primarily on domain-general rather than domain-specific working memory resources. In this work, we simultaneously evaluate all three of these claims using naturalistic comprehension in fMRI. In each participant, we functionally localize (a) a language-selective network and (b) a  multiple-demand network that supports working memory across domains, and we analyze the responses in these two networks of interest during naturalistic story listening with respect to a range of theory-driven predictors of working memory demand under rigorous surprisal controls. Results show robust surprisal-independent effects of word-by-word memory demand in the language network and no effect of working memory demand in the multiple demand network. Our findings thus support the view that language comprehension (1) entails word-by-word structure building using (2) computationally intensive memory operations that are not explained by surprisal. However, these results challenge (3) the domain-generality of the resources that support these operations, instead indicating that working memory operations for language comprehension are carried out by the same neural resources that store linguistic knowledge.

Significance StatementThis study uses fMRI to investigate signatures of working memory (WM) demand during naturalistic story listening, using a broad range of theoretically motivated estimates of WM demand. Results support a strong effect of WM demand in language-selective brain regions but no effect of WM demand in "multiple demand" regions that have previously been associated with WM in non-linguistic domains. We further show evidence that WM effects in language regions are distinct from effects of word predictability. Our findings support a core role for WM in incremental language processing, using WM resources that are specialized for language.

sentences: Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe. Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using Genotyping-by-Sequencing (GBS)) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g. Iberia). In addition, we tested multiple post-glacial re-colonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to an increased mammalian fauna on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.

sentences: Exposure to 1800 MHz LTE electromagnetic fields under proinflammatory conditions decreases the response strength and increases the acoustic threshold of auditory cortical neurons. Increased needs in mobile phone communications have raised successive generations (G) of wireless technologies, which could differentially affect biological systems. We have assessed how a single 2h head-only exposure to a 4G long term evolution (LTE)-1800 MHz electromagnetic field (EMF) impacts on the microglial space coverage and the electrophysiological neuronal activity in the primary auditory cortex (ACx) in rats submitted to an acute neuroinflammation induced by lipopolysaccharide. The mean specific absorption rate in the ACx was 0.5 W/kg. Multiunit recording revealed that LTE-EMF triggered reduction in the response strength to pure tones and to natural vocalizations, together with an increase in acoustic threshold in the low and medium frequencies. Iba1 immunohistochemistry showed no change in the area covered by microglia cell bodies and processes. In healthy rats, the same LTE-exposure induced no change in response strength and acoustic threshold. Our data indicate that an acute neuroinflammation sensitize neuronal responses to LTE-EMF, which leads to an altered processing of acoustic stimuli in the ACx.

sentences: Gap genes are involved in inviability in hybrids between Drosophila melanogaster and D. santomea Evolved changes within species lead to the inevitable loss of viability in hybrids. Inviability is also a convenient phenotype to genetically map and validate functionally divergent genes and pathways differentiating closely related species. Here we identify the Drosophila melanogaster form of the highly conserved essential gap gene giant (gt) as a key genetic determinant of hybrid inviability in crosses with D. santomea. We show that the coding region of this allele in D. melanogaster/D. santomea hybrids is sufficient to cause embryonic inviability not seen in either pure species. Further genetic analysis indicates that tailless (tll), another gap gene, is also involved in the hybrid defects. giant and tll are both members of the gap gene network of transcription factors that participate in establishing anterior-posterior specification of the dipteran embryo, a highly conserved developmental process. Genes whose outputs in this process are functionally conserved nevertheless evolve over short timescales to cause inviability in hybrids.

sentences: Breast cancer stem cell-derived tumors escape from γδ T cell immunosurveillance in vivo by modulating γδ T cell ligands Triple negative breast cancer (TNBC) lacks targeted therapy options. TNBC is enriched in breast cancer stem cells (BCSCs), which play a key role in metastasis, chemoresistance, relapse and mortality. {gamma}{delta} T cells hold great potential in immunotherapy against cancer, and might be an alternative to target TNBC. {gamma}{delta} T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor sensing, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. We show that patient-derived triple negative BCSCs are efficiently recognized and killed by ex vivo expanded {gamma}{delta} T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to {gamma}{delta} T cell immunotherapy. Mechanistically, we unraveled concerted differentiation and immune escape: xenografted BCSCs lost stemness, expression of {gamma}{delta} T cell ligands, adhesion molecules and pAgs, thereby evading immune recognition by {gamma}{delta} T cells. Indeed, neither pro-migratory engineered {gamma}{delta} T cells, nor anti-PD-1 checkpoint blockade significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the {gamma}{delta} T cells, and could be pharmacologically reverted by Zoledronate or IFN- treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.

sentences: A causal analysis of the effect of age and sex differences on brain atrophy in the elderly brain We study how brain volume loss at old age is affected by factors such as age, APOE gene, sex, and school level. The quantitative characterization of brain volume loss at old age relative to young age requires at least in principle two MRI scans performed at both young and old age. There is, however, a way to address the problem by having only one MRI scan at old age. We compute the total brain loss of elderly subjects as the ratio between the estimated brain volume and the estimated total intracranial volume. Magnetic resonance imaging (MRI) scans of 890 healthy subjects aged 70 to 85 were assessed. The causal analysis of factors affecting brain atrophy was performed using Probabilistic Bayesian Modeling and the Mathematics of Causal Inference. We find that healthy subjects get into their seventies with an average brain volume loss of 30% from their maximum brain volume at a young age. Both age and the sexes are causally related to brain atrophy, with women getting to elderly age with 1% larger brain volume relative to intracranial volume than men. How the brain ages and what are the reasons for sex differences in adult lifespan are causal questions that need to be addressed with causal inference and empirical data. The graphical causal modeling presented here can be instrumental in understanding a puzzling scientific inquiry -the biological age of the brain.

sentences: Defensive and offensive behaviors in social interaction settings in a Kleefstra syndrome mouse model Kleefstra syndrome in humans is characterized by general delay in development, intellectual disability and autistic features. The mouse model of this disease (Ehmt1+/-) expresses anxiety, autistic-like traits, and aberrant social interactions with non-cagemates. To investigate how Ehmt1+/- mice behave with unfamiliar conspecifics, we allowed adult, male animals to freely interact for 10 minutes in a neutral, novel environment within a host-visitor setting. In 17 out of 74 trials there were defensive and offensive behaviors. Our key finding was that Ehmt1+/- mice displayed defensive postures, attacking and biting; in contrast, wild-type (WT) interacting with other WT did not enact such behaviors. Further, if there was a fight between an Ehmt1+/- and a WT mouse, it was always the Ehmt1+/- who initiated these behaviors.

sentences: Network instability dynamics drive a transient bursting period in the developing hippocampus in vivo Spontaneous correlated activity is a universal hallmark of immature neural circuits. However, the cellular dynamics and intrinsic mechanisms underlying neuronal synchrony in the intact developing brain are largely unknown. Here, we use two-photon Ca2+ imaging to comprehensively map the developmental trajectories of spontaneous network activity in hippocampal area CA1 in vivo. We unexpectedly find that synchronized activity peaks after the developmental emergence of effective synaptic inhibition in the second postnatal week. We demonstrate that the enhanced network synchrony reflects an increased functional coupling of individual neurons to local population activity. However, pairwise neuronal correlations are low, and network bursts recruit CA1 pyramidal cells in a virtually random manner. Using a dynamic systems modeling approach, we reconcile these experimental findings and identify network bi-stability as a potential regime underlying network burstiness at this age. Our analyses reveal an important role of synaptic input characteristics and network instability dynamics for the emergence of neuronal synchrony. Collectively, our data suggest a mechanism, whereby developing CA1 performs extensive input-discrimination learning prior to the onset of environmental exploration.

sentences: Sex-specific Stress and Behavioural Responses to Human Experimenters in Rats The sex of the experimenter may cause stress in animal models and be a major confounding factor in preclinical research. We studied the effects of the sex of the experimenter on female and male rat anxiety behaviours using thigmotaxis in the open field test, anxiety-induced changes in brain and back temperature using infra-red thermography, and alterations in plasma concentrations of stress hormones, corticosterone and oxytocin. Female rats displayed consistently exacerbated anxiety-related behaviours along with increased infrared cutaneous temperature during repeated exposure to male experimenters. Experimental stress further intensified thermal responses to a male experimenter, especially in female rats. These behavioural responses to a male experimenter in females were associated with higher circulating corticosterone and lower oxytocin levels. Similar responses were induced by a T-shirt worn by a human male. These findings suggest that emotional and physiological responses of female rats to a male experimenter are influenced by visual and olfactory cues. These results emphasize the need to standardize and report experimenter sex throughout a study to avoid ambiguity in interpretation of the results.

sentences: runBioSimulations: an extensible web application that simulates a wide range of computational modeling frameworks, algorithms, and formats Comprehensive, predictive computational models have significant potential for science, bioengineering, and medicine. One promising way to achieve more predictive models is to combine submodels of multiple subsystems. To capture the multiple scales of biology, these submodels will likely require multiple modeling frameworks and simulation algorithms. Several community resources are already available for working with many of these frameworks and algorithms. However, the variety and sheer number of these resources make it challenging to find and use appropriate tools for each model, especially for novice modelers and experimentalists. To make these resources easier to use, we developed runBioSimulations (https://run.biosimulations.org), a single web application for executing a broad range of models. runBioSimulations leverages community resources, including BioSimulators, a new open registry of simulation tools. These resources currently enable runBioSimulations to execute nine frameworks and 44 algorithms, and they make runBioSimulations extensible to additional frameworks and algorithms. runBioSimulations also provides features for sharing simulations and interactively visualizing their results. We anticipate that runBioSimulations will foster reproducibility, stimulate collaboration, and ultimately facilitate the creation of more predictive models.

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NIMAA: an R/CRAN package to accomplish NomInal data Mining AnAlysis SummaryNominal data is data that has been "labeled" and can be designated into a number of non-overlapping unordered groups. The analysis of this type of data is often trivial because it is not feasible to conduct extensive numerical methods on this type of data. Graphs or networks, on the other hand, are comprised of sets of nodes and edges that can also be considered as nominal variables. By integrating graph theory and data mining approaches, we offer the R package NIMAA to define a nominal data-mining pipeline to explore more information. Using nominal variables in a dataset, NIMAA provides functions for constructing weighted and unweighted bipartite graphs, analysing the similarity of labels in nominal variables, clustering labels or categories to super-labels, validating clustering results, predicting bipartite edges by missing weight imputation, and providing a variety of visualization tools. Here, we also indicated the application of nominal data mining in a biological dataset with well-riched nominal variables.

AvailabilityNIMAAs official release and the beta update are available on CRAN and Github, respectively. URLs: https://CRAN.R-project.org/package=NIMAA and https://github.com/jafarilab/NIMAA

Contactmohieddin.jafari@helsinki.fi; jing.tang@helisnki.fi

ContributionsMJ conceived the study and developed the models, MJ and CC adopted and implemented the methods, MM improved the methods, JT provided the funding, MJ, CC, MM and JT wrote the paper.

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Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum The non-vascular plant Marchantia polymorpha has emerged as a valuable model for studying evolutionarily conserved microbial infection strategies and plant immune responses. However, only a handful of fungal pathogens of Marchantia have been described so far. Here we establish a new pathosystem using the root-infecting vascular wilt fungus Fusarium oxysporum. On angiosperms, this fungus exhibits exquisite adaptation to the plant vascular niche and host-specific pathogenicity, both of which are conferred by lineage-specific effectors secreted during growth in the xylem. We show that F. oxysporum isolates with different lifestyles - pathogenic or endophytic - are able to infect this non-vascular liverwort causing tissue maceration and plant cell killing. Similar to bacterial pathogens, F. oxysporum induces a PAMP-triggered immune response in M. polymorpha. Analysis of isogenic fungal mutants established that infection of Marchantia requires conserved fungal pathogenicity mechanisms such as mitogen activated protein kinases, transcriptional regulators and cell wall remodeling enzymes. Remarkably, lineage-specific virulence effectors are dispensable for infection, most likely due to the absence of xylem tissue in this non-vascular plant. The F. oxysporum - M. polymorpha system provides new insights into the mechanism and evolution of pathogenic and endophytic fungus-plant interactions.

Significance statementRoot-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they infect non-vascular plants, which diverged from vascular plants over 450 million years ago. Here we show that two strains of the fungus Fusarium oxysporum with opposed lifestyles, causing either wilting and death or beneficial protection on tomato, produce similar disease symptoms on the non-vascular plant Marchantia polymorpha. We define a set of core fungal pathogenicity factors required on both vascular and non-vascular plants and show that host-specific effectors contributing to disease on tomato are dispensable on Marchantia. These findings suggest that systemic wilt disease evolved in fungal pathogens after the emergence of vascular land plants.

sentences: Macrophage depletion blocks congenital SARM1-dependent neuropathy Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms result in amino acid substitutions, V98M and R232Q, which reduce NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survive to adulthood but develop progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes are all SARM1-dependent. Remarkably, macrophage depletion therapy blocks and reverses neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

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

sentences: Single-molecule imaging of microRNA-mediated gene silencing in cells MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying mRNA regulation at the single-molecule level with spatiotemporal information inside cells.

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From group to individual - Genotyping by pool sequencing eusocial colonies BackgroundEusocial insects play a central role in many ecosystems, and particularly the important pollinator honeybee (Apis mellifera). One approach to facilitate their study in molecular genetics, is to consider whole colonies as single individuals by combining DNA of multiple individuals in a single pool sequencing experiment. Such a technique comes with the drawback of producing data requiring dedicated analytical methods to be fully exploited. Despite this limitation, pool sequencing data has been shown to be informative and cost-effective when working on random mating populations. Here, we present new statistical methods for exploiting pool sequencing data of eusocial colonies in order to reconstruct the genotype of the colony founder, the queen. This leverages the possibility to monitor genetic diversity, perform genomic-based studies or implement selective breeding.

ResultsUsing simulations and honeybee real data, we show that the methods allow for a fast and accurate estimation of the genetic ancestry, with correlations of 0.9 with that obtained from individual genotyping, and for an accurate reconstruction of the queen genotype, with 2% genotyping error. We further validate the inference using experimental data on colonies with both pool sequencing and individual genotyping of drones.

ConclusionIn this study we present statistical models to accurately estimate the genetic ancestry and reconstruct the genotype of the queen from pool sequencing data from workers of an eusocial colony. Such information allows to exploit pool sequencing for traditional population genetics, association studies and selective breeding. While validated in Apis mellifera, these methods are applicable to other eusocial hymenoptera species.

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Before trilobite legs: Pygmaclypeatus daziensis reconsidered and the ancestral appendicular organization of Cambrian artiopods The Cambrian Stage 3 Chengjiang biota in South China is one of the most influential Konservat-Lagerstatten worldwide thanks to the fossilization of diverse non-biomineralizing organisms through pyritization. Despite their contributions to understanding the evolution of early animals, several Chengjiang species remain poorly known due to their scarcity and/or incomplete preservation. Here, we use micro-computed tomography to reveal in detail the ventral appendage organization of the enigmatic non-trilobite artiopod Pygmaclypeatus daziensis - one of the rarest euarthropods in Chengjiang - and explore its functional ecology and broader evolutionary significance. Pygmaclypeatus daziensis possesses a set of uniramous antennae and 14 pairs of post-antennal biramous appendages, the latter of which show an unexpectedly high degree of heteronomy based on the localized differentiation of the protopodite, endopodite and exopodite along the antero-posterior body axis. The small body size (less than 2 cm), presence of delicate spinose endites, and well-developed exopodites with multiple paddle-shaped lamellae on the appendages of P. daziensis indicate a nekto-benthic mode of life, and a scavenging/detritus feeding strategy. Pygmaclypeatus daziensis shows that appendage heteronomy is phylogenetically widespread within Artiopoda - the megadiverse clade that includes trilobites and their relatives with non-biomineralizing exoskeletons - and suggests that a single exopodite lobe with paddle-like lamellae is ancestral for this clade.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/456779v1_ufig1.gif" ALT="Figure 1">
View larger version (111K):
org.highwire.dtl.DTLVardef@b4ddaaorg.highwire.dtl.DTLVardef@44f686org.highwire.dtl.DTLVardef@1950095org.highwire.dtl.DTLVardef@1596739_HPS_FORMAT_FIGEXP  M_FIG C_FIG Cover image: Morphological reconstruction of the non-trilobite artiopod Pygmaclypeatus daziensis from the early Cambrian (Stage 3) Chengjiang biota in south China. Artwork by Holly Sullivan (https://www.sulscientific.com/).

sentences: IL-33 regulates age-dependency of long-term immune dysfunction induced by sepsis Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility to secondary infections. However, the long-term immune consequences of pediatric sepsis are unknown. Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. In contrast to adults, infant mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in post-septic adults but not infant mice. Impaired IL-33 production in post-septic infant mice was associated with increased DNA-methylation on lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in infant mice. Clinically, adults but not pediatric post-septic patients exhibited higher counts of Tregs and sera IL-33 levels. Hence, we describe a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression.

sentences: Ammonium regulates the development and architecture of pine roots through hormonal crosstalk and differential expression of transcription factors in the apex Ammonium is a prominent source of inorganic nitrogen for plant nutrition, but excessive amounts can be toxic for many species. However, most conifers are tolerant to ammonium, a relevant physiological feature of this ancient evolutionary lineage. For a better understanding of the molecular basis of this trait, ammonium-induced changes in the transcriptome of maritime pine (Pinus pinaster Ait.) root apex have been determined by laser capture microdissection and RNA sequencing. Ammonium promoted changes in the transcriptional profiles of multiple transcription factors, such as SHORT-ROOT, and phytohormone-related transcripts, such as ACO, involved in the development of the root meristem. Nano-PALDI-MSI and transcriptomic analyses showed that the distributions of IAA and CKs were altered in the root apex in response to ammonium nutrition. Taken together, the data suggest that this early response is involved in the increased lateral root branching and principal root growth, which characterize the long-term response to ammonium supply in pine. All these results suggest that ammonium induces changes in the root system architecture through the IAA-CK-ET phytohormone crosstalk and transcriptional regulation.

sentences: Priority effects among amphibian microbes lead to varying levels of Bd infection Priority effects in host-associated microbiomes can influence not only community composition and structure, but also community functions, such as disease resistance. However, evidence for these priority effects remains scarce. Past studies suggest that amphibian protection from chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is related to antifungal bacterial composition on host skin. Priority effects in these bacterial communities may influence susceptibility to Bd, but this possibility has not been tested. Using in vitro microcosms, we demonstrated that priority effects can influence interactions among amphibian-associated microbes. We observed strong priority effects irrespective of high antifungal ability such that the Bd-inhibitory potential of two strongly inhibitory bacterial species did not always produce higher levels of Bd-inhibition. This result suggests that interactions may be more complex than previously thought. Additionally, our results suggest that priority effects between commensal and pathogenic taxa can be either facilitatory or inhibitory, with the strength and direction of this effect dependent on the composition of the community. Thus, changes in assembly may lead to varying levels of Bd infection, influencing how we might augment amphibian-associated microbiomes to conserve taxa currently at risk of extinction.

sentences: NF90 Interacts with Components of RISC and Modulates Association of Ago2 with mRNA Nuclear Factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3 UTRs, NF90 association was increased upon loss of MOV10 and vice versa, suggesting that the two proteins may compete for the binding of common target mRNAs. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. These findings suggest a role for NF90 in the regulation of RISC-mediated silencing which stabilizes target mRNAs, such as VEGF, during cancer-induced hypoxia.

sentences: Non-cell autonomous OTX2 transcription factor regulates anxiety-related behaviors in the mouse The Otx2 homeoprotein transcription factor is expressed in the dopaminergic neurons of the ventral tegmental area, a mesencephalic nucleus involved in the control of complex behaviors through its projections to limbic structures, including the ventral hippocampus, amygdala, nucleus accumbens and medial prefrontal cortex. We find adult mice heterozygous for Otx2 show anxiolysis-like phenotype in light-dark box and elevated plus maze paradigms. However, the number of dopaminergic neurons, the integrity of their axons, their projection patterns in target structures, and the amounts of dopamine and dopamine metabolites in targets structures were not modified. Because OTX2 is expressed by the choroid plexus, secreted into cerebrospinal fluid and transferred to parvalbumin interneurons of the cortex, hippocampus, and amygdala, we investigated if this phenotype could result from the decreased synthesis of OTX2 in the choroid plexus. Indeed, the anxiolysis-like phenotype was partially recapitulated in the Otx2+/AA and scFvOtx2tg/0 choroid-plexus dependent non-cell-autonomous OTX2 loss of function mouse models. Furthermore, the phenotype was reversed by the overexpression of Otx2 specifically in choroid plexus of adult Otx2 heterozygous mice. Taken together, OTX2 synthesis by the choroid plexus followed by its secretion into the cerebrospinal fluid is an important regulator of the anxiety phenotype in the mouse.

sentences: Out-of-sight or out-of-site? Forays and detection probabilities in single-season occupancy models Occupancy models have become popular in wildlife survey analyses because they account for the frequent failure to detect individuals of targeted species. Those individuals sometimes move outside sampling sites, i.e. exhibit temporary emigration. In such cases, occupancy models may become difficult to interpret or even misleading either at the species or the individual level, because they confound presence at the site, availability for detection given presence, and actual detection by the observer. We quantified the probabilities of these three components with spruce grouse (Falcipennis canadensis) in southern Quebec, Canada. We conducted call-response surveys of 24 grouse monitored by radio-telemetry. We defined sites empirically as circular areas of 83 m radius centered on the observer, corresponding to the maximum detection distance obtained. Based on telemetry locations, grouse were present at the site during 42 % of the surveys. Six stationary grouse were present during surveys, but were never detected. Thus, only individuals that moved in the presence of the observer (89 %) were considered available for detection. Individuals available for detection were detected in 51 % of the cases. We simulated detection histories and built single-season occupancy models, based on the empirical relationship between detection probability and the distance measured between observers and grouse. When temporary emigration was ignored, site occupancy was{psi} = 0.89, and the associated probability of detection was p = 0.23. When instances of temporary emigration were dropped, estimates were{psi} = 0.88 and p = 0.41. Using only grouse available for detection, estimates were{psi} = 0.87 and p = 0.42. Disentangling the components of detection probabilities had little impact on occupancy estimates, but showed a major effect of temporary emigration on estimated detection probabilities.

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

sentences: Centriole-independent centrosome assembly in interphase mammalian cells AbstractThe major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein{-}mediated PCM transport, but the self-assembly properties of PCM in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based PCM transport and PCM self-clustering are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase PCM self-assembly depends on {gamma}-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152 or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor-and microtubule-based rearrangement.

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

sentences: Genetically Engineered Strains for Combinatorial Cardiovascular Optobiology Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage -specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (opto1AR) and cAMP (opto{beta}2AR), or Ca2+- permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in HCN4BAC-CatCh2/MHC-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

sentences: TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields Automated visual tracking of animals is rapidly becoming an indispensable tool for the study of behavior. It offers a quantitative methodology by which organisms sensing and decision-making can be studied in a wide range of ecological contexts. Despite this, existing solutions tend to be challenging to deploy in practice, especially when considering long and/or high-resolution video-streams. Here, we present TRex, a fast and easy-to-use solution for tracking a large number of individuals simultaneously using background-subtraction with real-time (60Hz) tracking performance for up to approximately 256 individuals and estimates 2D visual-fields, outlines, and head/rear of bilateral animals, both in open and closed-loop contexts. Additionally, TRex offers highly-accurate, deep-learning-based visual identification of up to approximately 100 unmarked individuals, where it is between 2.5-46.7 times faster, and requires 2-10 times less memory, than comparable software (with relative performance increasing for more organisms/longer videos) and provides interactive data-exploration within an intuitive, platform-independent graphical user-interface.

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Prediction of therapy outcomes of CLL using gene expression intensity, clustering, and ANN classification of single cell transcriptomes BackgroundSingle cell transcriptomics is a new technology that enables us to measure the expression levels of genes from an individual cell. The expression information reflects the activity of that individual cell which could be used to indicate the cell types. Chronic lymphocytic leukemia (CLL) is a malignancy of B cells, one of the peripheral blood mononuclear cells subtypes. We applied five analytical tools for the study of single cell gene expression in CLL course of therapy. These tools included the analysis of gene expression distributions - median, interquartile ranges, and percentage above quality control (QC) threshold; hierarchical clustering applied to all cells within individual single cell data sets; and artificial neural network (ANN) for classification of healthy peripheral blood mononuclear cell (PBMC) subtypes. These tools were applied to the analysis of CLL data representing states before and during the therapy.

ResultsWe identified patterns in gene expression that distinguished two patients that had complete remission (complete response), a patient that had a relapse, and a patient that had partial remission within three years of Ibrutinib therapy. Patients with complete remission showed a rapid decline of median gene expression counts, and the total number of gene counts below the QC threshold for healthy cells (670 counts) in 80% of more of the cells. These patients also showed the emergence of healthy-like PBMC cluster maps within 120 days of therapy and distinct changes in predicted proportions of PBMC cell types.

ConclusionsThe combination of basic statistical analysis, hierarchical clustering, and supervised machine learning identified patterns from gene expression that distinguish four CLL patients treated with Ibrutinib that experienced complete remission, partial remission, or relapse. These preliminary results suggest that new bioinformatics tools for single cell transcriptomics, including ANN comparison to healthy PBMC, offer promise in prognostics of CLL.

sentences: Relationship between monomer packing, receptor binding domain pocket status, and pH, in the spike trimer of SARS-CoV-2 variants Existence of a SARS-CoV-2 spike protein trimer form with closer packing between monomers when receptor binding domains (RBDs) are all down, locked as opposed to closed, has been associated with linoleic acid (LA) binding at neutral pH, or can occur at acidic pH in the absence of LA binding. The relationship between degree of closure of the LA binding pocket of the RBD, and monomer burial in the trimer, is examined for a range of spike protein structures, including those with D614G mutation, and that of the Delta variant (which also carries D614G). Some spike protein structures with this aspartic acid mutation show monomer packing approaching that of the locked form (at neutral pH, without LA binding) for two segments, a third (around the RBD) remains less closely packed. Mutations in the RBD are a focus for the Omicron variant spike protein. Structure reports suggest that these mutations are involved in increased RBD-RBD interactions, and also that they could lead to a closing of the LA pocket, both of which could impact on pH-dependence. One potential outcome is that the extent of pH-dependent conformational transitions of the pre-fusion SARS-CoV-2 spike trimer are reduced in the Omicron variant.

sentences: Multiplexed triage of candidate biomarkers in plasma using internal standard triggered-parallel reaction monitoring mass spectrometry Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered-parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex assay coupling immunodepletion and fractionation with IS-PRM was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

sentences: Local protein synthesis of mtIF3 regulates mitochondrial translation for axonal development Mitochondrial initiation factor 3 (mtIF3) binds to and dissociates mitochondrial ribosomes. The mtIF3-small subunit complex then recruits mtIF2, mRNA, and N-formylmethionine-tRNA to initiate mitochondrial translation. Intriguingly, transcripts of the nuclear-encoded mtIF3 gene have been shown present in axonal growth cones; however, the biological function of this compartmentalization remains largely unknown. Here, we demonstrate that brain-derived neurotrophic factor (BDNF) induces local translation of mtIF3 mRNA in axonal growth cones. Subsequently, mtIF3 protein is translocated into axonal mitochondria and promotes mitochondrial translation as assessed by our newly developed bimolecular fluorescence complementation sensor for the assembly of mitochondrial ribosomes. We further show that BDNF-induced axonal growth requires mtIF3-dependent mitochondrial translation in axons. These findings provide new insight into how neurons adaptively control mitochondrial physiology and axonal development via local mtIF3 translation.

sentences: Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm The circadian clock regulates extracellular matrix (ECM) homeostasis but whether altered ECM homeostasis regulates the clock was unknown. Thus, we generated mice with a Col1a2-Cre-ERT2 conditional deletion of matrix metalloproteinase-14 (Mmp14) to disrupt matrix homeostasis in postnatal mice. We show here that tamoxifen-treated Col1a2-Cre-ERT2::Mmp14F/F mice developed a severe tendon matrix phenotype with dorsiflexion of hind limbs, tissue thickening, and accumulation of hydroxylysine aldehyde (pyridinoline) crosslinked narrow-diameter collagen fibrils typically seen accumulating during embryonic development. Proteomic analysis of tamoxifen-treated postnatal mice showed [~]16% of detected proteins lost (176 proteins) or reversed (19 proteins) time-dependent rhythmicity. Ontological analysis identified  cytoskeleton as an affected system. Subsequent studies in CRISPR-Cas9 Mmp14 null cells showed decreased F-/G-actin ratio, decreased cell spreading, elevated levels of pyridinoline crosslink factor Plod2, nuclear translocation of transcription factor Pitx2, and loss of circadian rhythm. Thus, our study shows a bidirectional dependence of collagen-ECM homeostasis and the circadian clock.

sentences: Outer hair cell electro-mechanical power conversion Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function requires expression of the protein prestin in the OHC lateral membrane and mechano-electrical transduction in the apical hair bundle. In the present report, electrical power consumption and mechanical power output are determined using previously published voltage clamp data from isolated OHCs and membrane patches. Results reveal that power output by the prestin-motor complex peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90{degrees} shift in the phase of prestin-dependent electrical charge displacement, manifested electrically as emergence of imaginary-valued nonlinear capacitance.

sentences: Effect of different ambient temperatures on reproductive outcome and wellbeing of lactating females in two mouse strains Ambient temperature is an important non-biotic environmental factor influencing immunological and oncological parameters in laboratory mice. It is under discussion which temperature is more appropriate and whether the commonly used room temperature in rodent facilities of about 21{degrees}C represents a chronic cold stress or the 30{degrees}C of the thermoneutral zone constitutes heat stress for the animals. In this study we selected the physiological challenging period of lactation to investigate the influence of a cage temperature of 20{degrees}C, 25{degrees}C, and 30{degrees}C, respectively, on reproductive performance and stress hormone levels in two frequently used mouse strains. We found that more pups were weaned from B6D2F1 hybrids compared to C57BL/6N mothers and that the number of weaned pups was strongly reduced if mothers of both strains were kept at 30{degrees}C. Furthermore, at 30{degrees}C mothers and pups showed reduced body weight at weaning and offspring had longer tails. Despite pronounced temperature effects on reproductive parameters, we did not find any impact on adrenocortical activity in breeding and control mice. Independent of the ambient temperature however, we found that females raising pups showed elevated levels of fecal corticosterone metabolites (FCMs) compared to controls. Increased levels of stress hormone metabolites were measured specially around birth and during the third week of lactation. Our results provide no evidence for reduced or improved wellbeing of lactating mice at different ambient temperatures, but we found that a 30{degrees}C cage temperature impairs reproductive performance.

sentences: Single-synapse analyses of Alzheimers disease implicate pathologic tau, DJ1, CD47, and ApoE Synaptic molecular characterization is limited for Alzheimers disease (AD). We used mass cytometry to quantify 38 probes in approximately 17 million single synaptic events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), non-human primates (NHP), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, A{beta} was in PS/APP mice synaptic events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed novel findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue.

sentences: Strategies to assure optimal trade-offs among competing objectives for genetic improvement of soybean Plant breeding is a decision making discipline based on understanding project objectives. Genetic improvement projects can have two competing objectives: maximize rate of genetic improvement and minimize loss of useful genetic variance. For commercial plant breeders competition in the marketplace forces greater emphasis on maximizing immediate genetic improvements. In contrast public plant breeders have an opportunity, perhaps an obligation, to place greater emphasis on minimizing loss of useful genetic variance while realizing genetic improvements. Considerable research indicates that short term genetic gains from Genomic Selection (GS) are much greater than Phenotypic Selection (PS), while PS provides better long term genetic gains because PS retains useful genetic diversity during the early cycles of selection. With limited resources must a soybean breeder choose between the two extreme responses provided by GS or PS? Or is it possible to develop novel breeding strategies that will provide a desirable compromise between the competing objectives? To address these questions, we decomposed breeding strategies into decisions about selection methods, mating designs and whether the breeding population should be organized as family islands. For breeding populations organized into islands decisions about possible migration rules among family islands were included. From among 60 possible strategies, genetic improvement is maximized for the first five to ten cycles using GS, a hub network mating design in breeding populations organized as fully connected family islands and migration rules allowing exchange of two lines among islands every other cycle of selection. If the objectives are to maximize both short-term and long-term gains, then the best compromise strategy is similar except a genomic mating design, instead of a hub networked mating design, is used. This strategy also resulted in realizing the greatest proportion of genetic potential of the founder populations. Weighted genomic selection applied to both non-isolated and island populations also resulted in realization of the greatest proportion of genetic potential of the founders, but required more cycles than the best compromise strategy.

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Effect of levodopa on human brain connectome in Parkinsons disease ObjectiveLevodopa-based drugs are widely used for mitigating the complications induced by PD. Despite the positive effects, several issues regarding the way that levodopa changes brain activities have remained unclear.

MethodsA combined strategy using EEG data and graph theory was used for investigating how levodopa changed connectome and processing hubs of the brain during resting-state. Obtained results were subjected to ANOVA test and multiple-comparison post-hoc correction procedure.

ResultsResults showed that graph topology of PD patients was not significantly different with the healthy group during eyes-closed condition while in eyes-open condition statistical significant differences were found. The main effect of levodopa medication was observed for gamma-band activity of the brain in which levodopa changed the brain connectome toward a star-like topology. Considering the beta subband of EEG data, graph leaf number increased following levodopa medication in PD patients. Enhanced brain connectivity in gamma band and reduced beta band connections in basal ganglia were also observed after levodopa medication. Furthermore, source localization using dipole fitting showed that levodopa prescription suppressed the activity of collateral trigone.

ConclusionOur combined EEG and graph analysis showed that levodopa medication changed the brain connectome, especially in the high-frequency range of EEG (beta and gamma).

HighlightsO_LINo differences were found between graph features between ON and OFF PD cases in eyes-closed.
C_LIO_LILevodopa enhanced connectivity in gamma band in PD patients.
C_LIO_LILevodopa inhibited brain connectivity in beta band.
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Effect of RNA preservation methods on RNA quantity and quality of field collected avian whole blood A limitation of comparative transcriptomic studies of wild avian populations continues to be sample acquisition and preservation to achieve resulting high-quality RNA (i.e., ribonucleic acids that transfers, translates, and regulates the genetic code from DNA into proteins). Field sampling of wild bird samples provides challenges as RNA degradation progresses quickly and because cryopreservation is often not feasible at remote locations. We collected blood samples from songbirds, as avian blood is nucleated and provides sufficient transcriptionally active material in a small and non-lethal sample, to compare the efficacy of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at differing concentrations along with a dry ice-based flash freezing method (Isopropanol 99% and dry ice mixture, -109{degrees}C). Each blood sample was divided among five different preservation treatments (dry ice-based flash freezing, RNAlater with 1:5 or 1:10 dilution, or DNA/RNA Shield with 1:2 or 1:3 dilution). A new protocol was optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of small passerines. We quantified quality measures, RNA integrity numbers (RINe), rRNA ratios, and total RNA concentrations. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations, provide sample protection from RNA degradation. We suggest caution against using dry ice-based flash-freezing alone for samples preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial samples volumes and carryover across processing steps.

LAY SUMMARYO_LIPreserving RNA samples collected in field conditions, under extreme or variable conditions, remains a challenge and continues to limit the study of gene expression in wild birds.
C_LIO_LIWe optimized RNA extractions for small-volume whole avian blood samples.
C_LIO_LIWe compared commonly used RNA preservations methods, commercial RNA buffers at varying concentrations along with a dry ice-based flash freezing method, for field collected blood bird samples.
C_LIO_LIAll RNA buffers preserved samples resulted in high quality RNA extractions suitable for downstream RNA sequencing, while dry ice-based flash freezing resulted in less reliable sample preservation.
C_LIO_LIOur results support the feasibility of blood sampling as a method of non-lethal sampling that will enable the increased usage RNA sequencing in wild bird studies.
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KEY TERMSO_ST_ABSRNA-SeqC_ST_ABShigh-throughput sequencing which profiles RNA molecules in a sample at the collection timepoint. Provides content and abundance information and may identify novel genes and isoforms. Prior to sequencing, library preparation may either target mRNA or total RNA with the use of enrichment or depletion steps.

Transcriptomeall RNA transcripts, both coding and non-coding, in an individual.

rRNA ratio (28S/18S)commonly used ratio of 28S and 18S ribosomal RNA (rRNA) (band sizes or peak area ratio) that informs the integrity of RNA. Appropriate ratios vary by taxonomic groups and associated rRNA sizes. Acceptable ratios of 28S to 18S are approximately 2.0 for eukaryotes.

Electropherograma quantitative distribution of fragment size.

Qubit 4 Fluorometerfluorescence-based quantification of RNA molecule concentration using fluorescent intercalating dyes which bind to target molecules.

Agilent 4200 TapeStationhigh-throughput analyzer which uses microfluidics, microcapillary electrophoresis, and fluorescence detection that allows for size determination of the isolated molecules. Used for quality control and quantification of DNA and RNA samples for downstream methods. Reports metrics to assess sample (DNA, RNA, protein) integrity and quantification, which includes: electropherogram, gel-like image, concentration values, 28S/18S rRNA ratios, and an algorithmically determined integrity score (RINe). Provides a more complete characterization of the measured samples then previous analyzers for downstream analysis.

Fast zonearea between small RNAs and the 18S rRNA fragment (~200 bp to 1.8 kb). The fast zone is where the degradation of the 18S and 28S peaks accumulates.

RINeRNA integrity number (RIN equivalent) which is determined by quantitative measurements of total RNA degradation. Measurements are based on features of the electropherogram which assesses degradation or lack of degradation by measuring the ratio of area in the fast zone to the 18S peak signal. RIN of 10 signifies the highest quality intact RNA and zero is the worst score indicates a low-quality degraded sample.

sentences: Panzootic chytrid fungus exploits diverse amphibian host environments through plastic infection strategies While many pathogens are limited to a single host, others can jump from host to host, which likely contributes to the emergence of infectious diseases. Despite this threat to biodiversity, traits associated with overcoming eco-evolutionary barriers to achieve host niche expansions are not well understood. Here, we examined the case of Batrachochytrium dendrobatidis (Bd), a multi-host pathogen that infects the skin of hundreds of amphibian species worldwide. To uncover functional machinery driving multi-host invasion, we analyzed Bd transcriptomic landscapes across 14 amphibian hosts and inferred the origin and evolutionary history of pathogenic genes under a phylogenetic framework comprising 12 other early-divergent zoosporic fungi. Our results not only revealed a conserved basal genetic machinery, but also highlighted the ability of Bd to display plastic infection strategies when challenged under suboptimal host environments. We found that genes related to amphibian skin exploitation have arisen mainly via gene duplications. We argue that plastic gene expression can drive variation in Bd lifecycles with different mode and tempo of development. Our findings support the idea that host skin environments exert contrasting selective pressures, such that gene expression plasticity constitutes one of the evolutionary keys leading to the success of this panzootic multi-host pathogen.

sentences: Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

sentences: Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand--N-acetylgalactosamine (GalNAc)--which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.

sentences: Timing of water limitations affects source to sink differences in δ13C composition in apple Deficit irrigation is used to reduce vegetative vigor, increase fruit quality, and conserve water resources. However, physiological responses to deficit irrigation can vary depending on soil and environmental conditions. Although physiological measurements are often made at single points in time, responses are often longer lasting and a measurement that integrates responses over time would have greater value in assessing the effectiveness of deficit irrigation practices. Carbon isotope composition has long been used as a proxy measurement for water-use efficiency, stomatal conductance, and carbon dioxide exchange with the atmosphere and is heavily influenced by water status. Potentially, fruit, leaves, or other tissues could be used as samples for carbon isotope measurements. However, it is not well known how irrigation practices can influence both source and sink tissue carbon isotope composition in perennial systems. Here, we used two experiments to determine how irrigation timing affects both source and sink {delta}13C at the end of the season. Irrigation limitations were initiated after bloom for either the whole season or for early, middle, or late season and compared to a well-watered control. For both experiments, leaves were poor indicators of irrigation deficit treatments that were applied during the season. There were no significant differences in leaf {delta}13C between deficit treatments and the control for both experiments. However, all sink tissues including roots and stems in experiment one for both years and for fruits in experiment two for both years were significantly more enriched compared to the well-watered control. Environmental conditions during the season also appeared to influence the magnitude of difference in {delta}13C between deficit irrigation treatments and the control. These results indicate that the use of sink tissues are more sensitive for measuring signals associated with in-season water deficits. Carbon isotope composition can be an effective proxy to measure efficacy of irrigation treatments at the physiological level.

sentences: A Hybrid Communication Pattern in Brain Structural Network Revealed by Evolutionary Computation The human brain functional connectivity network (FCN) is constrained and shaped by the information communication processes in the structural connectivity network (SCN). The underlying communication model thus becomes a critical issue for understanding structure-function coupling in the human brain. A number of communication models featuring different point-to-point routing strategies have been proposed, with shortest path (SP), diffusion (DIF), and navigation (NAV) as the typical, respectively requiring network global knowledge, local knowledge, and their combination for path seeking. Yet these models all assumed the entire brain to use a uniform routing strategy, which ignored lumping evidence supporting the wide variety of brain regions in both terms of biological substrates and functional exhibitions. In this study, we developed a novel communication model that allowed each brain region to choose a routing strategy from SP, DIF, and NAV independently. A genetic algorithm was designed to uncover the underlying region-wise hybrid routing strategy (namely HYB) for maximizing the structure-function coupling. The HYB-based model outperformed the three typical models in terms of predicting FCN and supporting robust communication. In HYB, brain regions in lower-order functional modules inclined to choose the routing strategies requiring more global knowledge, while those in higher-order functional components preferred to choose DIF. Additionally, compared to regions using SP and NAV, regions using DIF had denser structural connections, participated in more functional modules, but were less dominant within them. Together, our findings revealed and evidenced the possibility and advantages of hybrid routing underpinning efficient SCN communication.

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Phage Cocktails can Prevent the Evolution of Phage-Resistant Enterococcus Antibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy is often deployed as a cocktail of phages, but there is little understanding of how to most effectively combine phages. Here we utilized a collection of 20 Enterococcus phages to test principles of phage cocktail design and determine the phenotypic effects of evolving phage resistance in Enterococcus isolates that were susceptible or resistant to antibiotics (e.g., Vancomycin Resistant Enterococcus (VRE)). We tested the ability of each phage to clear Enterococcus host cultures and prevent the emergence of phage resistant Enterococcus. We found that some phages which were ineffective individually were effective at clearing the bacterial culture when used in cocktails. To understand the dynamics within phage cocktails, we used qPCR to track which phages increased in abundance in each cocktail, and saw dynamics ranging from one dominant phage to even phage growth. Further, we isolated several phage-resistant mutants to test for altered Vancomycin sensitivity. We found that mutants tended to have no change or slightly increased resistance to Vancomycin. By demonstrating the efficacy of phage cocktails in suppressing growth of antibiotic susceptible and VRE clinical isolates when exposed to phages, this work will help to inform cocktail design for future phage therapy applications.

IMPORTANCEAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy in the form of cocktails is often suggested, with similar goals as the combination therapy that has been successful in the treatment of HIV infection, but there is little understanding about how to combine phages most effectively. Here we utilized a collection of 20 Enterococcus phages to test whether several phage cocktails could prevent the host from evolving resistance to therapy and to determine whether evolving resistance to phages affected host susceptibility to antibiotics. We showed that cocktails of two or three unrelated phages often prevented the growth of phage-resistant mutants, when the same phages applied individually were not able to.

sentences: Merkel cell polyomavirus regulates miR183 cluster and piR62011 in Merkel cell carcinoma Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer, a major subset of which is caused by the clonal integration of Merkel Cell Polyomavirus (MCV). Recent studies by Cheng et al. (2017) reported that virus-derived small T antigen protein-bound EP400 complex drives expression of genes essential for cellular transformation. On close analysis of their ChIP-Seq data, we uncovered that the complex binds to the promoter region of the microRNA-183 cluster. The miRNA183 cluster is a cluster of 3 miRNAs (miR183, 182 & 96) expressed and regulated together. These miRNAs are conserved across species, highly expressed in human embryonic stem cells and necessary for sensory/ mechanosensory organ development. We hypothesized that the MCV oncoproteins regulate host miRNA expression directly; an interaction novel in polyomaviruses. We tested miRNA expression via qPCR in both virus positive and negative MCC cell lines and found the former showed a much higher level. Further, fibroblasts expressing T antigens displayed an increase in miR182 expression in comparison to control. Knock-down of T antigens in MCC cells correspondingly decreased miR182 levels. To investigate its regulation we performed luciferase assays for the miRNA predicted promoter that showed increased activity in the presence of T antigens. Intriguingly, the seed sequence of miR182 completely matches to a piRNA called piR62011. Upon reanalysis of a MCC small RNA library, piR62011 emerged as the highest expressed. We found it expressed in MKL-1, a MCV positive cell line as well. Finally, to translate our findings into therapy for MCC, we screened small molecule (CMBL) library by performing surface plasmon resonance (SPR) assay and identified small molecules that binds to pre-miRNA182 and are testing them for their activity to kill MCC.

sentences: FDR control in GWAS with population structure We present a comprehensive statistical framework to analyze data from genome-wide association studies of polygenic traits, producing distinct and interpretable discoveries while controlling the false discovery rate. This approach leverages sophisticated multivariate models, correcting for linkage disequilibrium, and accounts for population structure and relatedness, adapting to the characteristics of the samples at hand. A key element is the recognition that the observed genotypes can be considered as a random sample from an appropriate model, encapsulating our knowledge of genetic inheritance and human populations. This allows us to generate imperfect copies (knockoffs) of these variables which serve as ideal negative controls; knockoffs are indistinguishable from the original genotypes in distribution, and independent from the phenotype. In sharp contrast with state-of-the-art methods, the validity of our inference in no way depends on assumptions about the unknown relation between genotypes and phenotype. We develop and leverage a model for the genotypes that accounts for arbitrary and unknown population structure, which may be due to diverse ancestries or familial relatedness. We build a pipeline that is robust to the most prominent possible confounders, facilitating the discovery of causal variants. Validity and effectiveness are demonstrated by extensive simulations with real data, as well as by the analysis of several phenotypes in the UK Biobank. Finally, fast software is made available for researchers to apply the proposed methodology to Biobank-scale data sets.

sentences: Eight week dietary inclusion of nitrite-containing frankfurter exacerbates colorectal cancer pathology, increases oxidative stress, alters metabolism and causes gut dybiosis in APCmin mice Colorectal cancer (CRC) is the second most prevelant malignancy in Europe and diet is an important modifiable risk factor. Processed meat consumption, including meats with preservative salts such as sodium nitrite, have been implicated in CRC pathogenesis. This study investigated how the CRC pathology and metabolic status of adenomatous polyposis coli (APC) multiple intestinal neoplasia (min) mice was perturbed following 8 weeks of pork meat consumption. Dietary inclusions (15%) of either nitrite-free pork, nitrite-free sausage or nitrite-containing sausage (frankfurter) were compared against a parallel control group (100% chow). Comprehensive studies investigated: gastrointestinal tract histology (tumours, aberant crypt foci (ACF) and mucin deplin foci (MDF), lipid peroxidation (urine and serum), faecal microbiota and serum metabolomics (599 metabolites). After 8 weeks mice consuming the frankfurter diet had 53% more (P=0.014) gastrointestinal tumours than control, although ACF and MDF did not differ. Urine and serum lipid peroxidation markers were 59% (P=0.001) and 108% (P=0.001) higher, respectively in the frankfurter group. Gut dysbiosis was evident in these mice with comparably fewer Bacteriodes and more Fermicutes. Fasting serum levels of trimethylamine N-oxide (TMAO) and numerous triglycerides were elevated. Various serum phosphotidylcholine species were decreased. These results demonstrate that nitrite-containing sausages may exaccerbate the development of CRC pathology in APCMin mice to a greater extent than nitrite-free sausages, and this is associated with greater lipid peroxidation, wide-ranging metabolic alternation and gut dysbiosis.

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Host-generalist fungal pathogens of seedlings may maintain forest diversity via host-specific impacts and differential susceptibility among tree species O_LIHost-specialized pathogens are credited with the maintenance of tropical forest diversity under the Janzen-Connell hypothesis. Yet, in diverse forests, selection may favor pathogens with broad host ranges given their passive dispersal and the relative rarity of tree species.
C_LIO_LIWe surveyed the host associations of potential pathogens isolated from symptomatic seedlings in the forests in Panama and used inoculations to assess: (i) the pathogenicity and host ranges of 27 fungal isolates, and (ii) differences among tree species in susceptibility.
C_LIO_LIThirty-one of the 33 non-singleton OTUs isolated from seedlings are multi-host. All 31 multi-host OTUs exhibit low to moderate specialization, and we observed phylogenetically overdispersed host use for 19 OTUs. The pathogenicity of 10 isolates was experimentally confirmed; nine caused disease in seedlings in multiple families. However, the outcome of infection differs among tree species susceptible to a given multi-host pathogen. Furthermore, some tree species were seemingly resistant to all fungi tested, while others were susceptible to multiple fungi. Tree species adapted to environments with lower disease pressure were most likely to exhibit disease.
C_LIO_LIOur results suggest that generalist pathogens contribute to the maintenance of local and regional forest diversity via host-specific impacts and the exclusion of disease-sensitive trees from disease-prone habitats.
C_LI

sentences: Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for ribosome biogenesis, and as an interactor with the PeBoW complex (PES1, BOP1, WDR12) in high-throughput affinity purifications. The PeBoW complex has been shown to be required for pre-28S rRNA processing. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in mammalian cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We also demonstrate that RSL24D1 is physically complexed with RNA polymerase I, revealing a connection between large ribosomal subunit biogenesis and rDNA transcription. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large subunit biogenesis factors in pre-rRNA transcription control.

sentences: ATPase activity of B. subtilis RecA affects the dynamic formation of RecA filaments at DNA double strand breaks RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed "threads", which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, and still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduces formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. While lowered ATP-binding activity only moderately affected RecA dynamics, loss of ATPase activity lead to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and non-specifically DNA-bound molecules into filaments upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Our data suggest that RecA/ssDNA filaments change in subcellular localization and length involving ATP-driven homology search.

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A technical pipeline for screening microbial communities as a function of substrate specificity through single cell fluorescent imaging The study of specific glycan uptake and metabolism has been shown to be an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards target. In this study, we successfully used the fluorescent label 2-aminobenzamide (2-AB), most commonly employed for enhancing the detection of protein anchored glycans, to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, were able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it was possible to comprehensively track the metabolism of the labelled glycans in both supernatants and at a single cell level. We envisage this combination of complimentary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

IMPORTANCEInformation on how bacterial consortia utilize polysaccharides at strain level, whilst progressing rapidly in recent years still lacks a suitable way to study the vast range of ornamentations and structural motifs found in the natural glycans we consume in everyday life. As multi-omic approaches commonly require complex and costly analysis, a screening platform, as described in our work, could be seen as both a complementary and essential new tool in the understanding of microbial polysaccharide metabolism. Our study demonstrates a fast and efficient glycan labelling technique composed of several integrated procedures and advanced analytical methodologies. Chromatography and mass spectrometry are applied in the tracking of metabolized labelled glycans whilst microscopy and flow cytometry are used in the visualization of labelled bacteria at a single cell level.

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Face familiarity detection with complex synapses Synaptic plasticity is a complex phenomenon involving multiple biochemical processes that operate on different timescales. We recently showed that this complexity can greatly increase the memory capacity of neural networks when the variables that characterize the synaptic dynamics have limited precision, as in biological systems. These types of complex synapses have been tested mostly on simple memory retrieval problems involving random and uncorrelated patterns. Here we turn to a real-world problem, face familiarity detection, and we show that also in this case it is possible to take advantage of synaptic complexity to store in memory a large number of faces that can be recognized at a later time. In particular, we show that the familiarity memory capacity of a system with complex synapses grows almost linearly with the number of the synapses and quadratically with the number of neurons. Complex synapses are superior to simple ones, which are characterized by a single variable, even when the total number of dynamical variables is matched. We further show that complex and simple synapses have distinct signatures that are testable in proposed experiments. Our results indicate that a memory system with complex synapses can be used in real-world tasks such as face familiarity detection.

SignificanceThe complexity of biological synapses is probably important for enabling us to remember the past for a long time and rapidly store new memories. The advantage of complex synapses in terms of memory capacity is significant when the variables that characterize the synaptic dynamics have limited precision. This advantage has been estimated under the simplifying assumption that the memories to be stored are random and uncorrelated. Here we show that synaptic complexity is important also in a more challenging and realistic face familiarity detection task. We built a simple neural circuit that can report whether a face has been previously seen or not. This circuit incorporates complex synapses that operate on multiple timescales. The memory performance of this circuit is significantly higher than in the case in which synapses are simple, indicating that the complexity of biological synapses can be important also in real-world memory tasks.

sentences: Linear B-cell epitope prediction for in silico vaccine design: a performance review of methods available via command-line interface Linear B-cell epitope prediction research has received a steadily growing interest ever since the first method was developed in 1981. B-cell epitope identification with the help of an accurate prediction method can lead to an overall faster and cheaper vaccine design process, a crucial necessity in the covid-19 era. Consequently, several B-cell epitope prediction methods have been developed over the past few decades, but without significant success. In this study, we review the current performance and methodology of some of the most widely used linear B-cell epitope predictors which are available via a command-line interface, namely BcePred, BepiPred, ABCpred, COBEpro, SVMTriP, LBtope, and LBEEP. Additionally, we attempted to remedy performance issues of the individual methods by developing a consensus classifier, which combines the separate predictions of these methods into a single output, accelerating the epitope-based vaccine design. While the method comparison was performed with some necessary caveats and individual methods might perform much better for specialized datasets, we hope that this update in performance can aid researchers towards the choice of a predictor, for the development of biomedical applications such as designed vaccines, diagnostic kits, immunotherapeutics, immunodiagnostic tests, antibody production, and disease diagnosis and therapy.

sentences: Simultaneous expression of UV and violet SWS1 opsins expands the visual palette in a group of freshwater snakes. Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths, with strong purifying selection acting on the UV-sensitive copy and dN/dS [~]1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually-guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.

sentences: Cap analysis of gene expression (CAGE) sequencing reveals alternative promoter usage in complex disease The role of alternative promoter usage in tissue specific gene expression has been well established, however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) tag sequencing from the left ventricle (LV) of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, the Brown Norway (BN) to understand role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites (TSS) in the rat LV, including 1,970 novel cardiac TSS resulting in new transcripts. We identified 27 genes with alternative promoter usage between SHR and BN which could lead to protein isoforms differing at the amino terminus between two strains. Additionally, we identified 475 promoter switching events where a shift in TSS usage was within 100bp between SHR and BN, altering length of the 5 UTR. Genomic variants located in the shifting promoter regions showed significant allelic imbalance in F1 crosses, confirming promoter shift. We found that the insulin receptor gene (Insr) showed a switch in promoter usage between SHR and BN in heart and liver. The Insr promoter shift was significantly associated with insulin levels and blood pressure within a panel of BXH/HXB recombinant inbred (RI) rat strains. This suggests that the hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.

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Population genetic analysis reveals the role of natural selection and phylogeography on genome-wide diversity in an extremely compact and reduced microsporidian genome The determinants of variation in a species genome-wide nucleotide diversity include historical, environmental, and stochastic aspects. This diversity can inform us about the species past and present evolutionary dynamics. In parasites, the mode of transmission and the interactions with the host might supersede the effects of these aspects in shaping parasite genomic diversity. We used genomic samples from ten populations of the microsporidian parasite Ordospora colligata to investigate present genomic diversity and how it was shaped by evolutionary processes, specifically, the role of phylogeography, co-phylogeography (with the host), natural selection, and transmission mode. Although very closely related microsporidia cause diseases in humans, O. colligata is specific to the freshwater crustacean Daphnia magna and has one of the smallest known eukaryotic genomes. We found an overlapping phylogeography between O. colligata and its host highlighting the long-term, intimate relationship between them. The observed geographic distribution reflects previous findings that O. colligata exhibits adaptations to colder habitats, which differentiates it from other microsporidian gut parasites of D. magna predominantly found in warmer areas. The co-phylogeography allowed us to calibrate the O. colligata phylogeny and thus estimate its mutation rate. We found patterns of more efficient purifying selection in O. colligata relative to other microsporidia sharing the same host, which likely allowed this parasite to maintain its very compact genome. We also identified regions under potential selection related to coevolution including the ribosomal protein L24, a leucyl-tRNA synthetase, and a putative ABC-like lipid transport protein. Our whole-genome study provides insights into the evolution of one of the most reduced eukaryotic genomes and shows how different processes shape genomic diversity of an obligate parasite.

Author summaryMicrosporidia are intracellular parasites that infect vertebrates, invertebrates, and even unicellular organisms. Due to their high variation in many aspects of life history and genomics, microsporidia have become a model clade for understanding evolutionary processes related to intracellular parasitism. However, the evolution of extreme genomic architectures in microsporidia and the coevolution with their hosts is still under-surveyed, especially given their role in human disease. Here, we study past and present evolutionary dynamics in a microsporidian species with one of the smallest known eukaryotic genomes, O. colligata. Close relatives of O. colligata cause death and disease in humans and agriculturally important animals. We show that purifying selection helped maintaining its reduced, compact genome and corroborate hypotheses about the evolution of different genome sizes in microsporidia. Importantly, we utilize the highly resolved phylogeny of its host to estimate the parasites mutation rate. This methodology allowed us to establish the first mutation rate estimate for a microsporidium, an estimate which is within the range of mutation rates estimated for phylogenetically related, non-parasitic fungi. Our study exemplifies how the combined knowledge about a species biology, ecology, and genomic diversity helps to resolve its evolutionary dynamics, in particular when phylogenomic information can be brought to bear for both host and parasite.

sentences: Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3-OH group of the primer strand to the -phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

sentences: E3 ligase TRIM47 positively regulates endothelial activation and pulmonary inflammation through potentiating the K63-linked ubiquitination Endothelial activation plays an essential role in the pathology of sepsis-induced acute lung injury, but the detailed regulatory mechanisms remain largely unknown. Here, we demonstrated that TRIM47, an ubiquitin E3 ligase of tripartite protein family, is highly expressed in vascular endothelial cells and is up-regulated during TNF-induced endothelial activation. Knockdown of TRIM47 in endothelial cells prevents the transcription of multiple pro-inflammatory cytokines, reduces monocyte adhesion and the expression of adhesion molecules, and inhibits the secretion of IL-1{beta} and IL-6 into the supernatant. By contrast, overexpression of TRIM47 promotes inflammatory response and monocyte adhesion upon TNF stimulation. TRIM47 modulates the activation of NF-{kappa}B and MAPK signaling pathways during endothelial activation. Further experiment confirmed that TRIM47 interacts with TRAF2 and mediates K63-linked ubiquitination. In addition, TRIM47-deficient mice are more resistant to lipopolysaccharide-induced acute lung injury and death, due to attenuated pulmonary inflammation. Taken together, our studies suggest that TRIM47 promotes pulmonary inflammation and injury at least partly through potentiating the K63-linked ubiquitination of TRAF2, which in turn activates NF-{kappa}B and MAPK signaling pathways to trigger inflammatory response in endothelial cells.

sentences: Efficacy of recombinant NP-M1 and NP-M1-CRT DNA vaccines against Influenza A viruses in mice C57/BL6 Effective vaccination against the influenza virus remains a challenge because of antigenic shift and drift in influenza viruses. Conservation is an important feature of the Nucleoprotein (NP) and Matrix protein 1(M1) qualifying them as potential candidates for developing a universal vaccine against the influenza A virus. Carliticulin (CRT), a member of heat shock protein (HSP) family, are conserved and widely distributed in many microorganisms and mammalian cells. In this study, a plasmid vector encoding the NP-M1-CRT sequence was constructed and compared with the NP-M1 sequence with respect to immunogenicity and protective efficacy in a murine model. The potency of the created construct for provoking humoral, cellular immune responses, and its protective immunity against the lethal influenza virus infection were then compared with commercial split vaccine and then evaluated in a murine model system. NP-M1-CRT as a DNA vaccine combined with in vivo electroporation could significantly improve the immunogenicity of constructed vectors. Serological evaluations demonstrated the potency of our approach to provoke strong anti-NP specific antibody responses. Furthermore, our strategy of immunization in prime-boost groups were able to provide protection against lethal viral challenge using H1N1 subtype. The ease of production of these types of vectors and the fact that they would not require annual updating and manufacturing may provide an alternative cost-effective approach to limit the spread of potential pandemic influenza viruses.

sentences: Explicit and implicit depth-cue integration: evidence of systematic biases with real objects In a previous series of experiments using virtual stimuli, we found evidence that 3D shape estimation agrees to a superadditivity rule of depth-cue combination. According to this rule, adding depth cues leads to greater perceived depth magnitudes and, in principle, to depth overestimation. The mechanism underlying the superadditivity effect can be fully accounted for by a normative theory of cue integration, through the adaptation of a model of cue integration termed the Intrinsic Constraint (IC) model. As for its nature, it remains unclear whether superadditivity is a byproduct of the artificial nature of virtual environments, causing explicit reasoning to infiltrate behavior and inflate the depth judgments when a scene is richer in depth cues, or the genuine output of the process of depth-cue integration. In the present study, we addressed this question by testing whether the IC models prediction of superadditivity generalizes beyond VR environments to real world situations. We asked participants to judge the perceived 3D shape of cardboard prisms through a matching task. To assay the potential influence of explicit control over those perceptual estimates, we also asked participants to reach and hold the same objects with their fingertips and we analyzed the in-flight grip size during the reaching. Using physical objects ensured that all visual information was fully consistent with the stimulis 3D structure without computer-generated artifacts. We designed a novel technique to carefully control binocular and monocular 3D cues independently from one another, allowing to add or remove depth information from the scene seamlessly. Even with real objects, participants exhibited a clear superadditivity effect in both explicit and implicit tasks. Furthermore, the magnitude of this effect was accurately predicted by the IC model. These results confirm that superadditivity is an inherent feature of depth estimation.

sentences: Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 [A] resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

sentences: Bioluminescent Genetically Encoded Glutamate Indicator for Molecular Imaging of Neuronal Activity Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor and overcomes several drawbacks of fluorescence imaging such as limited light penetration depth, excitation scattering, and tissue heating that are all associated with the external light needed for fluorescence imaging. Here we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes exhibit robust changes in light output in response to extracellular presentation of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

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Interferon induction and not replication interference mainly determines anti-influenza virus activity of defective interfering particles Defective interfering (DI) RNAs arise during influenza virus replication, can be packaged into particles (DIPs) and suppress spread of wildtype (WT) virus. However, the molecular signatures of DI RNAs and the mechanism underlying antiviral activity are incompletely understood. Here, we show that any central deletion is sufficient to convert a viral RNA into a DI RNA and that antiviral activity of DIPs is inversely correlated with DI RNA length when induction of the interferon (IFN) system is disfavored. When induction of the IFN system was allowed, it was found to be the major contributor to DIP antiviral activity. Finally, while both DIPs and influenza virus triggered expression of IFN-stimulated genes (ISG) only virus stimulated robust expression of IFN. These results suggest a key role of innate immune activation in DIP antiviral activity and point towards previously unappreciated differences in DIP- and influenza virus-mediated activation of the effector functions of the IFN system.

ImportanceDefective interfering (DI) RNAs naturally arise during RNA virus infection. They can be packaged into defective interfering particles (DIPs) and exert antiviral activity by suppressing viral genome replication and inducing the interferon (IFN) system. However, inhibition of influenza virus infection by DI RNAs has been incompletely understood. Here, we show that induction of the IFN system and not suppression of genome replication is the major determinant of DIP antiviral activity. Moreover, we demonstrate that DIPs induce IFN-stimulated genes (ISG) but not IFN with high efficiency. Our results reveal unexpected major differences in influenza virus and DIP activation of the IFN system, a key barrier against viral infection, and provide insights into how to design DIPs for antiviral therapy.

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Cortical-subcortical structural connections support transcranial magnetic stimulation engagement of the amygdala The amygdala processes valenced stimuli, influences affective states, and exhibits aberrant activity across anxiety disorders, depression, and PTSD. Interventions that modulate amygdala activity hold promise for treating transdiagnostic affective symptoms. We investigated (N=45) whether transcranial magnetic stimulation (TMS) elicits indirect changes in amygdala activity when applied to ventrolateral prefrontal cortex (vlPFC), a region important for affect regulation. Harnessing in-scanner interleaved TMS/functional MRI (fMRI), we reveal that vlPFC neurostimulation evoked acute, dose-dependent modulations of amygdala fMRI BOLD signal. Larger TMS-evoked changes in amygdala fMRI signal were associated with higher fiber density in a vlPFC-amygdala white matter pathway, suggesting this pathway facilitated stimulation-induced communication between cortex and subcortex. This work provides evidence of amygdala engagement by TMS, highlighting stimulation of vlPFC-amygdala circuits as a candidate treatment for affective psychopathology. More broadly, it indicates that targeting cortical-subcortical connections may enhance the impact of TMS on subcortical neural activity and, by extension, subcortex-subserved behaviors.

TeaserIndividualized, connectivity-guided transcranial magnetic stimulation modulates the amygdala, demonstrating therapeutic potential.

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DRDOCK: A Drug Repurposing platform integrating automated docking, simulations and a log-odds-based drug ranking scheme MotivationDrug repurposing, where drugs originally approved to treat a disease are reused to treat other diseases, has received escalating attention especially in pandemic years. Structure-based drug design, integrating small molecular docking, molecular dynamic (MD) simulations and AI, has demonstrated its evidenced importance in streamlining new drug development as well as drug repurposing. To perform a sophisticated and fully automated drug screening using all the FDA drugs, intricate programming, accurate drug ranking methods and friendly user interface are very much needed.

ResultsHere we introduce a new web server, DRDOCK, Drug Repurposing DOcking with Conformation-sampling and pose re-ranKing - refined by MD and statistical models, which integrates small molecular docking and molecular dynamic (MD) simulations for automatic drug screening of 2016 FDA-approved drugs over a user-submitted single-chained target protein. The drugs are ranked by a novel drug-ranking scheme using log-odds (LOD) scores, derived from feature distributions of true binders and decoys. Users can submit a selection of LOD-ranked poses for further MD-based binding affinity evaluation. We demonstrated that our platform can indeed recover one of the substrates for nsp16, a cap ribose 2'-O methyltransferase, and recommends that fluralaner, tegaserod and fenoterol could be repurposed for the COVID19 treatment with the latter two being confirmed in SARS-CoV2 suppression experiments. All the sampled docking poses and trajectories can be 3D-viewed and played via our web interface. This platform shall be easy-to-use for general scientists and medicinal researchers to carry out drug repurposing within a couple of days which should add value to our timely responses to, particularly, emergent disease outbreaks.

Availability and implementationDRDOCK can be freely accessed from https://dyn.life.nthu.edu.tw/drdock/. (Due to the hardware upgrade, the service is NOT available before 7/18, 2021)

sentences: Contrasting resistance and resilience of the coupled oxic and anoxic componentsof an experimental microbial ecosystem Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used phototrophic oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities is affected by light deprivation. Continuous measurements were performed to describe oxygen dynamics (mean/min/max/amplitude) and time-series of full-length 16S rRNA sequencing were used to quantify changes in alpha- and beta-diversity. In the top oxic layer, oxygen concentration decreased significantly under light deprivation, but showed resilience in mean, minimum and maximum after light conditions were restored. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha-diversity of the top oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient. In contrast, alpha-diversity of the anoxic bottom layer communities increased due to the stressor, but was resilient. Community composition changed significantly during light deprivation, showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha-diversity and the amplitude of oxygen within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function and structure, hence focusing on a single stability component could potentially be misleading.

sentences: Deep Learning Enables Design of Multifunctional Synthetic Human Gut Microbiome Dynamics Predicting the dynamics and functions of microbiomes constructed from the bottom-up is a key challenge in exploiting them to our benefit. Current ordinary differential equation-based models fail to capture complex behaviors that fall outside of a predetermined ecological theory and do not scale well with increasing community complexity and in considering multiple functions. We develop and apply a long short-term memory (LSTM) framework to advance our understanding of community assembly and health-relevant metabolite production using a synthetic human gut community. A mainstay of deep learning, the LSTM learns a high dimensional data-driven non-linear dynamical system model used to design communities with desired metabolite profiles. We show that the LSTM model can outperform the widely used generalized Lotka-Volterra model. We build methods decipher microbe-microbe and microbe-metabolite interactions from an otherwise black-box model. These methods highlight that Actinobacteria, Firmicutes and Proteobacteria are significant drivers of metabolite production whereas Bacteroides shape community dynamics. We use the LSTM model to navigate a large multidimensional functional landscape to identify communities with unique health-relevant metabolite profiles and temporal behaviors. In sum, the accuracy of the LSTM model can be exploited for experimental planning and to guide the design of synthetic microbiomes with target dynamic functions.

sentences: CPGL: Prediction of compound-protein interaction by integrating graph attention network with long short-term memory neural network Recent advancements of artificial intelligence based on deep learning algorithms have made it possible to computationally predict compound-protein interaction (CPI) without conducting laboratory experiments. In this manuscript, we integrated a graph attention network (GAT) for compounds and a long short-term memory neural network (LSTM) for proteins, used end-to-end representation learning for both compounds and proteins, and proposed a deep learning algorithm, CPGL (CPI with GAT and LSTM) to optimize the feature extraction from compounds and proteins and to improve the model robustness and generalizability. CPGL demonstrated an excellent predictive performance and outperforms recently reported deep learning models. Based on 3 public CPI datasets, C.elegans, Human and BindingDB, CPGL represented 1 - 5% improvement compared to existing deep-learning models. Our method also achieves excellent results on datasets with imbalanced positive and negative proportions constructed based on the above two datasets. More importantly, using 2 label reversal datasets, GPCR and Kinase, CPGL showed superior performance compared to other existing deep learning models. The AUC were substantially improved by 15% to 50% on the Kinase dataset, indicative of the robustness and generalizability of CPGL.

sentences: An open-source, ready-to-use and validated ripple detector plugin for the Open Ephys GUI Sharp wave-ripples (SWRs, 100-250 Hz) are oscillatory events extracellularly recorded in the CA1 subfield of the hippocampus during sleep and quiet wakefulness. SWRs are thought to be involved in the dialogue between the hippocampus and cortical regions to promote memory consolidation during sleep and memory-guided decision making. Many studies employed closed-loop strategies to either detect and abolish SWRs within the hippocampus or manipulate other relevant areas upon ripple detection. However, the code and schematics necessary to replicate the detection system are not always available, which hinders the reproducibility of experiments among different research groups. Furthermore, information about performance is not usually reported. Here, we present the development and validation of an open-source, real-time ripple detection plugin integrated into the Open Ephys GUI. It contains a built-in movement detector based on accelerometer or electromyogram data that prevents false ripple events (due to chewing, grooming, or moving, for instance) from triggering the stimulation/manipulation device. To determine the accuracy of the detection algorithm, we first carried out simulations in Matlab with synthetic and real ripple recordings. Using a specific combination of detection parameters (amplitude threshold of 5 standard deviations above the mean, time threshold of 10 ms, and RMS block size of 7 samples), we obtained a 97% true positive rate and 2.48 false positives per minute on the real data. Next, an Open Ephys plugin based on the same detection algorithm was developed, and a closed-loop system was set up to evaluate the round trip (ripple onset-to-stimulation) latency over synthetic data. The lowest latency obtained was 34.5 {+/-} 0.5 ms. Besides contributing to increased reproducibility, we anticipate that the developed ripple detector plugin will be helpful for many closed-loop applications in the field of systems neuroscience.

sentences: TXNRD3 supports male fertility via the redox control of spermatogenesis Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

sentences: Gene regulatory network analysis identifies key genes and regulatory mechanisms involved in acute myocardial infarction using bulk and single cell RNA-seq data Cardiovascular and cerebrovascular diseases are leading causes of death worldwide, accounting for more than 40% of all deaths in China. Acute myocardial infarction (AMI) is a common cardiovascular disease and traditionally divided into ST-segment (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), which are known with different prognoses and treatment strategies. However, key regulatory genes and pathways involved in AMI that may be used as potential biomarker for prognosis are unknown. In this study, we constructed weighted gene co-expression networks for differential expressed genes between STEMI and NSTEMI patients based on whole-blood RNA-seq transcriptomics. Network topological attributes (e.g., node degree, betweenness) were analyzed to identify key genes involved in different functional network modules. Furthermore, we used single-cell RNA-seq data to construct multilayer signaling network to infer regulatory mechanisms of the above key genes. PLAUR (receptor for urokinase plasminogen activator) was found to play a vital role in transducing inter-cellular signals from endothelial cells and fibroblast cells to intra-cellular pathways of myocardial cells, leading to gene expression involved in cellular response to hypoxia. Our study sheds lights on identifying molecular biomarkers for diagnosis and prognosis of AMI, and provides candidate key regulatory genes for further experimental validation.

sentences: GeneCup: mine PubMed for gene relationships using custom ontology and deep learning Interpreting and integrating results from omics studies typically requires a comprehensive and time consuming survey of extant literature. Here, we introduce GeneCup, an easy to use literature mining web service that searches all PubMed abstracts for user-provided gene symbols in conjunction with a set of custom keywords organized into a customized ontology, as well as results from human genome-wide association studies (GWAS). As an example, we organized over 300 keywords related to drug addiction into seven categories. The literature search is conducted by querying the NIH PubMed server using a programming interface, which is followed by retrieving abstracts from a local copy of the PubMed archive. The main results presented to the user are individual sentences containing the gene symbol, organized by the keywords they also contain. These sentences are presented through an interactive graphical interface or as tables. GWAS results are displayed using a similar method. All results are linked to the original abstract in PubMed. In addition, a convolutional neural network is employed to distinguish sentences describing systemic stress from those describing cellular stress. The automated and comprehensive search strategy provided by GeneCup facilitates the integration of new discoveries from omic studies with existing literature. GeneCup is free and open source software. The source code of GeneCup and the link to a running instance is available at https://github.com/hakangunturkun/GeneCup

sentences: Federated analysis of BRCA1 and BRCA2 variation in a Japanese cohort More than 40% of the germline variants in ClinVar today are variants of uncertain significance (VUS). These variants remain unclassified in part because the patient-level data needed for their interpretation is siloed. Federated analysis can overcome this problem by "bringing the code to the data": analyzing the sensitive patient-level data computationally within its secure home institution and providing researchers with valuable insights from data that would not otherwise be accessible. We tested this principle with a federated analysis of breast cancer clinical data at RIKEN, derived from the BioBank Japan repository. We were able to analyze these data within RIKENs secure computational framework, without the need to transfer the data, gathering evidence for the interpretation of several variants. This exercise represents an approach to help realize the core charter of the Global Alliance for Genomics and Health (GA4GH): to responsibly share genomic data for the benefit of human health.

sentences: Prefrontal cortical contributions to working memory loading, maintenance and recall are parsed by hippocampal-prefrontal oscillatory assembly dynamics Working memory enables incorporation of recent experience into subsequent decision-making. This processing recruits both prefrontal cortex and hippocampus, where neurons encode task cues, rules and outcomes. However, precisely which information is carried when, and by which neurons, remains unclear. Using population decoding of activity in rat medial prefrontal cortex (mPFC) and dorsal hippocampal CA1, we confirm that mPFC populations lead in maintaining sample information across delays of an operant nonmatch to sample task, despite individual neurons firing only transiently. During sample encoding, distinct mPFC subpopulations joined distributed CA1-mPFC cell assemblies hallmarked by 4-5Hz rhythmic modulation; CA1-mPFC assemblies re-emerged during choice episodes, but were not 4-5Hz modulated. Delay-dependent errors arose when attenuated rhythmic assembly activity heralded collapse of sustained mPFC encoding; pharmacological disruption of CA1-mPFC assembly rhythmicity impaired task performance. Our results map component processes of memory-guided decisions onto heterogeneous CA1-mPFC subpopulations and the dynamics of physiologically distinct, distributed cell assemblies.

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Apolipoprotein M attenuates doxorubicin cardiotoxicity by regulating transcription factor EB ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.

BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).

MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.

ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients. ApomTG mice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass. ApomTG attenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.

ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.

HighlightsO_LICirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathy
C_LIO_LIAnthracycline treatment reduces circulating ApoM in humans and mice
C_LIO_LIIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacy
C_LIO_LIAutophagic impairment is characteristic of human anthracycline cardiomyopathy
C_LI

sentences: Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2 HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitos. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.

sentences: Heterogeneity, turn-over rate and karyotype space shape susceptibility to missegregation-induced extinction The incidence of somatic copy number alterations (SCNAs) per base pair of the genome is orders of magnitudes larger than that of point mutations. This makes SCNAs phenotypically effective. One mitotic event stands out in its potential to significantly change a cells SCNA burden - a chromosome missegregation. We present a general deterministic framework for modeling whole chromosome missegregations and use it to evaluate the possibility of missegregation-induced population extinction (MIE). The model predicts critical curves that separate viable from non-viable populations as a function of their turnover- and mis-segregation rates. Missegregation- and turnover rates estimated for nine cancer types are then compared to these predictions. The majority of tumors across all nine cancer types had missegregation- and turnover rates that were within viable regions of the parameter space. When a dependency of mis-segregation rate on ploidy was introduced, ploidy states associated with low mis-segregation rates acted as a stabilizing refuge, rendering MIE impossible when turnover rates are low. Intra-tumor heterogeneity, including heterogeneity in mis-segregation rates, increases as tumors progress. Our predictions suggest that this intra-tumor heterogeneity hampers the chance of success of therapies aimed at MIE.

sentences: Milk-Way Algorithm applied in Imbalanced Dataset We wish to evaluate the algorithm Milk-Way, using a known dataset deposited in a public repository. The new algorithm, which converges various techniques from different areas of knowledge, can classify ligands and select potential new drugs. It was used a dataset of ligands, organized by 15 Bioassays and described by different fingerprints. Full details of the dataset architecture were already published in a public repository. Through the stratified feature selection, using the Milk-Way algorithm, the True Positive and False Positive Rates reached a higher performance compared to the published paper. Using all the features available for each Bioassay, we reached the lowest metrics in all of them. We demonstrated that adding more features have not made a significant impact on the performance. In all the Bioassays, the True Positives and False Positives reached 100% and 0%, respectively, only using 50% and 75% of the features available. The Milk-Way algorithm suggests a holistic approach, which will contribute to the machine-learning area, namely to classified ligands in the virtual screening.

sentences: Behaviour-driven Arc expression is greater in dorsal than ventral CA1 regardless of task or sex differences. Evidence from genetic, behavioural, anatomical, and physiological study suggests that the hippocampus functionally differs across its longitudinal (dorsoventral or septotemporal) axis. Although, how to best characterize functional and representational differences in the hippocampus across its long axis remains unclear. While some suggest that the hippocampus can be divided into dorsal and ventral subregions that support distinct cognitive functions, others posit that these regions vary in their granularity of representation, wherein spatial-temporal resolution decreases in the ventral (temporal) direction. Importantly, the cognitive and granular hypotheses make distinct predictions on cellular recruitment dynamics under conditions when animals perform tasks with qualitatively different cognitive-behavioural demands. The cognitive function account implies that dorsal and ventral cellular recruitment differs depending on relevant behavioural demands, while the granularity account suggests similar recruitment dynamics regardless of the nature of the task performed. Here, we quantified cellular recruitment with the immediate early gene (IEG) Arc across the entire longitudinal CA1 axis in female and male rats performing spatial- and fear-guided memory tasks. Our results show that recruitment is greater in dorsal than ventral CA1 regardless of task or sex. This experimentum crucis leads to the strong inference that the granularity hypothesis for functional differences across the longitudinal axis in the rodent hippocampus is correct.

sentences: Single-cell transcriptome reveals a testis-specific expression profile of TCEA in human spermatogenesis Transcription elongation factor A (TCEA) is a eukaryotic transcriptional molecule, required for a formation of initiation and elongation of gene transcription-mediated RNA polymerase II (RNAPII) complex, to promote transcription-coupled nucleotide excision repair (TC-NER) after RNAPII backtracking recovery. TCEA shares three isoforms in which TCEA1 is ubiquitously expressed among all eukaryotic cells. We found a spermatogenesis TCEA1 and TCEA2 expression profile has a unique transcriptional programme, compared with embryogenesis. Moreover, the testis-specific TCEA2 profile correlates with gene transcription, whereas TCEA1 specifically correlates with genes transcribed for Nuclear excision repair (NER) during human spermatogenesis. We also found that the expression activation of RNF20, a TCEA1 inhibitor, leads to expressional TCEA1 reduction, but having no direct impact on TCEA2 expression, implying the potential RNF20-dependent transcriptional switching of TCEA2 in transcriptional regulation during spermatogenesis. Our analysis defined a transcriptional bursting event where transcription-coupled repair (both Base excision repair and Nuclear excision repair) is a major pathway highly expressed in early spermatogenesis, supporting the transcriptional scanning hypothesis of which mutation of transcribed genes is effectively repaired as proposed by Xia B., et al. (2020).

sentences: The Biosynthetic Costs of Amino Acids at the Base of the Food Chain Determine Their Use in Higher-order Consumer Genomes Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited and for Drosophila melanogaster, this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumers exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. Here we update the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, we found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, we also found remarkable conservation of exome matched AA profiles across taxa and that the composition of these profiles could be explained by the metabolic costs of microbial AA synthesis. Thus, it appears that bioenergetic constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that that this constrains biomass composition.

sentences: Limited effects of age on the use of the ankle and counter-rotation mechanism in the sagittal plane Two mechanisms can be used to accelerate the center of mass (CoM) to control the CoM in relation to the base of support during standing. The first is applying ankle moments to shift the center of pressure (CoP), which has been coined the "CoP mechanism". The second is changing the angular momentum around the CoM to change the direction of the ground reaction force, i.e., the "counter-rotation mechanism". At both the beginning and the end of the lifespan, problems with postural control are common. In this study, we asessed anteroposterior balance performance and the related use of these postural control mechanisms in children, younger adults, and older adults. Sixteen pre-pubertal children (6-9y), 17 younger adults (18-24y) and eight older adults (65-80y) performed bipedal upright standing trials of 16 seconds on a rigid surface and on three balance boards that could freely move in the sagittal plane, varying in height (15-19 cm) of the board above the point of contact with the floor. Full body kinematics were measured. Performance related outcome measures, i.e., the number of trials with balance loss and the Root Mean Square (RMS) of the time series of the CoM acceleration were calculated. Additionally, the RMS of the time series of the CoM acceleration due to the CoP and counter-rotation mechanism and the contributions of the CoP and the counter-rotation mechanism to the CoM acceleration (in %) in the sagittal plane were calculated. Furthermore, selected kinematic measures, i.e., the orientation of the board and the head and the Mean Power Frequency of balance board orientation and of CoM acceleration were calculated. Compared to younger adults, children and older adults showed a poorer balance performance, reflected by a greater RMS of CoM accelerations and more balance loss in older adults. Across age groups and conditions, the contribution of the CoP mechanism to the total CoM acceleration was dominant, i.e., 95%-108%. The contribution of the counter-rotation mechanism ranged between 19%-31% (with totals higher than 100% indicating opposite effects of both mechanisms). We suggest that the contribution of the counter-rotation mechanism is limited, since the counter-rotation mechanism would conflict with stabilizing the orientation of the head in space. Furthermore, children did use the counter-rotation mechanism relatively more to accelerate the CoM compared to younger adults. Possibly this reflects that they are still learning to limit the contribution of the counter-rotation mechanism to the same extent as adults.

sentences: maxATAC: genome-scale transcription-factor binding prediction from ATAC-seq with deep neural networks Transcription factors read the genome, fundamentally connecting DNA sequence to gene expression across diverse cell types. Determining how, where, and when TFs bind chromatin will advance our understanding of gene regulatory networks and cellular behavior. The 2017 ENCODE-DREAM in vivo Transcription-Factor Binding Site (TFBS) Prediction Challenge highlighted the value of chromatin accessibility data to TFBS prediction, establishing state-of-the-art methods. Yet, while Assay-for-Transposase-Accessible-Chromatin (ATAC)-seq datasets grow exponentially, suboptimal motif scanning is commonly used for TFBS prediction from ATAC-seq. Here, we present "maxATAC", a suite of user-friendly, deep neural network models for genome-wide TFBS prediction from ATAC-seq in any cell type. With models available for 127 human TFs, maxATAC is the largest collection of state-of-the-art TFBS models to date. maxATAC performance extends to primary cells and single-cell ATAC-seq, enabling state-of-the-art TFBS prediction in vivo. We demonstrate maxATACs capabilities by identifying TFBS associated with allele-dependent chromatin accessibility at atopic dermatitis genetic risk loci.

sentences: HyperEx: A Tool to Extract Hypervariable Regions from 16S rRNA Sequencing Data The 16S ribosomal RNA gene is one of the most studied genes in biology. This 16S ribosomal RNA importance is due to its wide application in phylogenetics and taxonomic elucidation of bacteria and archaea. Indeed, 16S ribosomal RNA is present in almost all bacteria and archaea and has, among many other useful characteristics, a low mutation rate. The 16S ribosomal RNA is composed of nine hypervariable regions which are commonly targeted by high throughput sequencing technologies in identification or community studies like metabarcoding studies. Unfortunately, the hypervariable regions do not have the same taxonomic resolution among all bacteria taxa. This requires a preliminary in silico analysis to determine the best hypervariable regions to target in a particular study. Nevertheless, to the best of our knowledge, no automated primer-based open-source tool exists to extract hypervariable regions from complete or near-complete 16S rRNA sequencing data. Here we present HyperEx which efficiently extracts the hypervariable region of interest based on embedded primers or user-given primers. HyperEx implements the Myers algorithm for the exact pairwise sequence alignment. HyperEx is freely available under the MIT license as an operating system independent Rust command-line tool at https://github.com/Ebedthan/hyperex and https://crates.io.

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Gametogenesis in the green seaweed Ulva mutabilis coincides with massive transcriptional restructuring The molecular mechanism underlying sexual reproduction in land plants is well understood in model plants and is a target for crop improvement. However, unlike land plants, the genetic basis involved in triggering reproduction and gamete formation remains elusive in most seaweeds, which are increasingly viewed as an alternative source of functional food and feedstock for energy applications. Here, gametogenesis of Ulva mutabilis, a model organism for green seaweeds, is studied. We analyze transcriptome dynamics at different time points during gametogenesis following induction of reproduction by fragmentation and removal of sporulation inhibitors. Analyses demonstrate that 45% of the genes in the genome are differentially expressed during gametogenesis. We identified several transcription factors that potentially play a key role in the early gametogenesis of Ulva given the function of their homologs in higher plants and microalgae. In particular, the detailed expression pattern of an evolutionary conserved transcription factor containing an RWP-RK domain suggests a key role during Ulva gametogenesis. The identification of putative master regulators of gametogenesis provides a starting point for further functional characterization.

HighlightTranscriptomic analyses of gametogenesis in the green seaweed Ulva highlight the importance of a conserved RWP-RK transcription factor in induction of sexual reproduction.

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CHIPS: A Snakemake pipeline for quality control and reproducible processing of chromatin profiling data MotivationThe chromatin profile measured by ATAC-seq, ChIP-seq, or DNase-seq experiments can identify genomic regions critical in regulating gene expression and provide insights on biological processes such as diseases and development. However, quality control and processing chromatin profiling data involve many steps, and different bioinformatics tools are used at each step. It can be challenging to manage the analysis.

ResultsWe developed a Snakemake pipeline called CHIPS (CHromatin enrichment Processor) to streamline the processing of ChIP-seq, ATAC-seq, and DNase-seq data. The pipeline supports single- and paired-end data and is flexible to start with FASTQ or BAM files. It includes basic steps such as read trimming, mapping, and peak calling. In addition, it calculates quality control metrics such as contamination profiles, PCR bottleneck coefficient, the fraction of reads in peaks, percentage of peaks overlapping with the union of public DNaseI hypersensitivity sites, and conservation profile of the peaks. For downstream analysis, it carries out peak annotations, motif finding, and regulatory potential calculation for all genes. The pipeline ensures that the processing is robust and reproducible.

AvailabilityCHIPS is available at https://github.com/liulab-dfci/CHIPS

sentences: A comprehensive approach for artifact-free sample preparation and assessment of mitochondrial morphology in tissue and cultured cells Mitochondrial dynamics (fission, fusion, and the formation of nanotunnels) and morphology are very sensitive to the cellular environment and may be adversely affected by oxidative stress, changes in calcium levels, and hypoxia. Investigating the precise relationship between organelle structure and function requires methods that can adequately preserve mitochondria while providing accurate, quantitative measurements of morphological attributes. Here, we demonstrate a practical approach for preserving and measuring fine structural changes using two-dimensional, high-resolution electron micrographs and three-dimensional volume renderings, obtained using serial block-face and focused ion beam-scanning electron microscopy, highlighting the specific advantages of these techniques. Additionally, this study defines a set of quantifiable metrics that can be applied to measure mitochondrial architecture and other organellar structures. Finally, we validated specimen preparation methods that avoid the introduction of morphological artifacts that may interfere with mitochondrial appearance and do not require whole-animal perfusion.

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Ligand Binding Prediction using Protein Structure Graphs and Residual Graph Attention Networks MotivationComputational prediction of ligand-target interactions is a crucial part of modern drug discovery as it helps to bypass high costs and labor demands of in vitro and in vivo screening. As the wealth of bioactivity data accumulates, it provides opportunities for the development of deep learning (DL) models with increasing predictive powers. Conventionally, such models were either limited to the use of very simplified representations of proteins or ineffective voxelization of their 3D structures. Herein, we present the development of the PSG-BAR (Protein Structure Graph -Binding Affinity Regression) approach that utilizes 3D structural information of the proteins along with 2D graph representations of ligands. The method also introduces attention scores to selectively weight protein regions that are most important for ligand binding.

ResultsThe developed approach demonstrates the state-of-the-art performance on several binding affinity benchmarking datasets. The attention-based pooling of protein graphs enables identification of surface residues as critical residues for protein-ligand binding. Finally, we validate our model predictions against an experimental assay on a viral main protease (Mpro)- the hallmark target of SARS-CoV-2 coronavirus.

AvailabilityThe code for PSG-BAR is made available at https://github.com/diamondspark/PSG-BAR

Contactacherkasov@prostatecentre.com

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Disentangling object category representations driven by dynamic and static visual input Humans can label and categorize objects in a visual scene with high accuracy and speed--a capacity well-characterized with neuroimaging studies using static images. However, motion is another cue that could be used by the visual system to classify objects. To determine how motion-defined object category information is processed in the brain, we created a novel stimulus set to isolate motion-defined signals from other sources of information. We extracted movement information from videos of 6 object categories and applied the motion to random dot patterns. Using these stimuli, we investigated whether fMRI responses elicited by motion cues could be decoded at the object category level in functionally defined regions of occipitotemporal and parietal cortex. Participants performed a one-back repetition detection task as they viewed motion-defined stimuli or static images from the original videos. Linear classifiers could decode object category for both stimulus formats in all higher order regions of interest. More posterior occipitotemporal and ventral regions showed higher accuracy in the static condition and more anterior occipitotemporal and dorsal regions showed higher accuracy in the dynamic condition. Significantly above chance classification accuracies were also observed in all regions when training and testing the SVM classifier across stimulus formats. These results demonstrate that motion-defined cues can elicit widespread robust category responses on par with those elicited by luminance cues in regions of object-selective visual cortex. The informational content of these responses overlapped with, but also demonstrated interesting distinctions from, those elicited by static cues.

Significance StatementMuch research on visual object recognition has focused on recognizing objects in static images. However, motion cues are a rich source of information that humans might also use to categorize objects. Here, we present the first study to compare neural representations of several animate and inanimate objects when category information is presented in two formats: static cues or isolated dynamic cues. Our study shows that while higher order brain regions differentially process object categories depending on format, they also contain robust, abstract category representations that generalize across format. These results expand our previous understanding of motion-derived animate and inanimate object category processing and provide useful tools for future research on object category processing driven by multiple sources of visual information.

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Known mechanisms account for less than half of antimicrobial resistance in a diverse collection of non-aureus staphylococci. IntroductionNon-aureus staphylococci (NAS) are implicated in many healthcare-acquired infections and an understanding of the genetics of antimicrobial resistance in NAS is important in relation to both clinical intervention and the role of NAS as a reservoir of resistance genes.

Gap statementThe burden of antimicrobial resistance in NAS, particularly to clinically relevant antimicrobials, is understudied.

MethodologyWe sourced 394 NAS isolates from clinical samples, healthy human volunteers, animals and type cultures and subjected them to agar dilution susceptibility testing against eight antimicrobials. We performed whole genome sequencing on 316 isolates and analysed these genotypically for the presence of genetic mechanisms responsible for the phenotypic levels of reduced antimicrobial susceptibility.

ResultsCefoxitin is used to screen for methicillin resistance in S. aureus, as it stimulates expression of mecA. We observed 174 isolates with an MIC of at least 4 g/ml to cefoxitin, of which sequencing revealed 47.6% (80/168) did not harbour a known mec homologue. Seven clinical NAS isolates displayed high daptomycin minimum inhibitory concentrations (MICs) (>4 g/ml), with no known mechanism identified. Differences in MICs against erythromycin were attributable to the presence of different resistance genes (msrA and ermC). In total, 49% (187/394) of isolates displayed reduced susceptibility to three or more of the antimicrobials tested.

ConclusionsThe widespread presence of reduced antimicrobial susceptibility in NAS is a concern, with an increased likelihood of (1) harder to treat infections caused directly by NAS, and (2) resistance genes being passed on to other bacteria via horizontal gene transfer, both of which have clinical implications for treatment and management of patients.

sentences: Towards Compilation of Balanced Protein Stability Datasets: Flattening the ΔΔG Curve through Systematic Under-sampling Protein stability datasets contain neutral mutations that are highly concentrated in a much narrower {Delta}{Delta}G range than destabilizing and stabilizing mutations. Notwith-standing their high density, often studies analyzing stability datasets and/or predictors ignore the neutral mutations and use a binary classification scheme labeling only destabilizing and stabilizing mutations. Recognizing that highly concentrated neutral mutations would affect the quality of stability datasets, we have explored three protein stability datasets; S2648, PON-tstab and the symmetric Ssym that differ in size and quality. A characteristic leptokurtic shape in the {Delta}{Delta}G distributions of all three datasets including the curated and symmetric ones were reported due to concentrated neutral mutations. To further investigate the impact of neutral mutations on {Delta}{Delta}G predictions, we have comprehensively assessed the performance of eleven predictors on the PON-tstab dataset. Correlation and error analyses showed that all of the predictors performed the best on the neutral mutations while their performance became gradually worse as the {Delta}{Delta}G of the mutations departed further from the neutral zone regardless of the direction, implying a bias towards dense mutations. To this end, after unraveling the role of concentrated neutral mutations in biases of stability datasets, we described a systematic under-sampling approach to balance the {Delta}{Delta}G distributions. Before under-sampling, mutations were clustered based on their biochemical and/or structural features and then three mutations were systematically selected from every 2 kcal/mol of each cluster. Upon implementation of this approach by distinct clustering schemes, we generated five subsets varying in size and {Delta}{Delta}G distributions. All subsets notably showed amelioration of not only the shape of {Delta}{Delta}G distributions but also other pre-existing imbalances in the frequency distributions. We also reported differences in the performance of the predictors between the parent and under-sampled subsets due to the enrichment of previously under-represented mutations in the subsets. Altogether, this study not only elaborated the pivotal role of concentrated mutations in the dataset biases but also contemplated and realized a rational strategy to tackle this and other forms of biases. Under-sampling code is available on GitHub (https://github.com/narodkebabci/gRoR).

sentences: TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth RNA polymerase I (Pol I) products play pivotal roles in ribosomal assembly, protein synthesis and cell growth. Dysregulation of Pol I product synthesis closely correlates with tumourigenesis. However, the factors and pathways governing Pol I-dependent transcription remain to be identified. Here, we report that TFIIB-related factor 1 (BRF1), a subunit of TFIIIB required for Pol III-mediated transcription, is a nucleolar protein and modulates the rRNA synthesis directed by Pol I. We show that BRF1 can be co-localized with nucleolar protein markers in several human cell types. BRF1 expression positively correlates with Pol I product levels and tumour cell growth in vitro and in vivo. Mechanistically, BRF1 binds to the Pol I transcription machinery factors and is recruited to the rDNA promoter with them. Alteration of BRF1 expression affected the recruitment of Pol I transcription machinery at the rDNA promoter, the rDNA promoter activity and expression of TBP and TAF1A, indicating that BRF1 modulates Pol I-direct transcription by controlling the expression of selective factor 1 subunits. Collectively, we demonstrate that BRF1 acts as a positive factor to regulate Pol I-directed transcription, suggesting that BRF1 can concurrently modulate Pol I and Pol III transcription and acts as a key coordinator between them.

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Systemic circulating microRNA landscape in Lynch syndrome MicroRNAs (miRs) are non-coding RNA-molecules that regulate gene expression. Global circulating miR (c-miR) expression patterns (c-miRnome) change with carcinogenesis in various sporadic cancers. Therefore, aberrantly expressed c-miRs could have diagnostic, predictive and prognostic potential in molecular profiling of cancers. c-miR functions in carriers of inherited pathogenic mismatch-repair gene variants (path_MMR), also known as Lynch syndrome (LS), have remained understudied. LS cohort provides an ideal population for biomarker mining due to increased lifelong cancer risk and excessive cancer occurrence. Using high-throughput sequencing and bioinformatic approaches, we conducted an exploratory analysis to characterize systemic c-miRnomes of path_MMR carriers. Our discovery cohort included 81 healthy path_MMR carriers and 37 non-LS controls. Our analysis also included cancer cohort comprised of 13 path_MMR carriers with varying cancers and 24 sporadic rectal cancer patients. We showed for the first time that c-miRnome can discern healthy path_MMR carriers from non-LS controls but does not distinguish healthy path_MMR carriers from cancer patients with or without path_MMR. Our c-miR expression analysis combined with in silico tools suggest ongoing alterations of biological pathways shared in LS and sporadic carcinogenesis. We observed that these alterations can produce a c-miR signature which can be used to track oncogenic stress in cancer-free path_MMR carriers. Thus, c-miRs hold potential in monitoring which cancer patients would require more intensive surveillance or clinical management.

SignificanceC-miRnome can discern between healthy persons with or without path_MMR but does not distinguish healthy path_MMR carriers from cancer patients with or without path_MMR, indicating an ongoing alteration of biological pathways that can be used to track oncogenic stress at cancer-free state.

sentences: Diverse human astrocyte and microglial transcriptional responses to Alzheimer's pathology To better define roles that astrocytes and microglia play in Alzheimers disease (AD), we used single-nuclei RNA sequencing to comprehensively characterize transcriptomes in astrocyte and microglia nuclei isolated post mortem from neuropathologically-defined AD and control brains with a range of amyloid-beta and phospho-tau (pTau) pathology. Significant differences in glial gene expression (including AD risk genes expressed in astrocytes [CLU, MEF2C, IQCK] and microglia [APOE, MS4A6A, PILRA]) were correlated with tissue amyloid and pTau expression. Astrocytes were enriched for proteostatic, inflammatory and metal ion homeostasis pathways. Pathways for phagocytosis, proteostasis and autophagy were highly enriched in microglia and perivascular macrophages. Gene co-expression analyses revealed potential functional associations of soluble biomarkers of AD in astrocytes (CLU) and microglia (GPNMB). Our work highlights responses of both astrocytes and microglia for pathological protein clearance and inflammation, as well as glial transcriptional diversity in AD.

sentences: Isolation and Molecular Characterization of Pesticide Degrading Bacteria in Polluted Soil Pesticides are the substances for preventing, destroying, repelling any pest. Due to bulk handling or accidental release, they are accumulated in soil which leads to occasional entry into ecosystem that shows lethal effect on living system. An enrichment culture technique was used to isolate bacterial strains from organophosphate soil degrading high concentration of the selected pesticides. Five pure bacterial cultures were isolated. All five isolates were characterized on the basis of molecular and biochemical features like biodegradation test and substrate specificity, phosphate solubilization and screened for pesticide residue, pH, and extraction of DNA, quantity and quality check and salt tolerance. The organophosphate isolates were also tested for quantitative production. The screening of pesticide tolerance was done at for fungicides and insecticides.

sentences: Supergene degeneration opposes polymorphism: The curious case of balanced lethals Supergenes offer some of the most spectacular examples of long-term balancing selection in nature but their origin and maintenance remain a mystery. A critical aspect of supergenes is reduced recombination between arrangements. Reduced recombination protects adaptive multi-trait phenotypes, but can also lead to degeneration through mutation accumulation. Mutation accumulation can stabilize the system through the emergence of associative overdominance (AOD), destabilize the system, or lead to new evolutionary outcomes. One such outcome is the formation of balanced lethal systems, a maladaptive system where both supergene arrangements have accumulated deleterious mutations to the extent that both homozygotes are inviable, leaving only heterozygotes to reproduce. Here, we perform a simulation study to understand the conditions under which these different outcomes occur, assuming a scenario of introgression after allopatric divergence. We found that AOD aids the invasion of a new supergene arrangement and the establishment of a polymorphism. However, this polymorphism is easily destabilized by further mutation accumulation. While degradation may strengthen AOD, thereby stabilizing the supergene polymorphism, it is often asymmetric, which is the key disrupter of the quasi-equilibrium state of the polymorphism. Furthermore, mechanisms that accelerate degeneration also tend to amplify asymmetric mutation accumulation between the supergene arrangements and vice versa. As the evolution of a balanced lethal system requires symmetric degradation of both arrangements, this leaves highly restricted conditions under which such a system could evolve. We show that small population size and low dominance coefficients are critical factors, as these reduce the efficacy of selection. The dichotomy between the persistence of a polymorphism and degradation of supergene arrangements likely underlies the rarity of balanced lethal systems in nature.

sentences: Comparative analyses and phylogenetic relationships of 15 Trapa (Trapaceae) species based on Complete Chloroplast Genomes Trapa L. is floating-leaved aquatic plants with important economic and ecological values. However, species identification and phylogenetic relationship are still unresolved for Trapa. In this study, complete chloroplast genomes of 13 Trapa species/taxa were sequenced and annotated. Combined with released sequences of the other two species, comparative analysis of cp genomes was first performed on the 15 Trapa species/taxa. The 15 cp genomes exhibited quadripartite structures with medium size of 155, 453-155, 559 bp. IR/SC junctions were conservative with no obvious change found. Long repetitive repeats and SSRs were mostly detected in the intergenic and LSC regions, providing useful plastid markers for species and relationship identification. Three phylogenetic analyses (MP, ML and BI) consistently showed two clusters within Trapa, including large- and small-seed species/taxa, respectively. This study provided the baseline information for phylogeography of Trapa, which would facilitate the management and utilization of genetic resources of the genus.

sentences: Dichaete, a Sox2 homologue, prevents activation of cell death in multiple developmental contexts Precisely regulated cell death plays a critical role in normal development and is controlled by the balance of pro-apoptotic and anti-apoptotic signals. In Drosophila, transcription of the clustered cell death activators grim and reaper is turned on in the developing nervous system to eliminate neural stem cells at the end of embryonic development. This transcription is activated by a pulse of the Hox gene abdominal-A. We show here that the Sox2 homologue Dichaete inhibits neural stem cell death when overexpressed, and loss of Dichaete promotes premature neural stem cell death. The anti-apoptotic activity of Dichaete opposes the pro-apoptotic factors abdominal-A, as well as the transcription factor grainyhead. The function of all three genes impinge on an enhancer that regulates the transcription of grim and reaper. Furthermore, we find that the balance between abdominal-A and Dichaete is likely to regulate the death of other cells during development, including cells in the developing midline, the developing hindgut, and in the early abdominal epidermis. Loss of Dichaete results in premature death in these tissues. This death can be rescued by the deletion of the enhancer region between grim and reaper. These data suggest that Dichaete functions to inhibit cell death activated by abdominal-A in multiple developmental contexts.

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Do soil features condition seed germination of gypsum plants? An analysis of the effect of different natural soils Gypseous soils are widespread across arid and semiarid environments worldwide. They present remarkable challenges to plants and host a unique flora. We aimed to assess up to what point the specificity and distribution of species on gypsum might be driven by species-specific germination responses to soil gypsum availability. We analyzed the germination of six gypsum specialists and four closely related generalist plant species from the Iberian Peninsula and the Chihuahuan Desert in four different field soils with contrasting concentrations of gypsum, pH and soil texture. Plant restriction to gypsum was unrelated to the germinating ability of seeds on different substrates. Irrespective of their affinity for gypsum, most species germinated better on mixed gypsum-calcareous soil and worse in the acidic soil treatment. Our data suggest soil pH was the main soil feature driving seed germination, while the effect of gypsum content was generally not significant. The main exception was the Iberian gypsum specialist Helianthemum squamatum (L.) Dum. Cours., which showed increased germination on gypseous soils and higher germination in response to increased soil gypsum content. Except for this species, our findings indicate alkaline soils favor the germination of most of the species analyzed, irrespectively of their gypsum content.

HighlightsO_LIGypsum soils are one of the most widespread special substrates of the world
C_LIO_LIWe evaluated the effects of natural soils on the germination of gypsum plants
C_LIO_LISoil pH was the main soil feature driving seed germination
C_LIO_LIThe effect of gypsum content was not significant
C_LIO_LIWe suggest a potential origin of gypsophiles from plants adapted to alkaline soils
C_LI

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Impaired oligodendrocyte maturation is an early feature in SCA3 disease pathogenesis Spinocerebellar ataxia type 3 (SCA3), the most common dominantly inherited ataxia, is a polyglutamine neurodegenerative disease for which there is no disease-modifying therapy. The polyglutamine-encoding CAG repeat expansion in the ATXN3 gene results in expression of a mutant form of the ATXN3 protein, a deubiquitinase that causes selective neurodegeneration despite being widely expressed. The mechanisms driving neurodegeneration in SCA3 are unclear. Research to date, however, has focused almost exclusively on neurons. Here, using equal male and female age-matched transgenic mice expressing full-length human mutant ATXN3, we identified early and robust transcriptional changes in selectively vulnerable brain regions that implicate oligodendrocytes in disease pathogenesis. We mapped transcriptional changes across early, mid, and late stages of disease in two selectively vulnerable brain regions, the cerebellum and brainstem. The most significant disease-associated module through weighted gene co-expression network analysis revealed dysfunction in SCA3 oligodendrocyte maturation. These results reflect a toxic gain of function mechanism, as ATXN3 knockout mice do not exhibit any impairments in oligodendrocyte maturation. Genetic crosses to reporter mice revealed a marked reduction in mature oligodendrocytes in SCA3-disease vulnerable brain regions and ultrastructural microscopy confirmed abnormalities in axonal myelination. Further study of isolated oligodendrocyte precursor cells from SCA3 mice established that this impairment in oligodendrocyte maturation is a cell autonomous process. We conclude that SCA3 is not simply a disease of neurons and the search for therapeutic strategies and disease biomarkers will need to account for non-neuronal involvement in SCA3 pathogenesis.

SIGNIFICANCE STATEMENTDespite advances in SCA3 disease understanding, much remains unknown about how the disease gene causes brain dysfunction ultimately leading to cell death. We completed a longitudinal transcriptomic analysis of vulnerable brain regions in SCA3 mice to define the earliest and most robust changes across disease progression. Through gene network analyses followed up with biochemical and histological studies in SCA3 mice, we provide evidence for severe dysfunction in oligodendrocyte maturation early in SCA3 pathogenesis. Our results advance understanding of SCA3 disease mechanisms, identify additional routes for therapeutic intervention, and may provide broader insight into polyglutamine diseases beyond SCA3.

sentences: Liposome-assisted in-situ cargo delivery to artificial cells and cellular subcompartments We report on liposome-mediated targeted delivery of membrane-impermeable constituents into surface-adhered giant lipid compartments, employed as artificial cells. Soluble cargo compounds are delivered by means of an open-space microfluidic device, which perfuses selected lipid compartments with loaded small unilamellar vesicles (SUVs) composed of cationic lipids. The SUV membranes fuse with the surface-adhered containers, merging their contents. We monitored the fusion process via Forster resonance energy transfer (FRET) by labeling both the membranes of the SUVs and the target compartments with a fluorophore pair. We established that, upon fusion, water-soluble dyes, fluorescently labeled genetic polymers, sugars and proteins carried by the SUVs can be successfully internalized at high yield. Finally, by transferring carbonic anhydrase (CA) to the giant lipid compartments, enzymatic hydrolysis of the prefluorescent carboxyfluorescein diacetate (CFDA) is demonstrated by the emission intensity increase emanating from the product carboxyfluorescein (CF). Spontaneous subcompartmentalization occurred during liposomal delivery of the enzyme, leading to CF formation in an organelle-like subcompartment. The reported targeted delivery technique enables chemical reactions and cell-free gene expression in synthetic cell models with unprecedented ease and precision, and opens pathways to protocell architectures with distinct functional subcompartments in the context of origins of life research.

sentences: A conserved paint box underlies color pattern diversity in Estrildid finches Many animals exhibit typical color patterns that have been linked to key adaptive functions, yet the developmental mechanisms establishing these crucial designs remain unclear. Here, we surveyed color distribution in the plumage across a large number of passerine finches. Despite extreme apparent pattern diversity, we identified a small set of conserved color regions whose combinatory association can explain all observed patterns. We found these domains are instructed by early embryonic landmarks, and through profiling and comparative analyses produced a molecular map marking putative color domains in the developing skin. This revealed cryptic pre-patterning common to differently colored species, uncovering a simple molecular landscape underlying extensive color pattern variation.

sentences: Phospholipases D1 and D2 regulate cell cycling in primary prostate cancer cells and are differentially associated with the nuclear matrix Phospholipases D1 and D2 (PLD1/2) have been implicated in tumorigenesis. We previously detected higher expression of PLD in the nuclei of patient-derived prostate cancer (PCa) cells and prostate cancer cell lines. Here we have examined whether PLD1 or PLD2 are associated with the nuclear matrix and influence cell cycling. PLD1/PLD2 were detected by qualitative immunofluorescence in cultured PCa cells and extracted with a standardised protocol to reveal nuclear matrix-associated proteins. The effects of isoform-specific inhibition of PLD1or PLD2 on PCa cell cycle progression were analysed by flow cytometry. PLD2 mainly co-localised with the nucleolar marker fibrillarin in PCa cells. However, even after complete extraction, some PLD2 remained associated with the nuclear matrix. Inhibiting PLD2 effectively reduced PCa cell cycling into and through S phase. In contrast, PLD1 inhibition effects were weaker, and a subpopulation of cycling patient-derived PCa cells was unaffected by PLD1 inhibition. When associated with the nuclear matrix PLD2 could generate phosphatidic acid to regulate nuclear mTOR and control downstream transcriptional events. The association of PLD2 with the nucleolus also implies a role in stress regulation. The cell cycling results highlight the importance of PLD2 inhibition as a novel potential prostate cancer therapeutic mechanism by differential regulation of cell proliferation.

sentences: Higher phage virulence accelerates the evolution of host resistance Parasites and pathogens vary strikingly in their virulence and the resulting selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and as of now mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if resistance costs are outweighed by the benefits of avoiding infection. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of the filamentous phage, VALG{Phi}8, that differ in their virulence. The bacterial host exhibited two alternative defence strategies against future phage infection: (1) super infection exclusion (SIE) whereby phage-infected cells were immune to subsequent infection at a cost of reduced growth, and (2) surface receptor mutations (SRM) in genes encoding the MSHA type-IV pilus providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high virulence compared to the low virulence phage. Using a mathematical model of our system we show that increasing virulence strengthens selection for SRM due to the higher costs of infection suffered by SIE immune hosts. In both the experiments and the model, higher levels of SRM in the host population drove more rapid phage extinction. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

sentences: Generative neural networks separate common and specific transcriptional responses Genome-wide transcriptome profiling identifies genes that are prone to differential expression across contexts ("common DEGs"), as well as genes with changes specific to the experimental manipulation. Distinguishing common DEGs from those that are specifically changed in a context of interest allows more efficient prediction of which genes are specific to a given biological process under scrutiny. Currently, commonly differentially expressed genes or pathways can only be identified through the laborious manual curation of highly controlled experiments, an inordinately time-consuming and impractical endeavor. Here we pioneer an approach for identifying common patterns using generative neural networks. This approach produces a background set of transcriptomic experiments from which a null distribution of gene and pathway changes can be generated. By comparing the set of differentially expressed genes found in a target experiment against the generated background set, common results can be easily separated from specific ones. This "Specific cOntext Pattern Highlighting In Expression data" (SOPHIE) approach is broadly applicable to new platforms or any species with a large collection of gene expression data. We apply SOPHIE to diverse datasets including those from human, human cancer, and the bacterial pathogen Pseudomonas aeruginosa. SOPHIE identifies common DEGs in concordance with previously described, manually and systematically determined common DEGs. Further, molecular validation indicates that SOPHIE detects highly specific, but low magnitude, biologically relevant, transcriptional changes. SOPHIEs measure of specificity can complement log fold change values generated from traditional differential expression analyses. For example, by filtering the set of differentially expressed genes, one can identify those genes that are specifically relevant to the experimental condition of interest. Consequently, these results can inform future research directions.

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Epithelial-Mesenchymal Plasticity is regulated by inflammatory signalling networks coupled to cell morphology. Morphology dictates how cells sense physical and soluble cues in their environment; thus contributing to fate decisions. The differentiation of epithelial cells into mesenchymal forms, or epithelial-mesenchymal plasticity (EMP), is essential for metazoan development and homeostasis. Here we show that the decision to engage EMP is coupled to cell morphology by cell-cell adhesions by microtubule and nuclear organization (MTNO). Using an integrative  omic approach we identify Junctional Adhesion Molecule 3 (JAM3) as a new tumour suppressor in breast cancer patients. JAM3 depletion in epithelial cells alters MTNO and causes differentiation into mesenchymal forms. Soluble TGF{beta} also changes MTNO, and synergizes with JAM3 depletion to promote mesenchymal morphogenesis. Through systematic proteomic analysis we show that changes in MTNO lead to the upregulation of an inflammatory signalling network where YAP/TAZ, FOXO, IKK-NFKB, and JNK pathways are active; but where insulin signalling is suppressed. The actions of the MT-motor Kinesin-1 serve to both change MTNO and promote the upregulation of the core EMP network. Critically, the upregulation of the EMP network predicts the mesenchymal state across cancers.

Graphical Abstract

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Discovery of 10,828 new putative human immunoglobulin heavy chain IGHV variants The correct identification of immunoglobulin alleles in genome sequences is a challenge. Nevertheless, it can assist in the study of several human diseases associated with the antibody repertoire and in the development of new therapies using antibody engineering techniques. The advent of next-generation sequencing of human genomes and antibody repertoires enabled the development of several tools for the mapping and identification of new immunoglobulin (Ig) alleles. Some of these tools use 1,000 Genomes (G1K) data for new Ig alleles discovery. However, genome data from G1K present low coverage and variant call problems. Here, a computational screen of immunoglobulin alleles was carried out in the Genome Aggregation Database (gnomAD), the largest high-quality catalogue of variation from 125,748 exomes and 15,708 human genomes.

A total of 10,909 putative IGHV alleles were identified, in which 10,828 of them are new and 2,024 appear at least in 6 different alleles from genomes/exomes. The IGHV2-70 was the IGHV gene segment with the largest number of variants described. The majority of the variants were found in the framework 3 and most of them are missense. Interestingly, a large number of variants were found to be population exclusive. A database integrated with a web platform was created (YGL-DB) to store and make accessible the likely new variants found.

This available data can help the scientific community to validate new IGHV variants as well as it can shed light on the importance of variants in disease development and immunization protocols.

sentences: Adaptive Behavior and the Role of Primary Somatosensory Cortex Behavioral experience and flexibility are crucial for survival in a constantly changing environment. Despite evolutionary pressures to develop adaptive behavioral strategies in a dynamically changing sensory landscape, the underlying neural correlates have not been well explored. Here, we use genetically encoded voltage imaging to measure signals in primary somatosensory cortex (S1) during sensory learning and behavioral adaptation in the mouse. In response to changing stimulus statistics, mice adopt a strategy that modifies their detection behavior in a context dependent manner as to maintain reward expectation. Surprisingly, neuronal activity in S1 shifts from simply representing stimulus properties to transducing signals necessary for adaptive behavior in an experience dependent manner. Our results suggest that neuronal signals in S1 are part of an adaptive framework that facilitates flexible behavior as individuals gain experience, which could be part of a general scheme that dynamically distributes the neural correlates of behavior during learning.

sentences: LINE-1 retrotransposon activation intrinsic to interneuron development Transposable elements (TEs) are a reservoir of new transcription factor binding sites for protein-coding genes1-3. Developmental programs that activate TE-derived regulatory elements could, in principle, manifest in lineage-specific TE mobility. While somatic LINE-1 (L1) retrotransposon insertions have been detected in human neurons4-6, the impact of L1 insertions on neurodevelopmental gene regulation, and whether L1 mobility is restricted to certain neuronal lineages, is unknown. Here, we reveal programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV+) interneuron development7-9. PV+ neurons harbor unmethylated and euchromatic L1 promoters, express L1 mRNA, and permit L1 transgene mobilization in vivo. Elevated L1 expression in adult dentate gyrus PV+ neurons is however attenuated by environmental enrichment. Nanopore sequencing of PV+ neurons identifies unmethylated L1 loci providing alternative promoters to core PV+ neuron genes, such as CAPS2. These data depict SOX6-mediated L1 activation as an ingrained component of the mammalian PV+ neuron developmental program.

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Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization through Diminished Cell Spreading and Enhance Adipogenesis The disrupted surface of porous membranes, commonly used in tissue-chip and cellular co-culture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and sub-micron scale pores affect YAP localization and differentiation of adipose-derived stem cells (ADSCs). We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis, while decreasing osteogenesis.

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sentences: Correlated Functional Connectivity and Glucose Metabolism in Brain White Matter Revealed by Simultaneous MRI/PET Blood oxygenation level-dependent (BOLD) signals in white matter (WM) have usually been ignored or undetected, consistent with the lower vascular density and metabolic demands in WM than in gray matter (GM). Despite converging evidence demonstrating the reliable detection of BOLD signals in WM evoked by neural stimulation and in a resting state, few studies have examined the relationship between BOLD functional signals and tissue metabolism in WM. By analyzing simultaneous recordings of MRI and PET data, we found that the correlations between low frequency resting state BOLD signals in WM are spatially correlated with local glucose uptake, which also covaried with the amplitude of spontaneous low frequency fluctuations in BOLD signals. These results provide further evidence that BOLD signals in WM reflect variations in metabolic demand associated with neural activity, and suggest they should be incorporated into more complete models of brain function.

sentences: Cross-species Transcriptomic Network Analysis Reveals Links Between Ribosomal Protein Mutation and Cancer Ribosomal proteins (RPs) are critical to all cellular operations through their key roles in ribosome biogenesis and translation, as well as their extra-ribosomal functions. Leveraging previously identified RP mutants we characterised the RP macro transcriptome and sought to compare it with transcriptomes of pathologies associated with failures of ribosomal function, cancer and Zika virus (ZIKV). Single gene-based analysis revealed highly variable transcriptomes of RP mutations with little overlap in genes that were differentially expressed. In contrast, weighted gene co-expression network analysis revealed a highly conserved transcriptomic network pattern across all RP mutants studied. In addition, when we compared network changes in RP mutants, we observed similarities to transcriptome alterations in human cancer, and thus confirming the oncogenic role of RPs. Finally, it is known that ZIKV infection influences translational machinery, but this study shows infection network changes dissimilar to those of either the RP mutation or cancer.

sentences: Acclimation of phenology relieves leaf longevity constraints in deciduous forests Leaf phenology is key for regulating total growing season mass and energy fluxes. Long-term temporal trends towards earlier leaf unfolding are observed across Northern Hemisphere forests. Phenological dates also vary between years, whereby end-of-season (EOS) dates correlate positively with start-of-season (SOS) dates and negatively with growing season total net CO2 assimilation (Anet). These associations have been interpreted as the effect of a constrained leaf longevity or of premature carbon (C) sink saturation - with far-reaching consequences for long-term phenology projections under climate change and rising CO2. Here, we use multi-decadal ground and remote-sensing observations to show that the relationships between Anet and EOS are opposite at the interannual and the decadal time scales. A decadal trend towards later EOS persists in parallel with a trend towards increasing Anet - in spite of the negative Anet-EOS relationship at the interannual scale. This indicates that acclimation of phenology has enabled plants to transcend a constrained leaf longevity or premature C sink saturation over the course of several decades, leading to a more effective use of available light and a sustained extension of the vegetation CO2 uptake season over time.

sentences: Luminosity thresholds of colored surfaces are determined by their heuristic upper-limit luminances in the visual system We typically have a fairly good idea whether a given object is self-luminous or illuminated, but it is not fully understood how we make this judgement. This study aimed to identify determinants of the luminosity threshold, a luminance level at which a surface begins to appear self-luminous. We specifically tested a hypothesis that our visual system knows the maximum luminance level that a surface can reach under the physical constraint that a surface cannot reflect more light than any incident light and applies this prior to determine the luminosity thresholds. Observers were presented with a 2-degree circular test field surrounded by numerous overlapping colored circles and luminosity thresholds were measured as a function of (i) the chromaticity of the test field, (ii) the shape of surrounding color distribution and (iii) the color of the illuminant of the surrounding colors. We found that the luminosity thresholds peaked around the chromaticity of test illuminants and decreased as the purity of the test chromaticity increased. However, the loci of luminosity thresholds across chromaticities were nearly invariant to the shape of the surrounding color distribution and generally resembled the loci drawn from theoretical upper-limit luminances and upper-limit luminance boundaries of real objects. These trends were particularly evident for illuminants on the black-body locus and did not hold well under atypical illuminants such as magenta or green. These results support the idea that our visual system empirically internalizes the gamut of surface colors under natural illuminants and a given object appears self-luminous when its luminance exceeds this internalized upper-limit luminance.

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Jaagsiekte sheep retrovirus infection induces changes in microRNA expression in the ovine lung Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.

IMPORTANCEOvine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.

sentences: Experimental and Bioinformatic upgrade for genome-wide mapping of nucleosomes in Trypanosoma cruzi 1In Trypanosoma cruzi, as in every eukaryotic cell, DNA is packaged into chromatin by octamers of histone proteins that constitute nucleosomes. Besides compacting DNA, nucleosomes control DNA dependent processes by modulating the access of DNA binding proteins to regulatory elements on the DNA; or by providing the platform for additional layers of regulation given by histone variants and histone post-translational modifications. In trypanosomes, protein coding genes are constitutively transcribed as polycistronic units. Therefore, gene expression is controlled mainly post transcriptionally. However, chromatin organization and the histone code influence transcription, cell cycle progression, replication and DNA repair. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities of these processes in trypanosomes. Digestion of chromatin with micrococcal nuclease followed by deep sequencing has been widely applied for genome-wide mapping of nucleosomes in several organisms. Nonetheless, this parasite presents numerous singularities. On one hand, special growth conditions and cell manipulation are required. On the other hand, chromatin organization shows some uniqueness that demands a specially designed analytical approach. An additional entanglement is given by the nature of its genome harboring a large content of repetitive sequences and the poor quality of the genome assembly and annotation of many strains. Here, we adapted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we developed an exhaustive and thorough computational workflow for data analysis, highlighting the relevance of using its whole genome as a reference instead of the commonly used Esmeraldo-like haplotype. Moreover, the performance of two aligners, Bowtie2 and HISAT2 was tested to find the most appropriate tool to map any genomic read to reference genomes bearing this complexity.

sentences: Mosquito saliva sialokinin-dependent enhancement of arbovirus infection through endothelial barrier leakage Viruses transmitted by Aedes mosquitoes constitute an increasingly important global health burden. Defining common determinants of host susceptibility to this large group of heterogenous pathogens are key for informing the rational design of new pan-viral medicines. Infection of the vertebrate host with these viruses is enhanced by the presence of mosquito saliva, a complex mixture of salivary gland-derived factors and microbiota. We show that enhancement of infection by saliva was dependent on vascular function and was independent of most anti-saliva immune responses, including to salivary microbiota. Instead, the Aedes gene product sialokinin mediated enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having vertebrate-like tachykinin sequence and is absent from non-vector competent Anopheles mosquitoes, whose saliva was not pro-viral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have potential to limit disease severity following infection with Aedes mosquito-borne viruses.

sentences: Emergence of XDR high-risk Pseudomonas aeruginosa ST309 in South America: a global comparative genomic analysis Pseudomonas aeruginosa has been considered one of the major nosocomial pathogens associated with elevated morbidity and mortality worldwide. Outbreaks have been associated with few high-risk pandemic P. aeruginosa lineages, presenting a remarkable antimicrobial resistance. However, the biological features involved with the persistence and spread of such lineages among clinical settings remain to be unravel. This study reports the emergence of the ST309 P. aeruginosa lineage in South America/Brazil, more precisely, in the Amazon region. Global genomic analyses were performed with the Brazilian strain (PA834) and more 41 complete and draft ST309 genomes publicly available, giving insights about ST309 epidemiology and its resistome and mobilome. Antimicrobial susceptibility tests revealed that the Brazilian PA834 strain presented the XDR phenotype, which was mainly due to intrinsic resistance mechanisms. Genomic analyses revealed a heterogeneous distribution of acquired antimicrobial resistance genes among ST309 genomes, which included blaVIM-2, blaIMP-15 and qnrVC1, all of them associated with class 1 integrons. The mobilome mining showed the presence of Integrative and Conjugative Elements, transposons and genomic islands harbouring a huge arsenal of hevy metal resistance genes. Moreover, these elements also carried genes involved with virulence and adaptive traits. Therefore, the presence of such genes in ST309 lineage possibly accounted for the global spread and persistence of this emerging clone, and for its establishment as a pandemic lineage of clinical importance.

sentences: Results from a conservation initiative for parrotfishes in the Colombian Caribbean Parrotfish (Family Scaridae) are a family of herbivorous fishes crucial to coral reef health, particularly for Caribbean reefs due to their declining coral cover. However, despite parrotfish are fully protected in some countries, they are still heavy fished in most of their Caribbean range. The consequences of this targeted fishery in the Colombian Caribbean are not fully understood due to a lack of local conservation and management resources. This research aimed to evaluate and enhance the conservation status and protection of parrotfish among local communities in the National Natural Park Corales del Rosario and San Bernardo. Underwater visual census surveys (UVC) were undertaken to evaluate reef fish community structure, and participatory education campaigns and activities were carried out with local communities to raise awareness about parrotfish ecology and their functional role in conserving Caribbean coral reef ecosystems of Colombia. UVC showed parrotfish to be dominant in the fish community, yet there was evidence of exploitation of large adults by selective fishing. Conflicts exist between the community and environmental authorities because fishing regulations are not clear, and the level of enforcement is insufficient. Parrotfish are sold to tourists, as  red snapper to fulfil high seafood demand since commercially valuable fish are now scarce. However, following intensive awareness-raising activities developed as part of this study, the community has started to recognize the vital ecological role of parrotfish in coral reef systems, and are suggesting a redrafting of fishing legislation by the environmental authorities, in order to recognise and incorporate the traditional fishing rights of human communities living within the MPA. Lobbying for the protection of parrotfish and inclusion of local communities in decision-making will take time, but this research represents the crucial first steps towards sustainable practice and cooperative alliances in the Colombian Caribbean.

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Loss of the Long Non-coding RNA OIP5-AS1 Exacerbates Heart Failure in a Sex-Specific Manner BackgroundLong ncRNAs (lncRNAs) are known to influence numerous biological processes including cellular differentiation and tissue development. They are also implicated in the maintenance, health and physiological function of many tissues including the heart. Indeed, manipulating the expression of specific lncRNAs has been shown to improve pathological cardiac phenotypes such as heart failure. One lncRNA studied in various settings is OIP5-AS1 (also known as 1700020I14Rik and Cyrano), however its role in cardiac pathologies remains mostly uncharacterised.

MethodsWe used data generated from FACS sorted murine cardiomyocytes, human iPSC derived cardiomyocytes, as well as heart tissue from various animal models to investigate OIP5-AS1 expression in health and disease. Using CRISPR we engineered a global OIP5-AS1 knock out (KO) mouse model and performed cardiac pressure overload experiments to study heart failure in these animals. RNA-sequencing of left ventricles provided mechanistic insight between WT and KO mice.

ResultsWe demonstrate that OIP5-AS1 expression is regulated during cardiac development and cardiac specific pathologies in both rodent and human models. Moreover, we demonstrate that global female OIP5-AS1 KO mice develop exacerbated heart failure, but male mice do not. Transcriptomics and gene set enrichment analysis suggests that OIP5-AS1 may regulate pathways that impact mitochondrial function.

ConclusionsOIP5-AS1 is regulated in cardiac tissue and its deletion leads to worsening heart function under pressure overload in female mice. This may be due to impairments in mitochondrial function, highlighting OIP5-AS1 as a gene of interest in sex-specific differences in heart failure.

sentences: TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression through controlling chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes that, interestingly, differed in part from the one observed upon treatment with the hypomethylating agent decitabine. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Thus, an extensive cross-talk between TET2 and the oncogenic transcription factor MYC establishes a lysosomal storage disease-like state that contributes to an exacerbated sensitivity to autophagy inducers.

sentences: Inter-Subject Correlation during New Music Listening: A Study of Electrophysiological and Behavioral Responses to Steve Reich's Piano Phase Musical minimalism utilizes the temporal manipulation of restricted collections of rhythmic, melodic, and/or harmonic materials. One example, Steve Reichs Piano Phase, offers listeners readily audible formal structures containing unpredictable events at local levels. Pattern recurrences may generate strong expectations which are violated by small temporal and pitch deviations. A hyper-detailed listening strategy prompted by these minute deviations stands in contrast to the type of listening engagement typically cultivated around functional tonal Western music. Recent research has suggested that the inter-subject correlation (ISC) of electroencephalographic (EEG) responses to natural audio-visual stimuli objectively indexes a state of "engagement", demonstrating the potential of this approach for analyzing music listening. But can ISCs capture engagement with minimal music, which features less obvious expectation formation and has historically received a wide range of reactions? To approach this question, we collected EEG and continuous behavioral (CB) data while 30 adults listened to an excerpt from Steve Reichs Piano Phase, as well as three controlled manipulations and a popular-music remix of the work. Our analyses reveal that EEG and CB ISC are highest for the remix stimulus and lowest for our most repetitive manipulation. In addition, we found no statistical differences in overall EEG ISC between our most musically meaningful manipulations and Reichs original piece. We also found that aesthetic evaluations corresponded well with overall EEG ISC. Finally we highlight co-occurrences between stimulus events and time-resolved EEG and CB ISC. We offer the CB paradigm as a useful analysis measure and note the value of minimalist compositions as a limit case for studying music listening using EEG ISC. We show that ISC is less effective at measuring engagement with this minimalist stimulus than with popular music genres and argue that this may be due to a difference between the type of engagement measured by ISC and the particular engagement patterns associated with minimalism.

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Molecular epidemiology of peste des petits ruminants virus emergence in critically endangered Mongolian saiga antelope and other wild ungulates Peste des petits ruminants virus (PPRV) causes disease in domestic and wild ungulates, is the target of a global eradication programme and threatens biodiversity. Understanding the epidemiology and evolution of PPRV in wildlife is important, but hampered by the paucity of wildlife-origin PPRV genomes. In this study, full PPRV genomes were generated from three Mongolian saiga antelope, one Siberian ibex and one goitered gazelle from the 2016-2017 PPRV outbreak. Phylogenetic analysis showed that for Mongolian and Chinese PPRV since 2013, the wildlife and livestock-origin genomes were closely related and interspersed. There was strong phylogenetic support for a monophyletic group of PPRV from Mongolian wildlife and livestock, belonging to clade of lineage IV PPRV from livestock and wildlife from China since 2013. Discrete diffusion analysis found strong support for PPRV spread into Mongolia from China and phylogeographic analysis indicated Xinjiang Province as the most likely origin, although genomic surveillance for PPRV is poor and lack of sampling from other regions could bias this result. Times of most recent common ancestor (TMRCA) were June 2015 (95% HPD: August 2014 - March 2016) for all Mongolian PPRV genomes and May 2016 (95% HPD: October 2015 - October 2016) for Mongolian wildlife-origin PPRV. This suggests that PPRV was circulating undetected in Mongolia for at least six months before the first reported outbreak in August 2016, and that wildlife were likely infected before livestock vaccination began in October 2016. Finally, genetic variation and positively-selected sites were identified that might be related to PPRV emergence in Mongolian wildlife. This study is the first to sequence multiple PPRV genomes from a wildlife outbreak, across several host species. Additional full PPRV genomes and associated metadata from the livestock-wildlife interface are needed to enhance the power of molecular epidemiology, support PPRV eradication and safeguard the health of the whole ungulate community.

Author SummaryRecent mass mortality of critically endangered Mongolian saiga antelope due to peste des petits ruminants virus (PPRV) has dramatically highlighted the threat this viral disease represents for biodiversity. The genome of viruses such as PPRV evolve fast, so virus genetic data gathered from infected animals can be used to trace disease spread between livestock and wildlife, and to determine if the virus is adapting to infect wildlife more efficiently. Here we obtained PPRV virus genomes from Mongolian wildlife and compared them with other published PPRV genomes. Using a molecular clock, we estimated that the disease was circulating in Mongolia well before it was first reported. Genetic analyses support the hypothesis of virus spread from livestock to wildlife, with genetic changes potentially helping infection in Asian wild ungulates. However, more PPR virus genomes and epidemiology data are needed from disease outbreaks in areas shared between livestock and wildlife to confirm these results and take efficient actions to safeguard the health of the whole ungulate community.

sentences: Sequence structure organizes items in varied latent states of working memory neural network In memory experiences, events do not exist independently but are linked with each other via structure-based organization. Structure context largely influences memory behavior, but how it is implemented in the brain remains unknown. Here, we combined magnetoencephalogram (MEG) recordings, computational modeling, and impulse-response approaches to probe the latent states when subjects held a list of items in working memory (WM). We demonstrate that sequence context reorganizes WM items into distinct latent states, i.e., being reactivated at different latencies during WM retention, and the reactivation profiles further correlate with recency behavior. In contrast, memorizing the same list of items without sequence task requirements disrupts the recency effect and elicits comparable neural reactivations. Computational modeling further reveals a dominant function of sequence context, instead of passive memory decaying, in characterizing recency effect. Taken together, sequence structure context shapes the way WM items are stored in the human brain and essentially influences memory behavior.

sentences: Typical hippocampal transcriptional response across estrous is dysregulated by Cnih3 gene deletion The hippocampus is a critical brain region for coordinating learning, memory, and behavior. In females, the estrous cycle alters these functions through steroid hormone activity, with well-characterized effects on cellular physiology and behavior. However, the molecular basis of these outcomes has not been systematically explored. Therefore, we profiled the transcriptome of dorsal hippocampi from female mice in each estrous cycle stage, and contrasted it with that of males. We identify only subtle sex differences in gene expression between the sexes on average, yet comparing males to individual estrous stages reveals up to thousands of genes deviating from male expression patterns at specific estrous stages. These estrous-responsive genes are especially enriched in gene markers of oligodendrocytes and the dentate gyrus, and in functional gene sets relating to estrogen response, potassium channels, and synaptic gene splicing. Next we profiled Cnih3 knockouts across estrous to provide insight into their previously reported estrous-dependent phenotypes in hippocampal synaptic plasticity, composition, and learning and memory behaviors. Surprisingly, Cnih3 knockouts showed far broader transcriptomic differences between estrous cycle stages and males. Moreover, Cnih3 knockout drove subtle but extensive expression changes accenting sex differential expresssion at diestrus and estrus. Altogether, our profiling constitutes both a resource characterizing estrous-specific gene expression patterns in the adult hippocampus, which can provide insights into mechanisms of sex differential neuropsychiatric functions and dysfunctions, while also highlighting roles of Cnih3 as a buffer against transcriptional effects of estrous and providing insights into the molecular mechanisms that may underlie estrous-dependent phenotypes with its loss.

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Targeted redesign of suramin analogs for novel antimicrobial lead development The emergence of new viral infections and drug resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets is one potential way in which to accelerate this process. Suramin was initially developed as a successful anti-parasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous towards positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure activity relationship (SAR) studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramins target diversity, confirming that suramin preferentially binds to protein pockets which are both positively charged and enriched in aromatic or leucine residues. Further, suramins high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.

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sentences: AMPK regulates germline stem cell integrity and quiescence through a mir-1/tbc-7/rab-7 pathway in C. elegans. During periods of energetic stress, Caenorhabditis elegans can undergo a global quiescent stage known as "dauer". During this stage, all germline stem cells undergo G2 cell cycle arrest through an AMPK-dependent mechanism. In animals that lack AMPK signalling, the germ cells fail to arrest, undergo uncontrolled proliferation and lose their reproductive capacity. These germline defects are accompanied by an altered chromatin landscape and gene expression program. We identified an allele of tbc-7, a RabGAP protein that functions in the neurons, which when compromised, suppresses the germline hyperplasia in the dauer larvae, as well as the post-dauer sterility and somatic defects characteristic of AMPK mutants. This mutation also corrects the abundance and aberrant distribution of transcriptionally activating and repressive chromatin marks in animals that otherwise lack all AMPK signalling. We identified RAB-7 as one of the potential RAB proteins that is regulated by tbc-7 and show that the activity of RAB-7 is critical for the maintenance of germ cell integrity during the dauer stage. A singular small RNA, mir-1, was identified as a direct negative regulator of tbc-7 expression through the analysis of seed sequences on the 3UTR of tbc-7. Animals lacking mir-1 are post-dauer sterile, displaying a similar phenotype to AMPK mutants. Altogether, our findings describe a novel mir-1/tbc-7/rab-7 pathway occurring in the neurons that regulates the germ line in a cell non-autonomous manner.

sentences: Uncharted biosynthetic potential of the ocean microbiome Microbes are phylogenetically and metabolically diverse. Yet capturing this diversity, assigning functions to host organisms and exploring the biosynthetic potential in natural environments remains challenging. We reconstructed >25,000 draft genomes, including from >2,500 uncharacterized species, from globally-distributed ocean microbial communities, and combined them with [~]10,000 genomes from cultivated and single cells. Mining this resource revealed [~]40,000 putative biosynthetic gene clusters (BGCs), many from unknown phylogenetic groups. Among these, we discovered Candidatus Eudoremicrobiaceae as one of the most biosynthetically diverse microbes detected to date. Discrete transcriptional states structuring natural populations were associated with a potentially niche-partitioning role for BGC products. Together with the characterization of the first Eudoremicrobiaceae natural product, this study demonstrates how microbiomics enables prospecting for candidate bioactive compounds in underexplored microbes and environments.

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The impact of genetic risk for Alzheimers disease on the structural brain networks of young adults To facilitate pre-symptomatic diagnosis of late-onset Alzheimers disease, non-invasive imaging biomarkers could be combined with genetic risk information. In this work, we investigated the structural brain networks of young adults in relation to polygenic risk for Alzheimers disease, using magnetic resonance imaging (MRI) and genotype data for 562 19-year-old participants from the Avon Longitudinal Study of Parents and Children. Diffusion MRI data were acquired on a 3T scanner, and the data were used to perform whole-brain tractography. The resulting tractograms were used to generate structural brain networks, using the number of streamlines and the diffusion tensor fractional anisotropy as edge weights. This was done for the whole-brain connectome, and for the default mode, limbic and visual subnetworks. The mean clustering coefficient, mean betweenness centrality, characteristic path length, global efficiency and mean nodal strength were calculated for these networks, for each participant. The hubs of the networks were also identified, and the connectivity of the rich-club, feeder and local connections was also calculated. Polygenic risk scores (PRS), estimating the burden of genetic risk carried by an individual, were calculated both at genome-wide level and for nine specific disease pathways. The correlation coefficients were calculated between the PRS and a) the graph theoretical metrics of the structural networks and b) the rich-club, feeder and local connectivity of the whole-brain networks.

In the visual subnetwork, the mean nodal strength was negatively correlated with the genome-wide PRS including the APOE locus (r = -0.19, p = 1.3x10-5), the mean betweenness centrality was positively correlated with the pathway-specific PRS for plasma lipoprotein particle assembly including the APOE locus (r = 0.16, p = 9.2x10-4), and the mean clustering coefficient was negatively correlated with the tau protein binding PRS including the APOE locus (r = 0.16, p = 9.2x10-4). In the default mode network, the mean nodal strength was negatively correlated with the genome-wide PRS, including the APOE locus (r = -0.14, p=1.5x10-3). The rich-club and feeder connectivities were negatively correlated with the genome-wide PRS including the APOE locus (r = -0.16, p = 3.7x10-4; r = -0.15, p 8.8x10-4, respectively). Our results indicate small changes in the brain connectome of young adults at risk of developing Alzheimers disease in later life.

sentences: Whole-genome sequencing analysis reveals the population history of Mus musculus in Madagascar In Madagascar, the house mouse (Mus musculus) is thought to have colonized with human activities and is now one of the most abundant rodents on the island. In this study, we determined the whole-genome sequences of five Madagascar house mice captured from the wild. We examined the evolutionary history of the population by analyzing the mitochondrial and autosomal genomes. We confirmed that the mitochondrial genome lineages of Madagascar house mice formed a monophyletic clade placed at one of the most basal positions in the species. An analysis of autosomal genomic sequences indicates that the Madagascar house mice are genetically members of M. m. castaneus (CAS), but also contain genetic elements of M. m. domesticus (DOM) resulting from hybridization between subspecies. The signature of a strong population bottleneck 1000-3000 years ago was observed in both mitochondrial and autosomal genomic data. All samples showed strong genetic affinity to many CAS samples across a wide range of Indian Ocean coastal and island regions, with divergence time estimated around 4000 years ago. These findings support that the Madagascar house mice started to colonize the island with human agricultural activity, and experienced complex history for the establishment.

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Human neutrophil development and functionality are enabled in a humanized mouse model Mice with a functional human immune system serve as an invaluable tool to study the development and function of human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted (G-CSFR) in existing MISTRG mice. By targeting the GCSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

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

sentences: The genetic basis of interspecies cooperation and conflict in naturally-occurring isolates of a model plant symbiont Given the need to predict the outcomes of evolution in host-associated microbiomes, whether microbial and host fitnesses tend to trade off, generating conflict, remains a pressing question. Examining the relationships between host and microbe fitness proxies at both the phenotypic and genomic levels can illuminate the mechanisms underlying interspecies cooperation and conflict. We conducted genome-wide association studies (GWAS) to examined naturally-occurring genetic variation in 191 strains of the model microbial symbiont, Ensifer meliloti, paired with each of two host Medicago truncatula genotypes in single- or multi-strain experiments. Using this data, we determine how multiple proxies of microbial and host fitness were related to one another and test key predictions about mutualism evolution at the genomic scale, while also addressing the challenge of measuring microbial fitness. We found little evidence for interspecies fitness conflict; loci tended to have concordant effects on both microbe and host fitnesses, even in environments with multiple co-occurring microbes. Our results emphasize the importance of quantifying microbial relative fitness for understanding microbiome evolution and thus harnessing microbiomes to improve host fitness. Additionally, we find that mutualistic coevolution between hosts and microbes potentially acts to maintain, rather than erode, genetic diversity, thus, explaining why variation in mutualism traits persists in nature.

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Metallothionein 3-zinc axis suppresses caspase-11 inflammasome activation and impairs antibacterial immunity Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE- 4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (M{phi}) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), M{phi} increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1{beta}. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram- negative bacteria.

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sentences: Staphylococcus aureus β-toxin exerts anti-angiogenic effects by inhibiting re-endothelialization and neovessel formation Staphylococcus aureus is the causative agent of numerous severe human infections associated with significant morbidity and mortality worldwide. S. aureus often targets the vascular endothelium to interfere with proper host responses during invasive infections. In this study, we provide evidence that S. aureus {beta}-toxin inhibits wound repair mechanisms in human endothelial cells by preventing cell proliferation and migration. These findings were confirmed in a rabbit aortic explant model where {beta}-toxin impedes sprout formation. Decreased cell proliferation was accompanied by decreased production of the angiogenic proteins endothelin-1, IGFBP-3, thrombospondin-1, TIMP-1, and TIMP-4. Meanwhile, inhibited wound repair was marked by increased HGF secretion from endothelial cells, likely a marker of endothelial cell damage. Together, these findings establish a mechanistic role for {beta}-toxin where it inhibits proper tissue repair processes that likely promote S. aureus infective niche.

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High-performance Saccharomyces cerevisiae-based biosensor for heavy metal detection Heavy metals, i.e., Cu(II), are harmful to the environment. There is an increasing demand to develop inexpensive detection methods for heavy metals. Here, we developed a yeast biosensor with reduced-noise and improved signal output for potential on-site copper ion detection. The copper-sensing circuit was achieved by employing a secondary genetic layer to control the galactose-inducible (GAL) system in Saccharomyces cerevisiae. The reciprocal control of the Gal4 activator and Gal80 repressor under copper-responsive promoters resulted in a low-noise and ultrasensitive yeast biosensor for the copper ion detection. Furthermore, we developed a betaxanthin-based colorimetric assay, as well as 2-phenylethanol and styrene-based olfactory outputs for the copper ion detection. Notably, our engineered yeast sensor confers a narrow range switch-like behavior, which can give a "yes/no" response when coupled with betaxanthin-based visual phenotype. Taken together, we envision that the design principle established here might be applicable for developing other sensing systems for various chemical detections.

IMPORTANCEThe accumulation of heavy metals in the environment led to an imbalance with environmental and ecological impacts, and there is an increasing demand for inexpensive methods that permit on-site detection of heavy metals. In this study, a yeast biosensor with reduced-noise and improved signal output was developed. We used a betaxanthin-based colorimetric assay for monitoring the copper contamination in water samples, which confers a switch-like behavior with a "yes/no" response. In addition, we also explored the 2-phenylethanol and styrene-based outputs for copper detection, which might be useful for olfactory detection of heavy metals.

sentences: Using ddRAD-seq phylogeography to test for genetic effects of headwater river capture in suckermouth armored catfish (Loricariidae: Hypostomus) from the central Brazilian Shield River capture is a geological process of potentially great importance in shaping the genetic diversity, distributions, and community composition of freshwater taxa. Using phylogeographic analyses of ddRAD-seq data from suckermouth armored catfish (Hypostomus sp. 2) populations, we tested for predicted genetic effects of headwater river capture events in central Brazil, previously supported by geological and community ecological data. We analyzed 227 ddRAD tags (3829 SNP loci) across 42 samples. Molecular results strongly supported six Hypostomus genetic clusters/lineages, with the deepest divergence [~]1.25 million years ago in the early Pleistocene between a clade from the Upper Parana and Upper Sao Francisco river basins versus all other lineages. Consistent with the  Parana Capture Hypothesis, several lines of evidence supported mid-Pleistocene colonization and vicariant isolation of Hypostomus populations from an ancestral Upper Parana population, including: (1) significant phylogeographic structure, with predicted phylogenetic patterns, (2) higher Parana lineage diversity, (3) ancestral geographic locations reconstructed in the Parana basin, and (4) non-random interdrainage dispersal and vicariance events, indicating river captures primarily into the Tocantins and Upper Sao Francisco basins c. [~]220,000-145,500 years ago. Phylogeographic inference was complicated by lack of lineage monophyly across loci and lineages distributed in multiple basins, the latter of which lent support to the non-mutually exclusive  Frequent Interdrainage Dispersal Hypothesis. However, species tree and demographic modeling results suggested these were artefacts of incomplete sorting of alleles in large ancestral populations over a geologically recent timeframe of divergence. Qualitative and quantitative sensitivity analyses demonstrated that our downstream genetic results were robust to effects of varying ddRAD-seq assembly parameters, which heavily influenced the number of output loci. We predict that codistributed freshwater taxa in Central Brazil may not exhibit phylogeographic patterns similar to Hypostomus sp. 2 due to complex patterns of superimposed river capture events, or if smaller ancestral population sizes have allowed more complete lineage sorting in other taxa.

sentences: SYMBIOSIS: Synthetic Manipulable Biobricks via Orthogonal Serine Integrase Systems Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method that can robustly manipulate DNA parts in vivo and in vitro. First, we proposed a "Keys match Locks" model to demonstrate that three orthogonal serine integrases are able to irreversibly and stably switch on seven synthetic biobricks with high accuracy in vivo. Then, we demonstrated that purified integrases can facilitate the assembly of "Donor" and "Acceptor" plasmids in vitro to construct composite plasmids. Finally, we used SYMBIOSIS to assemble different chromoprotein genes and create novel colored Escherichia coli. We anticipate that our SYMBIOSIS strategy will accelerate synthetic biobricks manipulation, genetic circuit design, and multiple plasmids assembly for synthetic biology with broad potential applications.

sentences: Neural Readout of a Latency Code in the Active Electrosensory System The latency of spikes relative to a stimulus conveys sensory information across modalities. However, in most cases it remains unclear whether and how such latency codes are utilized by postsynaptic neurons. In the active electrosensory system of mormyrid fish, a latency code for stimulus amplitude in electroreceptor afferent nerve fibers (EAs) is hypothesized to be read out by a central reference provided by motor corollary discharge (CD). Here we demonstrate that CD enhances sensory responses in postsynaptic granular cells of the electrosensory lobe, but is not required for reading out EA input. Instead, diverse latency and spike count tuning across the EA population gives rise to graded information about stimulus amplitude that can be read out by standard integration of converging excitatory synaptic inputs. Inhibitory control over the temporal window of integration renders two granular cell subclasses differentially sensitive to information derived from relative spike latency versus spike count.

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Systems analysis shows a role of cytophilic antibodies in shaping innate tolerance to malaria The mechanism of acquisition and maintenance of natural immunity against Plasmodium falciparum malaria remains unclear. Although, clinical immunity develops over time with repeated malaria episodes, disease tolerance is more rapidly acquired compared to protective immunity. It remains unclear, how pre-existing immune responses impacts the mechanism responsible for disease tolerance. Here, we investigated a cohort of returning travelers treated for acute symptomatic P. falciparum malaria, either infected for the first time, or with a previous history of malaria. Through repeated sampling over one year in a malaria free setting, we were able to study the acute and longitudinal effects of the infection. We combined comprehensive immune cell and plasma protein profiling with integrated and data driven analysis, describing the immune landscape from acute disease to one year after infection. We identified a strong association between pro-inflammatory signatures and {gamma}{delta} T cell expansion. The association was significantly impacted by previous exposure to malaria, resulting in a dampened pro-inflammatory response, which translated to reduced V{delta}2+ {gamma}{delta} T cell expansion compared to primary infected individuals. The dampened inflammatory signal was associated with early expansion of Fc{gamma}RIII+ monocytes and parasite-specific antibodies of IgG1 and IgG3 isotypes.

Our data suggest that the interplay of Fc{gamma}RIII+ monocytes and a cytophilic parasite-specific IgG during the early blood stage infection lead to lower parasitemia and a dampened pro-inflammatory response with reduced {gamma}{delta} T cell expansion. This enhanced control and reduced inflammation points to a potential mechanism on how tolerance is established following repeated malaria exposure.

One Sentence SummaryA systems immunology analysis on natural malaria sheds light on disease tolerance mechanism associated with gamma delta T cell expansion

sentences: Effects of substrate stiffness and actin velocity on fibronectin fibril morphometry and mechanics Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also used the model to look at two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both tissue stiffness and contractile phenotype, with intermediate contractility and high tissue stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of tissue stiffness and cell contractility on FN fibril assembly.

sentences: HiCzin: Normalizing metagenomic Hi-C data and detecting spurious contacts using zero-inflated negative binomial regression High-throughput chromosome conformation capture (Hi-C) has recently been applied to natural microbial communities and revealed great potential to study multiple genomes simultaneously. Several extraneous factors may influence chromosomal contacts rendering the normalization of Hi-C contact maps essential for downstream analyses. However, the current paucity of metagenomic Hi-C normalization methods and the ignorance for spurious inter-species contacts weaken the interpretability of the data. Here, we report on two types of biases in metagenomic Hi-C experiments: explicit biases and implicit biases, and introduce HiCzin, a parametric model to correct both types of biases and remove spurious inter-species contacts. We demonstrate that the normalized metagenomic Hi-C contact maps by HiCzin result in lower biases, higher capability to detect spurious contacts, and better performance in metagenomic contig clustering. The HiCzin software and Supplementary Material are available at https://github.com/dyxstat/HiCzin.

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Endocytosis against high turgor pressure is made easier by partial protein coating and a freely rotating base During clathrin-mediated endocytosis, a patch of flat plasma membrane is deformed into a vesicle. In walled cells, such as plants and fungi, the turgor pressure is high and pushes the membrane against the cell wall, thus hindering membrane internalization. In this paper, we study how a patch of membrane is deformed against turgor pressure by force and by curvature-generating proteins. We show that a large amount of force is needed to merely start deforming the membrane and an even larger force is needed to pull a membrane tube. The magnitude of these forces strongly depends on how the base of the membrane is constrained and how the membrane is coated with curvature-generating proteins. In particular, these forces can be reduced by partially but not fully coating the membrane patch with curvature-generating proteins. Our theoretical results show excellent agreement with experimental data.

SIGNIFICANCEYeast cells have been widely used as a model system to study clathrin-mediated endocytosis. The mechanics of membrane during endocytosis has been extensively studied mostly in low turgor pressure condition, which is relevant for mammalian cells but not for yeast cells. It has been suggested that as a result of high turgor pressure in yeast cells, a large amount of force is needed to drive the progress of the membrane invagination. In this paper, we investigated biologically relevant mechanisms to reduce the force requirement. We highlight the role of boundary conditions at the membrane base, which is a factor that has been largely ignored in previous studies. We also investigate the role of curvature-generating proteins and show that a large protein coat does not necessarily reduce the force barrier for endocytosis.

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The developing kidney actively negotiates geometric packing conflicts to avoid defects The physiological functions of several organs rely on branched tubular networks, but little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between tubule tip duplication and limited area at the organ surface. Imaging, computational, and soft material modeling of tubule  families identifies six geometric packing phases, including two defective ones. Experiments in kidney explants show that a retrograde tension on tubule families is necessary and sufficient for them to avoid defects by switching to a vertical orientation that increases packing density. These results reveal developmental contingencies in response to physical limitations, and create a framework for classifying kidney defects.

One-Sentence SummaryEpithelial branching in the kidney causes a geometric packing conflict that is resolved through internally generated tensions

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Assessing extracellular vesicles from bovine mammary gland epithelial cells cultured in FBS-free medium AimMammary gland extracellular vesicles (EVs) are found in both human and livestock milk. Our knowledge of the role of EVs in the mammary gland development, breast cancer and mastitis derives mainly from in vitro cell culture models. However, a commonly shared limitation is the use of foetal bovine serum (FBS) as a supplement, which naturally contains EVs. For this reason, the purpose of the study is to establish a novel tool to investigate mammary gland EVs in vitro and in an FBS-free system.

MethodsPrimary bovine mammary epithelial cells (pbMECs) and a mammary gland alveolar epithelial cell line (MAC-T) were cultured in a chemically defined EV-free medium. To find a reliable EVs isolation protocol from a starting cell conditioned medium (10 mL), we compared eight different methodologies by combining ultracentrifugation (UC), chemical precipitation (CP), size exclusion chromatography (SEC), and ultrafiltration (UF).

ResultsThe medium formula sustained both pbMECs and MAC-T cell growth and did not alter MAC-T cell identity. Transmission electron microscopy revealed that we obtained EV-like particles in five out of eight protocols. The cleanest samples with the highest particles amount and detectable amounts of RNA were obtained by using UF-SEC-UC and UC-SEC-UC.

ConclusionOur chemically defined, EV-free medium sustains the growth of both pbMECs and MAC-T and allows the isolation of EVs that are free from any contamination by UF-SEC-UC and UC-SEC-UC. In conclusion, we propose a new culture system and EVs isolation protocols for further research on mammary epithelial EVs.

sentences: Endogenous retroviruses co-opted into divergently transcribed regulatory elements shape the regulatory landscape of embryonic stem cells Transposable elements are an abundant source of transcription factor binding sites and favorable genomic integration may lead to their recruitment by the host genome for gene regulatory functions. However, it is unclear how frequent co-option of transposable elements as regulatory elements is, to which regulatory programs they contribute and how they compare to regulatory elements devoid of transposable elements. Here, we report a transcription initiation-centric, in-depth characterization of the transposon-derived regulatory landscape of mouse embryonic stem cells. We demonstrate that a substantial number of transposable elements, in particular endogenous retroviral elements, carry open chromatin regions that are divergently transcribed into unstable RNAs in a cell-type specific manner, and that these elements contribute to a sizable proportion of active enhancers and gene promoters. We further show that transposon subfamilies contribute differently and distinctly to the pluripotency regulatory program through their repertoires of transcription factor binding sites, shedding light on the formation of regulatory programs and the origins of regulatory elements.

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Novel anti-inflammatory treatment for bradykinin-induced sore throat or pharyngitis BackgroundOften thought of as a minor health concern, sore throat or pharyngitis is an important public health issue. It is one of the most common symptoms of upper respiratory diseases including COVID-19 and is a leading cause of physician visits and antibiotic prescriptions. However, few over the counter medications are proven to heal sore throat inflammation.

MethodsAdenocarcinomic human alveolar basal epithelial cells (A549 cells) and three dimensional organotypic human respiratory tissues were used to study inflammation and various treatment effects on respiratory epithelia. The cells and tissues were studied both in the presence and absence of bradykinin, one of the first inflammatory mediators of pharyngitis. Inflammation was measured by analyzing levels of prostaglandin E2 (PGE2), interleukin 8 (IL-8), and leukotriene B4 (LTB4), transepithelial electrical resistance (TEER), and lactate dehydrogenase (LDH) release. Tissue morphology was analyzed by immunohistochemistry.

ResultsIn studying pharyngitis using organotypic human respiratory tissue stimulated with bradykinin, we saw an increase in prostaglandin E2 (PGE2) and interleukin-8 (IL-8) in response to bradykinin. Acetyl salicylic acid (ASA), a non-specific COX inhibitor, was able to mitigate a bradykinin-induced increase in PGE2 in our studies. However, ASA was inflammatory above its therapeutic window, increasing levels of PGE2 and IL-8 above those seen with bradykinin stimulation alone. We describe a novel, scientifically validated treatment for sore throat, that contains a low dose of aspirin and other anti-inflammatory ingredients.

ConclusionThis study elucidates the complex mechanisms involved in healing pharyngitis, an inflammatory condition of the upper respiratory epithelia. An ASA-based formula (Biovanta) mitigated bradykinin-induced inflammation more strongly than ASA alone in organotypic human respiratory tissues. Surprisingly, we found that many of the most common over the counter sore throat therapies exacerbate inflammation and IL-8 in organotypic human respiratory tissues, suggesting these common treatments may increase the likelihood of further respiratory complications.

Competing interest statementThis study was funded entirely by Applied Biological Laboratories, a private company that owns the Biovanta product. Some studies were conducted by third parties in a blind format, as indicated. All other experiments were performed at Applied Biological Laboratories research facility located at the SUNY Downstate Biotechnology Incubator, a part of StartUP NY. All of the authors were employees of Applied Biological Laboratories at the time the experiments were performed.

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Phellem translational landscape throughout secondary development in Arabidopsis roots O_LIThe phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana.
C_LIO_LIUsing specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ).
C_LIO_LIWe showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified.
C_LIO_LIThis strategy provided an unprecedented resolution of the transcriptome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms controlling cell differentiation in periderm tissues of the model plant Arabidopsis.
C_LI

Significance statementTo improve the understanding of phellem differentiation into a suberized protective layer, we followed the establishment of periderm in Arabidopsis roots and sequenced the phellem-specific translatome. We found that phellem suberization occurs shortly after pericycle cell divisions with the induction of pivotal suberin biosynthesis genes. In parallel, we detected the activation of three central genetic modules acting throughout the phellem differentiation. This study provides a unique and targeted genetic resource for further functional studies of phellem tissues.

sentences: The Influence of Irreversibly Electroporated Cells on Post Pulse Drug Delivery to Reversibly Electroporated Cells Electroporation can result in cell death in some proportion of a population of cells and, because the nature of the membrane disruption can vary significantly in irreversibly electroporated cells, there is uncertainty in the magnitude of and the transient behaviour of the associated permeability increases. This study numerically investigates the drug uptake by a population of cells that includes both reversibly and irreversibly electroporated cells. A theoretical continuum model is developed and simulations are conducted in conditions of low porosity (cells in tissues) and of high porosity (cells in suspension). This model estimates the permeability increases of electroporated cells using empirically based predictions of the dependence of long-lived electropore density on the local electric field magnitude. A parametric investigation investigates how the transmembrane transport characteristics of irreversibly electroporated cells (permeability and resealing rate) affect the drug uptake of the surviving cells. The results show that the magnitude and duration of the permeability increases of irreversibly electroporated cells is much more influential in low porosity tissues than in high porosity dilute suspensions. In applications of electroporation of cells in tissues, the uncertainty of irreversibly electroporated cells should be considered in regions of tissue experiencing field strengths for which the fraction of the total cells that are irreversibly electroporated exceeds about 0.1.

sentences: Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample Leukocyte Immunoglobulin (Ig)-like Receptors (LILR) LILRB1 and LILRB2 play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and non-classical Human Leukocyte Antigen (HLA) molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LIRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample comprising 528 healthy control individuals from Sao Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes with at least 1% of frequency. For LILRB2, we identified 41 SNVs arranged into 11 haplotypes with frequencies above 1%. We found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have shown that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Taken together, our findings demonstrate that LILRB1 and LILRB2 are highly polymorphic and provide strong evidence supporting the directional selection regime hypothesis.

sentences: An AI-guided signature reveals the nature of the shared proximal pathways of host immune response in MIS-C and Kawasaki disease A significant surge in cases of multisystem inflammatory syndrome in children (MIS-C, also called Pediatric Inflammatory Multisystem Syndrome - PIMS) has been observed amidst the COVID-19 pandemic. MIS-C shares many clinical features with Kawasaki disease (KD), although clinical course and outcomes are divergent. We analyzed whole blood RNA sequences, serum cytokines, and formalin fixed heart tissues from these patients using a computational toolbox of two gene signatures, i.e., the 166-gene viral pandemic (ViP) signature, and its 20-gene severe (s)ViP subset that were developed in the context of SARS-CoV-2 infection and a 13-transcript signature previously demonstrated to be diagnostic for KD. Our analyses revealed that KD and MIS-C are on the same continuum of the host immune response as COVID-19. While both the pediatric syndromes converge upon an IL15/IL15RA-centric cytokine storm, suggestive of shared proximal pathways of immunopathogenesis, they diverge in other laboratory parameters and cardiac phenotypes. The ViP signatures also revealed unique targetable cytokine pathways in MIS-C, place MIS-C farther along in the spectrum in severity compared to KD and pinpoint key clinical (reduced cardiac function) and laboratory (thrombocytopenia and eosinopenia) parameters that can be useful to monitor severity.

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BLight: Efficient exact associative structure for k-mers MotivationA plethora of methods and applications share the fundamental need to associate information to words for high throughput sequence analysis. Doing so for billions of k-mers is commonly a scalability problem, as exact associative indexes can be memory expensive. Recent works take advantage of overlaps between k-mers to leverage this challenge. Yet existing data structures are either unable to associate information to k-mers or are not lightweight enough.

ResultsWe present BLight, a static and exact data structure able to associate unique identifiers to k-mers and determine their membership in a set without false positive, that scales to huge k-mer sets with a low memory cost. This index combines an extremely compact representation along with very fast queries. Besides, its construction is efficient and needs no additional memory. Our implementation achieves to index the k-mers from the human genome using 8GB of RAM (23 bits per k-mer) within 10 minutes and the k-mers from the large axolotl genome using 63 GB of memory (27 bits per k-mer) within 76 minutes. Furthermore, while being memory efficient, the index provides a very high throughput: 1.4 million queries per second on a single CPU or 16.1 million using 12 cores. Finally, we also present how BLight can practically represent metagenomic and transcriptomic sequencing data to highlight its wide applicative range.

AvailabilityWe wrote the BLight index as an open source C++ library under the AGPL3 license available at github.com/Malfoy/BLight. It is designed as a user-friendly library and comes along with code usage samples.

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Paracrine factors of stressed peripheral blood mononuclear cells activate pro-angiogenic and anti- proteolytic processes in whole blood cells and protect the endothelial barrier Tissue regenerative properties have been attributed to secreted paracrine factors derived from stem cells and other cell types. Especially, the secretome of {gamma}-irradiated peripheral blood mononuclear cells (PBMCsec) has been shown to possess high tissue-regenerative and pro-angiogenic capacities in a variety of preclinical studies. In the light of future therapeutic intravenous applications of PBMCsec, we investigated possible effects of PBMCsec on circulating white blood cells and endothelial cells lining the vasculature.

MethodsTo identify changes in the transcriptional profile of white blood cells treated with PBMCSec, whole blood was drawn from healthy individuals and stimulated with PBMCsec for 8 hours ex vivo before further processing for single cell RNA sequencing (scRNAseq). In addition, we performed in vitro assay to confirm findings arising from the transcriptional profiling.

ResultsAddition of PBMCsec to whole blood significantly altered the gene signature of granulocytes (17 genes), T-cells (45 genes), B-cells (72 genes) and most prominently monocytes (322 genes). We detected a strong upregulation of several tissue-regenerative and pro-angiogenic cyto- and chemokines in monocytes, including VEGFA, CXCL1 and CXCL5. Intriguingly, inhibitors of endopeptidase activity, such as SERPINB2, were also strongly induced. Measurement of the trans-endothelial electrical resistance of primary human microvascular endothelial cells revealed a strong barrier-protective effect of PBMCsec after barrier disruption.

ConclusionTogether, we show that PBMCsec induces angiogenic and proteolytic processes in the blood and is able to attenuate endothelial barrier damage. These regenerative properties suggest that systemic application of PBMCsec might be a promising novel strategy to restore damaged organs.

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Evolving social behaviour through selection of single-cell adhesion in Dictyostelium discoideum The social amoeba Dictyostelium discoideum commonly forms chimeric fruiting bodies by aggregation of different strains. Genetic variants that produce a higher proportion of spores are predicted to undercut multicellular organization unless cooperators assort positively. Cell adhesion is considered a primary factor driving such assortment, but evolution of adhesion has not been experimentally connected to changes in social performance. We modified by experimental evolution the properties of individual cells, selecting for higher and lower adhesion to a surface. We then quantified the effects of these changes on fitness (reproductive success), on development (timing and patterns of aggregation) for clonal and chimeric aggregates. In binary chimeras, both evolved lines produce a smaller fraction of spores than the ancestor. Surprisingly, evolution of a single-cell trait thus appears to have produced social cooperators irrespective of whether stronger or weaker adhesion was selected. Examination of development and cell-cell adhesion however revealed two distinct social behaviours, whereby less adhesive cells behave as cheaters in the multicellular bodies. Such differences are captured by a metric for social success that generalizes the classical measure of spore bias by taking into account clonal spore production. Our work shows that cell mechanical interactions can constrain evolution of development and assortment in chimeras, and that elucidation of proximate mechanisms is necessary in order to understand the ultimate emergence of multicellular organization.

Author summaryThe multicellular life cycle of Dictyostelium discoideum is as amazing as puzzling. Genetically different cells commonly co-aggregate, so that collective functions - that repose on cooperation among cells - are constantly threatened by the presence of exploitative variants, commonly called  cheaters. The evolution of cheating and cooperative behaviours is expected to depend on cell adhesion, which influences assortment and thus the capacity of cells to interact preferentially with those having the same social strategy. Here, we use experimental evolution to explore how selection acting on single-cell adhesion impacts social behaviour. Higher levels of cooperation appear to evolve as a result of both increased and decreased adhesion of single cells to a surface. However, such phenotypes are associated to opposite developmental dynamics in chimeric aggregates. Differences in social behaviour are captured if spore production of isolated strains is also considered, confirming the expectation that within the multicellular body of chimeric aggregates cooperative behaviour is associated to stronger adhesion, while early cell segregation undermines the success of less adhesive cells.

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Ca2+ activity is required for injury-induced migration of microglia ObjectivesMicroglia are the resident immune cells in the brain. Brain injury can activate the microglia and induce its directional migration towards injury sites for exerting immune functions. While extracellular ATP released from the injury site mediates the directionality of activated microglias migration, what endows activated microglia with migration capability remains largely unexplored.

MethodsIn the present study, we used the larval zebrafish as an in vivo model to visualize the dynamics of both morphology and Ca2+ activity of microglia during its migration evoked by local brain injury.

ResultsWe found that, in response to local injury, activated microglia exhibited an immediate Ca2+ transient and later elevated Ca2+ bursts frequency during its migration towards the local injury site (P < 0.01). Furthermore, suppression of Ca2+ activities significantly retarded microglial migration (P < 0.05).

ConclusionThus, our study suggests that intracellular Ca2+ activity is required for activated microglias migration.

sentences: Global and network functional connectivity of nucleus basalis of Meynert is strengthened in blind individuals Plasticity in the brain is differentially affected by age of blindness onset. One possible, but not yet identified mechanism is that the cholinergic signals originating from the nucleus basalis of Meynert may underlie differential extent of plasticity in early and late blind individuals. This prospect is based on the fact that the nucleus basalis of Meynert modulates cortical processes such as plasticity and sensory encoding and that the degree of cross-modal plasticity varies depending on the age of blindness onset. However, this question yet remains largely unclear. Here, we tested whether the early and late blind individuals develop dissociable plasticity in the nucleus basalis of Meynert using multi-parametric magnetic resonance imaging. We found the relatively preserved volumetric size and cerebrovascular reactivity, but significant disruption in the white matter integrity of the nucleus basalis of Meynert in both early and late blind individuals. Critically, despite its reduction in the white matter integrity, the nucleus basalis of Meynert of early blind individuals presented greater global and network functional connectivity including visual, language, and default-mode networks. Such changes in the functional connectivity were not observed in the late-blind individuals. Further, less duration of the visual experience was associated with greater global and network functional connectivity. These results indicate that the nucleus basalis of Meynert is differentially involved in the plasticity of early and late blind individuals - a similar amount of reduction in microstructural integrity in early and late blind individuals, but stronger and more widespread functional connectivity of the NBM in the early blind individuals. Our findings suggest that the nucleus basalis of Meynert may develop greater cholinergic influence on the cortex of early blind individuals. Such change may explain why early blind individuals present stronger and more widespread cross-modal plasticity during non-visual tasks compared to late blind individuals.

sentences: An End-to-End Deep Learning Sound Coding Strategy for Cochlear Implants Cochlear implant (CI) users struggle to understand speech in noisy conditions. To address this problem, we propose a deep learning speech denoising sound coding strategy that estimates the CI electric stimulation patterns out of the raw audio data captured by the micro-phone, performing end-to-end CI processing. To estimate the relative denoising performance differences between various approaches, we compared this technique to a classic Wiener filter and to a convTasNet. Speech enhancement performance was assessed by means of signal-to-noise-ratio improvement and the short-time objective speech intelligibility measure. Additionally, 5 CI users were evaluated for speech intelligibility in noise to assess the potential benefits of each algorithm. Our results show that the proposed method is capable of replacing a CI sound coding strategy while preserving its general use for every listener and performing speech enhancement in noisy environments, without sacrificing algorithmic latency.

sentences: Molecular Mechanism of Hyperactivation Conferred by a Truncated TRPA1 Disease Mutant Suggests New Gating Insights The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons of the pain pathway, where it is activated by diverse chemical irritants. A direct role for TRPA1 in human health has been highlighted by the discovery of genetic variants associated with severe pain disorders. One such TRPA1 mutant was identified in a father-son pair with cramp fasciculation syndrome (CFS) and neuronal hyperexcitability-hypersensitivity symptoms that may be caused by aberrant channel activity, though the mechanism of action for this mutant is unknown. Here, we show the CFS-associated R919* TRPA1 mutant is functionally inactive when expressed alone in heterologous cells, which is not surprising since it lacks the 201 C-terminal amino acids that house critical channel gating machinery including the pore-lining transmembrane helix. Interestingly, the R919* mutant confers enhanced agonist sensitivity when co-expressed with wild type (WT) TRPA1. This channel hyperactivation mechanism is conserved in distant TRPA1 species orthologues and can be recapitulated in the capsaicin receptor, TRPV1. Using a combination of ratiometric calcium imaging, immunostaining, surface biotinylation, pulldown assays, fluorescence size exclusion chromatography, and proximity biotinylation assays, we show that the R919* mutant co-assembles with WT subunits into heteromeric channels. Within these heteromers, we postulate that R919* TRPA1 subunits contribute to hyperactivation by lowering energetic barriers to channel activation contributed by the missing regions. Additionally, we show heteromer activation can originate from the R919* TRPA1 subunits, which suggests an unexpected role for the ankyrin repeat and coiled coil domains in concerted channel gating. Our results demonstrate the R919* TRPA1 mutant confers gain-of-function thereby expanding the physiological impact of nonsense mutations, reveals a novel and genetically tractable mechanism for selective channel sensitization that may be broadly applicable to other receptors, and uncovers new gating insights that may explain the molecular mechanism of temperature sensing by some TRPA1 orthologues.

sentences: Sequential functional differentiation of luminal epithelial cells regulated by maternal and embryonic signaling in the mouse endometrium Embryo implantation requires temporospatial maternal-embryo dialog to regulate the interactions between an activated blastocyst and a receptive endometrium; however, the details of the related cell-specific coordination is largely unknown because of the cellular complexity and dynamic developmental processes of both the embryo and endometrium during peri-implantation. By comparing single-cell RNA (scRNA) data obtained from entire uteri of pregnant and pseudo-pregnant mice with embryos that were in the oviduct (dpc2.5 days post-coitum [dpc]) or had entered the uterus (after 3.5 dpc), we found a maternal estrogen and progesterone signaling-dependent functional differentiation process in which progesterone induced estrogen-responsive luminal epithelial cells to differentiate into two new types of epithelial cells: adhesion epithelial cells (AECs) and supporting epithelial cells (SECs) on 2.5 dpc. In addition to maternal signaling data, the uterine scRNA and corresponding embryo bulk sequencing data were obtained, and analyses revealed that embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, enhancing the attachment of embryos to the endometrium after implantation. Specifically, embryonic signaling induced additional transformation of AECs by preventing adjacent AECs, but not AECs away from the embryo in the implantation chamber, from undergoing apoptosis. Furthermore, we demonstrated that epithelial cell differentiation and related regulatory signaling were largely conserved in humans and mice. In the human endometrium, developmental defects of SOX9-positive epithelial cells similar to luminal epithelial cells were related to thin endometrium. Our work provides comprehensive and systematic information on endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is important for endometrial receptivity and embryo implantation.

sentences: Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution The large-scale fluorescence microscopy has enabled the observation of dynamic physiological activities at the single cellular level across the mouse cortex, such as distributed neuronal population representations. However, video-rate high-resolution microscopy at sophisticated biological surfaces in nature keeps a challenging task for the tradeoff between the speed, resolution, and field of view. Here we propose Spinning Disk Multifocal Microscopy (SDiM) for arbitrarily shaped surfaces, which enables imaging at centimeter field-of-view, micrometer resolution and up to 30 frames per second across the depth range of 450 {micro}m. We apply this technique in various microscopic systems, including customized macroscopic systems and the Real-time Ultra-large-Scale imaging at High resolution macroscopy (RUSH), in both the reflective mode and the fluorescence mode, and in the study of cortex-wide single-neuron imaging and immune cell tracking. SDiM provides an opportunity for studying the cortex-wide multi-scale cellular interactions in biological tissues.

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Phylogenetic and Ecological Trends in Specialization: Disentangling the Drivers of Ectoparasite Host Specificity O_LIEcological specialization reflects both evolutionary and ecological processes. For parasitic taxa, ecological specialization can be assessed as the degree to which a parasite species will associate with certain host species, a property known as host specificity.
C_LIO_LIEctoparasitic bat flies have been previously reported as highly host specific, presumably due to a history of coevolution with their bat hosts. However, there is conflicting evidence of coevolution between bats and bat flies. Resource-driven competition between parasite individuals and between species may also be important in explaining patterns of bat fly specificity.
C_LIO_LITo test the importance of evolutionary and ecological factors on bat fly specificity, we collected and identified 21 bat fly species from 16 host bat species from the State of Rio de Janeiro in Brazil. We generated a bat fly species phylogeny from molecular data, estimated d specialization values (a metric of specificity), and used linear and cophylogenetic models to compare the importance of various drivers of parasite ecological specialization.
C_LIO_LIWe found that bat fly co-occurrence frequency (a proxy for interspecific competition) and mean infection intensity (a proxy for intraspecific competition) best predicted patterns of bat fly specialization. Co-occurrence frequency had a significantly negative association with specialization, while mean infection intensity has a significantly positive association with specialization. Coevolutionary congruence had a small effect size and did not significantly predict parasite specialization.
C_LIO_LIWe found multiple shifts toward more generalized host niches across the bat fly phylogeny. Our results suggest that ecological processes such as resource-driven competition may be more important than evolutionary processes in shaping bat fly host specialization networks.
C_LIO_LIBat flies showed variable degrees of host specialization, parasitized phylogenetically distant host species, and showed low phylogenetic congruence to their hosts. This suggests that as a group, bat flies may show flexibility in their host preference phenotypes and may change their host associations in the face of environmental disturbance.
C_LI

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Interleukin-6 mediates PSAT1 expression and serine metabolism in TSC2-deficient cells Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2. Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking upregulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed upregulation of IL-6 transcription and secretion compared to wildtype cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wildtype cells. Treatment with anti-IL-6 (aIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2+/- mice, while reducing PSAT1 expression. These data reveal a novel mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.

ClassificationMajor category: Biological Sciences Minor category: Cell Biology

sentences: Attention Samples Items in Visual Working Memory Rhythmically Attention allows us to selectively enhance the processing of specific locations or features in our external environment while simultaneously filtering out momentarily irrelevant information. It is currently hypothesized that this is achieved through the boosting of relevant sensory signals which biases the competition between competing neural representations. Recent neurophysiological and behavioral studies have revealed that attention is a fundamentally rhythmic process, tightly linked to neural oscillations in fronto-parietal networks. Instead of continuously highlighting a single object or location, attention rhythmically alternates between multiple relevant representations at a frequency of 3 - 6 Hz. However attention can not only be directed towards the external world but also towards internal visual working memory (VWM) representations, e.g. when selecting one of several search templates to find corresponding objects in the external world. Two recent studies have revealed that objects in VWM are attended in a similarly rhythmic fashion as perceived objects. We add to the current literature by showing that retro-cues towards multi-feature gratings in VWM initiate a similar theta-rhythmic competition, modulating reaction times in an anti-phasic manner. Our findings add to the converging body of evidence that external and internal visual representations are accessed by highly similar, rhythmic attentional mechanisms.

sentences: Soil, senescence and exudate utilisation: Characterisation of the Paragon var. spring bread wheat root microbiome Conventional methods of agricultural pest control and crop fertilisation are contributing to a crisis of biodiversity loss, biogeochemical cycle dysregulation, and ecosystem collapse. Thus, we must find ecologically responsible means to control disease and promote crop yields. The root-associated microbiome may contribute to this goal as microbes can aid plants with disease suppression, abiotic stress relief, and nutrient bioavailability. We applied 16S rRNA gene & fungal 18S rRNA gene (ITS2 region) amplicon sequencing to profile the diversity of the bacterial, archaeal & fungal communities associated with the roots of UK elite spring bread wheat variety Triticum aestivum var. Paragon in different soils and developmental stages. This revealed that community composition shifted significantly for all three groups across compartments. This shift was most pronounced for bacteria and fungi, while we observed weaker selection on the ammonia oxidising archaea-dominated archaeal community. Across multiple soil types we found that soil inoculum was a significant driver of endosphere community composition, however several bacterial families were identified as core enriched taxa in all soil conditions. The most abundant of these were Streptomycetaceae and Burkholderiaceae. Moreover, as the plants senesce, both families were reduced in abundance, indicating that input from the living plant was required to maintain their abundance in the endosphere. To understand which microbes are using wheat root exudates in the rhizosphere, root exudates were labelled in a 13CO2 DNA stable isotope probing experiment. This shows that bacterial taxa within the Burkholderiaceae family among other core enriched taxa, such as Pseudomonadaceae, were able to use root exudates but Streptomycetaceae were not. Overall, this work provides a better understanding of the wheat microbiome, including the endosphere community. Understanding crop microbiome formation will contribute to ecologically responsible methods for yield improvement and biocontrol in the future.

sentences: Single and complex spikes relay distinct frequency-dependent circuit information in the hippocampus Hippocampal neurons generate either single spikes or stereotyped bursts of spikes known as complex spikes. Although single and complex spikes co-occur in the same neuron, their contribution to information processing remains unclear. We analyzed hippocampal CA1 neurons in awake mice and in behaving rats, combining cellular membrane voltage imaging with optogenetics and extracellular recordings. We found that network-driven subthreshold membrane rhythms in the theta versus gamma frequencies preferably entrained complex versus single spikes in individual neurons. Optogenetic membrane perturbation revealed a causal link between subthreshold theta and gamma power and the initiation of complex versus single spikes. Further, single and complex spikes exhibited different place field properties and frequency-dependent coding during spatial navigation. Thus, individual hippocampal neurons do not integrate theta and gamma rhythms into a combined spike timing code, but instead, transmit frequency-specific information as distinct output modes of single versus complex spikes during spatial cognition.

sentences: The antique genetic plight of the Mediterranean monk seal (Monachus monachus) Disentangling the impact of Late Quaternary climate change from human activities can have crucial implications on the conservation of endangered species. We investigated the population genetics and demography of the Mediterranean monk seal (Monachus monachus), one of the worlds most endangered marine mammals, through an unprecedented dataset encompassing historical (extinct) and extant populations from the eastern North Atlantic to the entire Mediterranean Basin. We show that Cabo Blanco (Western Sahara/Mauritania), Madeira, Western Mediterranean (historical range), and Eastern Mediterranean regions segregate into four populations. This structure is likely the consequence of recent drift, combined with long-term isolation by distance (R2 = 0.7), resulting from prevailing short-distance (< 500 km) and infrequent long-distance dispersal (< 1,500 km). All populations (Madeira especially), show high levels of inbreeding and low levels of genetic diversity, seemingly declining since historical time, but surprisingly not being impacted by the 1997 massive die-off in Cabo Blanco. Approximate Bayesian Computation analyses support scenarios combining local extinctions and a major effective population size decline in all populations during Antiquity. Our results suggest that the early densification of human populations around the Mediterranean Basin coupled with the development of seafaring techniques were the main drivers of the decline of Mediterranean monk seals.

sentences: Breathing on Chip: Biomechanical forces change airway epithelial cell biology ina human Airway Lung-Chip Human lung function is intricately linked to the mechanics of breathing; however, it remains unknown whether and how these mechanical cues shape human lung cellular biology. While respiration-related strains and fluid flows have been suggested to promote alveolar epithelial cell function, the study of such fundamental mechanisms in the conducting airway epithelium has been hindered by the lack of suitable in vitro airway models. Here, we developed a model of human bronchial airway epithelium using well-differentiated primary cell cultures on a commercial Organs-on-Chips platform that enables the application of breathing-associated airflow and cyclic strain. It furthermore features optional endothelial cell co-culture to allow for crosstalk with the vascular compartment. Using this model, we evaluated the impact of airflow and physiological levels of cyclic strain on airway epithelial cell differentiation and function. Our findings suggest that breathing-associated mechanical stimulation changes epithelial composition, reduces secretion of IL-8, and downregulates gene expression of matrix metalloproteinase 9, fibronectin, and other extracellular matrix (ECM) factors. These results indicate that breathing-associated forces are important modulators of airway epithelial cell biology and that their fine-tuned application could generate models of specific epithelial phenotypes and pathologies.

sentences: Glial activation in prion diseases is strictly nonautonomous and requires neuronal PrPSc Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons, astrocytes or oligodendrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

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The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells BackgroundDNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESC) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss.

ResultsWe determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 {micro}M or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within two days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole genome bisulfite sequencing (WGBS) showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862, similar to the methylation level observed in Dnmt1 deficient mESCs.

ConclusionsGSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.

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Reconstructing contact network structure andcross-immunity patterns from multiple infection histories Interactions within a population shape the spread of infectious diseases but contact patterns between individuals are difficult to access. We hypothesised that key properties of these patterns can be inferred from multiple infection data in longitudinal follow-ups. We developed a simulator for epidemics with multiple infections on networks and analysed the resulting individual infection time series by introducing the concept of infection barcodes. We find that, depending on infection multiplicity and network sampling, infection barcode summary statistics can recover network properties such as degree distribution. Furthermore, we show that by mining infection barcodes for multiple infection patterns, one can detect immunological interference between pathogens (i.e. the fact that past infections in a host condition future probability of infection). The combination of individual-based simulations and barcode analysis of infection histories opens promising perspectives to infer and validate transmission networks and immunological interference for infectious diseases from longitudinal cohort data.

Author summaryInfectious disease dynamics are constrained both by between-host contacts and pathogen interactions within a host. Furthermore, multiple parasites circulate such that hosts are infected (sometimes simultaneously) by a variety of strains or species. We hypothesise that multiple infection history can inform us about the networks on which parasites are transmitted, but also on within-host interactions such as immunological interference. We developed a simulator for multiple infections on networks. By combining intuitive novel metrics for multiple infection events and established tools from computational data analysis, we show that similarity in infection history between two hosts correlates with their proximity in the contact network. By analysing pathogens co-occurrence patterns within hosts, we also recover immunological interference at the population level. The demonstrated robustness of our results in terms of observability, network clustering, and pathogen diversity opens new perspectives to extract host contact and between-pathogen immunity information from longitudinal cohort data.

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pySuStaIn: a Python implementation of the Subtype and Stage Inference algorithm Progressive disorders are highly heterogeneous. Symptom-based clinical classification of these disorders may not reflect the underlying pathobiology. Data-driven subtyping and staging of patients has the potential to disentangle the complex spatiotemporal patterns of disease progression. Tools that enable this are in high demand from clinical and treatment-development communities. Here we describe the pySuStaIn software package, a Python-based implementation of the Subtype and Stage Inference (SuStaIn) algorithm. SuStaIn unravels the complexity of heterogeneous diseases by inferring multiple disease progression patterns (subtypes) and individual severity (stages) from cross-sectional data. The primary aims of pySuStaIn are to enable widespread application and translation of SuStaIn via an accessible Python package that supports simple extension and generalization to novel modelling situations within a single, consistent architecture.

Code metadataCurrent code version v1.0

Permanent link to code/repository used of this code version https://github.com/ucl-pond/pySuStaIn

Legal Code License MIT

Code versioning system used git

Software code languages, tools, and services used Python

Compilation requirements, operating environments & dependencies Linux, Mac, Windows

Support email for questions leon.aksman@loni.usc.edu, p.wijeratne@ucl.ac.uk, alexandra.young@kcl.ac.uk

sentences: Functional architecture of cerebral cortex during naturalistic movie-watching Characterizing the functional organization of cerebral cortex is a fundamental step in understanding how different kinds of information are processed in the brain. Neuroimaging studies over the past decades have uncovered the function of many cortical areas in response to selected stimulus categories. However, it is still unclear how cortical areas are organized during naturalistic visual and auditory stimulation. Here we used high-resolution functional MRI data from 176 human subjects to map the macro-architecture of the entire cerebral cortex based on responses to a 60-minute audiovisual movie stimulus. A data-driven clustering approach revealed a map of 24 functional areas/networks, each related to a specific aspect of sensory or cognitive processing. The map included three distinct executive control (domain-general) networks which showed a strong push-pull interaction with domain-specific regions in visual, auditory, and language cortex. The cortical parcellation scheme presented here provides a comprehensive and unified map of functionally defined areas, which could replace a large set of functional localizer maps.

sentences: Proteome-wide landscape of solubility limits in a bacterial cell Proteins are prone to aggregate when they are expressed above their solubility limits, a phenomenon termed supersaturation. Aggregation may occur as proteins emerge from the ribosome or after they fold and accumulate in the cell, but the relative importance of these two routes remain poorly known. Here, we systematically probed the solubility limits of each Escherichia coli protein upon overexpression using an image-based screen coupled with machine learning. The analysis suggests that competition between folding and aggregation from the unfolded state governs the two aggregation routes. Remarkably, the majority (70%) of insoluble proteins have low supersaturation risks in their unfolded states and rather aggregate after folding. Furthermore, a substantial fraction ([~]35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our proteome-wide study provides empirical insights into the molecular determinants of protein aggregation routes in a bacterial cell.

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Emergence and maintenance of different sized actin filaments in a common pool of building blocks Actin is one of the key structural components of the eukaryotic cytoskeleton that regulates cellular architecture and mechanical properties. Dynamic regulation of actin filament length and organization is essential for the control of many physiological processes including cell adhesion, motility and division. While previous studies have mostly focused on the mechanisms controlling the mean length of individual actin filaments, it remains poorly understood how distinct actin filament populations in cells maintain different size using the same set of molecular building blocks. Here we develop a theoretical model for the length regulation of multiple actin filaments by nucleation and growth rate modulation by actin binding proteins in a limiting pool of monomers. We first show that spontaneous nucleation of actin filaments naturally leads to heterogeneities in filament length distribution. We then investigate the effects of filament growth inhibition by capping proteins and growth promotion by formin proteins on filament length distribution. We find that filament length heterogeneity can be increased by growth inhibition, whereas growth promoters do not significantly affect length heterogeneities. Interestingly, a competition between filament growth inhibitors and growth promoters can give rise to bimodal filament length distribution as well as a highly heterogeneous length distribution with large statistical dispersion. We quantitatively predict how heterogeneity in actin filament length can be modulated by tuning F-actin nucleation and growth rates in order to create distinct filament subpopulations with different lengths.

SIGNIFICANCEActin filaments organize into different functional network architectures within eukaryotic cells. To maintain different actin network architectures, actin filaments of different lengths are often required. How cells regulate the length heterogeneity in actin filaments is not well understood. Here we show that the modulation of filament growth rates and nucleation rate by binding partners like formin and capping proteins can regulate the emergent length heterogeneity in the filament population and can create distinct sub-populations with different lengths. Our study sheds light into how actin binding proteins can control various aspects of filament size distribution by tuning their concentration or expression levels in the cell.

sentences: Impaired Cx43 gap junction endocytosis causes cardiovascular defects in zebrafish Gap junction proteins, termed connexins (Cx), mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting exchange of molecules, ions, and electrical impulses. The most ubiquitously expressed gap junction protein, connexin43 (Cx43) has been implicated in cardiovascular diseases including arrhythmias, cardiomyopathies, hypertension and diabetes. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43{Delta}256-289) and zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired cardiovascular development. These findings suggest that normal, unimpaired Cx43 gap junction endocytosis and turnover is an essential aspect of gap junction function as demonstrated here for cardiovascular development that when impaired can give rise to arrhythmias, heart malformations and aberrant vasculature structure and function.

sentences: Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 [A]. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C-terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.

sentences: PRC1 Sustains the Memory of Neuronal Fate Independent of PRC2 Function Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We unexpectedly found that PRC2 is dispensable to preserve the morphogen-induced positional fates of spinal motor neurons (MNs), while PRC1 is essential for the specification of segmentally-restricted subtypes. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox networks by derepressed caudal Hox genes. These results indicate that PRC1 can function independently of de novo PRC2-dependent histone methylation to maintain chromatin topology and transcriptional memory at the time of neuronal differentiation.

sentences: Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues During embryonic development, cellular forces synchronize in space and time to generate functional tissue shapes. Apical constriction is one of these force-generating processes, and it is necessary to modulate epithelial curvature in fundamental morphogenetic events, such as neural tube folding. The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues currently hinders the progress of the field. Here we report the development of "OptoShroom3", a new optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination of individual cells in an epithelial cell sheet reduced their apical surface while illumination of groups of cells caused deformation in the adjacent regions. By using OptoShroom3, we further manipulated 3D tissue shapes. Light-induced apical constriction provoked the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids led to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

sentences: Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma.

sentences: Web-based Histology Reference Atlas for the Freshwater Crustacean Daphnia magna Daphnia, an important model system for the study of evolution, development, phenotypic plasticity, and environmental health, lacks a modern reference atlas for microanatomy. To facilitate the comprehensive assessment of phenotypic effects of genes and environment, we created the Daphnia histology reference atlas (http://daphnia.io/anatomy/), a tractable, interactive web-based tool that provides insight into normal phenotype through vectorized annotations overlaid onto digital histology sections imaged at 40X magnification. Guided by our expert-curated and multimodal informed hierarchical anatomical ontology, we show that this resource can be used to elucidate sex-specific differences between female and male Daphnia magna in each of 3 orthogonal planes, providing new insight for the study of sex-specific traits. This atlas is a new, open-source resource for the Daphnia community to facilitate education and research collaboration, and as a precursor for the 3D atlas. It is also our intention that this atlas aids in phenotypic anchoring of large-scale biomolecular (multi-omics) data from comparative toxicological studies. Greater access to high-quality histological data may clarify cross-correlations between microanatomic and multi-omic phenotypes caused by genetic variation, environment, and disease across phylogeny.

sentences: Neutrophil extracellular traps impair regeneration Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound induced hair follicle regeneration (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We have previously demonstrated that TLR3 promotes WIHN through binding dsRNA, but the source of which is still unclear. Here, we demonstrate that multiple distinct contexts of high WIHN all show a strong neutrophil signature, and given the likelihood of nuclear dsRNA release during the production of neutrophil extracellular trap (NETs), we hypothesized that mature neutrophils and NETs might promote WIHN. Consistent with this, in addition to the presence of mature neutrophils shortly after wounding, neutrophils remain within the wound after the barrier is reestablished, where they produce extracellular traps (NETs) that likely release spliceosomal U1 dsRNA. Contrary to our hypothesis, genetic models of neutrophil depletion show enhanced WIHN. Pad4 null mice that are defective in NET production also augment WIHN. Finally, using single-cell RNA sequencing, we identified a dramatic increase in mature neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder WIHN.

sentences: A promiscuity locus confers Lotus burttii nodulation with rhizobia from five different genera Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil dwelling organisms and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, where the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity QTL to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for host-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions.

sentences: Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

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Robust, data-driven bioregionalizations emerge from diversity concordance AimDespite the increasing interest in developing new bioregionalizations and assessing the most widely accepted biogeographic frameworks, no study to date has sought to systematically define a system of small bioregions nested within larger ones that better reflect the distribution and patterns of biodiversity. Here, we examine how an algorithmic, data-driven model of diversity patterns can lead to an ecologically interpretable hierarchy of bioregions.

LocationAustralia.

Time periodPresent.

Major taxa studiedTerrestrial vertebrates and vascular plants.

MethodsWe compiled information on the biophysical characteristics and species occupancy of Australias geographic conservation units (bioregions). Then, using cluster analysis to identify groupings of bioregions representing optimal discrete-species areas, we evaluated what a hierarchical bioregionalization system would look like when based empirically on the within-and between-site diversity patterns across taxa. Within an information-analytical framework, we then assessed the degree to which the World Wildlife Funds (WWF) biomes and ecoregions and our suite of discrete-species areas are spatially associated and compared those results among bioregionalization scenarios.

ResultsInformation on biodiversity patterns captured was moderate for WWFs biomes (50- 58% for birds beta, and plants alpha and beta diversity, of optimal discrete areas, respectively) and ecoregions (additional 4-25%). Our plants and vertebrate optimal areas retained more information on alpha and beta diversity across taxa, with the two algorithmically derived biogeographic scenarios sharing 86.5% of their within- and between-site diversity information. Notably, discrete-species areas for beta diversity were parsimonious with respect to those for alpha diversity.

Main conclusionsNested systems of bioregions must systematically account for the variation of species diversity across taxa if biodiversity research and conservation action are to be most effective across multiple spatial or temporal planning scales. By demonstrating an algorithmic rather than subjective method for defining bioregionalizations using species-diversity concordances, which reliably reflects the distributional patterns of multiple taxa, this work offers a valuable new tool for systematic conservation planning.

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Pharmacologic analysis of non-synonymous coding 5-HT2A SNPs reveals alterations psychedelic drug potencies and efficacies Serotonin (5-Hydroxytryptamine; 5-HT) 2A receptor (5-HT2AR) signaling is essential for the actions of classical psychedelic drugs. In this study, we examined whether random sequence variations in the gene (single nucleotide polymorphisms, SNPs) encoding the 5-HT2AR affect the signaling of four commonly used psychedelic drugs. We examined the in vitro pharmacology of seven non-synonymous SNPs, which give rise to S12N, T25N, D48N, I197V, A230T, A447V, and H452Y variant 5-HT2A serotonin receptors. We found that these non-synonymous SNPs exert statistically significant, although modest, effects on the efficacy and potency of four therapeutically relevant hallucinogens. Significantly, the in vitro pharmacological effects of the SNPs drug actions at 5-HT2AR are drug specific.

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sentences: 'Phytopathological strolls' in the dual context of COVID-19 lockdown and IYPH2020: transforming constraints into an opportunity for public education about plant pathogens The experience presented here relates to 2020, a particularly timely year for plant disease-related communication ( International Year of Plant Health IYPH2020), but also a unique year because of the COVID-19 pandemic. Our goal was to illustrate the diversity and beauty of fungal plant pathogens through a naturalist approach that could be followed by any amateur. We achieved this end through  phytopathological strolls, in which we observed and determined the origin of symptoms on diseased plants found in our garden, in the local streets, in nearby open spaces, and sharing this matter with a broad public. The lockdown imposed in France created an additional motivation to take up the challenge, and to involve our children, even under strong constraints, such as movement restrictions. We observed and described fungal pathogens through hundreds of photographs, shared our findings with a large audience on Twitter, and received feedback. The material used was deliberately simple and transportable: a digital reflex camera, an old microscope, a mobile phone, some books and an Internet connexion. Between March 17, 2020 and June 20, 2021 we found 196 plant pathogens, including 97 rusts, 27 powdery mildews and 28 septoria-like diseases. We discuss here the importance of promoting searches for plant pathogens, their description and conservation, through a combination of classical approaches and digital tools in tune with the times, such as Twitter, by treating pathogen identification like a detective game and, more surprisingly, by making use of the addictive nature of collection approaches, drawing a parallel with Pokemon Go.

sentences: Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions further. They found upon resequencing DNA from the cell lines used in their study that non-CRISPR edited genomes dominated the cultures. They believed they had confirmed complete editing in single-cell clones at the outset. Therefore, they are not confident based on their current experiments that the virologic effect of HIV-1 Nef is independent of SERINC3 and SERINC5 in T cells of the leukemic CEM line as they originally claimed. They are reengineering the cells to resolve this and apologize for any inconvenience that this has caused. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

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Type 1 interferon supports B cell responses to polysaccharide antigens but is not required for MPL/TDCM adjuvant effects on innate B cells We previously described monophosphoryl lipid A (MPL) and synthetic cord factor, trehalose-6,6-dicorynomycolate (TDCM) significantly increases antibody (Ab) responses to T cell independent type 2 antigens (TI-2 Ags) in a manner dependent on B cell-intrinsic TLR4 expression as well as MyD88 and TRIF adapter proteins. Given the requirement for TRIF in optimal MPL/TDCM adjuvant effects and the capacity of MPL to drive type I IFN production, we aimed to investigate the extent to which adjuvant effects on TI-2 Ab responses depend on type I IFN receptor (IFNAR) signaling. We found IFNAR-/- mice had impaired early TI-2 Ag-induced B cell activation and expansion and that B cell-intrinsic type I IFN signaling on B cells was essential for normal antibody responses to TI-2 Ags, including haptenated Ficoll and the pneumococcal vaccine, Pneumovax23. However, MPL/TDCM significantly increased TI-2 IgM and IgG responses in IFNAR-/- mice. MPL/TDCM enhanced TI-2 Ab production primarily by activating innate B cells (B-1b and splenic CD23- B cells) as opposed to CD23+ enriched follicular B cells. In summary, our study highlights an important role for type I IFN in supporting early B cell responses to TI-2 Ags through B cell-expressed IFNAR, but nonetheless demonstrates an MPL/TDCM adjuvant significantly increases TI-2 Ab responses independently of type I IFN signaling and does so by predominantly supporting increased polysaccharide-specific Ab production by innate B cell populations.

Key pointsO_LIB cell-intrinsic IFNAR expression promotes TI-2 Ab responses.
C_LIO_LIMPL/TDCM adjuvant effects are independent of type 1 IFN.
C_LIO_LIMPL/TDCM promotes TI-2 Ab responses by innate B cells.
C_LI

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Early embryonic heat shock induces long-term epigenetic memory by affecting the transition to zygotic independence Early-life stress can generate persistent life-long effects that impact adult health and disease risk, but little is known of how such programming is established and maintained. Previous use of the Drosophila strain wm4h show that an early embryonic heat shock result in stable epigenetic alteration in the adult fly. To investigate the potential role of small non-coding RNA (sncRNA) in the initiation of such long-term epigenetic effects, we here generated a fine timeline of sncRNA expression during the first 5 stages of Drosophila embryogenesis in this strain. Building on this, we show that (1) miRNA is increased following early embryonic heat shock, and (2) the increased miRNA is coming from two separate sources, maternal and zygotic. By performing long RNA sequencing on the same single embryo, we found that a subgroup of miRNA with maternal origin, had a strong negative correlation with a group of early zygotic transcripts. Critically, we found evidence that one such early zygotic transcript, the insulator binding factor Elba1, is a Su(var) for wm4h. The findings provide insights of the dynamics and stress-sensitivity of sncRNA during the first embryonic stages in Drosophila and suggest an interplay between miRNA, Elba1 and long-term epigenetic alteration.

HIGHLIGHTSO_LIWe provide a high-resolution timeline for sncRNA for Drosophila stage 1-5 embryos
C_LIO_LIHeat shock before midblastula transition (MBT) results in a massive upregulation of miRNA at cellularization
C_LIO_LIHeat shock-induced miRNAs negatively associate with downregulation of a specific subset of pre-MBT genes
C_LIO_LIElba1 is a position-effect-variegation (PEV) modifier for wm4h
C_LIO_LIHeat shock-induces an "leaky" expression of genes that overlap with Elba 1-3 binding sites
C_LI

sentences: Invasive Earthworms Alter Forest Soil Microbiomes and Nitrogen Cycling Northern hardwood forests in formerly glaciated areas had been free of earthworms until exotic European earthworms were introduced by human activities. The invasion of exotic earthworms is known to dramatically alter soil physical, geochemical, and biological properties, but its impacts on soil microbiomes are still unclear. Here we show that the invasive earthworms alter soil microbiomes and ecosystem functioning, especially for nitrogen cycling. We collected soil samples at different depths from three sites across an active earthworm invasion chronosequence in a hardwood forest in Minnesota, USA. We analyzed the structures and the functional potentials of the soil microbiomes by using amplicon sequencing, high-throughput nitrogen cycle gene quantification (NiCE chip), and shotgun metagenomics. Both the levels of earthworm invasion and soil depth influenced the microbiome structures. In the most recently and minimally invaded soils, Nitrososphaera and Nitrospira as well as the genes related to nitrification were more abundant than in the heavily invaded soils. By contrast, genes related to denitrification and nitrogen fixation were more abundant in the heavily invaded than the minimally invaded soils. Our results suggest that the N cycling in forest soils is mostly nitrification driven before earthworm invasion, whereas it becomes denitrification driven after earthworm invasion.

sentences: Multi-omics approach reveals dysregulation of protein phosphorylation correlated with lipid metabolism in mouse fatty liver Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites (with localization probability > 0.75), of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.

sentences: Characterization and functional interrogation of SARS-CoV-2 RNA interactome Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies.

sentences: {-}Multivariable Association Discovery in Population-scale Meta-omics Studies It is challenging to associate features such as human health outcomes, diet, environmental conditions, or other metadata to microbial community measurements, due in part to their quantitative properties. Microbiome multi-omics are typically noisy, sparse (zero-inflated), high-dimensional, extremely non-normal, and often in the form of count or compositional measurements. Here we introduce an optimized combination of novel and established methodology to assess multivariable association of microbial community features with complex metadata in population-scale observational studies. Our approach, MaAsLin 2 (Microbiome Multivariable Associations with Linear Models), uses general linear models to accommodate a wide variety of modern epidemiological studies, including cross-sectional and longitudinal designs, as well as a variety of data types (e.g. counts and relative abundances) with or without covariates and repeated measurements. To construct this method, we conducted a large-scale evaluation of a broad range of scenarios under which straightforward identification of meta-omics associations can be challenging. These simulation studies reveal that MaAsLin 2s linear model preserves statistical power in the presence of repeated measures and multiple covariates, while accounting for the nuances of meta-omics features and controlling false discovery. We also applied MaAsLin 2 to a microbial multi-omics dataset from the Integrative Human Microbiome (HMP2) project which, in addition to reproducing established results, revealed a unique, integrated landscape of inflammatory bowel disease (IBD) across multiple time points and omics profiles.

sentences: Adaptation in cone photoreceptors contributes to an unexpected insensitivity of On parasol retinal ganglion cells to spatial structure in natural images Neural circuits are constructed from nonlinear building blocks, and not surprisingly overall circuit behavior is often strongly nonlinear. But neural circuits can also behave near linearly, and some circuits shift from linear to nonlinear behavior depending on stimulus conditions. Such control of the linearity or nonlinearity of circuit behavior is fundamental to neural computation. Here we study a surprising stimulus dependence of the responses of On (but not Off) parasol retinal ganglion cells: these cells respond nonlinearly to spatial structure in temporally-modulated grating stimuli but linearly to spatial structure in flashed gratings and natural visual inputs. We show that this unexpected response linearity can be explained by a shift in the balance of excitatory and inhibitory synaptic inputs that originates at least in part from adaptation in the cone photoreceptors. More generally, this highlights how subtle asymmetries in signaling - here in the cone signals - can qualitatively alter circuit computation.

sentences: A fast and objective hidden Markov modeling for accurate analysis of biophysical data with numerous states The hidden Markov model (HMM) is widely used to analyze biophysical chronological data with discrete states, such as binding/detachment of biomolecules, protein/nucleotide conformational changes and step-like movement of single proteins. Despite its usefulness, classical HMM fitting has practical drawbacks that it requires the determination of the number of hidden states and fine initialization of many parameters before fitting. To overcome these drawbacks, several HMM pre-analyses have been reported, but do not provide enough accuracy when data have unknown kinetics and/or low signal-to-noise ratio. Therefore, in many cases, HMM fitting needs trial-and-error manual process that can impair the objectivity of the analysis. Moreover, for data composed of numerous hidden states, such as stepping data of cytoskeletal motors, there has been difficulty in HMM analysis because the large number of parameters were hardly properly initialized. Here, by combining a statistical step-finding method and the Gaussian mixture model clustering, we developed a new algorithm for more objective HMM analysis. Our algorithm can execute accurate state number estimation and parameter optimization with fully automated way. Simulation analysis demonstrated that our algorithm accurately fit both fast- and slow-transition trajectories. Compared with the previous method, the speed of our algorithm was 10-20 times faster for standard size data. Our algorithm also showed the accurate fit of the simulated motor-stepping data with more than 10 transition states, suggesting the applicability of the method to the data with numerous states. Furthermore, the algorithm is flexible enough to cope with cases where some kinetics are known in advance. Some available prior information, such as the dwell time of each state, can be integrated into the algorithm via two user-tunable parameters. In summary, our method enables fast, accurate and objective HMM analysis, and broadens the application range of HMM fitting that can provide more accurate interpretation of a wide variety of biophysical data.

sentences: Mutational Changes and Enhanced Cell Migration during Transformation of Breast Non-tumorigenic Cells to Tumorigenic Cells Although cancer stem cells (CSCs) play a major role in tumor initiation and progression, their mechanisms in invasiveness and metastasis are unclear. Tumor microenvironment signals, such as extracellular matrix (ECM) composition, significantly influence cell and tissue behaviors. Unfortunately, these signals are often lost in in vitro cell culture. The current study determines putative CSC populations and contrasts genomic changes in human breast epithelial tumorigenic versus non-tumorigenic cells (both derived from the same normal parental stem cells) and investigates the single-cell migration properties on ECM-mimetic platforms. Whole exome sequencing data indicate that tumorigenic cells have a higher mutation burden than non-tumorigenic cells, and mutations exclusive to tumorigenic cells exhibit higher predictive deleterious scores. ECM-mimetic topography selectively enhances migration speed of tumorigenic cells but not of non-tumorigenic cells. The enhanced migratory ability of tumorigenic cells is lost in conventional two-dimension culture conditions. A wide distribution of tumorigenic single-cell migration speeds suggests heterogeneity in cellular sensing of contact guidance cues. This study identifies mutations acquired during breast tumorigenesis, which can be associated with enhanced invasiveness of breast tumorigenic cells. Results demonstrate that the nanotopographically defined platform can be applied to recapitulate an ECM structure for investigating cellular migration in the simulated tumor microenvironment.

sentences: Investigation of extramammary sources of Group B Streptococcus reveals its unusual ecology and epidemiology in camels Camels are vital to food production in the drylands of the Horn of Africa, with milk as their main contribution to food security. A major constraint to camel milk production is mastitis, inflammation of the mammary gland. The condition negatively impacts milk yield and quality as well as household income. The leading cause of mastitis in dairy camels is Streptococcus agalactiae, group B Streptococcus (GBS), which is also a commensal and pathogen of humans. It has been suggested that extramammary reservoirs for this pathogen may contribute to the occurrence of mastitis in camels. We explored the molecular epidemiology of GBS in camels using a cross-sectional study design for sample collection and phenotypic, genomic and phylogenetic analysis of isolates. Among 88 adult camels and 93 calves from six herds in Laikipia County, Kenya, GBS was detected in 20% of 50 milk samples, 25% of 152 nasal swabs, 8% of 90 oral swabs and 3% of 90 rectal swabs, but not in vaginal swabs. Per camel herd, two to four sequence types (ST) were present. More than half of the isolates belonged to ST617 or its single-locus variant, ST1652, with these STs found across all sample types. Serotype VI was detected in 30 of 58 isolates. In three herds, identical STs were detected in milk and swab samples, suggesting that extramammary sources of GBS may contribute to the maintenance and spread of GBS within camel herds. This needs to be considered when developing prevention and control strategies. In addition, the high nasal carriage rate, low recto-vaginal carriage rate, and high prevalence of serotype VI for GBS in camels are in stark contrast to the distribution of GBS in humans and reveal hitherto unknown ecological and molecular features of this bacterial species.

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Patterns of gene content and co-occurrence constrain the evolutionary path toward animal association in CPR bacteria Candidate Phyla Radiation (CPR) bacteria are small, likely episymbiotic organisms found across Earths ecosystems. Despite their prevalence, the distribution of CPR lineages across habitats and the genomic signatures of transitions amongst these habitats remain unclear. Here, we expand the genome inventory for Absconditabacteria (SR1), Gracilibacteria, and Saccharibacteria (TM7), CPR bacteria known to occur in both animal-associated and environmental microbiomes, and investigate variation in gene content with habitat of origin. By overlaying phylogeny with habitat information, we show that bacteria from these three lineages have undergone multiple transitions from environmental habitats into animal microbiomes. Based on co-occurrence analyses of hundreds of metagenomes, we extend the prior suggestion that certain Saccharibacteria have broad bacterial host ranges and constrain possible host relationships for Absconditabacteria and Gracilibacteria. Full-proteome analyses show that animal-associated Saccharibacteria have smaller gene repertoires than their environmental counterparts and are enriched in numerous protein families, including those likely functioning in amino acid metabolism, phage defense, and detoxification of peroxide. In contrast, some freshwater Saccharibacteria encode a putative rhodopsin. For protein families exhibiting the clearest patterns of differential habitat distribution, we compared protein and species phylogenies to estimate the incidence of lateral gene transfer and genomic loss occurring over the species tree. These analyses suggest that habitat transitions were likely not accompanied by large transfer or loss events, but rather were associated with continuous proteome remodeling. Thus, we speculate that CPR habitat transitions were driven largely by availability of suitable host taxa, and were reinforced by acquisition and loss of some capacities.

IMPORTANCEStudying the genetic differences between related microorganisms from different environment types can indicate factors associated with their movement among habitats. This is particularly interesting for bacteria from the Candidate Phyla Radiation because their minimal metabolic capabilities require symbiotic associations with microbial hosts. We found that shifts of Absconditabacteria, Gracilibacteria, and Saccharibacteria between environmental ecosystems and mammalian mouths/guts probably did not involve major episodes of gene gain and loss; rather, gradual genomic change likely followed habitat migration. The results inform our understanding of how little-known microorganisms establish in the human microbiota where they may ultimately impact health.

sentences: Kinetic modelling of β-cell metabolism reveals control points in the insulin-regulating pyruvate cycling pathways Insulin, a key hormone in the regulation of glucose homeostasis, is secreted by pancreatic {beta}-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in {beta}-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP as well as other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. In this work we developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. We successfully validated our model against recent experimental observations and performed local and global sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier (DIC) and pyruvate transporter (PYC) are the most important regulators of pyruvate cycling and NADPH production. In contrast, our analysis showed that variation in the pyruvate carboxylase (PC) flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for treatment of type 2 diabetes.

sentences: Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain - especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

sentences: Crystal structure of a bacterial CNNM magnesium transporter CBS-pair domain divalent metal cation transport mediators (CNNMs) are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. CNNM proteins are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determined the crystal structure of an archaeal CNNM protein with Mg2+-ATP bound. The structure reveals a novel transmembrane fold for the DUF21 domain, the largest family of domains of unknown function. The protein has a negatively charged cavity that penetrates halfway through the membrane suggesting it functions as a cation transporter. The cytosolic portion of the protein is comprised of highly charged four-helix bundle and a CBS-pair domain. HDX-MS experiments, molecular dynamics, and additional crystal structures show that the cytosolic domains undergo large conformational changes upon nucleotide binding suggesting a mechanism of regulation shared between human and bacterial orthologs. The molecular characterization of CNNM proteins has profound implications for understanding their biological functions in human diseases, including cancer, and in animals, bacteria and plants.

sentences: Local-scale virome depiction supports significant differences between Aedes aegypti and Aedes albopictus Aedes spp. comprise the primary group of mosquitoes that transmit arboviruses such as dengue, Zika, and chikungunya viruses to humans, and thus these insects pose a significant burden on public health worldwide. Advancements in next-generation sequencing and metagenomics have expanded our knowledge on the richness of RNA viruses harbored by arthropods such as Ae. aegypti and Ae. albopictus; increasing evidence suggests that vectorial competence can be modified by the microbiome (comprising both bacteriome and virome) of mosquitoes present in endemic zones. Using an RNA-seq-based metataxonomic approach, this study determined the virome structure of field-caught Ae. aegypti and Ae. albopictus mosquitoes in Medellin, Colombia, a municipality with a high incidence of mosquito-transmitted arboviruses. The two species are sympatric, but their core viromes differed considerably in richness, diversity, and abundance; the viromes were dominated by a few viruses. BLAST searches of assembled contigs suggested that at least 17 virus species (16 of which are insect-specific viruses [ISVs]) infect the Ae. aegypti population. Dengue virus 3 was detected in one sample. In Ae. albopictus, up to 11 ISVs and one plant virus were detected. Therefore, the virome composition was species-specific. The bacterial endosymbiont Wolbachia was identified in all Ae. albopictus samples and in some Ae. aegypti samples collected after 2017. The presence of Wolbachia sp. in Ae. aegypti was not related to significant changes in the richness, diversity, or abundance of this mosquitos virome, although it was related to an increase in the abundance of Aedes aegypti To virus 2 (unclassified). The mitochondrial diversity of these mosquitoes suggested that the Ae. aegypti population underwent a change that started in the second half of 2017, which coincides with the release of Wolbachia-infected mosquitoes in Medellin, indicating that the population of wMel-infected mosquitoes has expanded. However, additional studies are required on the dispersal speed and intergenerational stability of wMel in Medellin and nearby areas as well as on the introgression of genetic variants in the native mosquito population.

sentences: Comprehensive profiling of genomic and transcriptomic  differences between risk groups of lung adenocarcinoma and lung squamous cell carcinoma Lung cancer is the second frequently diagnosed cancer type and responsible for the highest number of cancer deaths worldwide. Lung adenocarcinoma and lung squamous cell carcinoma are subtypes of non-small cell lung cancer which has the highest frequency of lung cancer cases. We aimed to analyze genomic and transcriptomic variations including simple nucleotide variations (SNVs), copy number variations (CNVs) and differential expressed genes (DEGs) in order to find key genes and pathways for diagnostic and prognostic prediction for lung adenocarcinoma and lung squamous cell carcinoma. We performed univariate cox model and then lasso regularized cox model with leave-one-out cross-validation using TCGA gene expression data in tumor samples. We generated a 35-gene signature and a 33-gene signature for prognostic risk prediction based on the overall survival time of the patients with LUAD and LUSC, respectively. When we clustered patients into high-risk and low-risk groups, the survival analysis showed highly significant results with high prediction power for both training and test datasets. Then we characterized the differences including significant SNVs, CNVs, DEGs, active subnetworks, and the pathways. We described the results for the risk groups and cancer subtypes separately to identify specific genomic alterations between both high-risk groups and cancer subtypes. Both LUAD and LUSC high-risk groups have more down-regulated immune pathways and upregulated metabolic pathways. On the other hand, low-risk groups have both upregulated and downregulated genes on cancer-related pathways. Both LUAD and LUSC have important gene alterations such as CDKN2A and CDKN2B deletions with different frequencies. SOX2 amplification occurs in LUSC and PSMD4 amplification in LUAD. EGFR and KRAS mutations are mutually exclusive in LUAD samples. EGFR, MGA, SMARCA4, ATM, RBM10, and KDM5C genes are mutated only in LUAD but not in LUSC. CDKN2A, PTEN, and HRAS genes are mutated only in LUSC samples. Low-risk groups of both LUAD and LUSC, tend to have a higher number of SNVs, CNVs, and DEGs. The signature genes and altered genes have the potential to be used as diagnostic and prognostic biomarkers for personalized oncology.

sentences: Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. Here we report isolation and characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7[A] cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, implying that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

sentences: Involvement of ASIC1a channels in the spinal processing of pain information by deep projection neurons Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but which exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of spinal neurons with computational modelling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a/ASIC2 heteromeric channels. A computational model of WDR neuron supplemented with heteromeric ASIC1a/ASIC2 channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using either a combination of calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin), or the Texas coral snake ASIC-activating toxin (MitTx). This study demonstrates a possible dual contribution to windup of calcium permeable ASIC1a/ASIC2 channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases.

sentences: Global protein responses of multi-drug resistant plasmid containing Escherichia coli to ampicillin, cefotaxime, imipenem and ciprofloxacin. Antimicrobial resistance (AMR) and multi-drug resistance (MDR) in pathogenic bacteria are frequently mediated by plasmids. However, plasmids do not exist in isolation but rather require the bacterial host interaction in order to produce the AMR phenotype. This study aimed to utilise mass spectrometry-based proteomics to reveal the plasmid and chromosomally derived protein profile of Escherichia coli under antimicrobial stress. This was achieved by comparing the proteomes of E. coli containing the MDR pEK499 plasmid, under ampicillin, cefotaxime, imipenem or ciprofloxacin stress with the proteomes of these bacteria grown in the absence of antimicrobial. Our analysis identified statistically significant differentially abundant proteins common to groups exposed to the {beta}-lactam antimicrobials but not ciprofloxacin, indicating a {beta}-lactam stress response to exposure from this class of drugs, irrespective of {beta}-lactam resistance or susceptibility. These include ecotin and free methionine-R-sulfoxide reductase. These data also identified distinct differences in the cellular response to each {beta}-lactam. Data arising from comparisons of the proteomes of ciprofloxacin-treated E. coli and controls detected an increase in the relative abundance of proteins associated with ribosomes, translation, the TCA-cycle and several proteins associated with detoxification and a decrease in the relative abundances of proteins associated with stress response, including oxidative stress. We identified changes in proteins associated with persister formation in the presence of ciprofloxacin but not the {beta}-lactams. The plasmid proteome differed across each treatment and did not follow the pattern of antimicrobial - AMR protein associations. For example, a relative increase in the amount of blaCTX-M-15 in the presence of cefotaxime and ciprofloxacin but not the other {beta}-lactams, suggesting regulation of the blaCTX-M-15 protein production. The proteomic data from the this study provided novel insights into the proteins produced from the chromosome and plasmid under different antimicrobial stresses. These data also identified novel proteins not previously associated with AMR or antimicrobials responses in pathogens, which may well represent potential targets of AMR inhibition.

sentences: Prolonged dexamethasone exposure enhances zebrafish lateral-line regeneration but disrupts mitochondrial homeostasis and hair cell function The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 hours. Consistent with previously work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed more negatively charged mitochondrial membrane potentials ({Delta}{Psi}m) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.

sentences: Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

sentences: Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis Photo-inhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional upregulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light-induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of PSII D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

sentences: The Mycobacterium tuberculosis protein O-phosphorylation landscape Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of [~]30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

sentences: Life history strategies explain bacterial activity in the soil carbon cycle Soil microorganisms determine the fate of soil organic matter (SOM), and their activities comprise a major component of the global carbon (C) cycle. We employed a multi-substrate DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interact within soil. We identified and tracked 1,286 bacterial taxa that assimilated 13C in an agricultural soil over a period of 48 days. Overall 13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the 13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds comprised of taxa that had similar 13C assimilation dynamics. We show that C source bioavailability explained significant variation in both C mineralization dynamics and guild structure. In addition, guild structure explained significant variation in bacterial growth dynamics. We demonstrate that the observed guild structure is consistent with predictions made by bacterial life history theory. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C-cycling.

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Sensing hand position in Ehlers-Danlos Syndrome PurposeTo explore the effect of joint hypermobility on acuity, and plasticity, of hand proprioception.

Materials and MethodsWe compared proprioceptive acuity between EDS patients and controls. We then measured any changes in their estimate of hand position after participants adapted their reaches in response to altered visual feedback of their hand. The Beighton Scale was used to quantify the magnitude of joint hypermobility.

ResultsThere were no differences between the groups in the accuracy of estimates of hand location, nor in the visually-induced changes in hand location. However, EDS patients estimates were less precise when based purely on proprioception and could be partially predicted by Beighton score.

ConclusionsEDS patients are less precise at estimating their hands location when only afferent information is available, but the presence of efferent signalling may reduce this imprecision. Those who are more hypermobile are more likely to be imprecise.

sentences: Reevaluating claims of ecological speciation in Halichoeres bivittatus Understanding the role of ecological processes in speciation has become one of the most active areas of research in marine population biology in recent decades. The traditional view was that allopatry was the primary driver of speciation in marine taxa, but the geography of the marine environment and the dispersal capabilities of many marine organisms render this view somewhat questionable. One of the earliest and most highly cited empirical examples of ecological speciation with gene flow in marine fishes is that of the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep north-south divergence in a single mitochondrial locus, combined with a finding that these two haplotypes were associated with different habitat types in the Florida Keys and Bermuda, where they overlap. Here we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study, and find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations. These results severely undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. We argue that future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.

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SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1 macrophage polarisation. Background and aimsTLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesis in vitro and demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages.

MethodsAdult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFN{gamma}, S1/IFN{gamma} or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2.

ResultsTLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid(R)), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore(R), suggesting ACE2 expression is mediated by the alternative endosomal/{beta}-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-{gamma} treatment of THP-1-derived macrophages induced pro-inflammatory M1 polarisation as shown by an increase in IL-1{beta} and IL-6 mRNA.

ConclusionsThese results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1 macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid(R)) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.

sentences: Dairy vs. beef production - expert views on welfare Consumers views and concerns about the welfare of farm animals may play an important role in their decision to consume dairy, meat and/or plants as their primary protein source. As animals are killed prematurely in both dairy and beef industries, it is important to quantify and compare welfare compromise in these two sectors before the point of death. Seventy world-leading bovine welfare experts based in 23 countries, were asked to evaluate the likelihood of a bovine to experience 12 states of potential welfare concern, inspired by the Welfare Quality(R) protocol. The evaluation focused on the most common beef and dairy production systems in the experts country, and was carried out separately for dairy/beef calves raised for red-meat, dairy/beef calves raised for veal, dairy/beef calves raised as replacement, and for dairy/beef cows. The results show experts rated the overall likelihood of a negative welfare state (i.e. welfare risk) to be higher in animals from dairy herds than from beef herds, for all animal categories, regardless of whether they were used to produce milk, red-meat or veal. These findings suggest that consuming food products derived from common dairy production systems (dairy or meat), may be more harmful to the welfare of animals than consuming products derived from common beef production systems (i.e. from animals solely raised for their meat). Raising awareness about the linkage between dairy and meat production, and the toll of milk production on the welfare state of animals in the dairy industry, may encourage a more sustainable and responsible food consumption.

sentences: Rethinking protein drug design with highly accurate structure prediction of anti-CRISPR proteins Protein therapeutics play an important role in controlling the functions and activities of disease-causing proteins in modern medicine. Despite protein therapeutics having several advantages over traditional small-molecule therapeutics, further development has been hindered by drug complexity and delivery issues. However, recent progress in deep learning-based protein structure prediction approaches such as AlphaFold opens new opportunities to exploit the complexity of these macro-biomolecules for highly-specialised design to inhibit, regulate or even manipulate specific disease-causing proteins. Anti-CRISPR proteins are small proteins from bacteriophages that counter-defend against the prokaryotic adaptive immunity of CRISPR-Cas systems. They are unique examples of natural protein therapeutics that have been optimized by the host-parasite evolutionary arms race to inhibit a wide variety of host proteins. Here, we show that these Anti-CRISPR proteins display diverse inhibition mechanisms through accurate structural prediction and functional analysis. We find that these phage-derived proteins are extremely distinct in structure, some of which have no homologues in the current protein structure domain. Furthermore, we find a novel family of Anti-CRISPR proteins which are structurally homologous to the recently-discovered mechanism of manipulating host proteins through enzymatic activity, rather than through direct inference. Using highly accurate structure prediction, we present a wide variety of protein-manipulating strategies of anti-CRISPR proteins for future protein drug design.

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A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency TET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, whether and how TET1 exerts catalytic activity-independent functions in regulating bivalent genes is not well understood. Using a proteomics approach, we mapped the TET1 interactome in mouse ESCs and identified PSPC1 as a novel TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2) complex. We establish that PSPC1 and TET1 repress, and Neat1, the PSPC1 cognate lncRNA, activates the bivalent gene expression. In ESCs, Neat1 tethers the TET1-PSPC1 pair with PRC2 at bivalent promoters. During the ESC-to-formative epiblast-like stem cell (EpiLC) transition, PSPC1 and TET1 promote PRC2 chromatin occupancy at bivalent gene promoters while restricting Neat1 functions in facilitating PRC2 binding to bivalent gene transcripts. Our study uncovers a novel TET1-PSPC1-Neat1 molecular axis that modulates PRC2 binding affinity to chromatin and bivalent gene transcripts in controlling stem cell bivalency.

In BriefTET1 is a transcriptional repressor for bivalent genes in pluripotent stem cells, but its mechanistic action on stem cell bivalency is unclear. Huang et al. use proteomics and genetic approaches to reveal that catalytic activity-independent functions of TET1, coordinated with the paraspeckle components PSPC1 and its cognate lncRNA Neat1, dynamically regulates stem cell bivalency by modulating PRC2 binding affinity to chromatin and bivalent gene transcripts in pluripotent state transition.

HighlightsO_LIThe TET1 interactome identifies PSPC1 as a novel partner in ESCs
C_LIO_LITET1 and PSPC1 repress bivalent genes by promoting PRC2 chromatin occupancy
C_LIO_LINeat1 facilitates bivalent gene activation by promoting PRC2 binding to their mRNAs
C_LIO_LINeat1 bridges the TET1-PSPC1 and PRC2 complexes in regulating bivalent gene transcription
C_LI

sentences: A partially nested cortical hierarchy of neural states underlies event segmentation in the human brain A fundamental aspect of human experience is that it is segmented into discrete events. This may be underpinned by transitions between distinct neural states. Using an innovative data-driven state segmentation method, we investigate how neural states are organized across the cortical hierarchy and where in the cortex neural state boundaries and perceived event boundaries overlap. Our results show that neural state boundaries are organized in a temporal cortical hierarchy, with short states in primary sensory regions, and long states in lateral and medial prefrontal cortex. State boundaries are shared within and between groups of brain regions that resemble well-known functional networks. Perceived event boundaries overlap with neural state boundaries across large parts of the cortical hierarchy, particularly when those state boundaries demarcate a strong transition or are shared between brain regions. Taken together, these findings suggest that a partially nested cortical hierarchy of neural states forms the basis of event segmentation.

sentences: Protein disulphide isomerase (PDI) is protective against several types of DNA damage, including that induced by amyotrophic lateral sclerosis-associated mutant TDP-43 in neuronal cells/ in vitro models Protein disulphide isomerase (PDI) is a chaperone that catalyses the formation of thiol-disulphide bonds during protein folding. Whilst up-regulation of PDI is a protective mechanism to regulate protein folding, an increasingly wide range of cellular functions have been ascribed to PDI. Originally identified in the endoplasmic reticulum (ER), PDI has now been detected in many cellular locations, including the nucleus. However, its role in this cellular compartment remains undefined. PDI is implicated in multiple diseases, including amyotrophic lateral sclerosis (ALS), a fatal and rapidly progressing neurodegenerative condition affecting motor neurons. Loss of essential proteins from the nucleus is an important feature of ALS. This includes TAR DNA-binding protein-43 (TDP-43), a DNA/RNA binding protein present in a pathological form in the cytoplasm in almost all (97%) ALS cases, that is also mutated in a proportion of familial cases. PDI is protective against disease-relevant phenotypes associated with dysregulation of protein homeostasis (proteostasis) in ALS. DNA damage is also increasingly linked to ALS, which is induced by pathological forms of TDP-43 by impairment of its normal function in the non-homologous end-joining (NHEJ) mechanism of DNA repair. However, it remains unclear whether PDI is protective against DNA damage in ALS. In this study we demonstrate that PDI was protective against several types of DNA damage, induced by either etoposide, hydrogen peroxide (H2O2), or ALS-associated mutant TDP-43M337V in neuronal cells. This was demonstrated using widely used DNA damage markers, phosphorylated H2AX and 53BP1, which is specific for NHEJ. Moreover, we also show that PDI translocates into the nucleus following DNA damage. Here PDI is recruited directly to sites of DNA damage, implying that it has a direct role in DNA repair. This study therefore identifies a novel role of PDI in the nucleus in preventing DNA damage.

sentences: Genome of the parasitoid wasp Dinocampus coccinellae reveals extensive duplications, accelerated evolution, and independent origins of thelytokous parthenogeny and solitary behavior Dinocampus coccinellae (Hymenoptera: Braconidae) is a generalist parasitoid wasp that parasitizes >50 species of predatory lady beetles (Coleoptera: Coccinellidae), with thelytokous parthenogeny as its primary mode of reproduction. Here we present the first high quality genome of D. coccinellae using a combination of short and long read sequencing technologies, followed by assembly and scaffolding of chromosomal segments using Chicago+ HiC technologies. We also present a first-pass ab initio genome annotation, and resolve timings of divergence and evolution of (1) solitary behavior vs eusociality, (2) arrhenotokous vs thelytokous parthenogenesis, and (3) rates of gene loss and gain among Hymenopteran lineages. Our study finds (1) at least two independent origins of eusociality and solitary behavior among Hymenoptera, (2) two independent origins of thelytokous parthenogenesis from ancestral arrhenotoky, and (3) accelerated rates of gene duplications, loss, and gain along the lineages leading to D. coccinellae. Our work both affirms the ancient divergence of Braconid wasps from ancestral Hymenopterans and accelerated rates of evolution in response to adaptations to novel hosts, including polyDNA viral co-evolution.

sentences: Connectome-wide association analysis identified significant brain functional alteration in adults with attention-deficit/hyperactivity disorder Adults with attention-deficit/hyperactivity disorder (ADHD), as an extreme-phenotype of ADHD, is still facing problems of inconsistency and undeciphered mechanisms for its neuropathology. To address this matter, our present study performed connecotome-wide voxel-based analyses with the resting-state fMRI data of 84 adults with ADHD and 89 healthy controls. We found that functional connectivity patterns of the left precuneus and the left middle temporal significantly altered in ADHD populations serving as potential neural biomarkers to distinguish ADHD with healthy controls, with subsequent seed-based analysis revealing the dysfunction of functional connections both intra- and inter-default mode and attention networks, among which middle temporal gyrus plays the key role of  bridge linking the default mode and attention networks. After cognitive behavioral therapy, two of these ADHD-altered functional connections ameliorated accompanied with improvement of ADHD core symptoms. Additionally, imaging genetic analyses also revealed close relationships between the observed brain functional alterations and ADHD-risk genes. Taken together, our findings suggested the interference of default mode on attention networks in adults with ADHD, which would be severing as a potential biomarker for both ADHD pathogenesis and treatment effects.

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Scaling of subcellular structures with cell length through decelerated growth How cells tune the size of their subcellular parts to scale with cell size is a fundamental question in cell biology. Until now, most studies on the size control of organelles and other subcellular structures have focused on scaling relationships with cell volume, which can be explained by limiting pool mechanisms. Here, we uncover a distinct scaling relationship with cell length rather than volume, revealed by mathematical modeling and quantitative imaging of yeast actin cables. The extension rate of cables decelerates as they approach the rear of the cell, until cable length matches cell length. Further, the deceleration rate scales with cell length. These observations reveal a new mode of scaling that senses the linear dimensions of a cell.

One Sentence SummaryAs actin cables in yeast cells grow longer, their extension rate decelerates, enabling the cable length to match cell length.

sentences: Disrupting ACE2 Dimerization Mitigates the Infection by SARS-COV-2 The coronavirus disease 2019 (COVID-19) pandemic has caused over 6 million death and 460 million reported cases globally. More effective antiviral medications are needed to curb the continued spread of this disease. The infection by SARS-COV-2 virus is initiated via the interaction between the receptor binding domain (RBD) of the viral glycoprotein Spike (S protein) and the N-term peptidase domain (PD) of the angiotensin-converting enzyme 2 (ACE2) expressed on host cell membrane. ACE2 forms protein homodimer primarily through its ferredoxin-like fold domain (aka. Neck-domain). We investigated whether the dimerization of ACE2 receptor plays a role in SARS-COV-2 virus infection. We report here that the ACE2 receptor dimerization enhances the recognition of SARS-COV-2 S protein. A 43 amino acid peptide based on the N-term of Neck-domain could block the ACE2 dimerization and the interaction between RBD and ACE2, and mitigate the SARS-COV-2/host cell interaction. Our study illustrated a new route to develop potential therapeutics for the prevention and treatment of SARS-COV-2 viral infection.

sentences: YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri The intracellular pathogen Shigella flexneri, which is the causative agent of bacillary dysentery, significantly influences the worldwide implication of diarrheal infections, consequentially causing about 1.1 million deaths each year. Due to a non-availability of an authorized vaccine and the upsurge of multidrug resistance amongst Shigella strains, there has been a huge demand for further genetic analyses which could help in the advancement of new/improved drugs, and finding vaccine candidates against the pathogen. Whilst many features about the invasion of colonic cells by Shigella have been identified, fundamental gaps in information concerning in what way the bacteria transit, survive, and control gene expression, remain. The present study aims to illustrate the role of yfiB gene in Shigella virulence, which is a part of the periplasmic YfiBNR tripartite signaling system. This system is involved in the regulation of cyclic-di-GMP levels inside the bacterial cells, which is a vital messenger molecule impacting varied cellular processes such as biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms like adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect biofilm formation, bacterial invasion, host-surface attachment, and the overall virulence of Shigella. This study eventually improves our understanding of the in-vivo persistence and survival of Shigella, brings light to the c-di-GMP lead regulation of Shigella virulence, and provides a prospective new target to design anti-infective drugs and vaccines against S. flexneri and other bacterial pathogens.

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Covid19Risk.ai: An open source repository and online calculator of prediction models for early diagnosis and prognosis of Covid-19 ObjectiveThe current pandemic has led to a proliferation of predictive models being developed to address various aspects of COVID-19 patient care. We aimed to develop an online platform that would serve as an open source repository for a curated subset of such models, and provide a simple interface for included models to allow for online calculation. This platform would support doctors during decision-making regarding diagnoses, prognoses, and follow-up of COVID-19 patients, expediting the models transition from research to clinical practice.

MethodsIn this proof-of-principle study, we performed a literature search in PubMed and WHO database to find suitable models for implementation on our platform. All selected models were publicly available (peer reviewed publications or open source repository) and had been validated (TRIPOD type 3 or 2b). We created a method for obtaining the regression coefficients if only the nomogram was available in the original publication. All predictive models were transcribed on a practical graphical user interface using PHP 8.0.0, and published online together with supporting documentation and links to the associated articles.

ResultsThe open source website https://covid19risk.ai/ currently incorporates nine models from six different research groups, evaluated on datasets from different countries. The website will continue to be populated with other models related to COVID-19 prediction as these become available. This dynamic platform allows COVID-19 researchers to contact us to have their model curated and included on our website, thereby increasing the reach and real-world impact of their work.

ConclusionWe have successfully demonstrated in this proof-of-principle study that our website provides an inclusive platform for predictive models related to COVID-19. It enables doctors to supplement their judgment with patient-specific predictions from externally-validated models in a user-friendly format. Additionally, this platform supports researchers in showcasing their work, which will increase the visibility and use of their models.

sentences: Stochastic analysis of frequency bandwidth and noise attenuation in neurotransmission Action potential (AP)-triggered neurotransmitter release forms the key basis of inter-neuronal communication. We present a stochastic hybrid system model that captures the release of neurotransmitter-filled vesicles from a presynaptic neuron. More specifically, vesicles arrive as a Poisson process to attach at a given number of docking sites, and each docked vesicle has a certain probability of release when an AP is generated in the presynaptic neuron. The released neurotransmitters enhance the membrane potential of the postsynaptic neuron, and this increase is coupled to the continuous exponential decay of the membrane potential. The buildup of potential to a critical threshold level results in an AP firing in the postsynaptic neuron, with the potential subsequently resetting back to its resting level. Our model analysis develops formulas that quantify the fluctuations in the number of released vesicles and mechanistically connects them to fluctuations in both the postsynaptic membrane potential and the AP firing times. Increasing the frequency of APs in the presynaptic neuron leads to saturation effects on the postsynaptic side, resulting in a limiting frequency range of neurotransmission. Interestingly, AP firing in the postsynaptic neuron becomes more precise with increasing AP frequency in the presynaptic neuron. We also investigate how noise in AP timing varies with different parameters, such as the probability of releases, the number of docking sites, the voltage threshold for AP firing, and the timescale of voltage decay. In summary, our results provide a systematic understanding of how stochastic mechanisms in neurotransmission enhance or impinge the precision of AP fringing times.

sentences: ARG1 could be expressed by human, but not represent for repair Arginase 1 (ARG1), typically expressed under stress or stimulation rather than in the healthy condition in humans. However, more and more studies claim ARG1 could be expressed by human and represents healing process. Including THP-1 monocyte shift to M2 macrophages rendered a higher expression of ARG1 than that with bone-graft has an ideal effect on anti-inflammation, resulting in tissue healing. With the higher expression of ARG1 seemingly induced by the constructed material and represented by the anti-inflammatory situation. For these reasons, this study focuses on whether traditional markers of macrophages could be express by human or not during clinical healing process. Through harvesting bone tissue samples during bone healing process and confirm the expression of markers at transcriptome and protein levels. The results showed the expression of ARG1 relatively low by bulk-RNA sequencing, quantitative polymerase chain reaction (qPCR), immunofluorescence (IF) and flow cytometry or could not be detected during process of healing of healthy donor. Taken together, results indicate that the ARG1 is not chosen for a regenerative marker in human bone tissue.

sentences: Refinement of corticospinal neuron activity during skilled motor learning The learning of motor skills relies on plasticity of the primary motor cortex as task acquisition drives the remodeling of cortical motor networks1,2. Large scale cortical remodeling of evoked motor outputs occurs in response to the learning of skilled, corticospinal-dependent behavior, but not simple, unskilled tasks1. Here we determine the response of corticospinal neurons to both skilled and unskilled motor training and assess the role of corticospinal neuron activity in the execution of the trained behaviors. Using in vivo calcium imaging, we found that refinement of corticospinal activity correlated with the development of skilled, but not unskilled, motor expertise. Animals that failed to learn our skilled task exhibited a limited repertoire of dynamic movements and a corresponding absence of network modulation. Transection of the corticospinal tract and aberrant activation of corticospinal neurons show the necessity for corticospinal network activity patterns in the execution of skilled, but not unskilled, movement. We reveal a critical role for corticospinal network modulation in the learning and execution of skilled motor movements. The integrity of the corticospinal tract is essential to the recovery of voluntary movement after central nervous system injuries and these findings should help to shape translational approaches to motor recovery.

sentences: PeakVI: A Deep Generative Model for Single Cell Chromatin Accessibility Analysis AO_SCPLOWBSTRACTC_SCPLOWSingle-cell ATAC sequencing (scATAC-seq) is a powerful and increasingly popular technique to explore the regulatory landscape of heterogeneous cellular populations. However, the high noise levels, degree of sparsity, and scale of the generated data make its analysis challenging. Here we present PeakVI, a probabilistic framework that leverages deep neural networks to analyze scATAC-seq data. PeakVI fits an informative latent space that preserves biological heterogeneity while correcting batch effects and accounting for technical effects such as library size and region-specific biases. Additionally, PeakVI provides a technique for identifying differential accessibility at a single region resolution, which can be used for cell-type annotation as well as identification of key cis-regulatory elements. We use public datasets to demonstrate that PeakVI is scalable, stable, robust to low-quality data, and outperforms current analysis methods on a range of critical analysis tasks. PeakVI is publicly available and implemented in the scvi-tools framework: https://docs.scvi-tools.org/.

sentences: Exact distribution of threshold-crossing times for protein concentrations: Implication for biological timekeeping Stochastic transcription and translation dynamics of protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold-crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance and skewness. The distribution is asymmetric and its skewness non-monotonically varies with the threshold. We study lysis times of E-coli cells for holin gene mutants of bacteriophage-{lambda} and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted.

sentences: Multi-state Modeling of G-protein Coupled Receptors at Experimental Accuracy The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the human genome. GPCRs transduct chemical signals from extracellular to intracellular regions via a conformational switch between active and inactive states upon ligand binding. While experimental structures of GPCRs remain limited, high-accuracy computational predictions are now possible with AlphaFold2. However, AlphaFold2 only predicts one state and is biased towards either the active or inactive conformation depending on the GPCR class. Here, a multi-state prediction protocol is introduced that extends AlphaFold2 to predict either active or inactive states at very high accuracy using state-annotated templated GPCR databases. The predicted models accurately capture the main structural changes upon activation of the GPCR at the atomic level. For most of the benchmarked GPCRs (10 out of 15), models in the active and inactive states were closer to their corresponding activation state structures. Median RMSDs of the transmembrane regions were 1.12 [A] and 1.41 [A] for the active and inactive state models, respectively. The models were more suitable for protein-ligand docking than the original AlphaFold2 models and template-based models. Finally, our prediction protocol predicted accurate GPCR structures and GPCR-peptide complex structures in GPCR Dock 2021, a blind GPCR-ligand complex modeling competition. We expect that high accuracy GPCR models in both activation states will promote understanding in GPCR activation mechanisms and drug discovery for GPCRs. At the time, the new protocol paves the way towards capturing the dynamics of proteins at high-accuracy via machine-learning methods.

sentences: Gene flow influences the genomic architecture of local adaptation in six riverine fish species Understanding how gene flow influences adaptive divergence is important for predicting adaptive responses. Theoretical studies suggest that when gene flow is high, clustering of adaptive genes in fewer genomic regions would protect adaptive alleles from among-population recombination and thus be selected for, but few studies have tested this hypothesis with empirical data. Here, we used RADseq to generate genomic data for six fish species with contrasting life histories from six reaches of the Upper Mississippi River System, USA. We then conducted genome scans for genomic islands of divergence to examine the distribution of adaptive loci and investigated whether these loci were found in inversions. We found that gene flow varied among species, and adaptive loci were clustered more tightly in species with higher gene flow. For example, the two species with the highest overall FST (0.03 - 0.07) and therefore lowest gene flow showed little evidence of clusters of adaptive loci, with adaptive loci spread uniformly across the genome. In contrast, nearly all adaptive loci in the species with the lowest FST (0.0004) were found in a single large putative inversion. Two other species with intermediate gene flow (FST ~ 0.004) also showed clustered genomic architectures, with most islands of divergence clustered on a few chromosomes. These results provide important empirical evidence to support the hypothesis that increasingly clustered architectures of local adaptation are associated with high gene flow. Our study utilized a unique system with species spanning a large gradient of life histories to highlight the importance of gene flow in shaping adaptive divergence.

sentences: Sex differences in choice-based thermal nociceptive tasks in adult rats Interest in the role of sex as a biological variable continues to increase, including a mandate for the study of both sexes in NIH-funded research. Choice-based thermal nociceptive tests allow for the study of a more spontaneous response to thermal stimuli and avoidance behavior compared to traditional nociceptive assays, and their usage has been increasing in recent years. However, to date no comparison of naive male and female responses to such tests has been published. As sex differences are known to exist in both human chronic pain conditions and rodent models of nociception, it is critical to understand the impact of sex on any nociceptive assay. Herein, we examined the effect of sex on two choice-based thermal nociceptive tests, the thermal gradient test and the temperature place preference test, in adult rats. We report that marked sex differences exist in responses to these tests. Namely, the activation of a 10{degrees} C-to-47{degrees} C thermal gradient results in an increase in time spent in the 10{degrees} C zone in females, compared to a reduction in males. In a temperature place preference test pairing a surface temperature of 22{degrees} C with either 5{degrees} C, 10{degrees} C, 47{degrees} C, or 50{degrees} C, males spent less than 50% of their time in every non-22{degrees} C zone, but in females this was only observed when testing 50{degrees} C. Together, these results suggest that male rats show more avoidance behavior to non-ambient temperatures when given free access to multiple zones, including at temperatures which are milder than those typically used to evoke a nociceptive response in traditional hot and cold plate tests.

sentences: Dynamic Ins2 gene activity defines β cell maturity states Heterogeneity within specific cell types is common and increasingly apparent with the advent of single cell transcriptomics. Transcriptional and functional cellular specialization has been described for insulin-secreting {beta} cells of the endocrine pancreas, including so-called extreme {beta} cells exhibiting >2-fold higher insulin gene activity. However, it is not yet clear whether {beta} cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments employing FACS and single cell RNA sequencing, in {beta} cells from Ins2GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, [~]25% of {beta} cells exhibited significantly higher activity at the conserved insulin gene Ins2. Live cell imaging captured Ins2 gene activity dynamics in single {beta} cells over time. Autocorrelation analysis indicated that cells displaying fluctuations in Ins2 gene activity most commonly exhibited a frequency of 17 hours. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single cell RNA sequencing determined that Ins2(GFP)HIGH {beta} cells were enriched for markers of {beta} cell maturity and had reduced expression of anti- oxidant genes. Ins2(GFP)HIGH {beta} cells were also significantly less viable at all glucose concentrations and in the context of ER stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human {beta} cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of {beta} cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.

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Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect.

This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.

sentences: Causal Inference for Heritable Phenotypic Risk Factors Using Heterogeneous Genetic Instruments Over a decade of genome-wide association studies (GWAS) have led to the finding of extreme polygenicity of complex traits. The phenomenon that "all genes affect every complex trait" complicates Mendelian Randomization (MR) studies, where natural genetic variations are used as instruments to infer the causal effect of heritable risk factors. We reexamine the assumptions of existing MR methods and show how they need to be clarified to allow for pervasive horizontal pleiotropy and heterogeneous effect sizes. We propose a comprehensive framework GRAPPLE to analyze the causal effect of target risk factors with heterogeneous genetic instruments and identify possible pleiotropic patterns from data. By using GWAS summary statistics, GRAPPLE can efficiently use both strong and weak genetic instruments, detect the existence of multiple pleiotropic pathways, determine the causal direction and perform multivariable MR to adjust for confounding risk factors. With GRAPPLE, we analyze the effect of blood lipids, body mass index, and systolic blood pressure on 25 disease outcomes, gaining new information on their causal relationships and the potential pleiotropic pathways.

sentences: The cardiopharyngeal mesoderm contributes to lymphatic vessel development Lymphatic vessels are crucial for tissue homeostasis and immune responses in vertebrates. Recent studies have demonstrated that lymphatic endothelial cells (LECs) arise from both venous sprouting (lymphangiogenesis) and de novo production from non-venous origins (lymphvasculogenesis), which is similar to blood vessel formation through angiogenesis and vasculogenesis. However, the contribution of LECs from non-venous origins to lymphatic networks is considered to be relatively small. Here, we identify the Islet1 (Isl1)-expressing cardiopharyngeal mesoderm (CPM) as a non-venous origin of craniofacial and cardiac LECs. Genetic lineage tracing with Isl1-Cre and Isl1-MerCreMer mice suggested that a subset of CPM cells gives rise to LECs. These CPM-derived LECs are distinct from venous-derived LECs in terms of their developmental processes and anatomical locations. Later, they form the craniofacial and cardiac lymphatic vascular networks in collaboration with venous-derived LECs. Collectively, our results demonstrate that there are two major sources of LECs, the cardinal vein and the CPM. As the CPM is evolutionarily conserved, these findings may improve our understanding of the evolution of lymphatic vessel development across species. Most importantly, our findings may provide clues to the pathogenesis of lymphatic malformations, which most often develop in the craniofacial and mediastinal regions.

sentences: Arctic evergreening - encroaching allelopathy in a management blind spot Management blind spots, i.e., influential processes not included in management models, may undermine management objectives and reduce system resilience. Here we address one such blind spot in the management of Rangifer in Norway. The current model is based on regulation of pastures and population vital rates through reindeer density, but lacks validation of the underlying assumption of forage stability, which is a critical blind spot. We report pasture changes over 18 growing seasons, under variable reindeer densities and a changing climate. We show a strong increase of the native evergreen, allelopathic crowberry, contrasted by smaller increases of deciduous dwarf-shrubs and stagnation in herbaceous species. We found no evidence of reindeer density influencing these vegetation changes. Our results unveil a trajectory of Arctic evergreening and increasing allelopathy, and a shift towards lower biodiversity and ecosystem functioning. It exemplifies an ongoing change in a management blind spot that greatly impacts social-ecological systems.

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ELIMINATOR: Essentiality anaLysIs using MultIsystem Networks And inTeger prOgRamming A gene is considered as essential when it is indispensable for cells to grow and replicate under a certain environment. However, gene essentiality is not a structural property but rather a contextual one, which depends on the specific biological conditions affecting the cell. This circumstantial essentiality of genes is what brings the attention of scientist since we can identify genes essential for cancer cells but not essential for healthy cells. This same contextuality makes their identification extremely challenging. Huge experimental efforts such as Project Achilles where the essentiality of thousands of genes is measured in over one thousand cell lines together with a plethora of molecular data (transcriptomics, copy number, mutations, etc.) can shed light on the causality behind the essentiality of a gene in a given environment by associating the measured essentiality to molecular features of the cell line.

Here, we present an in-silico method for the identification of patient-specific essential genes using constraint-based modelling (CBM). Our method expands the ideas behind traditional CBM to accommodate multisystem networks, that is a biological network that focuses on complex interactions within several biological systems. In essence, it first calculates the minimum number of non-expressed genes required to be active by the cell to sustain life as defined by a set of requirements; and second, it performs an exhaustive in-silico gene knockout to find those that lead to the need of activating extra non-expressed genes.

We validated the proposed methodology using a set of 452 cancer cell lines derived from the Cancer Cell Line Encyclopedia where an exhaustive experimental large-scale gene knockout study using CRISPR (Achilles Project) evaluates the impact of each removal. We also show that the integration of different essentiality predictions per gene, what we called Essentiality Congruity Score, (derived from multiple pathways) reduces the number of false positives. Finally, we explored the gene essentiality predictions for a breast cancer patient dataset, and our results showed high concordance with previous publications.

These findings suggest that identifying genes whose activity are fundamental to sustain cellular life in a patient-specific manner is feasible using in-silico methods. The patient-level gene essentiality predictions can pave the way for precision medicine by identifying potential drug targets whose deletion can induce death in tumour cells.

sentences: The N501Y spike substitution enhances SARS-CoV-2 transmission Beginning in the summer of 2020, a variant of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the United Kingdom (UK). This B.1.1.7 variant increased rapidly in prevalence among sequenced strains, attributed to an increase in infection and/or transmission efficiency. The UK variant has 19 nonsynonymous mutations across its viral genome including 8 substitutions or deletions in the spike protein, which interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that, of the 8 individual spike protein substitutions, only N501Y exhibited consistent fitness gains for replication in the upper airway in the hamster model as well as primary human airway epithelial cells. The N501Y substitution recapitulated the phenotype of enhanced viral transmission seen with the combined 8 UK spike mutations, suggesting it is a major determinant responsible for increased transmission of this variant. Mechanistically, the N501Y substitution improved the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil and South Africa, our results indicate that N501Y substitution is a major adaptive spike mutation of major concern.

sentences: Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogens gradients and to generate hypotheses that are difficult to study experimentally.

sentences: A Probabilistic Approach For Registration Of Multi-Modal Spatial Transcriptomics Data Observing the spatial characteristics of gene expression by image-based spatial transcriptomics technology allows studying gene activity across different cells and intracellular structures. We present a probabilistic approach for the registration and analysis of transcriptome images and immunostaining images. The method is based on particle filters and jointly exploits intensity information and image features. We applied our approach to synthetic data as well as real transcriptome images and immunostaining microscopy images of the mouse brain. It turns out that our approach accurately registers the multi-modal images and yields better results than a state-of-the-art method.

sentences: Automated assembly of high-quality diploid human reference genomes The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has greatly benefited society1, 2. However, it still has many gaps and errors, and does not represent a biological human genome since it is a blend of multiple individuals3, 4. Recently, a high-quality telomere-to-telomere reference genome, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a duplicate genome, and is thus nearly homozygous5. To address these limitations, the Human Pangenome Reference Consortium (HPRC) recently formed with the goal of creating a collection of high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and automated assembly approaches yields the most complete, accurate, and cost-effective diploid genome assemblies with minimal manual curation. Approaches that used highly accurate long reads and parent-child data to sort haplotypes during assembly outperformed those that did not. Developing a combination of all the top performing methods, we generated our first high- quality diploid reference assembly, containing only [~]4 gaps (range 0-12) per chromosome, most within + 1% of CHM13s length. Nearly 1/4th of protein coding genes have synonymous amino acid changes between haplotypes, and centromeric regions showed the highest density of variation. Our findings serve as a foundation for assembling near-complete diploid human genomes at the scale required for constructing a human pangenome reference that captures all genetic variation from single nucleotides to large structural rearrangements.

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AI for predicting chemical-effect associations at the chemical universe level - deepFPlearn Many chemicals are out there in our environment, and all living species are exposed. However, numerous chemicals pose risks, such as developing severe diseases, if they occur at the wrong time in the wrong place. For the majority of the chemicals, these risks are not known. Chemical risk assessment and subsequent regulation of use require efficient and systematic strategies. Lab-based methods - even if high throughput - are too slow to keep up with the pace of chemical innovation. Existing computational approaches are designed for specific chemical classes or sub-problems but not usable on a large scale. Further, the application range of these approaches is limited by the low amount of available labeled training data.

We present the ready-to-use and stand-alone program deepFPlearn that predicts the association between chemical structures and effects on the gene/pathway level using a combined deep learning approach. deepFPlearn uses a deep autoencoder for feature reduction before training a deep feedforward neural network to predict the target association. We received good prediction qualities and showed that our feature compression preserves relevant chemical structural information. Using a vast chemical inventory (unlabeled data) as input for the autoencoder did not reduce our prediction quality but allowed capturing a much more comprehensive range of chemical structures. We predict meaningful - experimentally verified-associations of chemicals and effects on unseen data. deepFPlearn classifies hundreds of thousands of chemicals in seconds.

We provide deepFPlearn as an open-source and flexible tool that can be easily retrained and customized to different application settings at https://github.com/yigbt/deepFPlearn.

Supplementary informationSupplementary data are available at bioRxiv online.

Contactjana.schor@ufz.de

sentences: Acetyltransferases GCN5 and PCAF are required for B lymphocyte maturation in mice B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacks both GCN5 and PCAF in B cells. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We conclude that both GCN5 and PCAF are required for B cell development in vivo.

sentences: Functional correlates of immediate early gene expression in mouse visual cortex During visual development, response properties of layer 2/3 neurons in visual cortex are shaped by experience. Both visual and visuomotor experience are necessary to coordinate the integration of bottom-up visual input and top-down motor-related input. Whether visual and visuomotor experience engage different plasticity mechanisms, possibly associated with the two separate input pathways, is still unclear. To begin addressing this, we measured the expression level of three different immediate early genes (IEG) (c-fos, egr1 or Arc) and neuronal activity in layer 2/3 neurons of visual cortex before and after a mouses first visual exposure in life, and subsequent visuomotor learning. We found that expression levels of all three IEGs correlated positively with neuronal activity, but that first visual and first visuomotor exposure resulted in differential changes in IEG expression patterns. In addition, IEG expression levels differed depending on whether neurons exhibited primarily visually driven or motor-related activity. Neurons with strong motor-related activity preferentially expressed EGR1, while neurons that developed strong visually driven activity preferentially expressed Arc. Our findings are consistent with the interpretation that bottom-up visual input and top-down motor-related input are associated with different IEG expression patterns and hence possibly also with different plasticity pathways.

sentences: Phenotypic reconstruction of the last universal common ancestor reveals a complex cell A fundamental concept in evolutionary theory is the last universal common ancestor (LUCA) from which all living organisms originated. While hypotheses regarding the phenotype of LUCA do exist, most are at best based on gene presence or absence. However, despite recent attempts to link genes and phenotypic traits in prokaryotes, it is still inherently difficult to predict phenotype based on the presence or absence of genes alone. Here, we apply a novel approach based on phylogenetically informed ancestral character reconstruction with 22 fundamental descriptors of prokaryotic cells to reconstruct LUCAs phenotype. We infer that the last universal common ancestor of all living organisms was likely an ovoid cell with a large genome, which probably had a cell wall and was actively motile. Potentially obtaining energy from the oxidation of inorganic compounds, tolerant of saltwater and thriving at temperatures above 70{degrees}C, LUCA was probably found living freely in anaerobic conditions in water of neutral pH. Our results depict LUCA as likely to be a far more complex cell than has previously been proposed, challenging the evolutionary model of increased complexity through time in prokaryotes. Given current estimates for the emergence of LUCA we suggest that early life very rapidly evolved considerable cellular complexity.

sentences: Preconditioning by voluntary wheel running attenuates later neuropathic pain via Nrf2 antioxidant signaling in rats Animal and human studies have shown that exercise prior to nerve injury prevents later chronic pain, but the mechanisms of such preconditioning remain elusive. Given that exercise acutely increases formation of free radicals, triggering antioxidant compensation, we hypothesized that voluntary running preconditioning would attenuate neuropathic pain by supporting redox homeostasis after sciatic nerve injury in male and female rats. We show that 6 weeks of voluntary wheel running suppresses neuropathic pain development induced by chronic constriction injury (CCI) across both sexes. This protection was associated with reduced nitrotyrosine immunoreactivity--a marker for peroxynitrite--at the sciatic nerve injury site. Our data suggest that prior voluntary wheel running does not reduce production of peroxynitrite precursors, as expression levels of inducible nitric oxide synthase and NADPH oxidase 2 were unchanged. Instead, voluntary wheel running increased superoxide scavenging by elevating expression of superoxide dismutases 1 and 2. Prevention of neuropathic pain was further associated with activation of the master transcriptional regulator of the antioxidant response, nuclear factor E2-related factor 2 (Nrf2). Six weeks of prior voluntary wheel running increased Nrf2 nuclear translocation at the sciatic nerve injury site; in contrast, 3 weeks of prior wheel running, which failed to prevent neuropathic pain, had no effect on Nrf2 nuclear translocation. The protective effects of prior voluntary wheel running were mediated by Nrf2, as suppression was abolished across both sexes when Nrf2 activation was blocked during the running phase. This study provides insight into the mechanisms by which physical activity may prevent neuropathic pain.

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Bacterial Normalized Binary Fission Growth Model Mathematical models have traditionally been used to facilitate the interpretation of bacterial growth curves in order to more accurately understand and identify variations in bacterial proliferation. Here, a binary fission growth model was developed to normalize starting bacterial levels, allowing for the identification of changes in bacterial growth and the separation of a bacterial population as it correlates to size. This normalized binary fission model (NBF) relies on a multi-bin growth mode, where each bin is associated with a size range during a growth cycle. The proposed NBF model allows for a determination of the percentage of treated bacteria eradicated compared to a control sample, either generally across all bacterial binary fission sizes or specific to a size range or bin. Comparisons between the NBF model and experimental observations demonstrates that bacterial growth curves, and the ratio of sample growth to a control, can be used to both determine and normalize initial variations in bacterial size, and quantity, among test samples, as well as identify final nutrient levels and the percentage of bacteria affected by treatment.

Significance StatementIt is difficult to determine the effectiveness of selective bacterial eradication based upon a bacterias characteristics size, related to acoustic resonance. Here we develop a binary fission model to analyze effect on growth curves of size depend eradications. Understanding the effect on growth curves provides a method to extract the eradication percentage and initial bacterial level differences between treated sample and control sample, by using the ratio of treated growth curve to control growth curve.

sentences: Binary Interactome Models of Inner- Versus Outer-Complexome Organization The interactome is often conceived of primarily as a collection of hundreds of multimeric machines, collectively referred to as the "complexome". However, a large proportion of the interactome exists outside of the complexome, or in the "outer-complexome", and may account for most of the functional plasticity exhibited by cellular systems. To compare features of inner- versus outer-complexome organization, we systematically generated a yeast all-by-all binary interactome map, integrated it with previous binary maps, and compared the resulting interactome "atlas" with systematic co-complex association and functional similarity network datasets. Direct protein-protein interactions in the inner-complexome tend to be readily detected in multiple assays and exhibit high levels of coherence with functional similarity relationships. In contrast, pairs of proteins connected by relatively transient, harder-to-detect binary interactions in the outer-complexome, exhibit higher levels of functional heterogeneity. Thus, a small proportion of the interactome corresponds to a stable, functionally homogeneous, inner-complexome, forming quaternary structure, while a much greater proportion consists of transient interactions between pairs of functionally heterogeneous proteins in the outer-complexome, forming quinary structure.

sentences: The epidemiology and genomics of a virulent emerging fungal pathogen in an Australian reptile Emerging infectious fungal diseases (EIFDs) represent a major conservation concern worldwide. Here, we provide early insights into the potential threat that Nannizziopsis barbatae (Nb), a novel EIFD, poses to Australian herpetological biodiversity. First known to the reptile pet trade as a primary pathogen causing untreatable severe dermatomycosis, since 2013, Nb has emerged in a growing number of phylogenetically and ecologically distant free-living reptiles across Australia. Observing its emergence in a long-term study population of wild eastern water dragons (Intellagama lesueurii), we demonstrate the pathogens virulence-related genomic features, within-population spatiotemporal spread, and survival costs, all of which imply that Nb could pose a threat to Australian reptiles in the future. Our findings highlight the need to closely monitor this pathogen in Australian ecosystems.

sentences: Helical ultrastructure of the oncogenic metalloprotease meprin α in complex with a small molecule hydroxamate inhibitor The zinc-dependent metalloprotease meprin  is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin  performs key roles in inflammation, immunity, and extracellular matrix remodelling. The latter activity is furthermore important for driving aggressive metastasis in the context of certain cancers such as colorectal carcinoma. Accordingly, meprin  is the target of drug discovery programs. In contrast to meprin {beta}, meprin  is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin , as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin  forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin {beta} reveal unique features of the active site of meprin , and helical assembly more broadly.

sentences: A2A Receptor contributes to tumor progression in P2X7 null mice ATP and adenosine are key constituents of the tumor niche where they exert opposite and complementary roles. ATP promotes tumor growth but also immune eradicating responses mainly via the P2X7 receptor (P2X7R), while adenosine acts as a potent immune suppressor and facilitates neovascularization thanks to A2A receptor (A2AR) activity. However, studies exploring the interplay between P2X7R and A2AR in the tumor microenvironment are as yet missing. Here we investigated tumor growth in C57/bl6 P2X7 null mice inoculated with B16-F10 melanoma cells, showing that several pro-inflammatory cytokines (IL1-{beta}, TNF-, IL-6, IL-12, IL-17, IFN-{gamma}) were significantly decreased while the immune suppressant TGF-{beta} was almost three-fold increased. Interestingly, tumors growing in P2X7-null mice also upregulated tumor-associated and splenic A2AR, suggesting that immunosuppression associated to lack of the P2X7R might depend upon A2AR overexpression. Immunohistochemical analysis showed that tumor cells A2AR expression was increased, especially around necrotic areas, and that VEGF and the endothelial marker CD31 were also upregulated. The A2AR antagonist SCH-58261 reduced tumor growth similarly in the P2X7 WT, or null mice strain. However, SCH-58261only reduced VEGF in the P2X7-KO mice, thus supporting the hypothesis of an A2AR mediated increase in vascularisation in P2X7-null host. SCH-58261 administration also significantly reduced intratumor TGF-{beta}, thus supporting a key immune suppressive role of A2AR in our model. This study shows a novel direct correlation between P2X7R and A2AR in oncogenesis and paves the way for new combined therapies promoting anti-cancer immune responses and reducing tumor vascularization.

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A simple, inexpensive and multi scale 3D fluorescent test sample for optical sectioning microscopies Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-{micro}m to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes.

Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective x20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this "primitive" and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities.

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- standard tissue paper presents features ranging from 400 nm to centimetres
- our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast
- it is easy to prepare, versatile, photostable and inexpensive

sentences: Physachenolide C induces complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17{beta}-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.18 - 1.7 {micro}M. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17{beta}-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

sentences: Expression and protein sequence analyses of zebrafish impg2a and impg2b, two proteoglycans of the interphotoreceptor matrix Photoreceptor outer segments projecting from the surface of the neural retina toward the retinal pigment epithelium (RPE) are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix (IPM) [1,2]. This extracellular compartment is necessary for physiological retinal function. However, specific roles for molecules characterizing the IPM have not been clearly defined [3]. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene in patients affected by autosomal recessive Retinitis Pigmentosa (arRP) [4,5] and autosomal dominant and recessive vitelliform macular dystrophy (VMD) [6,7]. The gene encodes for a proteoglycan synthesized by photoreceptors and secreted in the IPM. However, little is known about the function and structure of this protein. We used the teleost zebrafish (D.rerio) as a model to study IMPG2 expression both during development and in adulthood, as its retina is very similar in humans [8]. In zebrafish, there are two IMPG2 proteins, IMPG2a and IMPG2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among different vertebrate species, showing a significant similarity despite the evolutionary distance between humans and teleosts. In fact, human IMPG2 and D.rerio IMPG2a and IMPG2b share conserved SEA and EGF-like domains. Homology models of these domains were obtained by using the iTasser server. Finally, expression analyses of impg2a and impg2b during development and in the adult fish showed expression of both mRNAs starting from 3 days post fertilization (dpf) in the outer nuclear layer of zebrafish retina that continues throughout adulthood. This data lays the groundwork for the generation of novel and most needed animal models for the study of IMPG2-related inherited retinal dystrophies.

sentences: Locally adaptive temperature response of vegetative growth in Arabidopsis thaliana We investigated early vegetative growth of natural Arabidopsis thaliana accessions in cold, non-freezing temperatures, similar to temperatures these plants naturally encounter in fall at northern latitudes. We found that accessions from northern latitudes produced larger seedlings than accessions from southern latitudes, partly as a result of larger seed size. However, their subsequent vegetative growth when exposed to colder temperatures was slower. The difference was too large to be explained by random population differentiation, and is thus suggestive of local adaptation, a notion that is further supported by substantial transcriptome and metabolome changes in northern accessions. We hypothesize that the reduced growth of northern accessions is an adaptive response, and a consequence of reallocating resources towards cold acclimation and winter survival.

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Rule-based and stimulus-based cues bias auditory decisions via different computational and physiological mechanisms Expectations, such as those arising from either learned rules or recent stimulus regularities, can bias subsequent auditory perception in diverse ways. However, it is not well understood if and how those diverse effects depend on the source of the expectations. Further, it is unknown whether these different sources of bias use the same or different computational and physiological mechanisms. We examined how rule-based and stimulus-based expectations influenced behavior and pupil-linked arousal, a marker of certain forms of expectation-based processing, of human subjects performing an auditory frequency-discrimination task. Rule-based cues biased choices and response times (RTs) toward the more-probable stimulus. In contrast, stimulus-based cues had a complex combination of effects, including choice and RT biases toward and away from the frequency of recently presented stimuli. These different behavioral patterns also had: 1) distinct computational signatures, including different modulations of key components of a novel form of a drift-diffusion decision model and 2) distinct physiological signatures, including substantial bias-dependent modulations of pupil size in response to rule-based but not stimulus-based cues. These results imply that different sources of expectations can modulate auditory processing via distinct mechanisms: one that uses arousal-linked, rule-based information and another that uses arousal-independent, stimulus-based information to bias the speed and accuracy of auditory perceptual decisions.

Author summaryPrior information about upcoming stimuli can bias our perception of those stimuli. Whether different sources of prior information bias perception in similar or distinct ways is not well understood. We compared the influence of two kinds of prior information on tone-frequency discrimination: rule-based cues, in the form of explicit information about the most-likely identity of the upcoming tone; and stimulus-based cues, in the form of sequences of tones presented before the to-be-discriminated tone. While both types of prior information biased auditory decision-making, they demonstrated distinct behavioral, computational, and physiological signatures. Our results suggest that the brain processes prior information in a form-specific manner rather than utilizing a general-purpose prior. Such form-specific processing has implications for understanding decision biases real-world contexts, in which prior information comes from many different sources and modalities.

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Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in Idiopathic Pulmonary Fibrosis Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF.

PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-{beta}/PGE2 balance in vitro in control and IPF fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR dependent manner in control ones.

PRRX1 inhibition decreased fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-{beta} driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-{beta} response global decrease.

Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using precision-cut lung slices.

Our results identified PRRX1 as a mesenchymal transcription factor driving lung fibrogenesis.

Brief SummaryInhibition of a single fibroblast-associated transcription factor, namely paired-related homeobox protein 1, is sufficient to dampen lung fibrogenesis.

sentences: Cochlear nucleus small cells use olivocochlear collaterals to encode sounds in noise Understanding communication signals, especially in noisy environments, is crucial to social interactions. Yet, as we age, acoustic signals can be disrupted by cochlear damage and the subsequent auditory nerve fiber degeneration. The most vulnerable--medium and high-threshold--auditory nerve fibers innervate various cell types in the cochlear nucleus, among which, the small cells are unique in receiving this input exclusively. Furthermore, small cells project to medial olivocochlear (MOC) neurons, which in turn send branched collaterals back into the SCC. Here, we use single-unit recordings to characterise small cell firing characteristics and demonstrate superior intensity coding in this cell class. We show converse effects when activating/blocking the MOC system, demonstrating that small-cell unique coding properties are facilitated by direct cholinergic input from the MOC system. Small cells also maintain tone-level coding in the presence of background noise. Finally, small cells precisely code low-frequency modulation more accurately than other ventral CN (VCN) cell types, demonstrating accurate envelope coding that may be important for vocalisation processing. These results highlight the small cell-olivocochlear circuit as a key player in signal processing in noisy environments, which may be selectively degraded in aging or after noise insult.

sentences: Genome sequence data reveal at least two distinct incursions of the tropical race 4 (TR4) variant of Fusarium wilt into South America The global banana industry is threatened by one of the most devastating diseases: Fusarium wilt (FWB). FWB is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), which almost annihilated the banana production in the late 1950s. A new strain of Foc, known as tropical race 4 (TR4), attacks a wide range of banana varieties including Cavendish clones which are the source of 99% of banana exports. In 2019, Foc TR4 was reported in Colombia, and more recently (2021) in Peru. In this study, we sequenced three fungal isolates identified as Foc TR4 from La Guajira (Colombia) and compared them against 19 whole-genome sequences of Foc TR4 publicly available, including four genome sequences recently released from Peru. To understand the genetic relatedness of the Colombian Foc TR4 isolates and those from Peru, we conducted a phylogenetic analysis based on a genome-wide set of single nucleotide polymorphisms (SNPs). Additionally, we compared the genomes of the 22 available Foc TR4 isolates looking for the presence-absence of gene polymorphisms and genomic regions. Our results reveal that (i) the Colombian and Peruvian isolates are genetically distant, which could be better explained by independent incursions of the pathogen to the continent, and (ii) there is a high correspondence between the genetic relatedness and geographic origin of Foc TR4. The profile of present/absent genes and the distribution of missing genomic regions showed a high correspondence to the clades recovered in the phylogenetic analysis, supporting the results obtained by SNP-based phylogeny.

sentences: Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany, 2020 SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with many new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August to October 2020, indicating the major cause of the second wave was the introduction of new variants.

sentences: Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis The rapid-growing and genetically tractable methanogen Methanococcus maripaludis is a promising host organism for the biotechnological conversion of carbon dioxide and renewable hydrogen to fuels and value-added products. Expansion of its product scope through metabolic engineering necessitates reliable and efficient genetic tools, particularly for genome edits to the primary metabolism that affect cell growth. Here, we have designed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery endogenously present in M. maripaludis. Remarkably, this toolbox can knock out target genes with a success rate of up to 95%, despite the hyper-polyploidy of M. maripaludis. For the purposes of demonstrating a large-sized deletion, we have replaced the flagellum operon (ca. 8.9 kbp) by the {beta}-glucuronidase gene. To facilitate metabolic engineering and flux balancing in M. maripaludis, the relative strength of 15 different promoters were quantified in the presence of the two common growth substrates, formate or carbon dioxide and hydrogen. This CRISPR/LbCas12a toolbox can be regarded as a reliable and fast method for genome editing in a methanogen.

sentences: Collagen (I) homotrimer potentiates the osteogenesis imperfecta (oim) mutant allele and reduces survival in male mice Type I collagen is the major structural component of bone where it exists as an (1)2(2)1 heterotrimer in all vertebrates. The osteogenesis imperfecta (oim) mouse model comprising solely homotrimeric (1)3 type I collagen, due to a dysfunctional 2 chain, has a brittle bone phenotype implying that the heterotrimeric form is required for physiological bone function. However, humans with rare null alleles preventing synthesis of the 2 chain have connective tissue and cardiovascular abnormalities (cardiac valvular Ehlers Danlos Syndrome), without evident bone fragility. Conversely a prevalent human single nucleotide polymorphism leading to increased homotrimer synthesis is associated with osteoporosis. Whilst the oim line is well-studied, whether homotrimeric type I collagen is functionally equivalent to the heterotrimeric form in bone has not been demonstrated. Col1a2 null and oim mouse lines were used in this study and bones analysed by microCT and 3-point bending. RNA was also extracted from heterozygote tissues and allelic discrimination analyses performed using qRT-PCR. Here we comprehensively show for the first time that mice lacking the 2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However Mendelian inheritance was affected in male mice of both lines and male mice null for the 2 chain exhibited age-related loss of condition. The brittle bone phenotype of oim homozygotes could result from detrimental effects of the oim mutant allele, however, the phenotype of oim heterozygotes is known to be less severe. We used allelic discrimination to show that the oim mutant allele is not downregulated in heterozygotes. We then tested whether gene dosage was responsible for the less severe phenotype of oim heterozygotes by generating compound heterozygotes. Data showed that compound heterozygotes had impaired bone structural properties as compared to oim heterozygotes, albeit to a lesser extent than oim homozygotes. Hence, we concluded that the presence of heterotrimeric collagen-1 in oim heterozygotes alleviates the effect of the oim mutant allele but a genetic interaction between homotrimeric collagen-1 and the oim mutant allele leads to bone fragility.

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Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function. N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction.

HIGHLIGHTSO_LISpinophilin bidirectionally regulates GluN2B-containing NMDAR function.
C_LIO_LILoss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker.
C_LIO_LILoss of spinophilin in vivo decreases measures of spatial cognitive flexibility.
C_LI

Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks.

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Biosynthesis of aurodox, a Type III secretion system inhibitor from Streptomyces goldiniensis The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid Polyketide Synthase/Non-Ribosomal Peptide Synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus T{upsilon} 365; however, the genetic organisation of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data support a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis follow that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus T{upsilon} 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique Type III Secretion System inhibitory properties to aurodox.

ImportanceEnterohaemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the Type III Secretion System, whilst circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we have characterised the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and have established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.

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NOMA: a high-throughput microchip for robust, sequential measurements of secretions from the same single-cells Despite recent advances in single-cell analysis technologies, lacking simple methods to keep the live single-cells traceable for longitudinal detection reliably poses a significant obstacle in single-cell secretion analysis. Here we reported the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of[≥] 97% of trapped single cells in dedicated spatial locations during repetitive detection procedures, verified with both adherent and suspension cells by two researchers independently. We applied it in monitoring single-cell protein secretions sequentially from the same single cells, and we found the digital protein secretion patterns dominate the protein secretion. We also demonstrated the microchip for longitudinally tracking IL-8 and the CD81+EV secretions from the same single-cells over days, which revealed the presence of "super secretors" within the cell population be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for tracking sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.

Table of Contents Graphic

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sentences: Genes used together are more likely to be fusedtogether in evolution by mutational mechanisms: Abioinformatic test of the used-fused hypothesis Cases of parallel or recurrent gene fusions, whether in evolution or in cancer and genetic disease, are difficult to explain, as they require multiple of the same or similar breakpoints to repeat. The used-together-fused-together hypothesis holds that genes that are used together repeatedly and persistently in a certain context are more likely than otherwise to undergo a fusion mutation in the course of evolution--reminiscent of the Hebbian learning rule where neurons that fire together wire together. This mutational hypothesis offers to explain both evolutionary parallelism and recurrence in disease of gene fusions under one umbrella. Here, we test this hypothesis using bioinformatic data. Various measures of gene interaction, including co-expression, co-localization, same-TAD presence and semantic similarity of GO terms show that human genes whose homologs are fused in one or more other organisms are significantly more likely to interact together than random genes, controlling for genomic distance between genes. In addition, we find a statistically significant overlap between pairs of genes that fused in the course of evolution in non-human species and pairs that undergo fusion in human cancers. These results provide support for the used-together-fused-together hypothesis over several alternative hypotheses, including that all gene pairs can fuse by random mutation, but among pairs that have thus fused, those that have interacted previously are more likely to be favored by selection. Multiple consequences are discussed, including the relevance of mutational mechanisms to exon shuffling, to the distribution of fitness effects of mutation and to parallelism.

sentences: Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level.

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Transcriptomic, epigenetic and metabolic characterization of the pluripotency continuum in rabbit preimplantation embryos Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit pre-implantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic, and metabolic map of the pluripotency continuum in rabbit preimplantation embryos and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with that of humans and non-human primates.

Summary StatementRabbit preimplantation embryos share characteristics with human and monkey embryos with respect to timing of early lineage segregation and expression of marker genes for naive and primed pluripotency.

sentences: Parallel processing by distinct classes of principal neurons in the olfactory cortex Understanding the specific roles that different neuron types play within a neural circuit is essential for understanding what that circuit does and how it does it. Primary olfactory (piriform, PCx) cortex contains two main types of principal neurons: semilunar (SL) and pyramidal (PYR). SLs and PYRs have different morphologies, connectivity, biophysical properties, and downstream projections, predicting distinct roles in cortical odor processing. The prevailing model is that odor processing in PCx occurs in two stages, where SLs are the primary recipients of olfactory bulb (OB) input, and PYRs receive and transform information from SLs. Here we recorded from opto-genetically-identified SLs and PYRs in awake, head-fixed mice. We found differences in SL and PYR odor-evoked activity that reflect their different connectivity profiles. But SL responses did not precede PYR responses and suppressing SL activity had little effect on PYR odor responses. These results suggest that SLs and PYRs form parallel odor processing channels.

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Perception of effort during an isometric contraction is influenced by prior muscle lengthening or shortening PurposeFollowing a shortening or lengthening muscle contraction, the torque produced in the isometric steady state is distinctly lower (residual torque depression; rTD) or higher (residual torque enhancement; rTE), respectively, compared to a purely isometric contraction at the same final muscle length and level of activation. This is referred to as the history dependence of force. When matching a given torque level, there is greater muscle activation (electromyography; EMG) following shortening and less activation following lengthening. Owing to these differences in neuromuscular activation, it is unclear whether perception of effort is altered by the history dependence of force.

MethodsExperiment 1 tested whether perception of effort differed between the rTD and rTE state when torque was matched. Experiment 2 tested whether perception of effort differed between the rTD and rTE state when EMG was matched. Finally, experiment 3 tested whether EMG differed between the rTD and rTE state when perception of effort was matched.

ResultsWhen torque was matched, both EMG and perception of effort were higher in the rTD compared to rTE state. When EMG was matched, torque was lower in the rTD compared to rTE state while perception of effort did not differ between the two states. When perception of effort was matched, torque was lower in the rTD compared to rTE state and EMG did not differ between the two states.

ConclusionThe combined results from these experiments indicate that the history dependence of force alters ones perception of effort, dependent on the level of motor command.

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Comparison of cerebrospinal fluid biomarkers relevant to neurodegenerative diseases in healthy cynomolgus and rhesus macaque monkeys Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSNon-human primates are important translational models of neurodegenerative disease. We characterized how species, sex, age, and site of sampling affected concentrations of key biomarkers of neurodegeneration.

METHODSAmyloid-beta (A{beta}40, A{beta}42), tau (tTau, pTau), and neurofilament light (NFL) in CSF were measured in 82 laboratory-housed naive cynomolgus and rhesus macaques of both sexes.

RESULTSA{beta}40, A{beta}42, and NFL were significantly higher in rhesus compared with cynomolgus macaques. tTau and NFL were higher in males. pTau was not affected by species or sex. Site of acquisition only affected NFL, with NFL being higher in CSF acquired from lumbar compared with cisterna magna puncture.

DISCUSSIONNormative values for key neurodegeneration biomarkers were established for laboratory housed cynomolgus and rhesus macaque monkeys. Differences were observed as a function of species, sex and site of CSF acquisition that should be considered when employing primate models.

Research In ContextO_LISystematic review: We reviewed reports characterizing CSF biomarkers of neurodegenerative diseases in non-human primates - an increasingly important model of disease - revealing that studies with laboratory housed macaque monkeys were of small sample size, with a paucity of data about how biomarkers varied as a function of species, sex, age, and site of acquisition.
C_LIO_LIInterpretation: To address this gap, we collected CSF from 82 naive laboratory housed male and female macaques of two species and measured A{beta}40, A{beta}42, tTau, pTau, and NFL. In addition to providing normative statistics for concentrations of these biomarkers, we revealed various species and sex differences.
C_LIO_LIFuture directions: Establishing normative values of biomarkers is an important step to the efficient development of cynomolgus and rhesus macaques as models of neurodegenerative disorders such as Alzheimers disease. Reference values reduce the need for large control groups by which to compare with disease model animals.
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sentences: Trapped Ion Mobility Spectrometry Reduces Spectral Complexity in Mass Spectrometry Based Workflow In bottom-up mass spectrometry based proteomics, deep proteome coverage is limited by high cofragmentation rates. This occurs when more than one analyte is isolated by the quadrupole and the subsequent fragmentation event produces fragment ions of heterogeneous origin. One strategy to reduce cofragmentation rates is through effective peptide separation techniques such as chromatographic separation and, the more recently popularized, ion mobility (IM) spectrometry which separates peptides by their collisional cross section. Here we investigate the capability of the Trapped Ion Mobility Spectrometry (TIMS) device to effectively separate peptide ions and quantify the separation power of the TIMS device in the context of a Parallel Accumulation-Serial Fragmentation (PASEF) workflow. We found that TIMS IM separation increases the number of interference-free MS1 features 9.2-fold, while decreasing the average peptide density in precursor spectra 6.5 fold. In a Data Dependent Acquisition (DDA) PASEF workflow, IM separation increased the number of spectra without cofragmentation by a factor of 4.1 and the number of high quality spectra 17-fold. This observed decrease in spectral complexity results in a substantial increase in peptide identification rates when using our data-driven model. In the context of a Data Independent Acquisition (DIA), the reduction in spectral complexity resulting from IM separation is estimated to be equivalent to a 4-fold decrease in isolation window width (from 25Da to 6.5Da). Our study shows that TIMS IM separation dramatically reduces cofragmentation rates leading to an increase in peptide identification rates.

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Optical and computational dissection of emergent prefrontal rewiring to encode fear memory Associative learning is crucial for adapting to environmental changes. The encoding of associative learning involves the dorso-medial prefrontal cortex (dmPFC), and is underpinned by interactions within the resident neuronal population. However, the nature of this population coding is poorly understood. Here we developed a pipeline for computational dissection and longitudinal two-photon imaging of neural population activities in the mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC topology. Through regularized regression methods and graphical modeling, we found fear conditioning organized neuronal ensembles encoding conditioned responses (CR), with enhancing their coactivity, functional connectivity, and association with conditioned stimuli (CS). This suggests that fear conditioning drives dmPFC reorganization to generate novel associative circuits for CS-to-CR transformation. Importantly, neurons strongly responding to unconditioned stimuli (US) during conditioning anterogradely became a hub of the CR ensemble. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on an activity-dependent hub-network formation within the dmPFC.

TeaserOptical and computational dissection uncovered how prefrontal cortical networks are rewired to encode new associative memory

Significance statementAnimals learn to adapt to changing environments. Associative learning is one of the simplest types of learning that has been intensively studied over the past century. Recent development in molecular, genetic, and optogenetic methods has enabled the identification of a neural population encoding the associative memory in the brain. However, it remains unclear how information is stored and processed by the neural population to encode and retrieve the associative memory. To investigate the nature of this population coding, we developed an optical and computational dissection method, demonstrating how associative learning drives reorganization of the neural network in the dorso-medial prefrontal cortex and generates novel circuits for associative memory and signal transformation.

sentences: Thymic macrophages consist of two populations with distinct localization and origin Tissue-resident macrophages are essential for protection from pathogen invasion and maintenance of organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and found out that they express typical tissue-resident macrophage genes yet also exhibit organ-specific features. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Using shield chimeras, transplantation of embryonic thymuses, and fate mapping, we found that three distinct waves of precursors generate thymic macrophages. Moreover, some of them proliferated in situ. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations with distinct localization and origin. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

sentences: Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

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Study on gene knockout mice and human mutant individual reveals absence of CEP78 causes photoreceptor and sperm flagella impairments BackgroundCone-rod dystrophy (CRD) is a genetically inherited retinal disease characterized by photoreceptor degeneration. In some rare cases, CRD and hearing loss can be associated with male fertility, while the underlying mechanism is not well known.

MethodsUsing CRISPR/Cas9 system, we generated Cep78-/- mice. And electroretinogram (ERG), immunofluorescence staining and transmission electron microscopy (TEM) were used to analyze visual function and photoreceptor ciliary structure changes in Cep78-/- mouse. HE/PAS staining, scanning-electron microscopy (SEM) were conducted to Cep78-/- mice and human CRD patient with CEP78 protein loss to illustrate male infertility and multiple morphological abnormalities of the sperm flagella (MMAF) caused by CEP78 deficiency. TEM and immunofluorescence staining were performed to characterize morphological and molecular changes of sperm flagella microtubule arrangement, centriole development and spermatid head shaping in Cep78-/- mice. Mass-spectrometry analyses were conducted to identify protein abnormalities after Cep78 deletion and Cep78 interacting proteins in spermiogenesis. Co-immunoprecipitation was used to show the Cep78-Ift20-Ttc21a trimer. The role of Cep78-Ift20-Ttc21a trimer in cilliogenesis and centriole elongation was assessed by cilia induction assay.

ResultsCep78 knockout mice exhibited impaired function and morphology of photoreceptors, typified by reduced electroretinogram amplitudes, disrupted translocation of cone arrestin, attenuated and disorganized photoreceptor outer segments (OS) disks and widen OS bases, as well as interrupted cilia elongations and structures. Cep78 deletion also caused male infertility and MMAF, with disordered "9 + 2" structure and triplet microtubules in sperm flagella. CEP78 forms a trimer with intraflagellar transport (IFT) proteins IFT20 and TTC21A essential for sperm flagella formation, is essential for their interaction and stability, and recruits IFT20 to centrosome. Insufficiency of any component in the trimer causes centriole elongation and cilia shortening. Additionally, absence of CEP78 protein in human leaded to similar phenotypes in vision and MMAF as Cep78-/- mice.

ConclusionsWe found CEP78 as the causative gene of CRD with MMAF in human and mouse. Cep78 forms a trimer with Ift20 and Ttc21a, and regulate the interaction, stability and localization of the trimer proteins, which regulate cilliogenesis, centriole length, and sperm flagella formation.

FundingThis work was supported by the National Key R&D Program (2021YFC2700200 to X.G); National Natural Science Foundation of China (82020108006, 81730025 to C.Z, 81971439, 81771641 to X.G, 82070974 to X.C, 82060183 to X.S); Shanghai Outstanding Academic Leaders (2017BR013 to C.Z); and Six Talent Peaks Project in Jiangsu Province (YY-019 to X.G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

sentences: Protein structure prediction using AI and quantum computers This work seeks to combine the combined advantage of leveraging these emerging areas of Artificial Intelligence and quantum computing in applying it to solve the specific biological problem of protein structure prediction using Quantum Machine Learning algorithms. The CASP dataset from ProteinNet was downloaded which is a standardized data set for machine learning of protein structure. Its large and standardized dataset of PDB entries contains the coordinates of the backbone atoms, corresponding to the sequential chain of N, C_alpha, and C atoms. This dataset was used to train a quantum-classical hybrid Keras deep neural network model to predict the structure of the proteins. To visually qualify the quality of the predicted versus the actual protein structure, protein contact maps were generated with the experimental and predicted protein structure data and qualified. Therefore this model is recommended for the use of protein structure prediction using AI leveraging the power of quantum computers.

sentences: Fenbendazole resistance in Heterakis gallinarum, the vector of Histomonas meleagridis, the causative agent of Blackhead Disease in poultry. Due to their ubiquity, management of parasites is a common and important factor for profitable production of poultry. Heterakis gallinarum, the cecal nematode, is the most common nematode parasite of poultry. While typically causing no pathology on its own, H. gallinarum is the vector of Histomonas meleagridis, a protozoan parasite that causes blackhead disease. Histomonas meleagridis is highly pathogenic in turkeys, potentially causing high mortality. In contrast, disease caused by H. meleagridis is much less severe in chickens, where it primarily reduces productivity without manifestations of clinical disease. There are no approved treatments for H. meleagridis, making control reliant on control of the helminth vector through the use of fenbendazole (FBZ) the only drug labeled for treatment of H. gallinarum in the United States We were contacted by an industry veterinarian regarding health-related concerns in a broiler-breeder house due to histomoniasis, despite frequent anthelmintic treatments. Since we had recently diagnosed resistance to FBZ in Ascaridia dissimilis, a closely related nematode of turkeys, we were interested to determine if H. gallinarum had also evolved resistance to FBZ. Heterakis gallinarum eggs were isolated from litter collected from the house and used to infect 108 chickens. Treatment groups included a non-treated control, a label-dose and a 2X-label dose of FBZ, with 36 birds per group divided into two replicate pens of 18 birds each. Birds were placed at 1-day post hatch, and at 3 weeks of age were infected with 150 embryonated eggs via oral gavage. Two weeks post infection, treated birds were administered a minimum of either a label- or 2X label-dose of FBZ in water for 5 days (SafeGuard(R) Aquasol, 1mg/kg BW). To ensure that all birds consumed the full intended dose at a minimum, the dosage was calculated using 1.25 times the average body weight. One-week post treatment, birds were euthanized, ceca removed, and parasites enumerated. Efficacy was calculated by comparing the total numbers of worms recovered from each treatment group to the numbers recovered in the non-treated control group. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.81). There also was no efficacy benefit to treatment with a 2X dose; H. gallinarum worm counts were reduced by 42.7% and 41.4%, for the label and 2X dosages, respectively. These data provide strong evidence that H. gallinarum has developed resistance to FBZ. Consequently, in houses infected with FBZ-resistant H. gallinarum, H. meleagridis will be able to cycle through the birds in an unrestricted manner. Further investigation is needed to determine the prevalence of resistance in H. gallinarum on chicken farms, but it is clear this has the potential to have a large-scale economic impact on the poultry industry. These data when viewed together with our recent findings of FBZ resistance in A. dissimilis, suggest that drug resistance in ascarid nematodes may be an important emerging problem on poultry operations.

sentences: Degeneracy and stability in neural circuits of dopamine and serotonin neuromodulators: A theoretical consideration Degenerate neural circuits perform the same function despite being structurally different. However, it is unclear whether neural circuits with interacting neuromodulator sources can themselves be degenerate while maintaining the same neuromodulatory function. Here, we address this by computationally modelling the neural circuits of neuromodulators serotonin and dopamine, local glutamatergic and GABAergic interneurons, and their possible interactions, under reward/punishment-based conditioning tasks. We show that a single parsimonious, sparsely connected neural circuit model can recapitulate several separate experimental findings, but not all, suggesting potentially multiple circuits acting in parallel. Using computational simulations and dynamical systems analysis, we demonstrate that several different stable circuit architectures can produce the same observed network activity profile. Further, simulating dopamine (D2) receptor agonist can distinguish among sub-groups of these degenerate networks; a testable model prediction. Overall, this theoretical work suggests the plausibility of degeneracy within neuromodulator circuitry and has important implication for the stable and robust maintenance of neuromodulatory functions.

sentences: Retroactive analysis of single cell transcriptome profiles using next-generation algorithms revised the identification of several resilient retinal ganglion cell types The clustering of single cell RNA-sequencing (scRNA-seq) data enables the classification of cell types, and the development of integration algorithms has enabled the tracing of altered-from-baseline transcriptomes to their respective cell type origins. Here, we developed an algorithm that removes sources of noise from scRNA-seq datasets and improves the accuracy of their integration. We found that this improvement of integration is further increased by an algorithm we developed for auto-determination of optimal weight-assignment to anchors. As a proof-of-concept, we show that our algorithm determined the type-origin of the 17% of injured retinal ganglion cells (RGCs) that an existing integration algorithm could not identify. As we found that the majority of the originally unassigned cells belonged to only several out of 45 RGC types, a consequent change in the proportions of the surviving types resulted in a moderately different ranking of resiliency to injury. This updated standard for RGC type resilience ranking improved the understanding of RGC type biology and the ranking-dependent prediction of genes for therapeutic neuroprotective targeting. Additional bioinformatic analyses revealed single-gene cluster-markers for RGC types and contributed new insights into the global characteristics of RGC types and how axonal injury affects them, showing how the extent of variance between transcriptomes of RGC types increases differentially during maturation and after injury. We also show, for the first time, that specific global properties of the transcriptome can predict the resilience to injury of certain cell types. Furthermore, we developed a website for cluster-by-cluster comparison of gene expression between uninjured and injured RGC types. The publicly available R library package, CellTools, for denoising and improving the integration of scRNA-seq datasets, as well as for associating cell types with the global properties of the transcriptome, will assist scRNA-seq studies of developing, injured, or diseased cell types across biological fields.

sentences: Microtubule inhibitors identified through non-biased screening enhance DNA transfection efficiency by delaying p62-dependent ubiquitin recruitment Ectopic gene expression is an indispensable tool in biology and medicine, but is often limited by the low efficiency of DNA transfection. We previously reported that depletion of the autophagy receptor p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target for drugs to increase transfection efficiency. To identify such drugs, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the p62-dependent mechanisms, the time required for p62-dependent ubiquitination, which is required for autophagosome formation, was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. Microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405 was markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings demonstrate for the first time that microtubule inhibitors suppress p62 activation as a mechanism for increasing DNA transfection efficiency and provide solutions to increase efficiency.

sentences: Fendrr synergizes with Wnt signalling to regulate fibrosis related genes during lung development Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via a RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and find that this FendrrBox is partially required for Fendrr function in vivo. We find that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs, associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in fibroblasts. We find that Fendrr with the Wnt signaling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signaling in lung fibrosis.

sentences: Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, acquisition of comprehensive transcriptomes of spatially- and phenotypically-defined cells in situ remains challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved spatial transcriptomes including isoforms with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues, circumventing the problem of significant RNA degradation during this time-consuming process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between the mouse small intestine lacteal cells at the tip versus the main capillary body. With the extensive repertoire of specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can now be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immunomarkers can be used to compare with the phenotypes derived from single-cell RNAseq of dissociated cells as well as applied to bead-based spatial transcriptomic approaches that require such information a priori for cell identification.

sentences: Transport Dynamics of MtrD: an RND multidrug efflux pump from Neisseria gonorrhoeae The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae, the causative agent of gonorrhea. Using free and directed Molecular Dynamics (MD) simulations, we analyzed the interactions between MtrD and Azithromycin, a transport substrate of MtrD and a last-resort clinical treatment for multidrug resistant gonorrhea. We then simulated the interactions between MtrD and Streptomycin, an apparent non-substrate of MtrD. Using known conformations of MtrD homologues, we simulated a potential dynamic transport cycle of MtrD using Targeted MD techniques (TMD), and we note that forces were not applied to ligands of interest. In these TMD simulations, we observed the transport of Azithromycin and the rejection of Streptomycin. In an unbiased, long-timescale simulation of AZY-bound MtrD, we observed the spontaneous diffusion of Azithromycin through the periplasmic cleft. Our simulations show how the peristaltic motions of the periplasmic cleft facilitate the transport of substrates by MtrD. Our data also suggest that multiple transport pathways for macrolides may exist within the periplasmic cleft of MtrD.

sentences: Antibody Activities in Hyperimmune Plasma Against the Rhodococcus equi Virulence -Associated Protein A or Poly-N-Acetyl Glucosamine are Associated with Protection of Foals Against Rhodococcal Pneumonia The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide {beta}-1[->]6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n=119) or subclinical pneumonia at Farm B (n=114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity [≤] the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities [≤] the population median for PNAG or C1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

sentences: Tight association between microbial eukaryote and Imitervirales communities in the Pacific Arctic Ocean Viruses are important regulatory factors of marine microbial community including microeukaryotes. However, little is known about their role in the northern Chukchi Sea of the Arctic basin, which remains oligotrophic conditions in summer. To elucidate linkages of microbial eukaryotic community with viruses as well as environmental variables, we investigated the community structures of microeukaryotes (3-144 {micro}m and 0.2-3 {micro}m size fractions) and Imitervirales (0.2-3 {micro}m size fraction), a major group of viruses infecting marine microeukaryotes. Surface water samples were collected at 21 ocean stations located in the northeastern Chukchi Sea (NECS), an adjacent area outside the Beaufort Gyre (Adjacent Sea; AS), and two melt ponds on sea ice in the summer of 2018. At the ocean stations, nutrient concentrations were low in most of the locations expect at the shelf in the AS. The community variations were significantly correlated between eukaryotes and Imitervirales, even within the NECS characterized by relatively homogeneous environmental conditions. The association of the eukaryotic community with the viral community was stronger than that with geographical and physicochemical environmental factors. These results suggest that Imitervirales are actively infecting their hosts even in cold and oligotrophic sea water in the Arctic Ocean.

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Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production -galactosidase (-GAL) and -N-acetylgalactosaminidase (-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki-diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important -GAL and -NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansonis -GAL and -NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative -GAL/-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for -GAL/-NAGAL substrate cleavage. Both -GAL and -NAGAL enzymatic activities were higher in females compared to males (p<0.05; -NAGAL > -GAL), which was consistent with smp_089290s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited -NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in -GAL activities were observed in the same extracts. Despite this, decreases in -NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an -NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Pharmacological inhibition of SmNAGAL may lead to the development of a novel anthelmintic class of compounds.

Author summarySchistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni -N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.

sentences: Unlocking the functional potential of polyploid yeasts Breeding and domestication have generated widely exploited crops, animals and microbes. However, many Saccharomyces cerevisiae industrial strains have complex polyploid genomes and are sterile, preventing genetic improvement strategies based on breeding. Here, we present a novel strain improvement approach based on the budding yeasts property to promote genetic recombination when meiosis is interrupted and cells return-to-mitotic-growth (RTG). We demonstrated that two unrelated sterile industrial strains with complex triploid and tetraploid genomes were RTG-competent and developed a visual screening for easy and high-throughput identification of recombined RTG clones based on colony phenotypes. Sequencing of the evolved clones revealed unprecedented levels of RTG-induced genome-wide recombination. We generated and extensively phenotyped a RTG library and identified clones with superior biotechnological traits. Thus, we propose the RTG-framework as a fully non-GMO workflow to rapidly improve industrial yeasts that can be easily brought to the market.

sentences: Endosperm turgor pressure both promotes and restricts seed growth and size Organ size depends on complex biochemical and mechanical interactions between cells and tissues. Here, we investigate the control of seed size, a key agronomic trait, by mechanical interactions between two compartments: the endosperm and the testa. By combining experiments with computational modelling, we tested an incoherent mechanical feedforward loop hypothesis in which pressure-induced stresses play two antagonistic roles; directly driving seed growth, but indirectly inhibiting it through mechanosensitive stiffening of the seed coat. We show that our model can recapitulate wild type growth patterns and explain the small seed phenotype of the haiku2 mutant. Our work further reveals that the developmental regulation of endosperm pressure is needed to prevent a precocious reduction of seed growth rate induced by force-dependent seed coat stiffening.

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Multi-scale brain simulation with integrated positron emission tomography yields hidden local field potential activity that augments machine-learning classification of Alzheimer's disease INTRODUCTIONComputational brain network modeling using The Virtual Brain (TVB) simulation platform acts synergistically with machine learning and multi-modal neuroimaging to reveal mechanisms and improve diagnostics in Alzheimers disease.

METHODSWe enhance large-scale whole-brain simulation in TVB with a cause-and-effect model linking local Amyloid {beta} PET with altered excitability. We use PET and MRI data from 33 participants of Alzheimers Disease Neuroimaging Initiative (ADNI3) combined with frequency compositions of TVB-simulated local field potentials (LFP) for machine-learning classification.

RESULTSThe combination of empirical neuroimaging features and simulated LFPs significantly outperformed the classification accuracy of empirical data alone by about 10% (weighted F1-score empirical 64.34% vs. combined 74.28%). Informative features showed high biological plausibility regarding the Alzheimers-typical spatial distribution.

DISCUSSIONThe cause-and-effect implementation of local hyperexcitation caused by Amyloid {beta} can improve the machine-learning-driven classification of Alzheimers and demonstrates TVBs ability to decode information in empirical data employing connectivity-based brain simulation.

RESEARCH IN CONTEXTO_LISYSTEMATIC REVIEW. Machine-learning has been proven to augment diagnostics of dementia in several ways. Imaging-based approaches enable early diagnostic predictions. However, individual projections of long-term outcome as well as differential diagnosis remain difficult, as the mechanisms behind the used classifying features often remain unclear. Mechanistic whole-brain models in synergy with powerful machine learning aim to close this gap.
C_LIO_LIINTERPRETATION. Our work demonstrates that multi-scale brain simulations considering Amyloid {beta} distributions and cause-and-effect regulatory cascades reveal hidden electrophysiological processes that are not readily accessible through measurements in humans. We demonstrate that these simulation-inferred features hold the potential to improve diagnostic classification of Alzheimers disease.
C_LIO_LIFUTURE DIRECTIONS. The simulation-based classification model needs to be tested for clinical usability in a larger cohort with an independent test set, either with another imaging database or a prospective study to assess its capability for long-term disease trajectories.
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sentences: SARS-CoV-2 variants do not evolve to promote further escape from MHC-I recognition SARS-CoV-2 variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that VOCs retain similar MHC-I downregulation capacity compared to the ancestral virus. However, VOCs exhibit a greater ability to suppress type I IFN than the ancestral virus. Although VOCs possess unique mutations within the ORF8 gene, which suppresses MHC-I expression, none of these mutations enhanced the ability of ORF8 to suppress MHC-I expression. Notably, MHC-I upregulation was strongly inhibited after the ancestral SARS-CoV-2 infection in vivo. Collectively, our data suggest that the ancestral SARS-CoV-2 already possesses an intrinsically potent MHC-I evasion capacity, and that further adaptation by the variants was not observed.

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Crystal structure of Sulfolobus solfataricus topoisomerase III reveals a novel carboxyl-terminal zinc finger domain essential for decatenation activity DNA topoisomerases are essential enzymes for a variety of cellular processes involved in DNA transactions. Mechanistic insights into type IA DNA topoisomerases have come principally from studies on bacterial and eukaryotic enzymes. A structural understanding of type IA topoisomerases in Archaea is lacking. Here, we present a 2.1-angstrom crystal structure of full-length Sulfolobus solfataricus topoisomerase III (Sso topo III), an archaeal member of type IA topoisomerases. The structure shows that Sso topo III adopts a characteristic torus-like architecture consisting of a four-domain core region and a novel carboxyl-terminal zinc finger domain (domain V). Structure-based mutation analyses reveal that a novel zinc-binding motif in domain V is essential for the DNA decatenation activity of Sso topo III. Our data indicate that Sso topo III represents a subclass of Type IA topoisomerases capable of resolving DNA catenates using a domain V-dependent mechanism.

IMPORTANCEType IA topoisomerases are omnipresent in all cellular life forms and serve pivotal roles in cellular processes involved in DNA transactions. While considerable insights have been gained into Type IA topoisomerases from bacteria and eukaryotes, a structural understanding of type IA topoisomerases in Archaea remains elusive. we first determined the crystal structure of full-length Sulfolobus solfataricus topoisomerase III (Sso topo III), an archaeal member of type IA topoisomerases. Our structure provides the first molecular view of this archaeal topoisomerase, which removes negative supercoils and decatenates DNA catenane. Our findings manifest that Sso topo III may serve as an alternative prototype of type IA topoisomerases, whose decatenation mechanism differs from that of known bacterial and eukaryotic topoisomerases III such as Escherichia coli topoisomerase III (EcTOP3).

sentences: Fatty sweet symphony: Decoding distinct ganglioside patterns of native and differentiated mesenchymal stem cells by a novel glycolipidomics profiling strategy Gangliosides are an indispensable glycolipid class concentrated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. In addition to the significant technological advance, we identified 263 ganglioside species including cell-state-specific markers and previously unreported gangliosides in native and differentiated human mesenchymal stem cells. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. By proving the predictive power of gangliosides as ubiquitous cell state-specific markers, we demonstrated the high throughput universal capability of our novel analytical strategy, which comes with new insights on the biological role of gangliosides in stem cell differentiation. Our analytical workflow will pave the way for new ganglioside- and glycolipid-based clusters of differentiation markers to determine stem cell phenotypes.

sentences: Free for all, or free-for-all? A content analysis of Australian university open access policies Recent research demonstrates that Australia lags in providing open access to research outputs. In Australia, while the two major research funding bodies require open access of outputs from projects they fund, these bodies only fund a small proportion of research conducted. The major source of research and experimental development funding in Australian higher education is general university, or institutional, funding, and such funds are not subject to national funder open access policies. Thus, institutional policies and other institutional supports for open access are important in understanding Australias OA position. The purpose of this paper is, therefore, to understand the characteristics of Australian institutional open access policies and to explore the extent they represent a coherent and unified approach to delivering and promoting open access in Australia. Open access policies were located using a systematic web search approach and then their contents were analysed. Only half of Australian universities were found to have an open access policy. There was a wide variation in language used, expressed intent of the policy and expectations of researchers. Few policies mention monitoring or compliance and only three mention consequences for non-compliance. While it is understandable that institutions develop their own policies, when language is used which does not reflect national and international understandings, when requirements are not clear and with consequences, policies are unlikely to contribute to understanding of open access, to uptake of the policy, or to ease of transferring understanding and practices between institutions. A more unified approach to open access is recommended.

sentences: Unsupervised Models of Mouse Visual Cortex Task-optimized deep convolutional neural networks are the most quantitatively accurate models of the primate ventral visual stream. However, such networks are implausible as models of the mouse visual system because mouse visual cortex has both lower retinal resolution and a shallower hierarchy than the primate. Moreover, the category supervision deep networks typically receive is neither ethologically relevant to the mouse in semantic content, nor realistic in quantity. As a result, standard supervised deep neural networks have proven quantitatively ineffective at modeling mouse visual data. Here, we develop and evaluate models that remedy these structural and functional gaps. We first demonstrate that shallow hierarchical architectures applied to lower resolution images improve match to neural responses, both in electro-physiological and calcium imaging data. We then show that networks trained using contrastive embedding methods, a recent unsupervised learning objective that requires no semantic labeling, achieve neural prediction performance that substantially exceed that of the same architectures trained in a supervised manner, across a wide variety of architecture types. Combining these better structural and functional priors yields models that are the most quantitatively accurate match to mouse visual responses to natural scenes, significantly surpassing that of prior attempts using primate-specific models, and approaching the inter-animal consistency level of the data itself. We further find that these shallow unsupervised models transfer to a wide variety of non-categorical visual tasks better than categorization-trained models. Taken together, our results suggest that mouse visual cortex is a low-resolution, shallow network that makes best use of the mouses limited resources to create a light-weight, general-purpose visual system - in contrast to the deep, high-resolution, and more task-specific visual system of primates.

sentences: SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells While there is clinical evidence of neurological manifestation in coronavirus disease-19, its unclear whether this is due to differential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake from blood by cells of the cerebrovasculature. SARS-CoV-2 and its spike protein (SP) interact with the endothelium but the roles of extracellular peptidase domain on angiotensin converting enzyme 2 receptors (ACE2) and ACE2 independent pathways (such as glycans) are not fully elucidated. In addition, for SARS-CoV-2 to enter the brain parenchyma from blood it has to cross several cell types, including the endothelium, pericytes and vascular smooth muscle. Since SARS-CoV-2 interacts with host cells via it SP at the entry point of it life cycle, we used fluorescently labelled SP (SP-555) (wild type and mutants) to model viral behaviour, in vitro, for these cell types (endothelial, pericytes and vascular smooth muscle) to explore pathways of viral entry into brain from blood. There was differential SP uptake by these cell types. The endothelial cells had the least uptake, which may limit SP uptake into brain from blood. Uptake was mediated by ACE2, but it was dependent on SP interaction with ganglioside GM1 in the lipid raft. Mutation sites, N501Yand E484K and D614G, as seen in variants of interest, were differentially taken up by these cell types. There was greater uptake but neutralization with anti-ACE2 and anti-GM1antibodies was less effective. Our data suggested that GM1/lipid raft is an important entry point of SARS-CoV-2 into these cells since inhibition of SP uptake with both anti-ACE2 and anti-GM1 together was similar to that with only anti-GM1, and both ACE2 and GM1 are within the lipid raft region of plasma membrane. Thus, GM1 is a potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

sentences: α-catenin links integrin adhesions to F-actin to regulate ECM mechanosensing and rigidity-dependence Both cell-cell and cell-matrix adhesions are regulated by mechanical signals, but the mechanobiological processes that mediate the crosstalk between these structures are poorly understood. Here we show that - catenin, a mechanosensitive protein that is classically associated with cadherin-based adhesions, directly interacts with and regulates integrin adhesions. -catenin is recruited to the edges of mesenchymal cells, where it interacts with F-actin. This is followed by mutual retrograde flow of -catenin and F-actin from the cell edge, during which -catenin interacts with vinculin within integrin adhesions. This interaction affects adhesion maturation, stress-fiber assembly, and force transmission to the matrix. In epithelial cells, -catenin is present in cell-cell adhesions and absent from cell-matrix adhesions. However, when these cells undergo epithelial-to-mesenchymal transition, -catenin transitions to cell-matrix adhesions, where it facilitates proper mechanosensing. This is highlighted by the ability of -catenin-depleted cells to grow on soft matrices. These results suggest a dual role of -catenin in mechanosensing, through both cell-cell and cell-matrix adhesions.

sentences: Specialization of actin isoforms derived from the loss of key interactions with regulatory factors A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently.

sentences: Automated systematic evaluation of cryo-EM specimens with SmartScope Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance in structural biology. Yet, finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure. Attaining the optimal specimen requires the evaluation of multiple grids in a microscope as conditions are varied. While automation has significantly increased the speed of data collection, optimization is still carried out manually. This laborious process which is highly dependent on subjective assessments, inefficient and prone to error, often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-electron microscopy. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-electron microscopy.

sentences: Surface marker expression in small and medium/large mesenchymal stromal cell-derived extracellular vesicles in naive or apoptotic condition using orthogonal techniques Extracellular vesicles released by mesenchymal stromal cells (MSC EVs) are a promising resource for regenerative medicine. In particular, small MSC EVs represent the active EV fraction for therapeutic applications. A bulk analysis is applied to characterize MSC EVs identity and purity, coupled with the assessment of single EV morphology, size and integrity using electron microscopy. We here applied different orthogonal methods to provide a quantitative analysis of size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC EVs obtained by sequential ultracentrifugations. Bone marrow, adipose tissue, and umbilical cord MSC EVs were compared, in naive and apoptotic conditions. The 100k EV size <100 nm, as detected by electron microscopy, was confirmed by super-resolution microscopy and ExoView. Quantitative single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanin expressions, being all MSC EV fractions single, double and triple positive, in variable proportions, for CD63, CD81 and CD9. Moreover, ExoView analysis allowed a comparative multiplex screening of single MSC EV tetraspanin and mesenchymal marker levels. Finally, a semiquantitative bead based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC EV fraction. Apoptotic MSC EVs were released in higher number, without significant differences from the naive fractions in surface marker expression. These results indicate that a consistent profile of MSC EV fractions among the different MSC sources, and a safer profile of the 100k MSC EV population for clinical application. Finally, our study identified suitable applications for different EV analytical techniques.

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DecentTree: Scalable Neighbour-Joining for the Genomic Era SummaryNeighbour-Joining is one of the most widely used distance-based phylogenetic inference methods. However, current implementations do not scale well for datasets with more than 10,000 sequences. Given the increasing pace of generating new sequence data, particularly in outbreaks of emerging diseases, and the already enormous existing databases of sequence data for which NJ is a useful approach, new implementations of existing methods are warranted.

Here we present DecentTree, which provides highly optimised and parallel implementations of Neighbour-Joining and several of its variants. DecentTree is designed as a stand-alone application and a header-only library easily integrated with other phylogenetic software (e.g. it is integral in the popular IQ-TREE software). We show that DecentTree shows similar or improved performance over existing software (BIONJ, Quicktree, FastME, and RapidNJ), especially for handling very large alignments. For example, DecentTree is up to 6-fold faster than the fastest existing Neighbour-Joining software (e.g. RapidNJ) when generating a tree of 64,000 SARS-CoV-2 genomes.

Availability and implementationDecentTree is open source and freely available at https://github.com/iqtree/decenttree.

ContactMinh Bui: m.bui@anu.edu.au; Robert Lanfear: rob.lanfear@anu.edu.au

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR mediated homologous recombination Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 50% of Drosophila genes lack suitable coding introns for integration of artificial exons. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

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A Regression Framework for Brain Network Distance Metrics Analyzing brain networks has long been a prominent research topic in neuroimaging. However, statistical methods to detect differences between these networks and relate them to phenotypic traits are still sorely needed. Our previous work developed a novel permutation testing framework to detect differences between two groups. Here we advance that work to allow both assessing differences by continuous phenotypes and controlling for confounding variables. To achieve this, we propose an innovative regression framework to relate distances between brain network features to functions of absolute differences in continuous covariates and indicators of difference for categorical variables. We explore several similarity metrics for comparing distances between connection matrices, and adapt several standard methods for estimation and inference within our framework: Standard F-test, F-test with individual level effects (ILE), Feasible Generalized Least Squares (FGLS), and Permutation. Via simulation studies, we assess all approaches for estimation and inference while comparing them with existing Multivariate Distance Matrix Regression (MDMR) methods. We then illustrate the utility of our framework by analyzing the relationship between fluid intelligence and brain network distances in Human Connectome Project (HCP) data.

HighlightsO_LIRelated distances between connection matrices to differences in covariates.
C_LIO_LIAdapted methods for estimation and inference in this framework.
C_LIO_LIAssessment of methods and distance metrics via simulation.
C_LIO_LICompared our methods to existing MDMR methods via simulation.
C_LIO_LIAnalysis of the HCP data with the best approach for each distance metric.
C_LI

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reComBat: Batch effect removal in large-scale, multi-source omics data integration MotivationWith the steadily increasing abundance of omics data produced all over the world under vastly different experimental conditions residing in public databases, a crucial step in many data-driven bioinformatics applications is that of data integration. The challenge of batch-effect removal for entire databases lies in the large number of batches and biological variation which can result in design matrix singularity. This problem can currently not be solved satisfactorily by any common batch-correction algorithm.

ResultsWe present reComBat, a regularized version of the empirical Bayes method to overcome this limitation and benchmark it against popular approaches for the harmonization of public gene expression data (both microarray and bulkRNAsq) of the human opportunistic pathogen Pseudomonas aeruginosa. Batch-effects are successfully mitigated while biologically meaningful gene expression variation is retained. reComBat fills the gap in batch-correction approaches applicable to large-scale, public omics databases and opens up new avenues for data-driven analysis of complex biological processes beyond the scope of a single study.

Contactmichael.adamer@bsse.ethz.ch

AvailabilityThe code is available at https://github.com/BorgwardtLab/reComBat, all data and evaluation code can be found at https://github.com/BorgwardtLab/batchCorrectionPublicData

sentences: Epitope scaffolding using alpha-synuclein cyclic peptides to generate oligomer-selective antibodies for Parkinson's disease Effectively scaffolding epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimers disease or Parkinsons disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we scaffolded epitopes in cyclic peptides by varying the number of flanking glycines, to best mimic a misfolding-specific conformation of an epitope of -synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of -synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model, and isolated monomer ensembles. We introduce a method for screening against structured off-pathway targets in the human proteome, by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines can have markedly different conformational ensembles. Ensemble comparison and overlap was quantified by the Jensen-Shannon Divergence, and a new measure introduced here--the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble, and which may be a more useful measure in sculpting the conformational-selectivity of an antibody.

sentences: Acoustic and vibrational signaling in true katydid Nesoecia nigrispina: three means of sound production in the one species The males of Mexican katydids Nesoecia nigrispina (Stal, 1873) produce calling songs and protest sounds using the same stridulatory apparatus as in most of the other Ensifera at the base of the elytra. It includes pars stridens on the upper elytron and plectrum on the lower. Calling sounds of two types (fast and slow) are 2-pulse series, repeated with a frequency of 1.2-4.5 s-1. Protest signals in the form of short trills from the same pulse duration males produce with tactile stimulation. The pulse repetition rate is more higher than that of the calling sounds - up to 10 (mean c. 8) s-1. The frequency spectra of these signals have maxima in the band of 14-15 kHz. However, in addition to the sounds described, both males and females are capable to produce protest signals of the second type with the help of another sound apparatus, namely with the help of the wings. Insects with removed elytra are unable to produce an audible sound. Thus, the sound is produced by the friction of the wings on the elytra, but there are no specialized stridulatory structures on them. In individuals of both sexes, in response mainly to tactile stimulation, short clicks are recorded, which they make, apparently, by the mandibles. Vibrational signals at tremulation are emitted by individuals of both sexes during courtship and males, completing the calling signal cycle and after copulation. It is possible that vibrational signals are an additional factor in reproductive isolation in sympatric species, since the calling sound signals in representatives of the genus Nesoecia are similar and exhibit significant variability.

sentences: Preservation of neural synchrony at peak alpha frequency via global synaptic scaling compensates for white matter structural decline over adult lifespan We propose that preservation of functional integration, estimated from measures of neural synchrony, is a key neurocompensatory mechanism associated with healthy human ageing. To support this proposal, we demonstrate how phase-locking at peak alpha frequency from Magnetoencephalography (MEG) data is invariant over lifespan in a large cohort of human participants, aged 18-88 years. Using empirically derived connection topologies from diffusion tensor imaging (DTI) data, we create an in-silico model of whole-brain alpha dynamics. We show that enhancing inter-areal coupling can cancel the effect of increased axonal transmission delay associated with age-related degeneration of white matter tracts and thus, preserve neural synchrony. Together with analytical solutions for non-biological all-to-all connection scenarios, our model establishes the theoretical principles by which frequency slowing with age, frequently observed in the alpha band in diverse populations, can be viewed as an epiphenomenon of the underlying neurocompensatory mechanisms.

sentences: Partners in space: Discordant population structure between legume hosts and rhizobium symbionts in their native range Coevolution is predicted to depend on how the genetic diversity of interacting species is geographically structured. Plant-microbe symbioses such as the legume-rhizobium mutualism are ecologically and economically important, but distinct life history and dispersal mechanisms for these host and microbial partners, plus dynamic genome composition in bacteria, present challenges for understanding spatial genetic processes in these systems. Here we study the model rhizobium Ensifer meliloti using a hierarchically-structured sample of 191 strains from 21 sites in the native range and compare its population structure to that of its host plant Medicago truncatula. We find high local genomic variation and minimal isolation by distance across the rhizobium genome, particularly at the two symbiosis elements pSymA and pSymB, which have evolutionary histories and population structures that are similar to each other but distinct from both the chromosome and the host. While the chromosome displays weak isolation by distance, it is uncorrelated with hosts. Patterns of discordant population structure among elements with the bacterial genome has implications for bacterial adaptation to life in the soil versus symbiosis, while discordant population genetic structure of hosts and microbes might restrict local adaptation of species to each other and give rise to phenotypic mismatches in coevolutionary traits.

sentences: HOW CONCERNING IS A SARS-COV-2 VARIANT OF CONCERN? COMPUTATIONAL PREDICTIONS AND THE VARIANTS LABELING SYSTEM We herein report a study to evaluate the use of computational prediction of SARS-CoV-2 genetic variations in improving the current variants labeling system. First, we reviewed the basis of the system developed by the World Health Organization (WHO) for the labeling of SARS-CoV-2 genetic variants and the adaptations made to it by the United States Center of Diseases Control (CDC). We observed that the labeling system is based upon the virus major attributes. However, we found that the labeling criteria of the SARS-CoV-2 variants derived from these attributes are not accurately defined and are used differently by the two health management agencies. Consequently, discrepancies exist between the labels given by WHO and CDC to same variants. Our observations suggest that giving the VOC label to a new variant is premature and might not be appropriate. Therefore, we carried out a comparative computational study to predict the effects of the mutations on the virus structure and functions of five VOCs. By linking these data to the criteria used by WHO and the CDC for variant labeling, we ascertained that comparative computational predictions of the impact of genetic variations are a better ground for rapid and more accurate labelling of SARS-CoV-2 variants. We propose to label all emergent variants VUM or VBM and to carry out computational predictive studies and thorough variants comparison, upon which more appropriate and informative labels can be attributed. Furthermore, harmonization of the variants labeling system would be globally beneficial to communicate about and fight COVID-19 pandemic.

sentences: Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse brachial MNs at embryonic and postnatal stages. We found that genes encoding homeodomain (HOX, LIM) transcription factors (TFs), previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this, we inactivated Hoxc8 at successive stages of MN development. We found that Hoxc8 is not only required to establish but also maintain expression of several MN terminal differentiation markers. Furthermore, we uncovered novel TFs with continuous MN expression, a Hoxc8 dependency for maintained expression of Iroquois (Irx) homeodomain TFs, and a new role for Irx2 in MN development. Our findings dovetail recent observations in C. elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation.

sentences: Dynamic Phosphorylation of RGS Provides Spatial Regulation of MAP Kinase And Promotes Completion of Cytokinesis during the Yeast Pheromone Response Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.

sentences: Spatial genomics maps the structure, character and evolution of cancer clones Subclonality is a universal feature of cancers yet how clones grow, are spatially organised, differ phenotypically or influence clinical outcome is unclear. To address this, we developed base specific in situ sequencing (BaSISS). In fixed tissues, transcripts harbouring clone-defining mutations are detected, converted into quantitative clone maps and characterised through multi-layered data integration. Applied to 8 samples from key stages of breast cancer progression BaSISS localised 1.42 million genotype informative transcripts across 4.9cm2 of tissue. Microscopic clonal topographies are shaped by resident tissue architectures. Distinct transcriptional, histological and immunological features distinguish coexistent genetic clones. Spatial lineage tracing temporally orders clone features associated with the emergence of aggressive clinical traits. These results highlight the pivotal role of spatial genomics in deciphering the mechanisms underlying cancer progression.

sentences: Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis Synucleinopathies including Parkinsons disease and dementia with Lewy bodies are neurodegenerative diseases characterized by the intracellular accumulation of the protein alpha-synuclein called "Lewy pathology." Alpha-synuclein within Lewy pathology is aggregated into protease resistant filamentous structures and is predominantly phosphorylated at serine 129 (PSER129). Lewy pathology has been hypothesized to spread throughout the nervous system as the disease progresses. Cross-sectional studies have shown the olfactory bulb and olfactory tract consistently bare LP for common synucleinopathies, making these structures likely starting points for the spreading process, and thus disease. Here we examined the distribution of PSER129 in non-diseased brain. To do this we used a sensitive tyramide signal amplification (TSA) technique to detect low abundance endogenous PSER129 under ideal antibody binding conditions. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb and several brain regions of the olfactory cortex across the neuroaxis (i.e., olfactory bulb to brain stem). PSER129 was particularly apparent in the mitral cell layer and the outer plexiform layer of the olfactory bulb where it was observed as cytosolic/nuclear puncta or fibers, respectively. PSER129 immunoreactivity in the healthy olfactory bulb was abolished by pretreatment of the tissue with proteinase K, pre-absorption of the primary antibody against the purified PSER129 peptide fragment, or the omission of the PSER129 antibody. Furthermore, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. Dual labeling for the PSER129 and the mitral cell marker TBX21 showed that PSER129 positive structures of the healthy OB were found in mitral cells. We found evidence of the same PSER129 positive structures in the olfactory bulb of non-diseased rats, non-human primates, healthy humans, but not individuals diagnosed with PD. Results suggest biological pathways responsible for alpha-synuclein phosphorylation are constitutively active in OB mitral cells and alpha-synuclein in these cells may be predisposed to pathological aggregation. Pathological seeds originating in mitral cells may act as a source for alpha-synuclein spread competent assemblies that spreads throughout the brain via fibers of the olfactory tract. Future studies should investigate the normal function of alpha-synuclein in the mitral cells of the olfactory bulb, which may give insight into synucleinopathy disease origins.

sentences: Robust effects of corticothalamic feedback during naturalistic visual stimulation Neurons in the dorsolateral geniculate nucleus (dLGN) of the thalamus receive a substantial proportion of modulatory inputs from corticothalamic (CT) feedback and brain stem nuclei. Hypothesizing that these modulatory influences might be differentially engaged depending on the visual stimulus and behavioral state, we performed in vivo extracellular recordings from mouse dLGN while optogenetically suppressing CT feedback and monitoring behavioral state by locomotion and pupil dilation. For naturalistic movie clips, we found CT feedback to consistently increase dLGN response gain and promote tonic firing. In contrast, for gratings, CT feedback effects on firing rates were mixed. For both stimulus types, the neural signatures of CT feedback closely resembled those of behavioral state, yet effects of behavioral state on responses to movies persisted even when CT feedback was suppressed. We conclude that CT feedback modulates visual information on its way to cortex in a stimulus-dependent manner, but largely independently of behavioral state.

sentences: Olfactory-driven beta band entrainment of limbic circuitry during neonatal development Cognitive processing relies on the functional refinement of the limbic circuitry during the first two weeks of life. During this developmental period, when most sensory systems are still immature, the sense of olfaction acts as "door to the world", providing the main source of environmental inputs. However, it is unknown whether early olfactory processing shapes the activity in the limbic circuitry during neonatal development. Here, we address this question by combining simultaneous in vivo recordings from the olfactory bulb (OB), lateral entorhinal cortex (LEC), hippocampus (HP), and prefrontal cortex (PFC) with opto- and chemogenetic manipulations of mitral/tufted cells (M/TCs) in the OB of non-anesthetized neonatal mice. We show that the neonatal OB synchronizes the limbic circuity in beta frequency range. Moreover, it drives neuronal and network activity in LEC, as well as subsequently, HP and PFC via long-range projections from mitral cells (MCs) to HP-projecting LEC neurons. Thus, OB activity shapes the communications within limbic circuits during neonatal development.

sentences: Astrocyte Ca2+ Signaling is Facilitated in an Scn1a+/- Mouse Model of Dravet Syndrome Dravet syndrome (DS) is an infantile-onset epileptic encephalopathy. More than 80% of DS patients have a heterozygous mutation in SCN1A, which encodes a subunit of the voltage-gated sodium channel, Nav1.1, in neurons. The roles played by astrocytes, the most abundant glial cell type in the brain, have been investigated in the pathogenesis of epilepsy; however, the specific involvement of astrocytes in DS has not been clarified. In this study, we evaluated Ca2+ signaling in astrocytes using genetically modified mice that have a loss-of-function mutation in Scn1a. We found that the slope of spontaneous Ca2+ spiking was increased without a change in amplitude in Scn1a+/- astrocytes. In addition, ATP-induced transient Ca2+ influx and the slope of Ca2+ spiking were also increased in Scn1a+/- astrocytes. These data indicate that perturbed Ca2+ dynamics in astrocytes may be involved in the pathogenesis of DS.

sentences: Mechanical control of innate immune responses against viral infection revealed in a human Lung Alveolus Chip Mechanical forces associated with breathing play a fundamental role in lung development and disease but the molecular pathways remain largely unknown. Here, we used a mechanically actuatable Human Lung Alveolus Chip that recapitulates human lung alveolar type I and type II cell differentiation, alveolar-capillary interface formation, and genome-wide gene expression profiles characteristic of the distal lung to investigate the role of physical forces associated with cyclic breathing motions in lung innate immune responses to viral infection. When the mechanically active Alveolus Chips are infected with the influenza H3N2 virus, a cascade of host responses is elicited on-chip, including increased production of cytokines and expression of inflammation-associated genes in pulmonary epithelial and endothelial cells, resulting in enhanced recruitment of circulating immune cells as occurs during viral infection in vivo. Surprisingly, studies carried out in parallel with static chips revealed that physiological breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells. This is mediated at least in part through upregulation of S100 calcium-binding protein A7 (S100A7), which binds to the Receptor for Advanced Glycation End Products (RAGE), an inflammatory mediator that is most highly expressed in the lung alveolus in vivo. This mechano-immunological control mechanism is further supported by the finding that existing RAGE inhibitor drugs can suppress the production of inflammatory cytokines in response to influenza virus infection in this model. S100A7-RAGE interactions and modulation of mechanical ventilation parameters could therefore serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

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SARS-CoV-2 RECoVERY: a multi-platform open-source bioinformatic pipeline for the automatic construction and analysis of SARS-CoV-2 genomes from NGS sequencing data BackgroundSince its first appearance in December 2019, the novel Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), spread worldwide causing an increasing number of cases and deaths (35,537,491 and 1,042,798, respectively at the time of writing, https://covid19.who.int). Similarly, the number of complete viral genome sequences produced by Next Generation Sequencing (NGS), increased exponentially. NGS enables a rapid accumulation of a large number of sequences. However, bioinformatics analyses are critical and require combined approaches for data analysis, which can be challenging for non-bioinformaticians.

ResultsA user-friendly and sequencing platform-independent bioinformatics pipeline, named SARS-CoV-2 RECoVERY (REconstruction of CoronaVirus gEnomes & Rapid analYsis) has been developed to build SARS-CoV-2 complete genomes from raw sequencing reads and to investigate variants. The genomes built by SARS-CoV-2 RECoVERY were compared with those obtained using other software available and revealed comparable or better performances of SARS-CoV2 RECoVERY. Depending on the number of reads, the complete genome reconstruction and variants analysis can be achieved in less than one hour. The pipeline was implemented in the multi-usage open-source Galaxy platform allowing an easy access to the software and providing computational and storage resources to the community.

ConclusionsSARS-CoV-2 RECoVERY is a piece of software destined to the scientific community working on SARS-CoV-2 phylogeny and molecular characterisation, providing a performant tool for the complete reconstruction and variants analysis of the viral genome. Additionally, the simple software interface and the ability to use it through a Galaxy instance without the need to implement computing and storage infrastructures, make SARS-CoV-2 RECoVERY a resource also for virologists with little or no bioinformatics skills.

Availability and implementationThe pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) is implemented in the Galaxy instance ARIES (https://aries.iss.it).

sentences: Zones of Cellular Damage Around Pulsed-Laser Wounds After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

sentences: Gaussian Network Model Revisited: Effects of Mutation and Ligand Binding on Protein Behavior The coarse-grained Gaussian Network model, GNM, considers only the alpha carbons of the folded protein. Therefore it is not directly applicable to the study of mutation or ligand binding problems where atomic detail is required. This shortcoming is improved by including the local effect of heavy atoms in the neighborhood of each alpha carbon into the Kirchoff Adjacency Matrix. The presence of other atoms in the coordination shell of each alpha carbon diminishes the magnitude of fluctuations of that alpha carbon. But more importantly, it changes the graph-like connectivity structure, i.e., the Kirchoff Adjacency Matrix of the whole system which introduces amino acid specific and position specific information into the classical coarse-grained GNM which was originally modelled in analogy with phantom network theory of rubber elasticity. With this modification, it is now possible to make predictions on the effects of mutation and ligand binding on residue fluctuations and their pair-correlations. We refer to the new model as  all-atom GNM. Using examples from published data we show that the all-atom GNM applied to in silico mutated proteins and to their laboratory mutated structures gives similar results. Thus, loss and gain of correlations, which may be related to loss and gain of function, may be studied by using simple in silico mutations only.

sentences: Uip4p modulates nuclear pore complex function in Saccharomyces cerevisiae A double membrane bilayer perforated by nuclear pore complexes (NPCs) governs the shape of the nucleus, the prominent distinguishing organelle of a eukaryotic cell. Despite the absence of lamins in yeasts, the nuclear morphology is stably maintained and shape changes occur in a regulated fashion. In a quest to identify factors that contribute to regulation of nuclear shape and function in Saccharomyces cerevisiae, we used a fluorescence imaging based approach. Here we report the identification of a novel protein, Uip4p, that is required for regulation of nuclear morphology. Loss of Uip4 compromises NPC function and loss of nuclear envelope (NE) integrity. Our localisation studies show that Uip4 localizes to the NE and endoplasmic reticulum (ER) network. Furthermore, we demonstrate that the localization and expression of Uip4 is regulated during growth, which is crucial for NPC distribution.

sentences: Inflammatory stress signaling via NF-kB alters accessible cholesterol to upregulate SREBP2 transcriptional activity in endothelial cells There is a growing appreciation that a tight relationship exists between cholesterol homeostasis and immunity in leukocytes, however, this relationship has not been deeply explored in the vascular endothelium. Endothelial cells (ECs) rapidly respond to extrinsic signals, such as tissue damage or microbial infection, by upregulating factors to activate and recruit circulating leukocytes to the site of injury and aberrant activation of ECs leads to inflammatory based diseases, such as multiple sclerosis and atherosclerosis. Here, we studied the role of cholesterol and its master regulator, SREBP2, in the EC responses to inflammatory stress. Treatment of ECs with pro-inflammatory cytokines upregulates SREBP2 cleavage and cholesterol biosynthetic gene expression within the late phase of the acute inflammatory response. Furthermore, SREBP2 activation was dependent on NF-{kappa}B DNA binding and canonical SCAP-SREBP2 processing. Mechanistically, inflammatory activation of SREBP was mediated by a reduction in accessible cholesterol, leading to heightened sterol sensing and downstream SREBP2 cleavage. Detailed analysis of NF-{kappa}B inducible genes that may impact sterol sensing resulted in the identification of a novel RELA-inducible target, STARD10, that mediates accessible cholesterol homeostasis in ECs. Thus, this study provides an in-depth characterization of the relationship between cholesterol homeostasis and the acute inflammatory response in EC.

sentences: Costs of choosiness can promote reproductive isolation in parapatry Species often replace each other spatially along contact zones, giving scope for parapatric speciation. In models of parapatric speciation driven by assortative mating, costs of female choosiness have so far be ignored. Yet, if females encounter only a limited number of males, those that are too choosy may remain unmated, and this should create direct sexual selection against choosiness. In our individual-based model of parapatric ecological speciation, disruptive viability selection leads to divergence of an ecological trait. Additionally, female choosiness (following a  matching mating rule based on the same ecological trait) can evolve at the risk of remaining unmated, and can limit gene flow between diverging populations. In line with previous litterature, out of the contact zone, the evolution of cost-free choosiness stops at intermediate values due to indirect selection against strong choosiness. Here we show that a weak cost of choosiness, by modifying genotypic frequencies on which viability selection acts, reduces this selection pressure, thus permitting the evolution of stronger choosiness than in the absence of costs. In strong contrast to sympatric models, costs of choosiness can therefore promote reproductive isolation in parapatry.

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Neonatal exposures to sevoflurane in rhesus monkeys alter synaptic ultrastructure in later life Early-life exposure to anesthesia in infant humans and monkeys increases the risk for cognitive and socioemotional impairments. However, the long-term effects of neonatal anesthesia on synaptic ultrastructure have not been thoroughly investigated in primates. We used electron microscopy with unbiased stereological sampling to assess synaptic ultrastructure in the CA1 of the hippocampus and the dorsolateral prefrontal cortex (dlPFC) of female and male rhesus macaques four years after three 4-hour exposures to sevoflurane during the first five postnatal weeks. We counted synapses and measured synapse areas for all synapses and those classified as perforated or nonperforated with spine or dendritic shaft targets. We measured numbers and shapes of mitochondria within presynaptic boutons and calculated vesicle docking rates. In monkeys exposed to anesthesia as infants, synapse areas were reduced in the largest 20% of synapses in CA1 and the largest 5% of synapses in dlPFC, with differential sex effects for the largest 10% of synapses in CA1. Synapse areas were reduced by 7.6% for perforated spinous synapses in CA1, and by 10.4% for nonperforated spinous synapses in dlPFC. Perforated and nonperforated dendritic synapse numbers in CA1 increased by 180% and 63% respectively. Curved mitochondria decreased 25% in CA1 after anesthesia exposure, and dlPFC boutons with 0 mitochondria showed an interaction of anesthesia and sex. These results demonstrate that exposure to anesthesia in infancy can cause long-term structural deficits in primates. These structural changes may be substrates for long-term alterations in the strength and efficiency of synaptic transmission in hippocampus and prefrontal cortex.

Key pointsO_LIExposure to anesthesia in early life causes lasting cognitive and socioemotional impairments in human and nonhuman primates, but the extent to which early-life exposure to anesthesia alters synaptic ultrastructure in later life has not been known.
C_LIO_LIFour years after rhesus monkeys were given multiple exposures to anesthesia in infancy, the area of spinous synapses was reduced in CA1 and dlPFC, dendritic synapse numbers were elevated in CA1, there were fewer curved presynaptic mitochondria in CA1, and numbers of presynaptic boutons without mitochondria were altered in dlPFC.
C_LIO_LIThe long-term ultrastructural changes to synapses and presynaptic mitochondria of rhesus monkeys that were exposed to anesthesia as infants could help explain their behavioral deficits later in life.
C_LI

sentences: Characterisation and chemometric evaluation of 17 elements in ten seaweed species from Greenland Several Greenland seaweed species have potential as foods or food ingredients, both for local consumption and export. However, knowledge regarding their content of beneficial and deleterious elements on a species specific and geographical basis is lacking. This study investigated the content of 17 elements (As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Na, Ni, P, Pb, Se and Zn) in 77 samples of ten species (Agarum clathratum, Alaria esculenta, Ascophyllum nodosum, Fucus distichus, Fucus vesiculosus, Hedophyllum nigripes, Laminaria solidungula, Palmaria palmata, Saccharina latissima and Saccharina longicruris). Element profiles differed between species but showed similar patterns within the same family. For five species, different thallus parts were investigated separately, and showed different element profiles. A geographic origin comparison of Fucus species indicated regional differences. The seaweeds investigated were especially good sources of macrominerals (K > Na > Ca > Mg) and trace minerals, such as Fe. Iodine contents were high, especially in macroalgae of the family Laminariaceae. None of the samples exceeded the EU maximum levels for Cd, Hg or Pb, but some exceeded the stricter French regulations, especially for Cd and I. In conclusion, these ten species are promising food items.

sentences: Neophobia and innovation in critically endangered Bali myna, Leucopsar rothschildi Cognition underlies animal behaviour, which is key to successful conservation strategies, yet largely under-utilised in conservation, though there are recent calls for closer integration. Conservation-relevant cognitive abilities can impact on adaptability and survival, such as neophobia, e.g., responses to novelty, and innovation e.g., problem-solving, particularly in todays changing world. Bali myna are a critically endangered endemic species, which are a focus of active conservation efforts, including reintroductions. Therefore, gathering cognitive data can aid in improving and developing conservation strategies, like pre-release training and individual selection for release. In 22 captive Bali myna, we tested neophobia (novel object, novel food, control conditions), innovation (bark, cup, lid conditions) and individual repeatability. We found effects of condition and social environment, including longer latencies to touch familiar food in presence than absence of novel items, and between problem-solving tasks, as well as in the presence of conspecifics, compared with being alone, or with conspecifics and competing heterospecifics. Individuals were repeatable in latency responses: 1) temporally in both experiments; 2) contextually in innovation experiment and between both experiments (and approach order), suggesting a stable behaviour trait. These findings are an important starting point for improving conservation strategies in Bali myna and other similarly threatened species.

sentences: A study of thermally-induced sex reversal in casque-headed lizards Non-avian reptiles, unlike mammals and birds, have undergone numerous sex determination turnovers. For example, casque-headed lizards replaced the ancestral XY system shared across pleurodonts with a new pair of XY chromosomes. However, the evolutionary forces that triggered this transition have remained unclear. An interesting evolutionary hypothesis suggests that species with intermediate states, with sex chromosomes but also thermal-induced sex reversal at specific incubation temperatures, could be more susceptible to sex determination turnovers. We contrasted genotypic data (presence/absence of the Y chromosome) against the histology of gonads of embryos from stages 35-37 incubated at various temperatures, including typical male-producing (26{degrees}C) and female-producing (32{degrees}C) temperatures. We observed perfect concordance between genotype and phenotype at all temperatures. However, analysis of transcriptomic data from embryos incubated at 26{degrees}C and 32{degrees}C identified transcript variants of the chromatin modifiers JARID2 and KDM6B that have been linked to temperature-dependent sex determination in other reptiles. Besides, our work reports for the first time to our knowledge the histology of gonads, including morphological changes, from stages 35-37 of development in the Corytophanidae family. We also observed that all embryos developed hemipenes, suggesting sex-linked developmental heterochrony.Our work tested the validity of a mixed sex determination system in the Corytophanidae family. We showed that XY chromosomes are dominant, however, our work supports the hypothesis of a conserved transcriptional response to incubation temperatures across non-avian reptiles that could be the reminiscence of an ancestral sex determination system.

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Critical Assessment of Metagenome Interpretation - the second round of challenges Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results.

Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

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Effect of a low concentration in chloride, sodium and potassium on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout. Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity, and more specifically of low water concentrations in sodium, chloride and potassium, during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species.

Fish from the same commercial strain were raised either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting higher concentration in these elements. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation.

Here we show that low water salinity (i.e., low water concentration in chloride, sodium and potassium) negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggered delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (i.e., aquaporins) and ion (i.e., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the overall ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.

HighlightsO_LILow water salinity impairs final oocyte maturation and egg quality in rainbow trout
C_LIO_LILow water salinity induces delayed ovulation and impaired oocyte hydration
C_LIO_LILow water salinity induces a dysregulation of several key ovulatory genes
C_LIO_LIMonitoring of final oocyte maturation can be performed using ultrasound staging
C_LI

sentences: Ribosomal protein L23 drives the metastasis of hepatocellular carcinoma via upregulating MMP9 Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally and tumor metastasis is one of the major causes of high mortality. To identify novel molecules contributing to HCC metastasis is critical to understanding the underlining mechanism of cancer metastasis. Here, combining the analyze based on published database and liver tissues from HCC patients, we identified that RNA binding protein L23 (RPL23) as a tumor metastasis driver in HCC. RPL23 was elevated in HCC and closely related to poor clinical outcomes. Furthermore, RPL23 depletion inhibited HCC cell proliferation, migration and invasion, while RPL23 overexpression promoted HCC cell metastasis. Mechanistically, RPL23 positively regulated MMP9 expression by stabilizing its mRNA. And increased MMP9 is involved in RPL23-mediated HCC metastasis. Importantly, RPL23 silencing reduced tumor growth and metastasis in vivo. In summary, we identified that RPL23 play an important role in HCC metastasis in an MMP9-dependent manner and may be a novel potential therapeutic target for HCC tumorigenesis and metastasis.

sentences: Natural Selection as the Sum Over All Histories. As evolution can be connected to the principle of least action, and if it is depicted in evolution-space versus time then it corresponds to the direction of ultimate causation. As an organism evolves and follows a path of proximate causation, if the vector is closely parallel to that of the Ultimate Causation then the changes will confer desirable attributes which will lead to further development. If, however, the variations do not occur in a direction close to that of the ultimate causation vector the evolved organism will quickly die out. Therefore Natural Selection may be viewed as similar to Feynmans "sum over all histories". This approach is compatible with both Neutral Theory and Selection, as it includes both positive and negative mutations and selection. Therefore, the principle of least action gives a direction, but not a purpose, to evolution.

sentences: The African mosquito-borne diseasosome: Geographical patterns and range expansion Mosquito-borne diseases (MBDs) such as malaria, dengue, and Rift Valley fever threaten public health and food security globally. Despite their cohesive nature, they are typically treated as distinct entities. Applying biological system analysis to the African MBDs from a One Health perspective, we provide the first biogeographic description of the African mosquito fauna corresponding with the pathogens they transmit. After compiling records accumulated over a century, we find that there are 677 mosquito species in Africa, representing 16 genera, and 151 mosquito-borne pathogens (MBPs) circulating primarily among wild tetrapods, dominated by viruses (95) and protozoans (47). We estimate that reported MBPs represent [~]1% of the actual number. Unlike mosquitoes, African arboviruses and mammalian plasmodia represent a higher share of the Worlds total based on the area - species richness relationship (P<0.0001), explaining the disproportional large share of global MBPs that originated from Africa. Species richness of African mosquitoes and MBPs are similarly concentrated along the equator, peaking in central Africa, with a secondary "ridge" along eastern Africa. Moderate diversity and low endemicity in mosquitoes across the Sahel reveals a fauna with high propensity for long-range migration. Regional differences in species richness, endemicity, and composition agreed with country-based results. The composition of mosquitoes and MBPs separates sub-Saharan Africa from north Africa, in accordance with the Palearctic and Afrotropical faunal realms, and west and central Africa are clustered together distinctly from the cluster of eastern and southern Africa. With [~]25% of the species occupying a single country, [~]50% in 1-3 countries and <5% found in >25 countries, the typical ranges of both mosquitoes and MBPs are surprisingly small. The striking similarity in diversity and especially in range distributions of mosquitoes and MBPs suggest that most MBPs are transmitted by one or few narrow-range mosquito vectors. Exceptionally widespread mosquito species (e.g., Ae. aegypti, Cx. quinquefasciatus, and 10 Anopheles species) feed preferentially on people and domestic animals, and nearly half are windborne migrants. Likewise, exceptionally widespread MBPs are transmitted between people or domestic animals and are vectored by one or more of the aforementioned widespread mosquitoes. Our results suggest that few MBPs have undergone a dramatic range expansion, after adapting to people or domestic animals as well as to exceptionally-widespread mosquitoes. During the intermediate phase of range expansion, MBPs extend their vector and vertebrate host ranges with a concomitant gradual increase in geographical range. Because range size may serve as a marker of the phase of range expansion, ranking the African MBPs according to range, we identified several MBPs that pose elevated risk for disease emergence (e.g., Wesselsbron virus). Taken together, our database, approach, and results can help improve MBD surveillance and lead to a better understanding of disease emergence. This knowledge has the potential to improve capacity to prevent and mitigate new and emerging MBD threats.

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Comparison of fitness effects in the earthworm Eisenia fetida after exposure to single or multiple anthropogenic pollutants Terrestrial ecosystems are exposed to many anthropogenic pollutants. Non-target effects of pesticides and fertilizers have put agricultural intensification in the focus as a driver for biodiversity loss. However, other pollutants, such as heavy metals, particulate matter, or microplastic also enter the environment, e.g. via traffic and industrial activities in urban areas. As soil acts as a potential sink for such pollutants, soil invertebrates like earthworms may be particularly affected by them. Under natural conditions soil invertebrates will likely be confronted with combinations of pollutants simultaneously, which may result in stronger negative effects if pollutants act synergistically.

Within this work we study how multiple pollutants affect the soil-dwelling, substrate feeding earthworm Eisenia fetida. We compared the effects of the single stressors, polystyrene microplastic fragments, polystyrene fibers, brake dust and soot, with the combined effect of these pollutants when applied as a mixture. Endpoints measured were survival, growth, reproductive fitness, and changes in three oxidative stress markers. We found that among single pollutant treatments, brake dust imposed the strongest negative effects on earthworms in all measured endpoints including increased mortality rates. Sub-lethal effects were found for all pollutants. Exposing earthworms to all four pollutants simultaneously led to effects on mortality and oxidative stress markers that were smaller than expected by the respective null models. These antagonistic effects are likely a result of the adsorption of toxic substances found in brake dust to the other pollutants. With this study we show that effects of combinations of pollutants cannot necessarily be predicted from their individual effects and that combined effects will likely depend on identity and concentration of the pollutants.

sentences: Long-read transcriptome sequencing analysis with IsoTools Long-read transcriptome sequencing (LRTS) holds the promise to boost our understanding of alternative splicing. Recent advances in accuracy and throughput have diminished the major limitations and enabled the direct quantification of isoforms. Considering the complexity of the data and the broad range of potential applications, it is clear that highly flexible, accurate analysis tools are crucial. Here, we present IsoTools, a comprehensive Python-based analysis package, for the improvement of alternative and differential splicing analysis. Iso-Tools provides a comprehensive data structure that integrates genomic information from LRTS transcripts together with the reference annotation, and enables broad functionality to quality control, visualize and analyze the data. Additionally, we implemented a graph-based method for the identification of alternative splicing events and a statistical approach based on the beta binomial distribution for the detection of differential events. To demonstrate our methods, we generated PacBio Iso-Seq data of human hepatocytes treated with the HDAC inhibitor valproic acid, a compound known to induce widespread transcriptional changes. Contrasted with short read RNA-Seq of the same samples, this analysis shows that LRTS provides valuable additional insights for a better understanding of alternative splicing, in particular with respect to complex novel and differential splicing events. IsoTools is made available for the community along with extensive documentation at https://github.com/MatthiasLienhard/isotools.

sentences: Naturally-acquired immunity in Syrian Golden Hamsters provides protection from re-exposure to emerging heterosubtypic SARS-CoV-2 variants B.1.1.7 and B.1.351 The ability of acquired immune responses against SARS-CoV-2 to protect after subsequent exposure to emerging variants of concern (VOC) such as B1.1.7 and B1.351 is currently of high significance. Here, we use a hamster model of COVID-19 to show that prior infection with a strain representative of the original circulating lineage B of SARS-CoV-2 induces protection from clinical signs upon subsequent challenge with either B1.1.7 or B1.351 viruses, which recently emerged in the UK and South Africa, respectively. The results indicate that these emergent VOC may be unlikely to cause disease in individuals that are already immune due to prior infection, and this has positive implications for overall levels of infection and COVID-19 disease.

sentences: Field margins as substitute habitat for the conservation of birds in agricultural wetlands Breeding birds in agricultural landscapes have declined considerably since the 1950s and the beginning of agricultural intensification in Europe. Given the increasing pressure on agricultural land, it is necessary to identify conservation measures that consume little productive land. We tested the hypothesis that field margins represent substitute habitats for bird species in agricultural wetlands. We monitored bird species in 86 crop fields in rice paddy landscapes of Camargue (southern France), a wetland of international importance for birds. We investigated whether the area of three types of field margins (grass strips, hedgerows and reed strips) within a 500 m buffer around each studied crop field had an effect on the abundance of three groups of birds defined based on their primary habitat (reedbeds, grasslands, and forest edge species). We controlled for the area of each semi-natural habitat (wetlands, grasslands, and woodlands), crop diversity and mean crop field size. Our study confirms that bird guilds are favored by the cover of their primary habitat but are also influenced by the cover of field margins. Reedbed birds are favored by the cover of wetlands and reed strips, grassland birds are favored by the cover of grasslands and wetlands and negatively impacted by the cover of woodlands and hedgerows, while forest edge birds are favored by the cover of hedgerows and negatively impacted by the cover of reed strips. These results suggest that field margins may represent substitute habitats for reedbed and forest edge bird species and highlight their importance for biodiversity conservation in wetland agricultural landscapes. However, our results also suggest that increasing the area of hedgerows and reed strips may have a negative effect on grassland birds and forest edge birds, respectively. Recommendations for field margin management in agricultural wetlands should therefore be tailored to local conservation priorities.

sentences: The connectome spectrum as a canonical basis for a sparse representation of fast brain activity The functional organization of neural processes is constrained by the brain s intrinsic structural connectivity. Here, we explore the potential of exploiting this structure in order to improve the signal representation properties of brain activity and its dynamics. Using a multi-modal imaging dataset (electroencephalography, structural MRI and diffusion MRI), we represent electrical brain activity at the cortical surface as a time-varying composition of harmonic modes of structural connectivity. The harmonic modes are termed connectome harmonics, and their representation is known as the connectome spectrum of the signal. We found that: first, the brain activity signal is more compactly represented by the connectome spectrum than by the traditional area-based representation; second, the connectome spectrum characterizes fast brain dynamics in terms of signal broadcasting profile, revealing different temporal regimes of integration and segregation that are consistent across participants. And last, the connectome spectrum characterises fast brain dynamics with fewer degrees of freedom than area-based signal representations. Specifically, we show that with the connectome spectrum representation, fewer dimensions are needed to capture the differences between low-level and high-level visual processing, and the topological properties of the signal. In summary, this work provides statistical, functional and topological evidence supporting that by accounting for the brain s structural connectivity fosters a more comprehensive understanding of large-scale dynamic neural functioning.

sentences: Characteristics of intestinal microflora and dysbiosis in relation to the disease duration in patients with Meniere's disease Menieres disease is an intractable disease characterized by paroxysms of intense rotatory dizziness, hearing loss, and other auditory symptoms. It is believed to be caused by endolymphatic edema of the vestibule and cochlea. The mechanism underlying endolymphatic edema is believed to be stress-related. Endolymphatic edema identification using cochlear contrast-enhanced MRI (hybrid of reversed image of positive endolymph signal and native image of positive perilymph signal [HYDROPS]) is an objective test for Menieres disease, enabling reliable diagnosis of this disease. Since dysbiosis of the intestinal microflora has been reported in stress-induced depression and irritable colitis, a similar abnormality may also occur in stress-triggered Menieres disease. Therefore, we investigated the gut microbiota of 10 patients (6 males and 4 females; mean age, 49.6 {+/-} 8.1 years) diagnosed with Menieres disease and marked endolymphatic edema on HYDROPS. These 10 patients underwent intestinal microbiota examination, and the results of the examination were evaluated in relation to the duration of the disease, the results of auscultation of the affected side, and Dizziness Handicap Inventory (DHI) scores. A significant negative correlation was found between the disease duration and Shannon diversity index and Faiths phylogenetic diversity, which indicated dysbiosis of the intestinal microflora. No correlation was found between the indicators of microbial diversity and the results of audiometry or the DHI on the affected side. Dysbiosis of the intestinal microbiota worsened with increasing duration of Menieres disease. Moreover, Akkermansia muciniphila was not detected in any patient with Menieres disease. Thus, despite the small number of cases in this study (n = 10), the findings indicate the possibility of abnormalities of the intestinal microflora in Menieres disease.

sentences: Dopamine differentially modulates the size of projection neuron ensembles in the intact and dopamine-depleted striatum Dopamine (DA) is a critical modulator of brain circuits that control voluntary movements, but our understanding of its influence on the activity of target neurons in vivo remains limited. Here, we use two-photon Ca2+ imaging to monitor the activity of direct and indirect-pathway spiny projection neurons (SPNs) simultaneously in the striatum of behaving mice during acute and prolonged manipulations of DA signaling. We find that increasing and decreasing DA biases striatal activity towards the direct and indirect pathways, respectively, by changing the overall number of SPNs recruited during behavior in a manner not predicted by existing models of DA function. This modulation is drastically altered in a model of Parkinsons disease. Our results reveal a previously unappreciated population-level influence of DA on striatal output and provide novel insights into the pathophysiology of Parkinsons disease.

sentences: A spatial and cellular distribution of neurotropic virus infection in the mouse brain revealed by fMOST and single cell RNA-seq Neurotropic virus infection can cause serious damage to the central nervous system (CNS) in both human and animals. The complexity of the CNS poses unique challenges to investigate the infection of these viruses in the brain using traditional techniques. In this study, we explore the use of fluorescence micro-optical sectioning tomography (fMOST) and single cell RNA sequencing (scRNA-seq) to map the spatial and cellular distribution of a representative neurotropic virus, rabies virus (RABV), in the whole brain. Mice were inoculated with a lethal dose of recombinant RABV expressing enhanced green fluorescent protein (EGFP) under different infection routes, and a three-dimensional view of the distribution of RABV in the whole mouse brain was obtained using fMOST. Meanwhile, we pinpointed the cellular distribution of RABV by utilizing scRNA-seq. Our fMOST data provide the first evidence that RABV can infect multiple nuclei related to fear independent of different infection routes. More surprisingly, scRNA-seq data indicate that besides neurons RABV can infect macrophages and NK cells in vivo. Collectively, this study draws a comprehensively spatial and cellular map of RABV infection in the mouse brain, providing a novel and insightful strategy to investigate the pathogenesis of neurotropic viruses.

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Alpha oscillations shape sensory representation and perceptual accuracy Alpha activity (8-14 Hz) is the dominant rhythm in the awake brain, and thought to play an important role in setting the brains internal state. Previous work has associated states of decreased alpha power with enhanced neural excitability. However, evidence is mixed on whether and how such excitability enhancement modulates sensory signals of interest versus noise differently, and what, if any, the consequences are for subsequent perception. Here, human subjects (male and female) performed a visual detection task in which we manipulated their decision criteria in a block-wise manner. While our manipulation led to substantial criterion shifts, these shifts were not reflected in pre-stimulus alpha-band changes. Rather, lower pre-stimulus alpha power in occipital-parietal areas improved perceptual sensitivity and enhanced information content decodable from neural activity patterns. Additionally, oscillatory alpha phase immediately before stimulus presentation modulated accuracy. Together, our results suggest that alpha-band dynamics modulate sensory signals of interest more strongly than noise.

Significance statementThe internal state of our brain fluctuates, giving rise to variability in perception and action. Neural oscillations, most prominently in the alpha-band, have been suggested to play a role in setting this internal state. Here, we show that ongoing alpha-band activity in occipital-parietal regions predicts the quality of visual information decodable in neural activity patterns, and subsequently human observers sensitivity in a visual detection task. Our results provide comprehensive evidence that visual representation is modulated by ongoing alpha-band activity, and advance our understanding on how, when faced with unchanging external stimuli, internal neural fluctuations influence perception and behavior.

sentences: Shared and distinct functional effects of patient-specific Tbr1 mutations on cortical development Predicted loss-of-function and missense heterozygous de novo mutations of TBR1 are strongly associated with intellectual disability and autism. The functional effects of these heterogeneous mutations on cortical development and genotype-phenotype relationships have yet to be explored. We characterized mouse models carrying patient mutations A136PfsX80 and K228E, finding convergent and discordant phenotypes. The A136PfsX80 mutation is loss-of-function and allelic to the Tbr1 knockout. In contrast, K228E causes significant upregulation of TBR1. Heterozygosity of either mutation produces axon defects, including reduction of the anterior commissure, and CTIP2 downregulation in adult cortex. While mice lacking TBR1 show extensive cortical apoptosis and inverted layering, K228E homozygotes show normal apoptosis levels and a complex layering phenotype--suggesting partial, yet abnormal, function of the allele. The construct and face validity of these Tbr1 patient mutation mice suggests they will be valuable translational models for studying the function of this essential brain transcription factor.

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The lncRNA APOLO interacts with the transcription factor WRKY42 to trigger root hair cell expansion in response to cold Plant long noncoding RNAs (lncRNAs) have emerged as important regulators of chromatin dynamics, impacting on transcriptional programs leading to different developmental outputs. The lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes multiple independent loci across the Arabidopsis genome and modulates their three-dimensional chromatin conformation, leading to transcriptional shifts. Here, we show that APOLO recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) and controls RHD6 transcriptional activity leading to cold-enhanced RH elongation through the consequent activation of the transcription factor gene RHD6-like RSL4. Furthermore, we demonstrate that APOLO interacts with the transcription factor WRKY42 and modulates its binding to the RHD6 promoter. WRKY42 is required for the activation of RHD6 by low temperatures and WRKY42 deregulation impairs cold-induced RH expansion. Collectively, our results indicate that a novel ribonucleoprotein complex involving APOLO and WRKY42 forms a regulatory hub which activates RHD6 by shaping its epigenetic environment and integrates signals governing RH growth and development.

SUMMARYThe lncRNA APOLO directly regulates the transcription of the root hair-master gene RHD6. In response to cold, APOLO is induced and it decoys the H3K27me3-binding protein LHP1 away from RHD6. In addition, APOLO modulates the binding of the transcription factor WRKY42 to the RHD6 promoter at low temperatures.

sentences: The benefits of herbivory outweigh the costs of bioerosion in a eutrophic coral community Herbivores play an integral part in maintaining the health of coral reefs by suppressing the growth of algae and accumulation of sediment and facilitating coral growth. However, in predator-depleted systems where densities of herbivores are unnaturally high, grazing can have detrimental effects on corals through excessive bioerosion. Yet, these benefits and costs are rarely investigated concurrently, especially in eutrophic systems where grazers may play a disproportionate role. We used a year-long exclusion experiment to elucidate the effect of natural densities of the dominant herbivore (the sea urchin Diadema setosum) on coral communities in a heavily fished and eutrophic system (Hong Kong, China). To assess benthic community response to grazing, we monitored the survival and growth of three locally abundant coral species (Pavona decussata, Platygyra carnosus and Porites sp.), algal and sediment accumulation, and bioerosion of coral skeletons across seasons. We found that urchins maintained our experimental coral assemblages, and when excluded, there was a 25 to 75-fold increase in algal-sediment matrix accumulation. Contrary to predictions, there was no general response of corals to urchin presence; Porites sp. survivorship increased while P. decussata was unaffected, and growth rates of both species was unchanged. Surprisingly, P. carnosus experienced higher mortality and bioerosion of up to 33% of their buoyant weight when urchins were present. Therefore, under natural densities, sea urchins clear substrate of algae and sediment, increase survival, maintain growth rates and health of coral assemblages, yet can accelerate the bioerosion of species with porous skeletons following mortality.

sentences: A comparison of methodological approaches to the study of young sex chromosomes: A case study in Poecilia Studies of sex chromosome systems at early stages of divergence are key to understanding the initial process and underlying causes of recombination suppression. However, identifying signatures of divergence in homomorphic sex chromosomes can be challenging due to high levels of sequence similarity between the X and the Y. Variations in methodological precision and underlying data can make all the difference between detecting subtle divergence patterns or missing them entirely. Recent efforts to test for X-Y sequence differentiation in the guppy have led to contradictory results. Here we apply different analytical methodologies to the same dataset to test for the accuracy of different approaches in identifying patterns of sex chromosome divergence in the guppy. Our comparative analysis reveals that the most substantial source of variation in the results of the different analyses lies in the reference genome used. Analyses using custom-made de novo genome assemblies for the focal species successfully recover a signal of divergence across different methodological approaches. By contrast, using the distantly related Xiphophorus reference genome results in variable patterns, due to both sequence evolution and structural variations on the sex chromosomes between the guppy and Xiphophorus. Changes in mapping and filtering parameters can additionally introduce noise and obscure the signal. Our results illustrate how analytical differences can alter perceived results and we highlight best practices for the study of nascent sex chromosomes.

sentences: Metacells untangle large and complex single-cell transcriptome networks The exponential scaling of scRNA-seq data represents an important hurdle for downstream analyses. Here we develop a coarse-graining framework where highly similar cells are merged into metacells. We demonstrate that metacells not only preserve but often improve the results of downstream analyses including visualization, clustering, differential expression, cell type annotation, gene correlation, imputation, RNA velocity and data integration. By capitalizing on the redundancy inherent to scRNA-seq data, metacells significantly facilitate and accelerate the construction and interpretation of single-cell atlases, as demonstrated by the integration of 1.46 million cells from COVID-19 patients in less than two hours on a standard desktop.

sentences: SequenceBouncer: A method to remove outlier entries from a multiple sequence alignment Phylogenetic analyses can take advantage of multiple sequence alignments as input. These alignments typically consist of homologous nucleic acid or protein sequences, and the inclusion of outlier or aberrant sequences can compromise downstream analyses. Here, I describe a program, SequenceBouncer, that uses the Shannon entropy values of alignment columns to identify and remove outlier entries in a manner responsive to overall alignment context. I demonstrate the utility of this software using alignments of mammalian reference mitochondrial genomes, bird cytochrome c oxidase-derived sequence barcodes, and COVID-19 sequences.

sentences: Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the hosts immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohns disease.

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An accelerated thrombosis model for computational fluid dynamics simulations in rotary blood pumps PurposeThrombosis is one of the major complications in blood-carrying medical devices and a better understanding to influence design of such devices is desirable. Over the past years many computational models of thrombosis have been developed. However, open questions remain about the applicability and implementation within a pump development process. The aim of the study was to develop and test a computationally efficient model for thrombus risk prediction in rotary blood pumps.

MethodsWe used a two-stage approach to calculate thrombus risk. At the first stage, the velocity and pressure fields were computed by computational fluid dynamic (CFD) simulations. At the second stage, platelet activation by mechanical and chemical stimuli was determined through species transport with an Eulerian approach. The model was implemented in ANSYS CFX and compared with existing clinical data on thrombus deposition within the HeartMate II.

ResultsOur model shows good correlation (R2>0.94) with clinical data and identifies the bearing and outlet stator region of the HeartMate II as the location most prone to thrombus formation. The calculation of platelet activation requires an additional 10-20 core hours of computation time.

DiscussionThe concentration of activated platelets can be used as a surrogate marker to determine risk regions of thrombus deposition in a blood pump. Model expansion, e.g. by including more chemical species can easily be performed. We make our model openly available by implementing it for the FDA benchmark blood pump.

DeclarationsO_ST_ABSFundingC_ST_ABSThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Open access funding enabled and organized by Projekt DEAL.

Conflict of interestAll of the authors have nothing to disclose.

Availability of data and materialThe raw data can be retrieved by request from the authors.

Code availabilityThe implementation of the thrombus model in the FDA benchmark blood pump geometry is available on https://doi.org/10.5281/zenodo.5116063.

Authors contributionsAll authors contributed to the study conception and design. CB developed the numerical model, performed the simulations, gathered, analysed and discussed the results. SGH, MN and US were involved in the analysis and discussion of the results. MN supervised the project. MN and CB wrote the manuscript based on the input of all co-authors. All co-authors read and approved the final version of the manuscript.

sentences: Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roths Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. However, only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins (SASP) in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

sentences: Long-term stability of neural activity in the motor system How an established behavior is retained and stably produced by a nervous system in constant flux remains a mystery. One possible solution is to fix the activity patterns of single neurons in the relevant circuits. Alternatively, activity in single cells could drift over time provided that the population dynamics are constrained to produce stable behavior. To arbitrate between these possibilities, we recorded single unit activity in motor cortex and striatum continuously for several weeks as rats performed stereotyped motor behaviors - both learned and innate. We found long-term stability in behaviorally-locked single neuron activity patterns across both brain regions. A small amount of neural drift observed over weeks of recording could be explained by concomitant changes in task-irrelevant behavioral output. These results suggest that stereotyped behaviors are generated by stable neural activity patterns.

sentences: Progressive enhancement of kinetic proofreading in T cell antigen discrimination from receptor activation to DAG generation T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tischer and Weiner, 2019). Here we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at downstream signaling events. Leveraging the ability of our system to rapidly disengage ligand binding, we measure slower reset rates for downstream signaling events. Our observations of distributed kinetic proofreading and slowed resetting of downstream steps suggest a basis of cooperativity between multiple active receptors with implications in tissue homeostasis, autoimmunity, and immunotherapy off-target effects.

sentences: A DROSOPHILA CIRCUIT FOR HABITUATION OVERRIDE Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila. Here we analyze how exposure to a novel stimulus causes override of gustatory (proboscis-extension reflex or "PER") habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. We show that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of TH (Tyrosine-Hydroxylase) positive neurons, a subset of which send projections to the subesophagial zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.

sentences: Multiple lineages of Streptomyces produce antimicrobials within passalid beetle galleries across eastern North America Some insects form symbioses in which actinomycetes provide defense against pathogens by making antimicrobials. The range of chemical strategies employed across these associations, and how these strategies relate to insect lifestyle, remains underexplored. We assessed subsocial passalid beetles of the species Odontotaenius disjunctus, and their frass (fecal material) which is an important food resource within their galleries, as a model insect/actinomycete system. Through chemical and phylogenetic analyses, we found that O. disjunctus frass collected across eastern North America harbored multiple lineages of Streptomyces and diverse antimicrobials. Metabolites detected in frass displayed synergistic and antagonistic inhibition of a fungal entomopathogen, Metarhizium anisopliae, and multiple streptomycete isolates inhibited this pathogen when co-cultivated directly in frass. These findings support a model in which the lifestyle of O. disjunctus accommodates multiple Streptomyces lineages in their frass, resulting in a rich repertoire of antimicrobials that likely insulates their galleries against pathogenic invasion.

sentences: Transfer of Knowledge from Model Organisms to Evolutionarily Distant Non-Model Organisms: The Coral Pocillopora damicornis Membrane Signaling Receptome With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the need to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins are therefore often in the gray zone or undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to be true human homologues. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.

sentences: Harnessing the central dogma for stringent multi-level control of gene expression Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided at all costs, but ideally without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple way to effectively address this challenge. By simultaneously regulating both transcription and translation, we show how relative basal expression of an inducible system can be greatly reduced, with minimal impact on the maximum induced expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction in vivo and up to a 350-fold change when used in a cell-free expression system. Furthermore, we find that multi-level regulation is able to suppress transcriptional noise and creates a digital-like switch when transitioning between  on and  off states. This work provides foundational knowledge and a genetic toolkit of parts to create multi-level gene expression controllers for those working with toxic genes or requiring precise regulation and propagation of cellular signals. It also demonstrates the value of exploring more complex and diverse regulatory designs for synthetic biology.

sentences: Asymmetric learning facilitates human inference of transitive relations Humans and other animals are capable of inferring never-experienced relations (e.g., A>C) from other relational observations (e.g., A>B and B>C). The processes behind such transitive inference are subject to intense research. Here, we demonstrate a new aspect of relational learning, building on previous evidence that transitive inference can be accomplished through simple reinforcement learning mechanisms. We show in simulations that inference of novel relations benefits from an asymmetric learning policy, where observers update only their belief about the winner (or loser) in a pair. Across 4 experiments (n=145), we find substantial empirical support for such asymmetries in inferential learning. The learning policy favoured by our simulations and experiments gives rise to a compression of values which is routinely observed in psychophysics and behavioural economics. In other words, a seemingly biased learning strategy that yields well-known cognitive distortions can be beneficial for transitive inferential judgments.

sentences: Structural Differences In 3C-like protease (Mpro) From SARS-CoV and SARS-CoV-2: Molecular Insights For Drug Repurposing Against COVID-19 Revealed by Molecular Dynamics Simulations. A recent fatal outbreak of novel coronavirus SARS-CoV-2, identified preliminary as a causative agent for series of unusual pneumonia cases in Wuhan city, China has infected more than 20 million individuals with more than 4 million mortalities. Since, the infection crossed geographical barriers, the WHO permanently named the causing disease as COVID-2019 by declaring it a pandemic situation. SARS-CoV-2 is an enveloped single-stranded RNA virus causing a wide range of pathological conditions from common cold symptoms to pneumonia and fatal severe respiratory syndrome. Genome sequencing of SARS-CoV-2 has revealed 96% identity to the bat coronavirus and 79.6% sequence identity to the previous SARS-CoV. The main protease (known as 3C-like proteinase/ Mpro) plays a vital role during the infection with the processing of replicase polyprotein thus offering an attractive target for therapeutic interventions. SARS-CoV and SARS-CoV-2 Mpro shares 97% sequence identity, with 12 variable residues but none of them present in the catalytic and substrate binding site. With the high level of sequence and structural similarity and absence of any drug/vaccine against SARS-CoV-2, drug repurposing against Mpro is an effective strategy to combat COVID-19. Here, we report a detailed comparison of SARS-CoV-2 Mpro with SARS-CoV Mpro using molecular dynamics simulations to assess the impact of 12 divergent residues on the molecular microenvironment of Mpro. A structural comparison and analysis is made on how these variable residues affects the intra-molecular interactions between key residues in the monomer and biologically active dimer form of Mpro. The present MD simulations study concluded the change in microenvironment of active-site residues at the entrance (T25, T26, M49 and Q189), near the catalytic region (F140, H163, H164, M165 and H172) and other residues in substrate binding site (V35T, N65S, K88R and N180K) due to 12 mutation incorporated in the SARS-CoV-2 Mpro. It is also evident that SARS-CoV-2 dimer is more stable and less flexible state compared to monomer which may be due to these variable residues, mainly F140, E166 and H172 which are involved in dimerization. This also warrants a need for inhibitor design considering the more stable dimer form. The mutation accumulated in SARS-CoV-2 Mpro indirectly reconfigures the key molecular networks around the active site conferring a potential change in SARS-CoV-2, thus posing a challenge in drug repurposing SARS drugs for COVID-19. The new networks and changes in microenvironment identified by our work might guide attempts needed for repurposing and identification of new Mpro inhibitors.

sentences: SARS-CoV-2 20I/501Y.V1 variant in the hamster model Late 2020, SARS-CoV-2 Alpha variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the G614 strains initially involved in the global spread of the pandemic. In this study, we used a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium.

sentences: Differential roles for DNAJ isoforms in HTT-polyQ and mutant FUS aggregation modulation revealed by chaperone network screens Protein aggregation is a hallmark of many neurodegenerative diseases1,2. In order to cope with misfolding and aggregation, cells have evolved an elaborate network of molecular chaperones, composed of different families3. But while chaperoning mechanisms for different families are well established, functional and regulatory diversification within chaperone families is still largely a mystery4,5. Here we decided to explore chaperone functional diversity, through the lens of pathological aggregation. We revealed that different naturally-occurring isoforms of DNAJ chaperones showed differential effects on different types of aggregates. We performed a chaperone screen for modulators of two neurodegeneration-related aggregating proteins, the Huntingtons disease-related HTT-polyQ, and the ALS-related mutant FUS (mutFUS). The screen identified known modulators of HTT-polyQ aggregation6,7, confirming the validity of our approach. Surprisingly, modulators of mutFUS aggregation were completely different than those of HTT-polyQ. Interestingly, different naturally-occurring isoforms of DNAJ chaperones had opposing effects on HTT-polyQ vs. mutFUS aggregation. We identified a complex of the full length (FL) DNAJB14 and DNAJB12 isoforms which substantially alleviated mutFUS aggregation, in an HSP70-dependent manner. Their naturally occurring short isoforms were unable to form the complex, nor to interact with HSP70, and lost their ability to reduce mutFUS aggregation. In contrast, the short isoform of DNAJB12 significantly alleviated HTT-polyQ aggregation, while DNAJB12-FL aggravated HTT-polyQ aggregation. Finally, we demonstrated that full-length DNAJB14 ameliorated mutFUS aggregation compared to DNAJB14-short in primary neurons. Together, our data unraveled distinct molecular properties required for aggregation protection in different neurodegenerative diseases, and revealed a new layer of complexity of the chaperone network elicited by naturally occurring J-protein isoforms, highlighting functional diversity among the DNAJ family.

sentences: An analytical solution simulating growth of Lewy bodies This paper reports a minimal model simulating the growth of a Lewy body (LB). To the best of our knowledge, this is the first model simulating Lewy body growth. The LB is assumed to consist of a central spherical core, which is composed of membrane fragments and various dysfunctional intracellular organelles, and a halo, which is composed of alpha-synuclein fibrils. Membrane fragments and alpha-synuclein monomers are assumed to be produced in the soma at constant rates. The growth of the core and the halo are simulated by the Finke-Watzky model. Analytical (closed-form) solutions describing the growth of the core and the halo are obtained. A sensitivity analysis in terms of model parameters is performed.

sentences: Rapid fluctuations in functional connectivity of cortical networks encode spontaneous behavior Experimental work across a variety of species has demonstrated that spontaneously generated behaviors are robustly coupled to variation in neural activity within the cerebral cortex 1-9. Indeed, functional magnetic resonance imaging (fMRI) data suggest that functional connectivity in cortical networks varies across distinct behavioral states, providing for the dynamic reorganization of patterned activity 5,7,10. However, these studies generally lack the temporal resolution to establish links between cortical signals and the continuously varying fluctuations in spontaneous behavior typically observed in awake animals. Here, we took advantage of recent developments in wide-field, mesoscopic calcium imaging 11 to monitor neural activity across the neocortex of awake mice. Applying a novel approach to quantifying time-varying functional connectivity, we developed a "graph of graphs" method to show that spontaneous behaviors are more accurately represented by fast changes in the correlational structure versus the magnitude of large-scale network activity. Dynamic functional connectivity reveals subnetworks that are not predicted by traditional anatomical atlas-based parcellation of the cortex. These results provide insight into how behavioral information is represented across the mammalian neocortex and demonstrate a new analytical framework for investigating time-varying functional connectivity in neural networks.

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Social hosts evade predation but have deadlier parasites Parasites exploit hosts to replicate and transmit, but overexploitation kills host and parasite (1): predators may shift this cost-benefit balance by consuming hosts (2-4) or changing host behavior, but the strength of these effects remains unclear. Modeling both, we find a primary, strong effect: hosts group to defend against predators (5), increasing parasite transmission, thus multiple infections, and therefore favoring more exploitative, virulent, parasites (6). Indeed, among 18 Trinidadian Gyrodactyus spp. parasite lines, those collected from high predation guppy populations were more virulent in common garden than those from low predation populations. Our model accurately predicted this result when parametrized with our experimentally demonstrated virulence-transmission trade-off, implicating the behavioral effects of predation. Broadly, our results indicate that reduced social contact selects against parasite virulence.

One-Sentence SummaryOur theory and data show predators cause increased host social grouping; the resulting transmission favors parasite virulence.

sentences: Droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show slower inner fluidity A series of amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43 and hnRNPA1 has an ability to be liquid-liquid phase separation, and their disease-related mutations cause the transition of their responsible liquid droplets to aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD) and Pagets disease of bone. SQSTM1/p62 protein forms liquid-droplets through the interaction with ubiquitinated proteins, and the droplet serves as a platform of autophagosome formation and anti-oxidative stress response via the LC3-interacting region (LIR) and Keap1-interacting region (KIR), respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in LIR and KIR form aberrant liquid droplets, cause defective autophagy and stress response or both. To evaluate the effects of ALS/FTD-related p62 mutations in LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway and the autophagic turnover, we developed systems that enable to monitor them with high sensitivity. These systems revealed that some mutants but not all have their less abilities on the Nrf2-activation and show the delayed turnover. By contrast, while the sufficient ability to form liquid droplets, all droplets consisting of p62 mutants showed slower inner fluidity. These results indicate that like other ALS-related mutant proteins, a primary defect in ALS/FTD with p62 missense mutations is a qualitative change of p62-liquid droplets.

sentences: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli Chemical modifications of RNA 5' ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates (NTPs) for use as initiating entities, providing an ab initio mechanism for altering the RNA 5' end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here we use massively systematic transcript end readout ("MASTER") to detect and quantify RNA 5' ends generated by primer-dependent initiation for ~410 (~1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position, but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

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Non-invasive Chromatin Deformation and Measurement of Differential Mechanical Properties in the Nucleus The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1{degrees}C /m) can lead to substantial intra-nuclear chromatin displacements (>1 m), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high rigidity and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Main FindingsO_LINovel non-invasive active micro-rheology method to probe spatial intranuclear material responses, unhindered by the nuclear lamina, using strongly localized temperature gradients
C_LIO_LIChromatin shows both elastic and viscous properties at the mesoscale with a retardation time of {tau} [~] 1s
C_LIO_LICompacted heterochromatin in a model of nuclear inversion shows high resistance to deformation, suggesting dominantly solid-like behavior
C_LIO_LIThe nucleus displays spatially distinct material properties for different compartments
C_LIO_LIThe nucleolus shows high resistance to deformation on the time scale of seconds
C_LIO_LIImmobile nucleoli appear solidly anchored to and retain the deformation of surrounding chromatin
C_LI

sentences: Complexity of brain dynamics as a correlate of consciousness in anaesthetized monkeys The use of anaesthesia is a fundamental tool in the investigation of consciousness. Anesthesia procedures allow to investigate different states of consciousness from sedation to deep anesthesia within controlled scenarios. In this study we use information quantifiers to measure the complexity of electrocorticogram recordings in monkeys. We apply these metrics to compare different stages of general anesthesia for evaluating consciousness in several anesthesia protocols. We find that the complexity of brain activity can be used as a correlate of consciousness. For two of the anaesthetics used, propofol and medetomidine, we find that the anaesthetised state is accompanied by a reduction in the complexity of brain activity. On the other hand we observe that use of ketamine produces an increase in complexity measurements. We relate this observation with increase activity within certain brain regions associated with the ketamine used doses. Our measurements indicate that complexity of brain activity is a good indicator for a general evaluation of different levels of consciousness awareness, both in anesthetized and non anesthetizes states.

sentences: Dendritic inhibition by Shh signaling-dependent stellate cell pool is critical for motor learning Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and non-motor functions. The molecular layer interneurons (MLI), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2+ immature interneurons which migrate from the prospective white matter to the ML of the cortex. However, little is known as to how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Shh signal. Conditional abrogation of Shh signaling inhibited proliferation of GABAergic progenitors and reduced the number of Pax2+ cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early-born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2+ cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.

sentences: Identifying charismatic bird species and traits with community science observations AO_SCPLOWBSTRACTC_SCPLOWIdentifying which species are perceived as charismatic can improve the impact and efficiency of conservation outreach, as charismatic species receive more conservation funding and have their conservation needs prioritized (9; 17; 13). Sociological experiments studying animal charisma have relied on stated preferences to find correlations between hypothetical "willingness to pay" or "empathy" for a species conservation and species size, color, and aesthetic appeal (51; 13; 16). Recognizing the increasing availability of digital records of public engagement with animals that reveal preferences, an emerging field of "culturomics" uses Google search results, Wikipedia article activities, and other digital modes of engagement to identify charismatic species and traits (46; 31; 10; 41). In this study, we take advantage of community science efforts as another form of digital data that can reveal observer preferences. We apply a multi-stage analysis to ask whether opportunistic birders contributing to iNaturalist engage more with larger, more colorful, and rarer birds relative to a baseline, from eBird contributors, approximating unbiased detection. We find that body mass, color contrast, and range size all predict over-representation in the opportunistic dataset. We also find evidence that, across 473 modeled species, 52 species are significantly overreported and 158 are significantly underreported, indicating a wide variety of species-specific effects. Understanding which birds are charismatic can aid conservationists in creating impactful outreach materials and engaging new naturalists. The quantified differences between two prominent community science efforts may also be of use for researchers leveraging the data from one or both of them to answer scientific questions of interest.

sentences: High temperature perception in leaves promotes vascular regeneration in distant tissues Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and developmental pathways affect wound healing but little is known about how external factors influence regeneration. To better understand how the environment affects regeneration, we investigated the effects of temperature using the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration of Arabidopsis thaliana and tomato (Solanum lycopersicum) grafts. Leaves were critical for this effect since blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations had no effect upon graft healing at ambient temperatures and mutations in PIF4 did not affect the temperature enhancement of callus formation or tissue adhesion, suggesting that leaf-derived auxin was specific for enhancing vascular regeneration in response to elevated temperatures. Tissue-specific perturbations of auxin response using a BODENLOS (BDL) mutant revealed an asymmetric effect of temperature upon regeneration: the presence of bdl above the cut prevented temperature enhancement whereas the presence of bdl below the cut prevented graft healing regardless of temperature. Promotion of tissue regeneration by elevated temperatures was not specific for graft healing and we found that elevated temperatures accelerated xylem formation between the parasite Phtheirospermum japonicum and host Arabidopsis thaliana, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway by which elevated temperatures accelerate vascular development which could be of relevance for improving regeneration and better understanding inter-plant vascular connections.

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Red Blood Cells Function as DNA Sensors Erythrocytes have long been mistaken as exclusively inert oxygen carriers lacking immune function. Here we show that red blood cells (RBCs) serve as immune sensors through surface expression of the nucleic acid-sensing toll-like receptor 9 (TLR9), a classically endosomal receptor that initiates immune responses following the detection of unmethylated CpG motifs present in pathogen and mitochondrial DNA. Mammalian RBCs express TLR9 on their surface and bind CpG-containing bacterial, malarial, and mitochondrial DNA. Erythrocyte-bound CpG DNA increases during infection, and CpG-carrying RBCs trigger accelerated erythrophagocytosis and innate immune activation characterized by RBC-TLR9 dependent local and systemic cytokine production. Thus, RBC nucleic acid detection and capture regulates red cell clearance and immune responses and provides evidence for RBCs as innate immune sentinels during pathologic states.

One Sentence SummaryThe ability of RBCs to detect and bind cell-free nucleic acids contributes to immunity during acute inflammatory states.

sentences: A new, Critically Endangered species of Impatiens (Balsaminaceae) from the coastal plain of the Republic of Congo Impatiens moutsambotei is described from a herbarium specimen collected at a waterfall in forest on the coastal plain, below the Mayombe Mts of the Republic of Congo. Sharing many distinctive characters with Impatiens floretii of the Doudou Mts of Gabon, it is abundantly distinguished inter alia by the leaf-blades which are lanceolate or narrowly elliptic, not ovate, their bases acute, not obtuse or broadly rounded to truncate; the reduced, peduncular bracts bearing long, filiform setae, and not entire; the proximal (upper) of the lateral united petals are entire, not bifid; the spur is curved at the base and overlaps the lower sepal, not curved through its length to describe a semi-circle and held below the lower sepal. Both species are placed in the Impatiens macroptera species aggregate. Impatiens moutsambotei is known from a single site, where it was collected nearly 30 years ago and not seen since. The new species is assessed as Critically Endangered due to threats of habitat clearance from mining and road widening, and may be already be extinct.

sentences: Reducing the efforts to create reproducible analysis code with FieldTrip The analysis of EEG and MEG data typically requires a lengthy and complicated sequence of analysis steps, often requiring large amounts of computations, which are ideally represented in analysis scripts. These scripts are often written by researchers without formal training in computer science, resulting in the quality and readability of these analysis scripts to be highly dependent on individual coding expertise and style. Even though the computational outcomes and interpretation of the results can be correct, the inconsistent style and quality of analysis scripts make reviewing the details of the analysis difficult for other researchers that are either involved in the study or not, and the quality of the scripts might compromise the reproducibility of obtained results. This paper describes the design and implementation of a strategy that allows complete reproduction of MATLAB-based scripts with little extra efforts on behalf of the user, which we have implemented as part of the FieldTrip toolbox. Starting from the researchers idiosyncratic pipeline scripts, this new functionality allows researchers to automatically create and publish analysis pipeline scripts in a standardized format, along with all relevant intermediate data. We demonstrate the functionality and validate its effectiveness by applying it to the analysis of a recently published MEG study.

sentences: Individual-based modeling of genome evolution in haplodiploid organisms Ants, bees, wasps, bark beetles, and other species have haploid males and diploid females. Although such haplodiploid species play key ecological roles and are threatened by environmental changes, no general framework exists for simulating their genetic evolution. Here, we use the SLiM simulation environment to build a novel model for individual-based forward simulation of genetic evolution in haplodiploid populations. We compare the fates of adaptive and deleterious mutations and find that selection is more effective in haplodiploid species than in diploid species. Our open-source model will help understand the evolution of sociality and how ecologically important species may adapt to changing environments.

sentences: Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-beta, Wnt and Hedgehog signalling pathways Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts. CLIC4 expression and activity are regulated by TGF-{beta} signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show Wnt3a/{beta}-catenin and Smoothened/GLI signalling cooperate with SMAD3 to regulate CLIC4 expression in normal dermal fibroblasts, and that inhibition of SMAD3 expression or activity abolishes Wnt and Hh-dependent CLIC4 induction. We further show that expression of the profibrotic marker -smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Our data highlight novel mechanisms that regulate CLIC4 expression that present targetable pathways to prevent fibroblast activation in SSc and other fibrotic conditions.

sentences: 16S rRNA gene sequences of Candidatus Methylumidiphilus (Methylococcales), a putative methanotrophic genus in lakes and ponds A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds of boreal and subarctic area. However, it has probably escaped detection in many previous studies using 16S rRNA gene amplicon sequencing due to insufficient database coverage, which is because Ca. Methylumidiphilus lacks cultured representatives and previously analysed metagenome assembled genomes (MAGs) affiliated with it do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG dataset. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we could find 5 MAGs each containing one 1532 bp long sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in two unique 16S rRNA gene sequences. These sequences represented two different putative species, i.e. Ca. Methylumidiphilus alinenensis (Genbank accession: OK236221) as well as another so far unnamed species of Ca. Methylumidiphilus (Genbank accession: OK236220). We suggest that including these two sequences in reference databases will enhance 16S rRNA gene - based detection of members of this genus from environmental samples.

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ProteinPrompt: a webserver for predictingprotein-protein interactions MotivationProtein-protein interactions play an essential role in a great variety of cellular processes and are therefore of significant interest for the design of new therapeutic compounds as well as the identification of side-effects due to unexpected binding. Here, we present ProteinPrompt, a webserver that uses machine-learning algorithms to calculate specific, currently unknown protein-protein interactions. Our tool is designed to quickly and reliably predict contacts based on an input sequence in order to scan large sequence libraries for potential binding partners, with the goal to accelerate and assure the quality of the laborious process of drug target identification.

MethodsWe collected and thoroughly filtered a comprehensive database of known contacts from several sources, which is available as download. ProteinPrompt provides two complementary search methods of similar accuracy for comparison and consensus building. The default method is a random forest algorithm that uses the auto-correlations of seven amino acid scales. Alternatively, a graph neural network implementation can be selected. Additionally, a consensus prediction is available. For each query sequence, potential binding partners are identified from a protein sequence database. The proteom of several organisms are available and can be searched for contacts.

ResultsTo evaluate the predictive power of the algorithms, we prepared a test dataset that was rigorously filtered for redundancy. No sequence pairs similar to the ones used for training were included in this dataset. With this challenging dataset, the random forest method achieved an accuracy rate of 0.88 and an area under curve of 0.95. The graph neural network achieved an accuracy rate of 0.86 using the same dataset. Since the underlying learning approaches are unrelated, comparing the results of random forest and graph neural networks reduces the likelihood of errors. The consensus reached an accuracy of 0.89. ProteinPrompt is available online at: http://proteinformatics.org/ProteinPrompt

The server makes it possible to scan the human proteome for potential binding partners of an input sequence within minutes. For local offline usage, we furthermore created a ProteinPrompt Docker image which allows for batch submission: https://gitlab.hzdr.de/Proteinprompt/ProteinPrompt. In conclusion, we offer a fast, accurate, easy-to-use online service for predicting binding partners from an input sequence.

sentences: A simple bypass assay for DNA polymerases shows hypermutating variants associated with cancer show mechanistic differences in vitro Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, to genome instability and its associated diseases. Recently the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants and the recognition that a subgroup of these variants have a hypermutation phenotype in tumours. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease defective and two hypermutating DNA polymerase e variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed different misincorporation characteristics to P286R, suggesting that these variants cause hypermutation by different mechanisms. Using this assay misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C to T transitions. This is consistent with the observation that C to T transitions are commonly seen in POLE mutant tumours.

sentences: Mechanism of transcription modulation by the transcription-repair coupling factor Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes distressed RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

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Hepatic MIR20B promotes nonalcoholic fatty liver disease by suppressing PPARA BackgroundNon-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation and imbalances in lipid metabolism in the liver. Although nuclear receptors (NRs) play a crucial role in hepatic lipid metabolism, the underlying mechanisms of NR regulation in NAFLD remain largely unclear.

MethodsUsing network analysis and RNA-seq to determine the correlation between NRs and microRNA in human NAFLD patients, we revealed that MIR20B specifically targets PPARA. MIR20B mimic and anti-MIR20B were administered to human HepG2 and Huh-7 cells and mouse primary hepatocytes as well as high fat diet (HFD)- or methionine-deficient diet (MCD)-fed mice to verify the specific function of MIR20B in NAFLD. We tested the inhibition of the therapeutic effect of a PPAR agonist, fenofibrate, by MIR20B and the synergic effect of combination of fenofibrate with anti-MIR20B.

ResultsWe revealed that MIR20B specifically targets PPARA through miRNA regulatory network analysis of nuclear receptor genes in NAFLD. The expression of MIR20B was upregulated in free fatty acid (FA)- treated hepatocytes and the livers of both obesity-induced mice and NAFLD patients. Overexpression of MIR20B significantly increased hepatic lipid accumulation and triglyceride levels. Furthermore, MIR20B significantly reduced FA oxidation and mitochondrial biogenesis by targeting PPARA. In MIR20B-introduced mice, the effect of fenofibrate to ameliorate hepatic steatosis was significantly suppressed. Finally, inhibition of MIR20B significantly increased FA oxidation and uptake, resulting in improved insulin sensitivity and a decrease in NAFLD progression. Moreover, combination of fenofibrate and anti-MIR20B exhibited the synergic effect on improvement of NAFLD in MCD-fed mice.

ConclusionsTaken together, our results demonstrate that the novel MIR20B targets PPARA, plays a significant role in hepatic lipid metabolism, and present an opportunity for the development of novel therapeutics for NAFLD.

FundingThis research was funded by Korea Mouse Phenotyping Project (2016M3A9D5A01952411), the National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1F1A1061267, 2018R1A5A1024340, NRF-2021R1I1A2041463, 2020R1I1A1A01074940), and the Future-leading Project Research Fund (1.210034.01) of UNIST.

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Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 {degrees}C, 500 mol photons m-2 s-1) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin-Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin-Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if [≤]21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of [≥]62%. Lastly, we discuss interactions between the OPPP and the cytosolic oxidation-reduction cycle in supplying leaf cytosolic NADPH.

Main ConclusionThe oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by {approx}5%.

sentences: The Grainyhead/LSF transcription factor GRH-1 is rhythmically required for molting Molting, that is, the synthesis and shedding of a cuticular exoskeleton, is a defining characteristic of ecdysozoa. In nematodes such as C. elegans, molts rhythmically terminate each of four larval stages. The molting cycle is tightly coupled to the rhythmic expression of thousands of genes. The mechanisms that support the regular molting cycle and oscillatory gene expression have remained largely elusive. Here, we performed an RNAi-based screen for transcription factors required for molting to identify potential components of a molting clock. We find that depletion of GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1 or BED-3 impairs progression through the molting cycle. We characterize GRH-1, a Grainyhead/LSF transcription factor whose functions in C. elegans development have remained largely unexplored, but whose orthologues in other animals are known to be key epithelial cell fate regulators. We show that GRH-1 depletion causes a dose-dependent extension of molt duration, with severe depletion causing defects in cuticle formation and shedding, and larval death. GRH-1 is required repetitively, during a specific time window prior to each larval molt. This rhythmic activity is consistent with the rhythmic accumulation of GRH-1 protein. These features are consistent with GRH-1 functioning as a key component, or high-level output, of the a gene regulatory network that controls molting. As its mammalian orthologues, as well as those of BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may be conserved beyond nematodes.

sentences: A Novel Oncogenic Function of PRC2 Heterogeneity in Medulloblastoma Intratumor epigenetic heterogeneity is emerging as a key mechanism underlying tumor evolution and drug resistance. Medulloblastomas, the most common childhood malignant brain tumor, are classified into four subtypes including SHH medulloblastomas, which are characterized by elevated SHH signaling and a cerebellum granule neuron precursor (CGNP) cell-of-origin. Medulloblastomas are highly associated with epigenetic abnormalities. We observed that the histone H3K27 methyltransferase polycomb repressor complex 2 (PRC2) is often heterogeneous within individual SHH medulloblastoma tumors. Using mouse models, we showed that while a complete deletion of the PRC2 core subunit EED inhibited medulloblastoma growth, a mosaic deletion of EED significantly enhanced tumor growth. EED is intrinsically required for CGNP maintenance by inhibiting both neural differentiation and cell death. Complete EED deletion led to CGNP depletion and reduced occurrence of medulloblastoma. Surprisingly, we found that medulloblastomas with mosaic EED levels grew faster than did control wildtype tumors and expressed increased levels of oncogenes such as Igf2. Igf2 is directly repressed by PRC2 and has been demonstrated to be both necessary and sufficient for SHH medulloblastoma progression. We showed that IGF2 mediated the oncogenic effects of PRC2 heterogeneity in tumor growth. Using a human medulloblastoma cell line, we generated clones with different EED levels and confirmed that EEDlow cells could stimulate the growth of EEDhigh cells through derepressed IGF2 signals. Thus, PRC2 heterogeneity controls medulloblastoma growth through both intrinsic growth competence and non-cell autonomous mechanisms in distinct tumor subclones. We reveal a novel oncogenic function of PRC2 heterogeneity in tumor development.

sentences: The cell adhesion molecule Sdk1 shapes assembly of a retinal circuit that detects localized edges. Nearly 50 different mouse retinal ganglion cell (RGC) types sample the visual scene for distinct features. RGC feature selectivity arises from its synapses with a specific subset of amacrine (AC) and bipolar cell (BC) types, but how RGC dendrites arborize and collect input from these specific subsets remains poorly understood. Here we examine the hypothesis that RGCs employ molecular recognition systems to meet this challenge. By combining calcium imaging and type-specific histological stains we define a family of circuits that express the recognition molecule Sidekick 1 (Sdk1) which include a novel RGC type (S1-RGC) that responds to local edges. Genetic and physiological studies revealed that Sdk1 loss selectively disrupts S1-RGC visual responses which result from a loss of excitatory and inhibitory inputs and selective dendritic deficits on this neuron. We conclude that Sdk1 shapes dendrite growth and wiring to help S1-RGCs become feature selective.

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Niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the cytosol, and the bacterial genes required for cytosolic colonization, remain unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes/sRNAs with cytosol-specific or vacuole-specific expression signatures. Using these genes/sRNAs as environmental biosensors, we defined that Salmonella is exposed to iron and manganese deprivation and oxidative stress in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS and sitA-mntH. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, had a cytosolic-specific expression profile. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. Therefore, this archetypical vacuole-adapted pathogen requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Author SummaryIntracellular pathogens reside either within a membrane-bound vacuole or are free-living in the cytosol and their virulence programs are tailored towards survival within a particular intracellular compartment. Some bacterial pathogens (such as Salmonella enterica) can successfully colonize both intracellular niches, but how they do so is unclear. Here we have exploited the parallel intracellular lifestyles of S. enterica in epithelial cells to identify the niche-specific bacterial expression profiles and environmental cues encountered by S. enterica. We have also discovered bacterial genes that are required for colonization of the cytosol, but not the vacuole. Our results advance our understanding of pathogen-adaptation to alternative replication niches and highlight an emerging concept in the field of bacteria-host cell interactions.

sentences: Screening coffee genotypes for Cercospora coffeicola resistance in Brazil Several efforts have been made by many researchers worldwide to develop coffee plants resistant to different Cercospora species; however, studies concerning C. coffeicola, specifically, are still incipient. In the present study, a blend of strains from this pathogen was inoculated into 18 Brazilian commercial cultivars, a coffee clone of Arabica, as well as into 41 accessions from the Germplasm Collection of Minas Gerais, to evaluate the genetic resistance ability within the population and select superior genotypes for the breeding program. After predicting genotypic values of the evaluated material, the most efficient way to select genotypes based on the data of severity to brown eye spot (BES) was also examined. Moreover, the action of defense mechanisms against C. coffeicola attacks was investigated by assessing the levels of total soluble phenolic compounds and soluble lignin in contrasting genotypes regarding disease susceptibility. Based on the results, the accession MG 1207 Sumatra demonstrated an intrinsic genetic capacity to maintain low levels of severity to brown eye spot. This genotype can thus substantially contribute to the development of new cultivars, which may lead to reduced use of pesticides. This study also evidenced that four evaluations of severity is enough to reach accuracy and efficiency for the severity of BES, thus providing expressive genetic gains. Finally, it is suggested that the levels of lignin and phenolic compounds are not associated with the resistance of coffee genotypes to brown eye spot.

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Cell growth model with stochastic gene expression helps understand the growth advantage of metabolic exchange and auxotrophy During cooperative growth, microbes often experience higher fitness, due to sharing of resources by metabolic exchange and herd protection through biofilm structures. However, the trajectory of evolution of competitive species towards cooperation is not known. Moreover, existing models (based on optimisation of steady-state resources or fluxes) are often unable to explain the growth advantage for the cooperating species, even for simple reciprocally cross-feeding auxotrophic pairs. We present an abstracted model of cell growth that considers the stochastic burst-like gene expression of biosynthetic pathways of limiting biomass precursor metabolites, and directly connects their cellular levels to growth and division using a "metabolic sizer/adder" rule. Our model recapitulates Monods law and yields the experimentally observed right-skewed long-tailed distribution of cell doubling times. The model further predicts the growth effect of secretion and uptake of metabolites, by linking it to changes in the internal metabolite levels. The model also explains why auxotrophs may grow faster when provided the metabolite they cannot produce, and why a pair of reciprocally cross-feeding auxotrophs can grow faster than prototrophs. Overall, our framework allows us to predict the growth effect of metabolic interactions in microbial communities and also sets the stage to study the evolution of these interactions.

ImportanceCooperative behaviours are highly prevalent in the wild, but we do not understand how it evolves. Metabolic flux models can demonstrate the viability of metabolic exchange as cooperative interactions, but steady-state growth models cannot explain why cooperators grow faster. We present a stochastic model that connects growth to the cells internal metabolite levels and quantifies the growth effect of metabolite exchange and auxotrophy. We show that a reduction in gene expression noise explains why cells that import metabolites or become auxotrophs can grow faster, and also why reciprocal cross-feeding of metabolites between complementary auxotrophs allow them to grow faster. Our framework can simulate the growth of interacting cells, which will enable us to understand the possible trajectories of the evolution of cooperation in silico.

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Widespread ripples synchronize human cortical activity Declarative memory encoding, consolidation, and retrieval require the integration of elements encoded in widespread cortical locations. The mechanism whereby such  binding of different components of mental events into unified representations occurs is unknown. The  binding-bysynchrony theory proposes that distributed encoding areas are bound by synchronous oscillations enabling enhanced communication. However, evidence for such oscillations is sparse. Brief high-frequency oscillations ( ripples) occur in the hippocampus and cortex, and help organize memory recall and consolidation. Here, using intracranial recordings in humans, we report that these ~70ms duration 90Hz ripples often couple (within {+/-}500ms), co-occur ([≥]25ms overlap), and crucially, phase-lock (have consistent phase-lags) between widely distributed focal cortical locations during both sleep and waking, even between hemispheres. Cortical ripple co-occurrence is facilitated through activation across multiple sites, and phaselocking increases with more cortical sites co-rippling. Ripples in all cortical areas co-occur with hippocampal ripples but do not phase-lock with them, further suggesting that cortico-cortical synchrony is mediated by cortico-cortical connections. Ripple phase-lags vary across sleep nights, consistent with participation in different networks. During waking, we show that hippocampo-cortical and cortico-cortical co-ripples increase preceding successful delayed memory recall, when binding between the cue and response is essential. Ripples increase and phase-modulate unit firing, and co-ripples increase high-frequency correlations between areas, suggesting synchronized unit-spiking facilitating information exchange. Co-occurrence, phasesynchrony, and high-frequency correlation are maintained with little decrement over very long distances (25cm). Hippocampo-cortico-cortical co-ripples appear to possess the essential properties necessary to support binding-by-synchrony during memory retrieval, and perhaps generally in cognition.

Significance StatementDifferent elements of a memory, or any mental event, are encoded in locations distributed across the cortex. A prominent hypothesis proposes that widespread networks are integrated with bursts of synchronized high-frequency oscillations called  ripples, but evidence is limited. Here, using recordings inside the human brain, we show that ripples occur simultaneously in multiple lobes in both cortical hemispheres, and the hippocampus, generally during sleep and waking, and especially during memory recall. Ripples phase-lock local cell firing, and phase-synchronize with little decay between locations separated by up to 25cm, enabling long-distance integration. Indeed, co-rippling sites have increased correlation of very high-frequency activity which reflects cell firing. Thus, ripples may help bind information across the cortex in memory and other mental events.

sentences: Trade-offs between antiseptic cytotoxicity and efficacy in a human ex vivo wound contamination model Wound cleansing agents are routine in wound care, even in the absence of signs of infection. Antiseptic activity prevents contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. Here, we used an ex vivo human skin excisional wound model to evaluate the cytotoxicity of five clinically-used wound cleaning agents (saline, povidone iodine, Dove(R) soap, Dial(R) soap, and chlorhexidine gluconate). We established a wound contamination model using [~]100 cells of Pseudomonas aeruginosa per wound to evaluate antiseptic efficacy and microbial biofilm spatial organization. We found that Dial(R) soap and chlorhexidine gluconate significantly reduced metabolic activity of the biopsies, while all treatments except saline affected local cellular viability. Within the contamination model, only chlorhexidine gluconate treatment resulted in significantly lower P. aeruginosa counts at 24 hours post-treatment, driven by sub-limit-of-detection counts immediately post-treatment. Later applications of chlorhexidine gluconate had no effects on microbial growth, with microscopy showing extensive surface colonization of the wound bed. We present a clinically-relevant model for evaluating antiseptic cytotoxicity and efficacy, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

sentences: Multidimensional MRI for characterization of subtle axonal injury accelerated using an adaptive nonlocal multispectral filter Multidimensional MRI is an emerging approach that simultaneously encodes water relaxation (T1 and T2) and mobility (diffusion) and replaces voxel-averaged values with subvoxel distributions of those MR properties. While conventional (i.e., voxel-averaged) MRI methods cannot adequately quantify the microscopic heterogeneity of biological tissue, using subvoxel information allows to selectively map a specific T1-T2-diffusion spectral range that corresponds to a group of tissue elements. The major obstacle to the adoption of rich, multidimensional MRI protocols for diagnostic or monitoring purposes is the prolonged scan time. Our main goal in the present study is to evaluate the performance of a nonlocal estimation of multispectral magnitudes (NESMA) filter on reduced datasets to limit the total acquisition time required for reliable multidimensional MRI characterization of the brain. Here we focused and reprocessed results from a recent study that identified potential imaging biomarkers of axonal injury pathology from the joint analysis of multidimensional MRI, in particular voxelwise T1-T2 and diffusion-T2 spectra in human Corpus Callosum, and histopathological data. We tested the performance of NESMA and its effect on the accuracy of the injury biomarker maps, relative to the co-registered histological reference. Noise reduction improved the accuracy of the resulting injury biomarker maps, while permitting data reduction of 35.7% and 59.6% from the full dataset for T1-T2 and diffusion-T2 cases, respectively. As successful clinical proof-of-concept applications of multidimensional MRI are continuously being introduced, reliable and robust noise removal and consequent acquisition acceleration would advance the field towards clinically-feasible diagnostic multidimensional MRI protocols.

sentences: Structure and Function of the Retina of Low-density Lipoprotein Receptor-related Protein 5 (Lrp5)-deficient Rats Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by staining for claudin-5 and measurement of permeability to Evans blue dye. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed, correlated with absence of claudin-5 expression in superficial vessels and increased Evans blue permeability. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.

sentences: Strigolactones are chemoattractants for host tropism in Orobanchaceae parasitic plants Parasitic plants are worldwide threats that damage major agricultural crops. To initiate infection, parasitic plants have developed the ability to locate hosts and grow towards them. This ability, called host tropism, is critical for parasite survival, but its underlying mechanism remains mostly unresolved. To characterize host tropism, we used the model facultative root parasite Phtheirospermum japonicum, a member of the Orobanchaceae. Here, we show that strigolactones (SLs) function as host-derived chemoattractants. Chemotropism to SLs is also found in Striga hermonthica, a parasitic member of the Orobanchaceae, but not in non-parasites. Intriguingly, chemotropism to SLs in P. japonicum is attenuated in ammonium ion-rich conditions, where SLs are perceived, but the resulting asymmetrical accumulation of the auxin transporter PIN2 is diminished. P. japonicum encodes putative receptors that sense exogenous SLs, whereas expression of a dominant-negative form reduces its chemotropic ability. We propose a new function for SLs as navigators for parasite roots.

sentences: De novo transcriptome sequencing of the northern fowl mite, Ornithonyssus sylviarum, shed light into parasitiform poultry mites evolution and its chemoreceptor repertoires The poultry red mite (PRM), Dermanyssus gallinae (De Geer), and the northern fowl mite (NFM), Ornithonyssus sylviarum (Canestrini and Fanzago), are the most serious pests of poultry, both of which have an expanding global prevalence. Research on NFM has been constrained by a lack of genomic and transcriptomic data. Here, we report and analyze the first transcriptome data for this species. A total of 28,999 unigenes were assembled, of which 19,750 (68.10%) were annotated using seven functional databases. The biological function of these unigenes was predicted using the GO, KOG, and KEGG databases. To gain insight into the chemosensory receptor-based system of parasitiform mites, we furthermore assessed the gene repertoire of gustatory receptors (GRs) and ionotropic receptors (IRs), both of which encode putative ligand-gated ion channel proteins. While these receptors are well characterized in insect model species, our understanding of chemosensory detection in mites and ticks is in its infancy. To address this paucity of data, we identified 9 IR/iGluRs and 2 GRs genes by analyzing transcriptome data in the NFM, while 9 GRs and 41 IR/iGluRs genes were annotated in the PRM genome. Taken together, the transcriptomic and genomic annotation of these two species provide a valuable reference for studies of parasitiform mites, and also helps to understand how chemosensory gene family expansion/contraction events may have been reshaped by an obligate parasitic lifestyle compared with their free-living closest relatives. Future studies should include additional species to validate this observation, and functional characterization of the identified proteins as a step forward in identifying tools for controlling these poultry pests.

sentences: Principles of sensor-effector organization in six-transmembrane ion channels Receptor proteins sense stimuli and generate downstream signals via sensor and effector domains. Presently, the structural constraints on sensor-effector organization across receptor protein superfamilies are not clear. Here, we perform statistical coupling analysis (SCA) on the transient receptor potential (TRP) and voltage-gated potassium (Kv) ion channel superfamilies to characterize the networks of coevolving residues, or protein sectors, that mediate their receptor functions. Comparisons to structural and functional studies reveal a conserved "core" sector that extends from the pore and mediates effector functions, including pore gating and sensor-pore coupling, while sensors correspond to family-specific "accessory" sectors and localize according to three principles: Sensors (1) may emerge in any region with access to the core, (2) must maintain contact with the core, and (3) must preserve the integrity of the core. This sensor-core architecture may represent a conserved and generalizable paradigm for the structure-function relationships underlying the evolution of receptor proteins.

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

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

sentences: Single-component optogenetic tools for inducible RhoA GTPase signaling We created optogenetic tools to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activating GEF effectors, were fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these effectors induced potent contractile signaling sufficient to separate adherens junctions in response to as little as one pulse of blue light. Cytoskeletal morphology changes were dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation induced YAP nuclear localization within minutes and subsequent mechanotransduction, verified by YAP- TEAD transcriptional activity. These single-component tools, which do not require protein binding partners, offer spatiotemporally precise control over RhoA signaling that will advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

sentences: Can DyeCycling break the photobleaching limit in single-molecule FRET? Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of [≤] 3 orders of magnitude. Here, we introduce  DyeCycling, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

sentences: Deep sampling of ancestral genetic diversity reveals Saccharomyces cerevisiae pre-domestication life histories The ecology and genetic diversity of model yeast Saccharomyces cerevisiae prior to human domestication remain poorly understood. Taiwan is regarded as part of this yeasts geographic birthplace where the most divergent natural lineage was discovered. Here, we deep sampled the broad-leaf forests across this continental island to probe the ancestral species diversity. We found that S. cerevisiae is distributed ubiquitously at low abundance in the forests. Whole-genome sequencing of 121 isolates revealed nine distinct lineages, the highest known in any region. Three lineages are endemic to Taiwan and six are widespread in Asia. Molecular dating placed the divergence of the Taiwanese and Asian lineages during the Pleistocene, when a transient continental shelf land bridge connected Taiwan to other major landmasses. Extensive historical and recent admixture events were detected between natural lineages. In particular, the genetic component from a lineage associated with fruits that spanned the widest geographical range was present in most admixed isolates. Collectively, Taiwanese isolates harbor genetic diversity comparable to that of the whole Asia continent, and different lineages have coexisted at a fine spatial scale even on the same tree. Patterns of variations within each lineage revealed that S. cerevisiae is highly clonal and predominantly reproduces asexually in nature. We detected prevalent purifying selection genome-wide, with lineage-specific signals of positive or directional selection independent between lineages. This study establishes that S. cerevisiae has rich natural diversity sheltered from human influences, making it a powerful model system in microbial ecology.

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Physically interacting beta-delta pairs in the regenerating pancreas revealed by single cell sequencing The endocrine pancreas is able to regenerate in response to insult, including by driving beta-cells into the cell division cycle. Until recently, communication between neighboring cells in islets of Langerhans was overlooked by single-cell genomic technologies, which require rigorous tissue dissociation into single cells. Here, we utilize sorting of physically interacting cells (PICs) with single-cell RNA-sequencing to systematically map cellular interactions in the regenerating endocrine pancreas. The cellular landscape of the regenerated pancreas features regeneration-associated endocrine populations.

We explore the unexpected heterogeneity of beta-cells in regeneration, including an interaction-specific program between paired beta and delta-cells. Our analysis suggests that the particular cluster of beta-cells that pair with delta-cells benefits from stress protection, implying that the interaction between beta and delta-cells safeguards against regeneration-associated challenges.

sentences: The analysis of epigenomic evolution While the investigation of the epigenome becomes increasingly important, still little is known about the long-term evolution of epigenetic marks and systematic investigation strategies are still withstanding. Here, we systematically demonstrate the transfer of classic phylogenetic methods such as maximum likelihood based on substitution models, parsimony, and distance-based to interval-scaled epigenetic data (available at Github). Using a great apes blood data set, we demonstrate that DNA methylation is evolutionarily conserved at the level of individual CpGs in promotors, enhancers and genic regions. Our analysis also reveals that this epigenomic conservation is significantly correlated with its transcription factor binding density. Binding sites for transcription factors involved in neuron differentiation and components of AP-1 evolve at a significantly higher rate at methylation than at nucleotide level. Moreover, our models suggest an accelerated epigenomic evolution at binding sites of BRCA1, CBX2, and factors of the polycomb repressor 2 complex in humans. For most genomic regions, the methylation-based reconstruction of phylogenetic trees is at par with sequence-based reconstruction. Most strikingly, phylogenetic reconstruction using methylation rates in enhancer regions was ineffective independently of the chosen model. We identify a set of phylogenetically uninformative CpG sites enriching in enhancers controlling immune-related genes.

sentences: Deciphering Multi-way Interactions in the Human Genome Chromatin architecture, a key regulator of gene expression, can be inferred using chromatin contact data from chromosome conformation capture, or Hi-C. However, classical Hi-C does not preserve multi-way contacts. Here we use long sequencing reads to map genome-wide multi-way contacts and investigate higher order chromatin organization in the human genome. We use hypergraph theory for data representation and analysis, and quantify higher order structures in neonatal fibroblasts, biopsied adult fibroblasts, and B lymphocytes. By integrating multi-way contacts with chromatin accessibility, gene expression, and transcription factor binding, we introduce a data-driven method to identify cell type-specific transcription clusters. We provide transcription factor-mediated functional building blocks for cell identity that serve as a global signature for cell types.

sentences: Effect of the Ecological Location of a Water Source on Entropy and other Spatio-temporal Behavioral Features: An extended and systematic replication The continuous analysis of spatial behavioral-dynamics under stimuli-schedules has been a scarcely studied field in experimental psychology. A recent study conducted in our laboratory suggest that the features embedded in the spatial dynamics of behavior are affected by stimulus-schedules, at least, as much as features embedded in discrete responses. In that study we compared the spatial behavioral dynamics under two time-based schedules (fixed vs variable time) of water delivery, and two different locations of water delivery (delivery in central zone vs. perimetral zone) on a Modified Open Field System (MOFS). The present work replicates those findings taking in consideration previously uncontrolled variables. In Experiment 1, three subjects were exposed to a Fixed Time 30s water-delivery schedule. In the first phase the water dispenser was located at the perimetral zone. In the second condition, the water dispenser was located at the central zone. Each location was presented for 20 sessions. In Experiment 2, conditions were the same, but a Variable Time schedule was used. Measures of entropy were used to describe the spatial behavioral dynamics. We found higher levels of entropy under central location of water delivery than in the perimetral location; and higher entropy under Fixed than Variable Time Schedule, confirming previous findings but under different sequences of dispenser locations. In general, a well-differentiated dynamic between experimental conditions was observed in terms of direction (distance to the dispenser) and variation (entropy) of spatial behavior. These findings are discussed under a systemic, parametric, ecological, and non-mediational framework.

sentences: A single domain shark antibody targeting the transferrin receptor 1 delivers a TrkB agonist antibody across the blood brain barrier to provide full neuroprotection in a mouse model of Parkinson's Disease Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells can be used to shuttle antibodies and other cargos across the blood brain barrier (BBB). We have fused one of these (TXB4) to differing regions of TrkB and TrkC neurotrophin receptor agonist antibodies (AAb) and determined the effect on agonist activity, brain accumulation and engagement with neurons in the mouse brain following systemic administration. The TXB4-TrkB and TXB4-TrkC fusion proteins retain agonist activity at their respective neurotrophin receptors and in contrast to their parental AAb they rapidly accumulate in the brain reaching nM levels following a single IV injection. Following SC administration, the most active TrkB fusion protein, TXB4-TrkB1, associates with and activates ERK1/2 signalling in TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc). When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinsons disease (PD) TXB4-TrkB1, but not the parental TrkB AAb or a TXB4-TrkC1 fusion protein, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 TfR1 binding module allows a TrkB AAb to reach neuroprotective concentrations in the brain parenchyma following systemic administration.

sentences: Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenic lineages The ventricular-subventricular zone (V-SVZ) is home to the largest neurogenic niche in the adult mouse brain. Previous work has demonstrated that resident stem cells in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. While great progress has been made in understanding the differences in regional stem cell potential using viral and genetic lineage tracing strategies, the core molecular heterogeneity that underlies these regional differences is largely unknown. Here we present single whole-cell and single nucleus sequencing datasets of microdissected adult mouse V-SVZ, and evidence for the existence of two broad populations of adult neural stem cells. By using spatially resolved microdissections in the single nucleus sequencing dataset as a reference, and mapping marker gene expression in the V-SVZ, we find that these two populations reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. Furthermore, we identified two subpopulations of newly born neurons that have gene expression consistent with dorsal or ventral origins. Finally, we identify genes expressed by both stem cells and the neurons they generate that specifically mark either the dorsal or ventral adult neurogenic lineage. These datasets, methods and findings will facilitate the study of region-specific regulation of adult neurogenesis.

sentences: Ablation of SAMD1 in Mice Causes Failure of Angiogenesis, Embryonic Lethality Pathological retention of LDL in the intima is involved in atherosclerosis, although the retention mechanisms are not well-understood. Previously, we reported Sterile Alpha Motif Domain Containing 1 (SAMD1), a protein secreted by intimal smooth muscle cells in atherosclerotic lesions, appears to bind LDL in extracellular matrix around intimal cells. Fab-fragment inhibitors of apparently irreversible SAMD1/LDL binding reduced LDL retention in carotid injury models, but did not have a significant effect on early spontaneous lesion development. Our histology of mouse atherosclerosis models revealed extensive SAMD1 expression, possibly related to cell phenotype modulation and antigen presentation. Although the normal function of SAMD1 is unknown, it may have multiple epigenetic roles. For this report, we generated SAMD1-/-, SAMD1-/+, and SAMD1-/+ apoE-/- mice to further explore SAMD1s role in atherosclerosis. SAMD1 was found in tissues throughout the SAMD1+/+ and SAMD1-/+ embryos. Homozygous loss of SAMD1 was embryonic lethal: at embryonic day 14, organs were partially developed and/or degraded; heads and brains were malformed; no blood vessels were observed; red blood cells were scattered and pooled, primarily near the embryo surface; and cell death was occurring. Development appeared normal in heterozygous SAMD1 embryos, but postnatal genotyping showed a reduced ability to thrive. Growth of atherosclerotic lesions in SAMD1-/+ apoE-/- after 35 weeks was not significantly less than in mice SAMD1+/+ apoE-/- mice.

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Germline ERBB3 mutation in familial non-small cell lung carcinoma: expanding the role of the ErbB family in oncogenesis. BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2.

MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined.

ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic.

ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

sentences: Chromatin reader Dido3 regulates the genetic network of B cell differentiation The development of hematopoietic lineages is based on a complex balance of transcription factors whose expression depends on the epigenetic signatures that characterize each differentiation step. The B cell lineage arises from hematopoietic stem cells through the stepwise silencing of stemness genes and balanced expression of mutually regulated transcription factors, as well as DNA rearrangement. Here we report the impact on B cell differentiation of the lack of DIDO3, a reader of chromatin status, in the mouse hematopoietic compartment. We found reduced DNA accessibility in hematopoietic precursors, leading to severe deficiency in the generation of successive B cell differentiation stages. The expression of essential transcription factors and differentiation markers is affected, as is the somatic recombination process.

sentences: Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane {beta} barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic {beta} hairpin immediately preceding the first barrel strand can insert into the {beta} barrel. We propose that these motions enlarge the channel and hoist the substrate into it for secretion. An anchor region at the interface of the {beta} barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

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An exploratory study of EEG connectivity during the first year of life in preterm and full-term infants AimTo evaluate electroencephalography (EEG) connectivity during the first year of age in healthy full-term infants and preterm infants with prenatal and perinatal risk factors for perinatal brain damage.

MethodsThree groups of infants were studied: healthy full-term infants (n = 71), moderate/late-preterm infants (n = 54), and very preterm infants (n = 56). All preterm infants had perinatal and/or perinatal risk factors for brain damage. EEG was obtained during phase II of natural nonrapid eye movement (NREM) sleep. EEG analysis was performed in 24 2.56-s artifact-free segments. For the calculation of EEG sources, spectral structured sparse Bayesian learning was used. Connectivity was computed by the phase-lag index.

ResultsIn healthy full-term infants, EEG interhemispheric connectivity in the different frequency bands followed similar trends with age to those reported in each frequency band: delta connectivity decreased, theta increased at the end of the year, the alpha band showed different trends based on the region studied, and beta interhemispheric connectivity decreased with age. EEG connectivity in preterm infants showed differences from the results of the term group.

DiscussionImportant structural findings may explain the differences observed in EEG connectivity between the term and preterm groups.

ConclusionThe study of EEG connectivity during the first year of age provides essential information on normal and abnormal brain development.

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Improving the time and space complexity of the WFA algorithm and generalizing its scoring MotivationModern genomic sequencing data is trending toward longer sequences with higher accuracy. Many analyses using these data will center on alignments, but classical exact alignment algorithms are infeasible for long sequences. The recently proposed WFA algorithm demonstrated how to perform exact alignment for long, similar sequences in O(sN) time and O(s2) memory, where s is a score that is low for similar sequences (Marco-Sola et al., 2021). However, this algorithm still has infeasible memory requirements for longer sequences. Also, it uses an alternate scoring system that is unfamiliar to many bioinformaticians.

ResultsWe describe variants of WFA that improve its asymptotic memory use from O(s2) to O(s3/2) and its asymptotic run time from O(sN) to O(s2 + N). We expect the reduction in memory use to be particularly impactful, as it makes it practical to perform highly multithreaded megabase-scale exact alignments in common compute environments. In addition, we show how to fold WFAs alternate scoring into the broader literature on alignment scores.

AvailabilityAll code is publicly available for use and modification at https://github.com/jeizenga/wfalm.

Contactjeizenga@ucsc.edu

Supplementary informationSupplementary data are available online.

sentences: Measuring the temporal quality of a biodiversity database In the context of climate change it is important to keep biodiversity databases up-to-date. This priority generates the need for a metric to assess the concept of up-to-dateness. The objective of this paper is to devise a measure of up-to-dateness for atlas-type biodiversity data. The data input into the algorithm consists of the species, date and grid cell allocation of all available records for a taxon in a region. First, for each grid cell in a region, the median of the date of the most recent record of each species is calculated. Secondly, the median of the median dates for each grid cell yields an overall measure of up-to-dateness. The performance of this algorithm is investigated in relation to databases for six taxa in southern Africa. In June 2021, the up-to-dateness of the databases varied from 41 years for the reptile database to two years for the bird database. The quality of a biodiversity database is a multidimensional concept; up-to-dateness is only one of several dimensions. The paper identifies a need to quantify the rate at which the "value" of a record decays as evidence that a species still occurs at a locality, and suggests an experimental process for doing this. The use of the up-to-dateness index to motivate citizen scientists is discussed.

sentences: Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method High throughput measurement of cell perturbation, by libraries of small molecules or gene knockouts, is a key step in functional genomics and pre-clinical drug development. However, it is difficult to perform viable, single-cell assays in 384-well plates, limiting many studies to simple well-average measurements (e.g. CellTiter-Glo(R)). Here we describe a public domain "Dye Drop" method in which sequential density displacement is used to perform multi-step assays for cell viability and EdU incorporation followed by immunofluorescence imaging. The method is rapid, reproducible, can be readily customized, and is compatible with either manual or automated laboratory equipment. We demonstrate Dye Drop in the collection of dose-response data for 67 drugs in 58 breast cancer cell lines and separate cytostatic and cytotoxic responses, thereby providing new insight into the effects of specific drugs on cell cycle progression and cell viability. Dye Drop substantially improves the tradeoff between data content and cost, enabling collection of large information-rich datasets.

sentences: Extrachromosomal Amplification of Human Papillomavirus Episomes as a Mechanism of Cervical Carcinogenesis Integration of Human Papillomaviruses (HPV) is an important mechanism of carcinogenesis but is absent in a significant fraction of HPV16+ tumors. We applied long-read whole-genome sequencing (WGS) to cervical cancer cell lines and tumors. In two HPV16+ cell lines, we identified large tandem arrays of full-length and truncated viral genomes integrated into multiple locations indicating formation as extrachromosomal DNA (HPV superspreading). An HPV16+ cell line with episomal DNA has tandem arrays of full-length, truncated, and rearranged HPV16 genomes (multimer episomes). WGS of HPV16+ cervical tumors revealed that 11/20 with only episomal HPV (EP) have intact monomer episomes. The remaining nine EP tumors have multimer and rearranged HPV genomes. Most HPV rearrangements disrupt the E1 and E2 genes, and EP tumors overexpress the E6 and E7 viral oncogenes. Tumors with both episomal and integrated HPV16 display multimer episomes and concatemers of human and viral sequences. One tumor has a recurrent deletion of an inhibitory site regulating E6 and E7 expression, and another has a recurrent duplication consistent with HPV superspreading. Therefore, HPV16 can cause cancer without integration through aberrant episomal replication, forming rearranged and multimer episomes.

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Noradrenaline and ATP regulate white adipocyte adiponectin exocytosis: disturbed adrenergic and purinergic signalling in obesity-associated diabetes. White adipocyte adiponectin exocytosis is triggered by cAMP and a concomitant increase of cytosolic Ca2+ potentiates its release. White adipose tissue is richly innervated by sympathetic nerves co-releasing noradrenaline (NA) and ATP that may act on receptors in the adipocyte plasma membrane to increase cAMP via adrenergic receptors and Ca2+ via purinergic receptors, respectively. Here we determine the importance of NA and ATP for the regulation of white adipocyte adiponectin exocytosis, at the cellular and molecular level, and we specifically detail the ATP signalling pathway. Immunohistochemical staining demonstrates that tyrosine hydroxylase (enzyme involved in catecholamine synthesis) is dramatically reduced in inguinal white adipose tissue (IWAT) isolated from mice with diet-induced obesity; this is associated with diminished levels of NA in IWAT and with lowered serum adiponectin. Adiponectin exocytosis (measured as increase in plasma membrane capacitance and as secreted product) is triggered by NA or ATP alone in cultured and primary mouse IWAT adipocytes, and enhanced by a combination of the two secretagogues. The ATP-induced adiponectin exocytosis is largely Ca2+-dependent and activated via P2Y2 receptors (P2Y2Rs) and the Gq11/PLC pathway. Adiponectin release induced by the nucleotide is abrogated in adipocytes isolated from obese/diabetic mice and this is associated with [~]70% reduced abundance of P2Y2Rs. The NA-triggered adiponectin exocytosis is likewise abolished in "obese adipocytes", concomitant with a 50% lower gene expression of beta 3 adrenergic receptors ({beta}3ARs). The NA-stimulated adiponectin secretion does not contain Ca2+-dependent components. Collectively, our data suggest that sympathetic innervation is a principal regulator of adiponectin exocytosis and that disruptions of this control are associated with the obesity-associated reduction of circulating levels of HMW adiponectin.

Key point listO_LIWhite adipose tissue is richly innervated by sympathetic nerves that co-release noradrenaline (NA) and ATP.
C_LIO_LIProtein levels of tyrosine hydroxylase and NA are dramatically decreased in white adipose tissue from obese/diabetic mice, concomitant with reduced serum levels of high-molecular weight (HMW) adiponectin.
C_LIO_LINA and ATP stimulate white adipocyte adiponectin exocytosis via beta adrenergic and purinergic receptors respectively. The ATP-induced adiponectin secretion is chiefly Ca2+-dependent and activated via the P2Y2/Gq11/PLC pathway.
C_LIO_LIThe purinergic signalling is abrogated in adipocytes from obese/diabetic mice, due to reduced abundance of P2Y2Rs. The response to NA is likewise abolished in "obese adipocytes", associated with lowered gene expression of beta 3 adrenergic receptors ({beta}3ARs).
C_LIO_LIWe propose that sympathetic innervation is central in regulation of adiponectin exocytosis via co-secretion of NA and ATP and that this control is disrupted in obesity-associated diabetes, leading to lower circulating levels of HMW adiponectin.
C_LI

sentences: Evolutionary jumps in bacterial GC content Genomic GC content is a fundamental molecular trait linked with many key genomic features such as codon and amino acid use. Across bacteria, GC content is surprisingly diverse and has been studied for many decades; yet its evolution remains incompletely understood. Since it is difficult to observe GC content evolve on laboratory time-scales, phylogenetic comparative approaches are instrumental; but this dimension is rarely studied systematically in the case of bacterial GC content. We applied phylogenetic comparative models to analyse GC content evolution in multiple bacterial groups across two major bacterial phyla. We find that GC content diversifies via a combination of gradual evolution and evolutionary "jumps". Surprisingly, unlike prior reports that solely focused on reductions in GC, we found a comparable number of jumps with both increased and decreased GC content. Overall, many of the identified jumps occur in lineages beyond the well-studied peculiar examples of endosymbiotic and AT-rich marine bacteria, and do not support the predicted role of oxygen dependence. Our analysis of rapid and large shifts in GC content thus identifies new clades and novel contexts to further understand the ecological and evolutionary drivers of this important genomic trait.

sentences: Ability of the Ash dieback pathogen to reproduce and to induce damage on its host are controlled by different environmental parameters Ash dieback, induced by an invasive ascomycete, Hymenoscyphus fraxineus, has emerged in the last decade as a severe disease threatening ash populations in Europe. Future prospects for Ash are improved by the existence of individuals with natural genetic resistance to the disease and by limited disease impact in many environmental conditions where ashes are frequent. Nevertheless, it was suggested that even in those conditions, ash trees are infected and enable pathogen transmission. We studied the influence of climate and local environment on the ability of H. fraxineus to infect, be transmitted and cause damage on its host. We showed that healthy carrier, i.e. asymptomatic individuals carrying H. fraxineus, exists and may play a significant role in ash dieback epidemiology. Environment strongly influenced H. fraxineus with different parameters being important depending on the life cycle stage. The ability of H. fraxineus to establish on ash leaves and to reproduce on the leaf debris in the litter (rachises) mainly depended on total precipitations in July-August and was not influenced by local tree cover. By contrast, damages to the host, and in particular shoot mortality was significantly reduced by high summer temperature in July-August and by high autumn average temperature. As a consequence, in many situations ash trees are infected and enable H. fraxineus transmission while showing limited or even no damages. We also observed a decreasing trend of severity (leaf necrosis and shoot mortality likelihood) with the time of disease presence in a plot that could be significant for the future of Ash dieback.

sentences: VASP mediated actin dynamics activate and recruit a filopodia myosin Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin binding protein, the state of the actin cytoskeleton and MF myosin activity.

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Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation. PremiseMany cultivated coffee varieties descend from Coffea canephora, commonly known as Robusta coffee. The Congo Basin has a century long history of Robusta coffee cultivation and breeding, and is hypothesized to be the region of origin of many of the cultivated Robusta varieties. Since little is known about the genetic composition of C. canephora in this region, we assessed the genetic diversity of wild and cultivated C. canephora shrubs in the Democratic Republic of the Congo.

MethodsUsing 18 microsatellite markers, we studied the genetic composition of wild and backyard-grown C. canephora shrubs in the Tshopo and Ituri provinces, and from the INERA Yangambi Coffee Collection. We assessed genetic clustering patterns, genetic diversity, and genetic differentiation between populations.

Key resultsGenetic differentiation was relatively strong between wild and cultivated C. canephora shrubs, and both gene pools harbored multiple unique alleles. Strong genetic differentiation was also observed between wild populations. The level of genetic diversity in wild populations was similar to that of the INERA Yangambi Coffee Collection, but local wild genotypes were mostly missing from that collection. Shrubs grown in the backyards were genetically similar to the breeding material from INERA Yangambi.

ConclusionsMost C. canephora that is grown in local backyards originated from INERA breeding programs, while a few shrubs were obtained directly from surrounding forests. The INERA Yangambi Coffee Collection could benefit from an enrichment with local wild genotypes, to increase the genetic resources available for breeding purposes, as well as to support ex situ conservation.

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Ketamine and sleep modulate neural complexity dynamics in cats There is increasing evidence that level of consciousness can be captured by neural informational complexity: for instance, complexity, as measured by the Lempel Ziv (LZ) compression algorithm, decreases during anesthesia and non-rapid eye movement (NREM) sleep in humans and rats, when compared to LZ in awake and REM sleep. In contrast, LZ is higher in humans under the effect of psychedelics, including subanesthetic doses of ketamine. However, it is both unclear how this result would be modulated by varying ketamine doses, and whether it would extend to other species. Here we studied LZ with and without auditory stimulation during wakefulness and different sleep stages in 5 cats implanted with intracranial electrodes, as well as under subanesthetic doses of ketamine (5, 10, and 15 mg/kg i.m.). In line with previous results, LZ was lowest in NREM sleep, but similar in REM and wakefulness. Furthermore, we found an inverted U-shaped curve following different levels of ketamine doses in a subset of electrodes, primarily in prefrontal cortex. However, it is worth noting that the variability in the ketamine dose-response curve across cats and cortices was larger than that in the sleep-stage data, highlighting the differential local dynamics created by two different ways of modulating conscious state. These results replicate previous findings, both in humans and other species, demonstrating that neural complexity is highly sensitive to capture state changes between wake and sleep stages while adding a local cortical description. Finally, this study describes the differential effects of ketamine doses, replicating a rise in complexity for low doses, and further fall as doses approach anesthetic levels in a differential manner depending on the cortex.

In briefPrevious studies have shown that Lempel Ziv complexity (LZ) decreases during anesthesia and non-rapid eyes movement (NREM) sleep in humans and rats whereas it increases in REM sleep and under the effect of psychedelics. In this work we show that in the cat, LZ is lowest in NREM sleep, but similar in REM and wakefulness. We also found a ketamine inverted U-shape dose-response curve only in the auditory and prefrontal cortex, with a much larger variability in the ketamine across cats and cortices when compared to the sleep cycle.

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sentences: An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy is largely unclear. Using quantitative RNA Interactome Capture analysis, we reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of cancer cells, with a network of nuclear proteins centered around Nucleolin displaying enhanced RNA-binding activity. We show that Nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal-RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This Nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced cancer cell proliferation, and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of Nucleolin, and highlight the crucial role of this process in RAS-mediated tumorigenesis.

sentences: Simultaneous pathway engagement of translation in astrocytes, circadian rhythm in GABAergic neurons and cytoskeleton in glutamatergic neurons precede electroencephalographic changes in neurodegenerative prion disease Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific brain regions and cell types. This selectivity may arise from cells possessing varying capacities to regain proteostasis when stressed by cytotoxic protein conformers. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite having a normal outward appearance. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated a sharply reduced synthesis of ribosomal and mitochondrial components, 2) excitatory neurons showed increased expression of cytoskeletal genes, and 3) inhibitory neurons revealed reduced expression of circadian rhythm network genes. Further assessment for the role of circadian rhythms using a jet lag paradigm modestly exacerbated disease. These data demonstrate that early translatome responses to neurodegeneration emerge prior to other signs of disease and are unique to different cell types. Therapeutic strategies may need to target multiple pathways, each in specific populations of cells, early in the disease process.

sentences: The importance of environmental parameters and mixing zone in shaping estuarine microbial communities along a freshwater-marine gradient. Microbial communities are important elements in the marine environment, contributing to nutrient cycling and biogeochemical processes. Estuaries comprise environments exhibiting characteristics from freshwater to marine, leading to distinct microbial communities across this environmental gradient. Here, we examine the spatial dynamics of microbial communities in Macquarie Harbour, an estuarine system on the West coast of Tasmania, Australia. Water was sampled along the estuary to explore the structure and composition of the microbial communities using 16S/18S rRNA gene amplicon sequencing. Multivariate analyses showed environmental variables and community compositions varying along a longitudinal (river to adjacent ocean) gradient at the surface. In the harbour, differences in the microbial community were observed between surface (0-1 m) and intermediate depths (4.5-11 m depth). The results of differential abundance, network and Partial Least Square analyses suggest that Macquarie Harbour is a mixing zone, where the distributions of archaeal, bacterial and eukaryotic communities are influenced by oceanic and riverine inputs. Coupled with the natural characteristics of the Harbour, the heterotrophic component of this microbial communities inhabiting the surface and intermediate waters may play important roles in the nutrient cycle in the studied area. These results provide critical insights into the Macquarie Harbour environment and the importance of understanding the role of microbial communities for similar systems elsewhere.

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Dissociable influences of reward and punishment on adaptive cognitive control To invest effort into any cognitive task, people must be sufficiently motivated. Whereas prior research has focused primarily on how the cognitive control required to complete these tasks is motivated by the potential rewards for success, it is also known that control investment can be equally motivated by the potential negative consequence for failure. Previous theoretical and experimental work has yet to examine how positive and negative incentives differentially influence the manner and intensity with which people allocate control. Here, we develop and test a normative model of control allocation under conditions of varying positive and negative performance incentives. Our model predicts, and our empirical findings confirm, that rewards for success and punishment for failure should differentially influence adjustments to the evidence accumulation rate versus response threshold, respectively. This dissociation further enabled us to infer how motivated a given person was by the consequences of success versus failure.

Author SummaryFrom the school to the workplace, whether someone achieves their goals is determined largely by the mental effort they invest in their tasks. Recent work has demonstrated both why and how people adjust the amount of effort they invest in response to variability in the rewards expected for achieving that goal. However, in the real world, we are motivated both by the positive outcomes our efforts can achieve (e.g., praise) and the negative outcomes they can avoid (e.g., rejection), and these two types of incentives can motivate adjustments not only in the amount of effort we invest but also the types of effort we invest (e.g., whether to prioritize performing the task efficiently or cautiously). Using a combination of computational modeling and a novel task that measures voluntary effort allocation under varying incentive conditions, we show that people should and do engage dissociable forms of mental effort in response to positive versus negative incentives. With increasing rewards for achieving their goal, they prioritize efficient performance, whereas with increasing penalties for failure they prioritize performing cautious performance. We further show that these dissociable strategies enable us to infer how motivated a given person was based on the positive consequences of success relative to the negative consequences of failure.

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Global and Gene-specific Transcriptional Responses to Acute Stress Nucleosomes may regulate transcription by controlling access to promoters by transcription factors and RNA polymerase II (Pol2). Potentially active genes display nucleosome depleted regions flanked by positioned -1 and +1 nucleosomes. On yeast genes, the transcription start site (TSS) is on the upstream face of the +1 nucleosome, but whether precise +1 nucleosome positioning controls Pol2 access to the TSS remains unclear. Here, using acute nutrient starvation to rapidly reprogramme the genome, we show highly dynamic upstream or downstream shifts in the position of +1 nucleosomes, coincident with levels of transcriptionally engaged Pol2 at 58% of genes. Transcript level changes broadly reflect Pol2 occupancy changes with a delay but can be further influenced by Pub1 or Puf3 dependent changes in transcript degradation rates. The response to acute stress has a second component as we also observed genome-wide changes in Pol2 distribution on genes, independent of changes in Pol2 occupancy, with Pol2 accumulating upstream of a +170 nt stalling site. Mathematical modelling supports a global increase in promoter-proximal early transcription termination as a major component of the global stress response. Thus, we uncover a two-component transcriptional response to stress, one focused on the +1 nucleosome, the second on Pol2 itself.

O_LIA two-component responses to acute stress involving a gene-specific response and a global response
C_LIO_LIDynamic shifting of +1 nucleosome position with transcriptional activation or repression.
C_LIO_LIGlobal targeting of Pol2 leading to early transcription termination on acute stress
C_LI

sentences: Predictability and parallelism in the contemporary evolution of hybrid genomes Hybridization between species is widespread across the tree of life. As a result, many species, including our own, harbor regions of their genome derived from hybridization. Despite the recognition that this process is widespread, we understand little about how the genome stabilizes following hybridization, and whether the mechanisms driving this stabilization tend to be shared across species. Here, we dissect the drivers of variation in local ancestry across the genome in replicated hybridization events between two species pairs of swordtail fish: Xiphophorus birchmanni x X. cortezi and X. birchmanni x X. malinche. We find unexpectedly high levels of repeatability in local ancestry across the two types of hybrid populations. This repeatability is attributable in part to the fact that the recombination landscape and locations of functionally important elements play a major role in driving variation in local ancestry in both types of hybrid populations. Beyond these broad scale patterns, we identify dozens of regions of the genome where minor parent ancestry is unusually low or high across species pairs. Analysis of these regions points to shared sites under selection across species pairs, and in some cases, shared mechanisms of selection. We show that one such region is a previously unknown hybrid incompatibility that is shared across X. birchmanni x X. cortezi and X. birchmanni x X. malinche hybrid populations.

sentences: Dimensionality reduction by UMAP reinforces sample heterogeneity analysis in bulk transcriptomic data Transcriptome profiling and differential gene expression constitute a ubiquitous tool in biomedical research and clinical application. Linear dimensionality reduction methods especially principal component analysis (PCA) are widely used in detecting sample-to-sample heterogeneity in bulk transcriptomic datasets so that appropriate analytic methods can be used to correct batch effects, remove outliers and distinguish subgroups. In response to the challenge in analysing transcriptomic datasets with large sample size such as single-cell RNA-sequencing (scRNA-seq), non-linear dimensionality reduction methods were developed. t-distributed stochastic neighbour embedding (t-SNE) and uniform manifold approximation and projection (UMAP) show the advantage of preserving local information among samples and enable effective identification of heterogeneity and efficient organisation of clusters in scRNA-seq analysis. However, the utility of t-SNE and UMAP in bulk transcriptomic analysis has not been carefully examined. Therefore, we compared major dimensionality reduction methods (linear: PCA; nonlinear: multidimensional scaling (MDS), t-SNE, and UMAP) in analysing 71 bulk transcriptomic datasets with large sample sizes. UMAP was found superior in preserving sample level neighbourhood information and maintaining clustering accuracy, thus conspicuously differentiating batch effects, identifying pre-defined biological groups and revealing in-depth clustering structures. We further verified that new clustering structures visualised by UMAP were associated with biological features and clinical meaning. Therefore, we recommend the adoption of UMAP in visualising and analysing of sizable bulk transcriptomic datasets.

sentences: Topological and Structural Plasticity of the single Ig fold and the double Ig fold present in CD19 The Ig-fold has had a remarkable success in vertebrate evolution, with a presence in over 2% of human genes. The Ig-fold is not just the elementary structural domain of antibodies and TCRs, it is also at the heart of a staggering 30% of immunologic cell surface receptors, making it a major orchestrator of cell-cell-interactions. While BCRs, TCRs, and numerous Ig-based cell surface receptors form homo or heterodimers on the same cell surface (in cis), many of them interface as ligand-receptors (checkpoints) on interacting cells (in trans) through their Ig domains. New Ig-Ig interfaces are still being discovered between Ig-based cell surface receptors, even in well known families such as B7. What is largely ignored however is that the Ig-fold itself is pseudo-symmetric, a property that makes the Ig-domain a versatile self-associative 3D structure and may in part explain its success in evolution, especially through its ability to bind in cis or in trans in the context of cell surface receptor-ligand interactions. In this paper we review the Ig domains tertiary and quaternary pseudo symmetries, with a particular attention to the newly identified double Ig fold in the solved CD19 molecular structure to highlight the underlying fundamental folding elements of Ig domains, i.e. Ig protodomains. This pseudosymmetric property of Ig domains gives us a decoding frame of reference to understand the fold, relate all Ig-domain forms, single or double, and suggest new protein engineering avenues.

sentences: Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade Anhydrobiosis is one of the most extensively studied forms of cryptobiosis that is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades response to desiccation. However, the molecular mechanisms underlying these protein functions remain to be fully elucidated. In this study, we perfomed in vitro and in vivo characterizations of the self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. Our in vitro observations showed that CAHS1 proteins homo-oligomerized via the C-terminal -helical region and formed a hydrogel as their concentration increased, and that these molecular assembling processes were reversible. Furthermore, our in vivo observations demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into the protective mechanisms involved in the anhydrobiosis of tardigrades.

sentences: RFU derived LRs for activity level assignments usingBayesian Networks A comparative study has been carried out, comparing two different methods to estimate activity level likelihood ratios (LRa) using Bayesian Networks. The first method uses the sub-source likelihood ratio (log10LR{phi}) as a  quality indicator. However, this has been criticised as introducing potential bias from population differences in allelic proportions. An alternative method has been introduced that is based upon the total RFU of a DNA profile that is adjusted using the mixture proportion (Mx) which is calculated from quantitative probabilistic genotyping software (EuroForMix). Bayesian logistic regressions of direct transfer data showed that the two methods were comparable. Differences were attributed to sampling error, and small sample sizes of secondary transfer data. The Bayesian approach facilitates comparative studies by taking account of sampling error; it can easily be extended to compare different methods.

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Active reconfiguration of cytoplasmic lipid droplets governs migration of nutrient-limited phytoplankton As open oceans continue to warm, modified currents and enhanced stratification exacerbate nitrogen and phosphorus limitation, constraining primary production. The ability to migrate vertically bestows motile phytoplankton a crucial-albeit energetically expensive-advantage toward vertically redistributing for optimal growth, uptake and resource storage in nutrient-limited water columns. However, this traditional view discounts the possibility that the phytoplankton migration strategy may be actively selected by the storage dynamics when nutrients turn limiting. Here we report that storage and migration in phytoplankton are coupled traits, whereby motile species harness energy storing lipid droplets (LDs) to biomechanically regulate migration in nutrient limited settings. LDs grow and translocate-directionally-within the cytoplasm to accumulate below the cell nucleus, tuning the speed, trajectory and stability of swimming cells. Nutrient reincorporation reverses the LD translocation, restoring the homeostatic migratory traits measured in population-scale millifluidic experiments. Combining intracellular LD tracking and quantitative morphological analysis of red-tide forming alga, Heterosigma akashiwo, along with a model of cell mechanics, we discover that the size and spatial localization of growing LDs govern the ballisticity and orientational stability of migration. The strain-specific shifts in migration which we identify here are amenable to a selective emergence of mixotrophy in nutrient-limited phytoplankton. We rationalize these distinct behavioral acclimatization in an ecological context, relying on concomitant tracking of the photophysiology and reactive oxygen species (ROS) levels, and propose a dissipative energy budget for motile phytoplankton alleviating nutrient limitation. The emergent resource acquisition strategies, enabled by distinct strain-specific migratory acclimatizing mechanisms, highlight the active role of the reconfigurable cytoplasmic LDs in guiding vertical movement. By uncovering the mechanistic coupling between dynamics of intracellular changes to physiologically-governed migration strategies, this work offers a tractable framework to delineate diverse strategies which phytoplankton may harness to maximize fitness and resource pool in nutrient-limited open oceans of the future.

One sentence summaryPhytoplankton harness reconfigurable lipid droplets to biomechanically tune migratory strategies in dynamic nutrient landscapes.

sentences: Evolutionary analysis of THAP9 transposase: conserved regions, novel motifs THAP9 is a transposable element-derived gene that encodes the THAP9 protein, which is homologous to the Drosophila P-element transposase (DmTNP) and can cut and paste DNA. However, the exact functional role of THAP9 is unknown. Here, we perform evolutionary analysis and extensive in silico characterization of THAP9, including predicting domains and putative post-translational modification sites. We predict previously unreported mammalian-specific post-translational modification sites that may play a role in the subcellular localization of THAP9. We also observe that although THAP9 has evolved under a strong pervasive purifying selection, yielding high conservation of THAP9, there are distinct class-specific conservation patterns of key functional residues in certain domains. Furthermore, investigation of THAP9 expression profiles in various cancer and matched normal datasets demonstrated underexpression and overexpression in testicular cancers and thymic epithelial tumors, respectively, thus suggesting a possible role of THAP9 in cancer.

sentences: Genomic environments scale the activities of diverse core promoters A classical model of gene regulation is that enhancers provide specificity while core promoters provide a modular site for the assembly of the basal transcriptional machinery. However, examples of core promoter specificity have led to an alternate hypothesis in which specificity is achieved by core promoters with different sequence motifs that respond differently to genomic environments containing different enhancers and chromatin landscapes. To distinguish between these models, we measured the activities of hundreds of diverse core promoters in four different genomic locations and, in a complementary experiment, six different core promoters at thousands of locations across the genome. While genomic locations had large effects on expression, the intrinsic activities of different classes of promoters were preserved across genomic locations, suggesting that core promoters are modular regulatory elements whose activities are independently scaled up or down by different genomic locations. This scaling of promoter activities is non-linear and depends on the genomic location and the strength of the core promoter. Our results support the classical model of regulation in which diverse core promoter motifs set the intrinsic strengths of core promoters, which are then amplified or dampened by the activities of their genomic environments.

sentences: Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is driven by differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network by defining cell-type-specific enhancers.

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Individualized precision targeting of dorsal attention and default mode networks with rTMS in traumatic brain injury-associated depression BackgroundAt the group level, antidepressant efficacy of rTMS targets is inversely related to their normative connectivity with subgenual anterior cingulate cortex (sgACC). Individualized connectivity may yield better targets, particularly in patients with neuropsychiatric disorders who may have aberrant connectivity. However, sgACC connectivity shows poor test-retest reliability at the individual level. Individualized resting-state network mapping (RSNM) can reliably map inter-individual variability in brain network organization.

ObjectiveTo identify individualized RSNM-based rTMS targets that reliably target the sgACC connectivity profile.

MethodsWe used RSNM to identify network-based rTMS targets in 10 healthy controls and 13 individuals with traumatic brain injury-associated depression (TBI-D). These "RSNM targets" were compared with consensus structural targets and targets based on individualized anti-correlation with a group-mean-derived sgACC region ("anti-group-mean sgACC targets"). The TBI-D cohort was randomized to receive active (n=9) or sham (n=4) rTMS to RSNM targets.

ResultsThe group-mean sgACC connectivity profile was reliably estimated by individualized correlation with default mode network (DMN) and anti-correlation with dorsal attention network (DAN). Individualized RSNM targets were then identified based on DAN anti-correlation and DMN correlation. Counterintuitively, anti-correlation with the group-mean sgACC connectivity profile was stronger and more reliable for RSNM-derived targets than for "anti-group-mean sgACC targets". Improvement in depression after RSNM-targeted rTMS was predicted by target anti-correlation with the portions of sgACC. Active treatment led to increased connectivity within and between several relevant regions.

ConclusionsRSNM may enable reliable individualized rTMS targeting, although further research is needed to determine whether this personalized approach can improve clinical outcomes.

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Epidermal basal domains organization highlights skin robustness to environmental exposure Adult interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here we elucidate the molecular and cellular states of basal IFE domains by marker expression and single cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that reflect in part the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Second, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains: adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Synopsis

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sentences: Prefrontal top-down projections control context-dependent strategy selection The rules governing behavior often vary with behavioral contexts. As a consequence, an action rewarded in one context may be discouraged in another. Animals and humans are capable of switching between behavioral strategies under different contexts and acting adaptively according to the variable rules, a flexibility that is thought to be mediated by the prefrontal cortex (PFC)1-4. However, how the PFC orchestrates context-dependent switch of strategies remains unclear. Here we show that pathway-specific projection neurons in the medial PFC (mPFC) differentially contribute to context-instructed strategy selection. In a decision-making task in which mice have been trained to flexibly switch between a previously established rule and a newly learned rule in a context-dependent manner, the activity of mPFC neurons projecting to the dorsomedial striatum encodes the contexts, and further represents decision strategies conforming to the old and new rules. Moreover, the activity of these neuron is required for context-instructed strategy selection. In contrast, the activity of mPFC neurons projecting to the ventral midline thalamus does not discriminate between the contexts, and represents the old rule even if mice have adopted the new one; furthermore, these neurons act to prevent the strategy switch under the new rule. Our results suggest that the mPFC[->]striatum pathway promotes flexible strategy selection guided by contexts, whereas the mPFC[->]thalamus pathway favors fixed strategy selection by preserving old rules. Balanced activity between the two pathways may be critical for adaptive behaviors.

sentences: Continuous Theta Burst Stimulation to the Secondary Visual Cortex at 80% Active Motor Threshold Does Not Impair Central Vision in Humans During a Simple Detection Task Continuous theta burst stimulation (cTBS) is a powerful form of repetitive transcranial magnetic stimulation capable of suppressing cortical excitability for up to 50 min. A growing number of studies have applied cTBS to the visual cortex in human subjects to investigate the neural dynamics of visual processing, but few have specifically examined its effects on central vision, which has crucial implications for safety and inference on downstream cognitive effects. The present study assessed the safety of offline, neuronavigated cTBS to V2 by examining its effects on central vision performance. In this single-blind, randomized sham-controlled, crossover study, 17 healthy adults received cTBS (at 80% active motor threshold) and sham to V2 1-2 weeks apart. Their central vision ([≤]8{degrees}) was tested at 1-min (T1) and again at 50-min (T50) post-stimulation. Effects of condition (cTBS vs. sham) and time (T1 vs. T50) on accuracy and reaction time were examined using Bayes factor. Bayes factor results suggested that cTBS did not impair stimulus detection over the entire central visual field nor subfields at T1 or T50. Our results offer the first explicit evidence supporting that cTBS applied to V2 does not create blind spots in the central visual field in humans during a simple detection task. Any subtler changes to vision and downstream visual perception should be investigated in future studies.

sentences: Molecular basis for Ras suppressor-1 recruitment to focal adhesions and stabilization of consensus adhesome complex Ras suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating fundamental cell adhesion processes and tumor development. Rsu-1 interacts with a zinc-finger type multi LIM domain-containing adaptor protein PINCH-1 involved in the integrin-mediated consensus adhesome but not with highly homologous isoform PINCH-2. However, the structural basis for such specific interaction and regulatory mechanism remains unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture with eight LRRs shielded by the N- and C-terminal capping modules. We show that a large conserved concave surface of the Rsu-1 LRR domain recognizes the PINCH-1 LIM5 domain, and that the C-terminal non-LIM region of PINCH-2 but not PINCH-1 sterically disfavors the Rsu-1 binding. We further show that Rsu-1 can be assembled, via PINCH-1-binding, into a tight hetero-pentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2 that constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Consistently, our mutagenesis and cell biological data consolidate the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading. Our results provide a crucial molecular insight into Rsu-1-mediated cell adhesion with implication on how it may regulate tumorigenic growth.

sentences: An RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans reveals the involvement of unexpected processes Aberration in nuclear morphology is one of the hallmarks of cellular transformation. However, the processes that, when mis-regulated, result aberrant nuclear morphology are poorly understood. In this study we carried out a systematic, high-throughput RNAi screen for genes that affect nuclear morphology in Caenorhabditis elegans embryos. The screen employed over 1700 RNAi constructs against genes required for embryonic viability. Nuclei of early embryos are typically spherical and their NPCs are evenly distributed. The screen was performed on early embryos expressing a fluorescently tagged component of the nuclear pore complex (NPC), allowing visualization of nuclear shape as well as the distribution of NPCs around the nuclear envelope. Our screen uncovered 182 genes whose down-regulation resulted in one or more abnormal nuclear phenotypes, including multiple nuclei, micronuclei, abnormal nuclear shape, anaphase bridges and abnormal NPC distribution. Many of these genes fall into common functional groups, including some that were not previously known to affect nuclear morphology, such as genes involved in mitochondrial function, the vacuolar ATPase and the CCT chaperonin complex. The results of this screen add to our growing knowledge of processes that affect nuclear morphology and that may be altered in cancer cells that exhibit abnormal nuclear shape.

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Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor, respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into [~]74-nm compartments where transferrin receptor and {beta}3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). {beta}3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix.

SummaryAn ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. reveals that the focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion, and integrin {beta}3 molecules become temporarily immobilized at the islands.

sentences: GAGAM: a genomic annotation-based enrichment of scATAC-seq data for Gene Activity Matrix Single-cell Assay for Transposase Accessible Chromatin using sequencing (scATAC-seq) is rapidly becoming a powerful technology to assess the epigenetic landscape of thousands of cells. However, the current great sparsity of the resulting data poses significant challenges to their interpretability and informativeness. Different computational methods are available, proposing ways to generate significant features from accessibility data and process them to obtain meaningful results. In particular, the most common way to interpret the raw scATAC-seq data is through peak-calling, generating the peaks as features. Nevertheless, this method is dataset-dependent because the peaks are related to the given dataset and can not be directly compared between different experiments. For this reason, this study wants to improve on the concept of the Gene Activity Matrix (GAM), which links the accessibility data to the genes, by proposing a Genomic-Annotated Gene Activity Matrix (GAGAM), which aims to label the peaks and link them to the genes through functional annotation of the whole genome. Using genes as features solves the problem of the feature dataset dependency allowing for the link of gene accessibility and expression. The latter is crucial for gene regulation understanding and fundamental for the increasing impact of multi-omics data. Results confirm that our method performs better than the previous GAMs.

sentences: In situ structures of membrane-assisted assembly and selective autophagy of enteroviruses Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle.

sentences: Out of the blue: the independent activity of sulfur-oxidizers and diatoms mediate the sudden color shift of a tropical river Rio Celeste ("Sky-Blue River") is a river located in the Tenorio National Park (Costa Rica) that has become an important hotspot for eco-tourism due to its striking sky-blue color. A previous study suggested that this color is not caused by dissolved chemical species, but by precipitation of lightscattering aluminosilicate particles at the mixing point of two colorless streams, the acidic Quebrada Agria and the neutral Rio Buenavista. We now present microbiological information on Rio Celeste and its two tributaries, as well as a more detailed characterization of the particles that occur at the mixing point. Our results overturn the previous belief that the light scattering particles are formed by the aggregation of smaller particles coming from Rio Buenavista, and rather point to chemical formation of hydroxyaluminosilicate colloids with Quebrada Agria as the main contributor to the phenomenon, with Rio Buenavista acting as a secondary source of silica to the reaction. We also show how the sky-blue color of Rio Celeste arises from the tight interaction between chemical and biological processes. Sulfur-oxidizing bacteria generate an acidic environment in Quebrada Agria, which in turn cause dissolution and mobilization of aluminum and other metals, while in Rio Buenavista the growth of diatoms transforms dissolved silicon into colloidal biogenic forms. The local interaction between these two well-known biological activities gives rise to the unique Rio Celeste phenomenon, in what constitutes a textbook example of emergent behavior in environmental microbiology.

sentences: Sequencing of individual barcoded cDNAs on Pacific Biosciences and Oxford Nanopore reveals platform-specific error patterns Long-read transcriptomics requires understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform comparison method that combined barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences and Oxford Nanopore. We compared these long reads pairs in terms of sequence content and splicing structure. Although individual read pairs show high similarity, we found differences in (i) aligned length, (ii) TSS and (iii) polyA-site assignment, and (iv) exon-intron structures. Overall 25% of read pairs disagreed on either TSS, polyA-site, or a splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison revealed that inconsistencies are often caused by sequencing-error induced inaccurate ONT alignments, especially to downstream GTNNGT donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and thus likely real. In both barcoded and non-barcoded ONT reads, we found that intron number and proximity of other GT/AGs better predict inconsistency with the annotation than read quality alone. We summarized these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

sentences: An alignment free approach confirms semantic properties of species proteomes Alignment-based methods dominate molecular biology. However, by primarily allowing one-to-one comparisons, these methods are focused on a gene-centered viewpoint and lack the broad context essential to analyze how complex biological systems function and evolve. In actuality, a gene is part of genome where more than one sequence contributes to the functional network and evolutionary trajectory of the cell. The need for conservation of established interactions, is arguably more important to the evolutionary success of species than conservation of individual function. To test whether such contextual information exists, a distributional semantics method - Latent Semantic Analysis (LSA), was applied to thousands of species proteomes. Using natural language processing, Latent Taxonomic Signatures (LTSs) were identified that outperformed existing alignment-based BLAST methods when random protein sequences were being mapped to annotated taxonomy according to GenBank. LTSs are a novel proteome distributed feature, suggesting the existence of evolutionary constraints imposed on individual proteins by their proteome context. Even orphan proteins are exhibiting LTSs, which makes their uniqueness linked to a specific taxonomic level questionable. Unlike more simple bias, LTSs represent a self-similarity pattern, where random sets of species proteins show the same statistical properties of a complete proteome at many scales. Natural language processing and machine learning provide insights not easily discernable using alignment based methods suggestive there is more to species related differences than just translational optimization.

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Garlic-derived organosulfur compounds regulate metabolic and immune pathways in macrophages and attenuate intestinal inflammation in mice ScopeGarlic is a source of bioactive phytonutrients that may have anti-inflammatory or immunomodulatory properties. The mechanism(s) underlying the bioactivity of these compounds and their ability to regulate responses to enteric infections remains unclear.

Methods and ResultsWe investigated if a garlic-derived preparation (PTSO-PTS) containing two organosulfur metabolites, propyl-propane thiosulfonate (PTSO) and propyl-propane thiosulfinate (PTS), regulated inflammatory responses in murine macrophages and intestinal epithelial cells (IEC) in vitro, as well as in a model of enteric parasite-induced inflammation. PTSO-PTS decreased lipopolysaccharide-induced secretion of TNF, IL-6 and IL-27 in macrophages. RNA-sequencing demonstrated that PTSO-PTS strongly suppressed pathways related to immune and inflammatory signaling. PTSO-PTS induced the expression of a number of genes involved in antioxidant responses in IEC during exposure to antigens from the parasite Trichuris muris. In vivo, PTSO-PTS did not affect T. muris establishment or intestinal T-cell responses but significantly altered caecal transcriptomic responses. Notably, a reduction in T. muris-induced expression of Tnf, Saa2 and Nos2 was observed.

ConclusionGarlic-derived organosulfur compounds exert anti-inflammatory effects in macrophages and IEC, and regulate gene expression during intestinal infection. These compounds and related organic molecules may thus hold potential as functional food components to improve gut health in humans and animals.

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

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

sentences: No Evidence for a Single Oscillator Underlying Discrete Visual Percepts Theories of perception based on discrete sampling posit that visual consciousness is reconstructed based on snapshot-like perceptual moments, as opposed to being updated continuously. According to a model proposed by Schneider (2018), discrete sampling can explain both the flash-lag and the Frohlich illusion, whereby a lag in the conscious updating of a moving stimulus alters its perceived spatial location in comparison to a stationary stimulus. The alpha-band frequency, which is associated with phasic modulation of stimulus detection and the temporal resolution of perception, has been proposed to reflect the duration of perceptual moments. The goal of this study was to determine whether a single oscillator (e.g., alpha) is underlying the duration of perceptual moments, which would predict that the point of subjective equality (PSE) in the flash-lag and Frohlich illusions are positively correlated across individuals. Although our displays induced robust flash-lag and Frohlich effects, virtually zero correlation was seen between the PSE in the two illusions, indicating that the illusion magnitudes are unrelated across observers. These findings suggest that, if discrete sampling theory is true, these illusory percepts either rely on different oscillatory frequencies or not on oscillations at all. Alternatively, discrete sampling may not be the mechanism underlying these two motion illusions or our methods were ill-suited to test the theory.

sentences: The importance of m6A topology in chicken embryo mRNA; a precise mapping of m6A at the conserved chicken β-actin zipcode N6-Methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken {beta}-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody independent assay. We also demonstrated that methylation of this site in the {beta}-actin zipcode enhances ZBP1 binding in vitro, whilst methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localised translation of {beta}-actin mRNA, and the ability of m6A to enhance or inhibit a reader proteins RNA binding highlights the importance of m6A detection at nucleotide resolution.

sentences: Inside-out tracking and projection mapping for robot-assisted transcranial magnetic stimulation Transcranial Magnetic Stimulation (TMS) is a neurostimulation technique based on the principle of electromagnetic induction of an electric field in the brain with both research and clinical applications. To produce an optimal neuro-modulatory effect, the TMS coil must be placed on the head and oriented accurately with respect to the region of interest within the brain. A robotic method can enhance the accuracy and facilitate the procedure for TMS coil placement. This work presents two system improvements for robot-assisted TMS (RA-TMS) application. Previous systems have used outside-in tracking method where a stationary external infrared (IR) tracker is used as a reference point to track the head and TMS coil position. This method is prone to losing track of the coil or the head if the IR camera is blocked by the robotic arm during its motion. To address this issue, we implemented an inside-out tracking method by mounting a portable IR camera on the robot end-effector. This method guarantees that the line of sight of the IR camera is not obscured by the robotic arm at any time during its motion. We also integrated a portable projection mapping device (PPMD) into the RA-TMS system to provide visual guidance during TMS application. PPMD can track the head via an IR tracker, and can project a planned contact point of the TMS coil on the head or overlay the underlying brain anatomy in real-time.

sentences: Phenotypic shifts of tumor associated macrophages and STAT3 mediated suppression of myeloid derived suppressor cells drive sensitization of HER2+ tumor immunity. Understanding how novel therapeutic combinations alter solid tumor microenvironments (TME) in immunosuppressive tumors such as breast cancer is essential to improve their responses to immune checkpoint inhibitors (ICIs). Entinostat, an oral histone deacetylase inhibitor (HDACi), has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs, but the precise alterations induced by entinostat and mechanisms of synergy with ICIs remain unknown. Here, we employ single-cell RNA-sequencing on HER2 overexpressing breast tumors from mice treated with entinostat + ICIs to characterize these changes across cell types in the TME. This analysis demonstrates that treatment with entinostat induces a shift from a pro-tumor to an anti-tumor TME signature characterized predominantly by changes in the myeloid cells. Notably, myeloid-derived suppressor cells (MDSCs) are shifted toward the less suppressive granulocytic phenotype in association with reduced signaling through the STAT3 pathway. In addition, tumor-associated macrophages are shifted toward an anti-tumor M1 phenotype by epigenetic reprogramming. Overall, these entinostat-induced TME changes reduce immunosuppression and increase mechanisms of tumor cell killing to improve ICI responses and broaden the population of patients who could potentially benefit from immunotherapy.

sentences: A multiscale atlas of the molecular and cellular architecture of the human kidney The human kidney is composed of over 26 cell types that actively coordinate with each other to form higher-order structures, such as the nephron. It is not yet understood how these structures vary throughout a single organ or amongst the same organs within the human population. We have developed an extensive lipid and cellular atlas of the human kidney consisting of over 3 million cells comprising 75,000 functional tissue units (i.e., glomeruli, proximal tubules, distal tubules, and collecting ducts) from 13 human subjects. This atlas was developed using spatially registered and integrated technologies consisting of imaging mass spectrometry, multiplexed immunofluorescence, stained microscopy, and autofluorescence microscopy, to comprehensively probe large (i.e., centimeter-sized) areas of tissue. The cellular organization and lipid profiles of glomeruli, proximal tubules, distal tubules, and collecting ducts were discovered through these multimodal imaging data as well as their intra- and inter-subject variance. Relating the lipid profiles obtained from imaging mass spectrometry to distinct cell types obtained from immunofluorescence allowed us to hypothesize the functional role of specific phospholipids that have not previously been described. These hypotheses include subject characteristics, such as BMI and sex. The integrated data from the aforementioned datasets provide a valuable reference for kidney researchers, are publicly available through the NIH Human Biomolecular Atlas Program (https://portal.hubmapconsortium.org/), and discussed below.

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Comparative genomics of Bordetella pertussis isolates from New Zealand, a country with an uncommonly high incidence of whooping cough 2.Whooping cough, the respiratory disease caused by Bordetella pertussis, has undergone a wide-spread resurgence over the last several decades. Previously, we developed a pipeline to assemble the repetitive B. pertussis genome into closed sequences using hybrid nanopore and Illumina sequencing. Here, this sequencing pipeline was used to conduct a more high-throughput, longitudinal screen of 66 strains isolated between 1982 and 2018 in New Zealand. New Zealand has a higher incidence of whooping cough than many other countries; usually at least twice as many cases per 100,000 people as the USA and UK and often even higher, despite similar rates of vaccine uptake. To the best of our knowledge, these strains are the first New Zealand B. pertussis isolates to be sequenced. The analyses here show that, on the whole, genomic trends in New Zealand B. pertussis isolates, such as changing allelic profile in vaccine-related genes and increasing pertactin deficiency, have paralleled those seen elsewhere in the world. At the same time, phylogenetic comparisons of the New Zealand isolates with global isolates suggest that a number of strains are circulating in New Zealand which cluster separately from other global strains, but which are closely related to each other. The results of this study add to a growing body of knowledge regarding recent changes to the B. pertussis genome, and are the first genetic investigation into B. pertussis isolates from New Zealand.

3. Impact statementSince the 1990s, whooping cough has been resurgent in many countries around the world, despite the wide availability of vaccines. New Zealand has often had a higher incidence of whooping cough than other countries such as the USA, UK and Australia, both during outbreak periods and in the intervening years. One potential reason for the resurgence of whooping cough is genetic changes to the causative bacterium, Bordetella pertussis, with several recently identified, ongoing global genetic trends. No B. pertussis isolates from New Zealand have previously been sequenced, however. Here, we used hybrid sequencing to investigate the genomes of 66 New Zealand B. pertussis isolates, collected between 1982 and 2018. This revealed that genetic trends in New Zealand B. pertussis match those observed elsewhere, but over the years a number of highly similar or identical strains appear to have circulated (or are currently circulating) in the country, a phenomenon not commonly noted elsewhere. This first study of B. pertussis isolates from New Zealand contributes to the global understanding of B. pertussis genomics, as well as providing the groundwork for any future whole genome sequencing of New Zealand B. pertussis isolates.

4. Data summaryO_LINanopore and Illumina fastq sequence files for all strains have been deposited in NCBIs Sequence Read Archive, BioProject PRJNA556977. A full list of accession numbers for all sequence read files is provided in Supplementary Table S1.
C_LIO_LIGenome sequences for 63 strains have been deposited in NCBIs GenBank, BioProject PRJNA556977, accession numbers in Supplementary Table S1.
C_LIO_LIGenome assemblies for 3 strains assembled using only nanopore data (NZ1, NZ5 and NZ29), which had a high number of pseudogenes, were not deposited in GenBank, but are available from Figshare: https://doi.org/10.6084/m9.figshare.12640463
C_LIO_LISource code and full commands used are available from Github: https://github.com/nataliering/Comparative-genomics-of-Bordetella-pertussis-isolates-from-New-Zealand
C_LI

The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

sentences: An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor, to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

sentences: Can offsetting the energetic cost of hibernation restore an active season phenotype in grizzly bears (Ursus arctos horribilis)? Hibernation is characterized by suppression of many physiological processes. To determine if this state is reversible in a non-food caching species, we fed hibernating grizzly bears (Ursus arctos horribilis) glucose for 10 days to replace 53% or 100% of the estimated minimum daily energetic cost of hibernation. Feeding caused serum concentrations of glycerol and ketones ({beta}-hydroxybutyrate) to return to active season levels irrespective of the amount of glucose fed. By contrast, free-fatty acids and indices of metabolic rate, such as general activity, heart rate, and strength of the daily heart rate rhythm and insulin sensitivity were restored to roughly 50% of active season levels. Body temperature was unaffected by feeding. To determine the contribution of adipose to these metabolic effects of glucose feeding we cultured bear adipocytes collected at the beginning and end of the feeding and performed metabolic flux analysis. We found a roughly 33% increase in energy metabolism after feeding. Moreover, basal metabolism before feeding was 40% lower in hibernation cells compared to fed cells or active cells cultured at 37{degrees}C, thereby confirming the temperature independence of metabolic rate. The partial suppression of circulating FFA with feeding likely explains the incomplete restoration of insulin sensitivity and other metabolic parameters in hibernating bears. Further suppression of metabolic function is likely an active process. Together, the results provide a highly controlled model to examine the relationship between nutrient availability and metabolism on the hibernation phenotype in bears.

sentences: isoformant: A visual toolkit for reference-free long-read isoform analysis at single-read resolution isoformant is an analytical toolkit for isoform characterization of Oxford Nanopore Technologies (ONT) long-transcript sequencing data (i.e. direct RNA and cDNA). Deployment of these tools using Jupyter Notebook enables interactive analysis of user-defined region-of-interest (ROI), typically a gene. The core module of isoformant clusters sequencing reads by k-mer density to generate isoform consensus sequences without the requirement for a reference genome or prior annotations. The inclusion of differential isoform usage hypothesis testing based on read distribution among clusters enables comparison across multiple samples. Here, as proof-of-principle, we demonstrate the utility of isoformant for analyzing isoform diversity of commercially-available isoform standard mixtures. isoformant is available here: https://github.com/danledinh/isoformant.

sentences: A large and diverse autosomal haplotype is associated with sex-linked colour polymorphism in the guppy Colour polymorphism provides a tractable trait that can be harnessed to explore the evolution of sexual selection and sexual conflict. Male colour patterns of the Trinidadian guppy (Poecilia reticulata) are governed by both natural and sexual selection, and are typified by extreme pattern colour variation as a result of negative frequency dependent selection. Since guppy colour patterns are often inherited faithfully from fathers to sons, it has been historically presumed that colour genes are physically linked to sex determining loci as a  supergene on the sex chromosome. Yet the actual identity and genomic location of the colour pattern genes has remained elusive. We phenotyped and genotyped four guppy  Iso-Y lines, where colour was inherited along the patriline, but backcrossed into the stock population every 2 to 3 generations for 40 generations, thereby homogenising the genome at regions unrelated to colour. Using an unbiased phenotyping method to proportion colour pattern differences between and among the Iso-Y lines, we confirmed that the breeding design was successful in producing four distinct colour patterns. Our analysis of genome resequencing data of the four Iso-Y lines uncovered a surprising genetic architecture for colour pattern polymorphism. Genetic differentiation among Iso-Y lines was repeatedly associated with a large and diverse haplotype ([~]5Mb) on an autosome (LG1), not the sex chromosome (LG12). Moreover, the LG1 haplotype showed elevated linkage disequilibrium and exhibited evidence of sex-specific diversity when we examined whole-genome sequencing data of the natural source population. We hypothesise that colour pattern polymorphism is driven by Y-autosome epistasis, and conclude that predictions of sexual conflict should focus on incorporating the effects of epistasis in understanding complex adaptive architectures.

sentences: CD20+ T cells are associated with inflammatory responses in experimental arthritis. CD20+ T cells comprise a small but highly inflammatory subset that has been implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterise the CD20+ T cell subset at the site of inflammation in murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice. In addition, compared to naive mice and those that did not develop clinical symptoms, CD20 expressing T cells of arthritic mice produced increased levels of pro-inflammatory cytokines (GM-CSF, TNF-a, IL-17, and INF-g). Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells of disease mice were enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells that have been implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Importantly, CD3+CD20+ T cells were detected in the inflamed regions in the lymph nodes and paws of arthritic mice. Our findings suggest that CD20+ T cells are associated with inflammatory responses in the arthritic joint and may exacerbate pathology by promoting inflammatory B cell responses.

sentences: Zoogeographic patterns on very small spatial scales in rock-dwellingPlectostoma snails from Borneo(Gastropoda: Caenogastropoda: Diplommatinidae) We mapped the fine-grained distribution patterns of Plectostoma microsnails on two small isolated limestone outcrops in Malaysian Borneo. On both hills, two species were present (P. simplex and P. concinnum on Tandu Batu; P. fraternum and P. concinnum on Tomanggong Besar), but the patterns we found were different. On Tomanggong Besar, the two species occupy different parts of the hill and meet along a narrow hybrid zone that is characterised by a significantly higher rate of predation by Atopos slugs. On Tandu Batu, the two species broadly overlap and do not form hybrids. The predation rate here is the same in both species, regardless of whether they occur in monospecific localities or in mixed localities. Our results show that even small limestone outcrops of a few hundred m in diameter cannot be considered to be uniformly populated by limestone dwelling snails, and a detailed analysis of intra-hill patterns may reveal information on species differences and maintenance.

sentences: Addressing Enzymatic-Independent Tumor-Promoting Function of NAMPT via PROTAC-Mediated Degradation The rate-limiting enzyme of salvage pathway for NAD+ synthesis, NAMPT, is aberrantly overexpressed in a variety of tumor cells and is a poor prognosis factor for patient survival. NAMPT plays a major role in tumor cell proliferation, acting concurrently as an NAD+ synthase and unexpectedly, an extracellular ligand for several tumor-promoting signaling pathways. While previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers a possibility to simultaneously disrupt NAMPTs enzyme activity and ligand capabilities. Here, we report the development of two highly selective NAMPT-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent NAMPT degradation in a cereblon-dependent manner in multiple tumor cell lines. Notably, both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a better strategy for targeting proteins like NAMPT with dual tumor-promoting functions, which are not easily achieved by conventional enzymatic inhibitors.

sentences: Spinosaurids as 'subaqueous foragers' undermined by selective sampling and problematic statistical inference Fabbri et al.1 claim that the huge sail-backed dinosaur Spinosaurus aegyptiacus and the spinosaurid Baryonyx were "subaqueous foragers," diving underwater in pursuit of prey, based on their measure of bone "compactness." Using thin-sections and computed tomographic (CT) scans of thigh bone (femur) and trunk rib from various living and extinct vertebrates, they claim to be able to distinguish taxa with "aquatic habits" from others. Their conclusions are undermined by selective bone sampling, inaccuracies concerning spinosaurid bone structure, faulty statistical inferences, and novel redefinition of the term "aquatic."

sentences: REPP: A robust cross-platform solution for online sensorimotor synchronization experiments Sensorimotor synchronization (SMS), the rhythmic coordination of perception and action, is a fundamental human skill that supports many behaviors, from daily repetitive routines to the most complex behavioural coordination, including music and dance (Repp 2005; Repp & Su, 2013). Research on SMS has been mostly conducted in the laboratory using finger tapping paradigms, where participants typically tap with their index finger to a rhythmic sequence of auditory stimuli. However, these experiments require equipment with high temporal fidelity to capture the asynchronies between the time of the tap and the corresponding cue event. Thus, SMS is particularly challenging to study with online research, where variability in participants hardware and software can introduce uncontrolled latency and jitter into recordings. Here we present REPP (Rhythm ExPeriment Platform), a novel technology for measuring SMS in online experiments that can work efficiently using the built-in microphone and speakers of standard laptop computers. The audio stimulus (e.g., a metronome or a music excerpt) is played through the speakers and the resulting signal is recorded along with participants responses in a single channel. The resulting recording is then analyzed using signal processing techniques to extract and align timing cues with high temporal accuracy. In this paper, we validate REPP through a series of calibration and behavioural experiments. We demonstrate that our technology achieves high temporal accuracy (latency and jitter within 2 ms on average), high test-retest reliability both in the laboratory (r = .87) and online (r = .80), and high concurrent validity (r = .94). We also show that REPP is fully automated and customizable, enabling researchers to monitor experiments in real time and to implement a wide variety of SMS paradigms. We discuss methods for ensuring high recruiting efficiency and data quality, including pre-screening tests and automatic procedures for quality monitoring. REPP can therefore open new avenues for research on SMS that would be nearly impossible in the laboratory, reducing experimental costs while massively increasing the reach, scalability and speed of data collection.

sentences: Range expansion shifts clonal interference patterns in evolving populations Increasingly, predicting and even controlling evolutionary processes is a sought after goal in fields ranging from agriculture, artificial intelligence, astrobiology, oncology, and infectious diseases. However, our ability to predict evolution and plan such interventions in real populations is limited in part by our understanding of how spatial structure modulates evolutionary dynamics. Among current clinical assays applied to predict drug response in infectious diseases, for instance, many do not explicitly consider spatial structure and its influence on phenotypic heterogeneity, despite it being an inextricable characteristic of real populations. As spatially structured populations are subject to increased interference of beneficial mutants compared to their well-mixed counter-parts, among other effects, this population heterogeneity and structure may non-trivially impact drug response. In spatially-structured populations, the extent of this mutant interference is density dependent and thus varies with relative position within a meta-population in a manner modulated by mutant frequency, selection strength, migration speed, and habitat length, among other factors. In this study, we examine beneficial mutant fixation dynamics along the front of an asexual population expanding its range. We observe that multiple distinct evolutionary regimes of beneficial mutant origin-fixation dynamics are maintained at characteristic length scales along the front of the population expansion. Using an agent-based simulation of range expansion with mutation and selection in one dimension, we measure these length scales across a range of population sizes, selection strengths, and mutation rates. Furthermore, using simple scaling arguments to adapt theory from well-mixed populations, we find that the length scale at the tip of the front within which  local mutant fixation occurs in a successive mode decreases with increasing mutation rate, as well as population size in a manner predicted by our derived analytic expression. Finally, we discuss the relevance of our findings to real cellular populations, arguing that this conserved region of successive mutant fixation dynamics at the wave tip can be exploited by emerging evolutionary control strategies.

sentences: Multiscale modeling of tumor adaption and invasion following anti-angiogenic therapy In order to promote continued growth, a tumor must recruit new blood vessels, a process known as tumor angiogenesis. Many therapies have been tested that aim to inhibit tumor angiogenesis, thus starving the tumor of nutrients and preventing tumor growth. However, many of these therapies have been unsuccessful and can paradoxically further tumor development by leading to increased local tumor invasion and metastasis. In this study, we use agent-based modeling to examine how hypoxic and acidic conditions following anti-angiogenic therapy can influence tumor development. Under these conditions, we find that cancer cells experience a phenotypic shift to a state of higher survival and invasive capability, spreading further away from the tumor into surrounding tissue. Although anti-angiogenic therapy alone promotes tumor cell adaptation and invasiveness, we find that augmenting chemotherapy with anti-angiogenic therapy improves chemotherapeutic response and delays the time it takes for the tumor to regrow. Overall, we use computational modeling to explain the behavior of tumor cells in response to anti-angiogenic treatment in the dynamic tumor microenvironment.

sentences: Optogenetic manipulation of individual or whole population Caenorhabditis elegans worms with an under hundred-dollar tool: the OptoArm AbstractOptogenetic tools have revolutionized the study of neuronal circuits in Caenorhabditis elegans. The expression of light-sensitive ion channels or pumps under specific promotors allows researchers to modify the behavior of excitable cells. Several optogenetic systems have been developed to spatially and temporally photoactivate light-sensitive actuators in C. elegans. Nevertheless, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, and adjustable optogenetics device for use on different microscopes and worm trackers, called the OptoArm. The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles and light-color. We demonstrate the OptoArms power in a population-based study on contributions of motor circuit cells to age-related motility decline. We find that functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit aging.

sentences: APRIL drives a co-ordinated but diverse response as a foundation for plasma cell longevity. Antibody secreting cells (ASCs) survive in niche microenvironments, but cellular responses driven by particular niche signals are incompletely defined. The TNF superfamily member APRIL provides a niche signal that can support the transition of transitory plasmablasts into long-lived plasma cells. Here we explore how APRIL helps to establish the biological programs that promote life in the niche, by studying the initial response of primary human plasmablast to APRIL. Under conditions allowing the maturation of ex vivo or in vitro generated plasmablasts, we find that APRIL drives activation of ERK, p38 and JNK. This is accompanied by a classical NF{kappa}B response. Under these conditions induction of AKT phosphorylation is also observed with similar kinetics, paralleled by FOXO1 phosphorylation and nuclear exclusion. Time course gene expression data resolve the downstream co-ordinated transcriptional response. The APRIL-signal propagates via immediate early genes and classical NF{kappa}B responsive targets to converge onto modules of MYC- and OCT2-regulated gene expression linked to cell growth, as well as leading to enhanced expression of ICAM1 and SQSTM1 associated with adhesion and metabolic/stress responses. Thus, APRIL drives a combination of multiple transcriptional programs that co-ordinate cell growth, stress response and adhesion in human ASCs, providing a broad foundation to support plasma cell longevity.

sentences: Peptidome profiling for the immunological stratification in sepsis: a proof of concept study Sepsis has been called "the graveyard of pharmaceutical companies" due to the numerous failed clinical trials. The lack of tools to monitor the immunological status in sepsis constrains the development of immunomodulatory therapies. Here, we evaluated a test based on whole plasma peptidome acquired in a MALDI-TOF-mass spectrometer used for bacterial biotyping and machine-learning algorithms to discriminate the different immunological phases of sepsis. In this proof of concept study, two discrete lipopolysaccharide-(LPS) induced murine models emulating the pro- and anti-inflammatory phases that occur during sepsis were evaluated. The LPS group was inoculated with a single high dose of LPS, recalling the proinflammatory phase, and the IS groups was subjected to increasing doses of LPS to induce the anti-inflammatory/immunosuppression phase. Unstimulated mice served as controls. Both experimental groups showed the hallmarks of pro- and anti- inflammatory phases respectively; the LPS group showed leukopenia and higher levels of cytokines and tissue damage markers, and the IS group showed neutrophilia, lymphopenia and significantly lower antibody titers upon immunization. Principal component analysis of the plasma peptidomes formed three discrete clusters that mostly coincided with the experimental groups. In addition, machine-learning algorithms discriminated the different experimental groups with a sensitivity and specificity of up to 95.7% and 90.9% respectively. Our data reveal the potential of plasma peptidome analysis by MALDI-TOF-mass spectrometry as a simple, speedy and readily transferrable method for sepsis patient stratification that would contribute to therapeutic decision-making based on their immunological status.

sentences: Between-subject correlation of heart rate variability predicts movie preferences We introduce a novel and simple method for assessing audiences emotional responses to audiovisuals (e.g. films). Viewers (N=21) watched movies and TV commercials from different genres while wearing photoplethysmography (PPG) optic sensors on their wrists. Heart rate variability (HRV) synchrony was observed among the audience. Based on this between-subject synchrony measure, we identified emotionally arousing segments from the materials. New participants (N=24; N=16) were then invited to watch these identified segments along with some randomly selected segments as control; they reported that the former was more engaging (effect size w=.67; w=.5). This finding was confirmed in an online study with a larger cohort (N=300). While some specific effects varied depending on movie genre or gender, HRV-based editing generally performed better than the control. These findings suggest that HRV synchrony can be used as a new tool for audience psychology, and potentially also for automatically creating short trailers out of movies in a principled manner while taking into account the human perspective.

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Virtual screening for small molecule pathway regulators by image profile matching Identifying chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual profile-based screening approach instead matches compounds to pathways based on phenotypic information in public cell image data, created using the Cell Painting assay. Our simple correlation-based computational strategy efficiently uncovered the expected small molecule regulators for 32% of positive control gene queries. In discovery mode, we identified compounds related to three of seven tested query genes, validated in subsequent gene-relevant assays. This image profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

One sentence summaryIf a genetic perturbation impacts cell morphology, a computational query can reveal compounds whose morphology "matches".

sentences: Trait-trait relationships and functional tradeoffs vary with genome size in prokaryotes We report genomic traits that have been associated with the life history of prokaryotes and highlight conflicting findings concerning earlier observed trait correlations and tradeoffs. In order to address possible explanations for these contradictions we examined trait-trait variations of 11 genomic traits from [~] 17,000 sequenced genomes. The studied trait-trait variations suggested: (i) the predominance of two resistance and resilience-related orthogonal axes, (ii) an overlap between a resilience axis and an axis of resource usage efficiency. These findings imply that resistance associated traits of prokaryotes are globally decoupled from resilience and resource use efficiencies associated traits. However, further inspection of pairwise scatterplots showed that resistance and resilience traits tended to be positively related for genomes up to roughly five million base pairs and negatively for larger genomes. This in turn precludes a globally consistent assignment of prokaryote genomic traits to the competitor - stress-tolerator -ruderal (CSR) schema that sorts species depending on their location along disturbance and productivity gradients into three ecological strategies and may serve as an explanation for conflicting findings from earlier studies. All reviewed genomic traits featured significant phylogenetic signals and we propose that our trait table can be applied to extrapolate genomic traits from taxonomic marker genes. This will enable to empirically evaluate the assembly of these genomic traits in prokaryotic communities from different habitats and under different productivity and disturbance scenarios as predicted via the resistance-resilience framework formulated here.

sentences: Technical advances in the development of zonation liver in vitro systems that incorporate localized Wnt activating signals A Wnt microenvironment sustained by the hepatic central vein is essential for the segregation of liver functions into zones. Current liver culture systems lack localized Wnt cues and as a consequence fail to maintain the hepatocyte functional heterogeneity that is observed in the intact organ. In this study, organoid models and 2D-culture systems were used to identify cellular sources and Wnt presentation methods that could support the future development of zonated liver in vitro systems. Using soluble ligands, we show that primary hepatocyte (PH)-derived organoids but not bile duct (BD)-derived organoids may be used to recapitulate the resting liver. We provide evidence that differentiation of PH-organoids in the presence of Wnt9b and Rspo3 induce pericentral maturation. Finally, we show that immobilization of Rspo3 onto beads in combination with soluble Wnt9b may be a valid strategy to recreate the central vein Wnt microenvironment in vitro.

sentences: BrainStat: A toolbox for brain-wide statistics and neuroscientific contextualization AO_SCPLOWBSTRACTC_SCPLOWAnalysis and interpretation of neuroimaging datasets has become a multidisciplinary endeavor, relying not only on statistical methods, but increasingly on associations with respect to other brain-derived features such as gene expression, histological data, and functional as well as cognitive architectures. Here, we introduce BrainStat - a toolbox for (i) univariate and multivariate linear models in volumetric and surface-based brain imaging datasets, and (ii) multidomain feature association of results with respect to spatial maps of post-mortem gene expression and histology, task-based fMRI meta-analysis, as well as resting-state fMRI motifs across several common surface templates. The combination of statistics and feature associations into a turnkey toolbox streamlines analytical processes and accelerates cross-modal research. The toolbox is implemented in both Python and MATLAB, two widely used programming languages in the neuroimaging and neuroinformatics communities. BrainStat is openly available and complemented by an expandable documentation.

sentences: Aberrant accumulation of NIK promotes tumorigenicity by dysregulating post-translational modifications in breast cancer Post-translational modifications and mRNA translation are frequently altered in human cancers. However, investigations to understand their roles in the cancer progression mechanism remain insufficient. In this research, we explored protein levels altered by translational or post-translational regulation by analyzing transcriptome and western blotting data of the highly malignant breast cancer cell lines. From these analyses, NIK was found to be upregulated at the protein level to predominantly activate the non-canonical NF-{kappa}B pathway in a breast cancer cell line. Furthermore, the increase in NIK protein production was attributed to the dysregulation of ubiquitin-proteasome system caused by a decrease in the translation of cIAP1. NIK upregulation contributed to tumorigenicity by regulating the expression of inflammatory response-related genes. Collectively, our study suggests that NIK is post-translationally modified and has the potential to be a therapeutic target and diagnostic marker for breast cancer.

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Nucleus accumbens neurons encode initiation and vigor of reward approach behavior The nucleus accumbens (NAc) is considered an interface between motivation and action, with NAc neurons playing an important role in promoting reward approach. However, the encoding by NAc neurons that contribute to this role remains unknown. Here, we trained male rats to find rewards in an 8-arm radial maze. The activity of 62 neurons, mostly in the shell of the NAc, were recorded while rats ran towards each reward place. General linear model (GLM) analysis showed that variables related to the vigor of the locomotor approach, like speed and acceleration, and the fraction of the approach run completed were the best predictors of the firing rate for most NAc neurons. Nearly 23% of the recorded neurons, here named locomotion-off cells, were inhibited during the entire approach run, suggesting that reduction in firing of these neurons promotes initiation of locomotor approach. Another 24% of the neurons presented a peak of activity during acceleration followed by a valley during deceleration (peak-valley cells). Together, these neurons accounted for most of the speed and acceleration encoding identified in the GLM analysis. Cross-correlations between firing and speed indicated that the spikes of peak-valley cells were followed by increases in speed, suggesting that the activity of these neurons drives acceleration. In contrast, a further 19% of neurons presented a valley during acceleration followed by a peak just prior to or after reaching reward (valley-peak cells). These findings suggest that these three classes of NAc neurons control the initiation and vigor of the locomotor approach to reward.

Significance StatementDeciphering the mechanisms by which the NAc controls the vigor of motivated behavior is critical to better understand and treat psychiatric conditions in which motivation is dysregulated. Manipulations of the NAc profoundly impair subjects ability to spontaneously approach reward-associated locations, preventing them from exerting effort to obtain reward. Here, we identify for the first time specific activity of NAc neurons in relation to spontaneous approach behavior. We discover three classes of neurons that could control initiation of movement and the speed vs. time trajectory during locomotor approach. These results suggest a prominent but heretofore unknown role for the NAc in regulating the kinematics of reward approach locomotion.

sentences: Zfp281 inhibits the pluripotent-to-totipotent state transition in mouse embryonic stem cells The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.

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Ecological memory of prior nutrient exposure in the human gut microbiome Many ecosystems retain an ecological memory of past conditions that affects responses to future stimuli. However, it remains unknown what mechanisms and dynamics may govern such a memory in microbial communities. Here, in both a human dietary intervention cohort and an artificial gut, we show that the human gut microbiome encodes a memory of past carbohydrate exposures. Changes in the relative abundance of primary degraders were sufficient to enhance metabolism, and these changes were mediated by transcriptional changes within hours of initial exposure. We further found that ecological memory of one carbohydrate species impacted metabolism of others. These findings demonstrate that the human gut microbiomes metabolic potential reflects dietary exposures over preceding days and changes within hours of exposure to a novel nutrient.

One Sentence SummaryRecent nutrient exposures are encoded into the structure and activity of human gut microbial communities, which enables more efficient future metabolic responses.

sentences: RNA-sequencing of human post-mortem hypothalamus and nucleus accumbens identifies expression profiles associated with obesity. Obesity, the accumulation of body fat to excess, may cause serious negative health effects, including increased risk of heart disease, type 2 diabetes, stroke and certain cancers. The biology of obesity is complex and not well understood, involving both environmental and genetic factors and affecting metabolic and endocrine mechanisms in tissues of the gut, adipose, and brain. Previous RNA sequencing studies have identified transcripts associated with obesity and body mass index in blood and fat, often using animal models, but RNA sequencing studies in human brain tissue related to obesity have not been previously undertaken. We conducted both large and small RNA sequencing of hypothalamus (207 samples) and nucleus accumbens (276 samples) from individuals defined as consistently obese (124 samples), consistently normal weight as controls (148 samples) or selected without respect to BMI and falling within neither case nor control definition (211 samples), based on longitudinal BMI measures. The samples were provided by three cohort studies with brain donation programs; the Framingham Heart Study (FHS), the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP). For each brain region and large/small RNA sequencing set, differential expression of obesity, BMI, brain region and sex was performed. Analyses were done transcriptome-wide as well as with a priori defined sets of obesity or BMI-associated mRNAs and microRNAs (miRNAs). There are sixteen mRNAs and five microRNAs that are differentially expressed (adjusted p < 0.05) by obesity or BMI in these tissues, several of which were validated with qPCR data. The results include many that are BMI-associated, such as APOBR and CES1, as well as many associated with the immune system and some with addiction, such as the gene sets "cytokine signaling in immune system" and "opioid signaling". In spite of the relatively large number of samples, our study was likely under-powered to detect other transcripts or miRNA with relevant but smaller effects.

sentences: Eukaryotic genomes from a global metagenomic dataset illuminate trophic modes and biogeography of ocean plankton Molecular and genomic approaches that target mixed microbial communities (e.g., metagenomics or metatranscriptomics) provide insight into the ecological roles, evolutionary histories, and physiological capabilities of the microorganisms and the processes in the environment. Computational tools that harness large-scale sequence surveys have become a valuable resource for characterizing the genetic make-up of the bacterial and archaeal component of the marine microbiome. Yet, fewer studies have focused on the unicellular eukaryotic fraction of the community. Here, we developed the EukHeist automated computational pipeline, to retrieve eukaryotic and prokaryotic metagenome assembled genomes (MAGs). We applied EukHeist to the eukaryote-dominated large-size fraction data (0.8-2000m) from the Tara Oceans survey to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of more than 900 eukaryotic MAGs representing environmentally-relevant microbial and multicellular eukaryotes in addition to over 4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs retrieved with EukHeist complement previous efforts by expanding the existing phylogenetic diversity through the increase in coverage of many likely particle- and host-associated taxa. We also demonstrate how the novel eukaryotic genomic content recovered from this study might be used to infer functional traits, such as trophic mode. By coupling MAGs and metatranscriptomic data, we explored ecologically-significant protistan groups, such as the Stramenopiles. A global survey of both eukaryotic and prokaryotic MAGs enabled the identification of ecological cohorts, driven by specific environmental factors, and putative host-microbe associations. Accessible and scalable computational tools, such as EukHeist, are likely to accelerate the identification of meaningful genetic signatures from large datasets, ultimately expanding the eukaryotic tree of life.

sentences: Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

sentences: Tyr is Responsible for the Cctq1a QTL and Links Developmental Environment to Central Corneal Thickness Determination Central corneal thickness is a quantitative trait with important associations to human health. In a phenotype-driven approach studying corneal thickness of congenic derivatives of C57BLKS/J and SJL/J mice, the critical region for a quantitative trait locus influencing corneal thickness, Cctq1a, was delimited to a 10-gene interval. Exome sequencing, RNAseq, and studying independent mutations eliminated multiple candidate genes and confirmed one. Though the causative gene, Tyr, has no obvious direct function in the transparent cornea, studies with multiple alleles on matched genetic backgrounds, both in isolation and genetic complementation crosses, confirmed allelism of Tyr-Cctq1a; albino mice lacking Tyr function had thin corneas. Albino mice also had increased axial length. Because albinism exposes eyes to increased light, the effect of dark-rearing was tested and found to rescue central corneal thickness. In sum, the results point to an epiphenomenon; developmental light exposure interacts with genotype as an important determinate of adult corneal thickness.

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The role of competition versus cooperation in microbial community coalescence New microbial communities often arise through the mixing of two or more separately assembled parent communities, a phenomenon that has been termed "community coalescence". Understanding how the interaction structures of complex parent communities determine the outcomes of coalescence events is an important challenge. While recent work has begun to elucidate the role of competition in coalescence, that of cooperation, a key interaction type commonly seen in microbial communities, is still largely unknown. Here, using a general consumer-resource model, we study the combined effects of competitive and cooperative interactions on the outcomes of coalescence events. In order to do so, we simulate coalescence events between pairs of communities with different degrees of competition for shared carbon resources and cooperation through cross-feeding on leaked metabolic by-products (facilitation). We also study how structural and functional properties of post-coalescence communities evolve when they are subjected to repeated coalescence events. We find that in coalescence events, the less competitive and more cooperative parent communities contribute a higher proportion of species to the new community, because this endows superior ability to deplete resources and resist invasions. Consequently, when a community is subjected to repeated coalescence events, it gradually evolves towards being less competitive and more cooperative, as well as more species rich, robust and efficient in resource use. Encounters between microbial communities are becoming increasingly frequent as a result of anthropogenic environmental change, and there is great interest in how the coalescence of microbial communities affects environmental and human health. Our study provides new insights into the mechanisms behind microbial community coalescence, and a framework to predict outcomes based on the interaction structures of parent communities.

Author summaryIn nature, new microbial communities often arise from the fusion of whole, previously separate communities (community coalescence). Despite the crucial role that interactions among microbes play in the dynamics of complex communities, our ability to predict how these affect the outcomes of coalescence events remains limited. Here, using a general mathematical model, we study how the structure of species interactions confers an advantage upon a microbial community when it encounters another, and how communities evolve after undergoing repeated coalescence events. We find that competitive interactions between species preclude their survival upon a coalescence event, while cooperative interactions are advantageous for post-coalescence survival. Furthermore, after a community is exposed to many coalescence events, the remaining species become less competitive and more cooperative. Ultimately, this drives the community evolution, yielding post-coalescence communities that are more species-rich, productive, and resistant to invasions. There are many potential environmental and health implications of microbial community coalescence, which will benefit from the theoretical insights that we offer here about the fundamental mechanisms underlying this phenomenon.

sentences: Novel method for the extraction of DNA from Lamniformes tooth and denticle enamel Suitable for PCR Extraction of high-quality genomic DNA from Lamniformes tooth fragment and dermal denticle enamel is discussed as a method of identifying individual sharks. We describe a procedure that permits isolation of genomic DNA of satisfactory size and quality for PCR analysis, as well as for most routine cloning applications. This method should allow for the non-invasive collection of genomic samples from Lamniformes.

sentences: Mapping Molecular Gene Signatures Among Respiratory Viruses Based on Large-Scale and Genome-wide Transcriptomics Analysis Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging RNA virus causing COVID-19 disease across the globe. SARS-CoV-2 infected patients exhibit acute respiratory distress syndrome which can be compounded by endemic respiratory viruses and thus highlighting the need to understand the genetic bases of clinical outcome under multiple respiratory infections. In this study, 42 individual datasets and a multi-parametric based selected list of over 12,000 genes against five medically important respiratory viruses (SARS-CoV-2, SARS-CoV-1, influenza A, respiratory syncytial virus (RSV) and rhinovirus were collected and analysed in an attempt to understand differentially regulated gene patterns and to cast genetic markers of individual and multiple co-infections. While a certain cohort of virus-specific genes were regulated (negatively and positively), notably results revealed a greatest correlation among gene regulation by SARS-CoV-2 and RSV. Furthermore, out of analysed genes, the MAP2K5 and NFKBIL1 were specifically and highly upregulated in SARS-CoV-2 infection in vivo or in vitro. In contrast, several genes including GPBAR1 and SC5DL were specifically downregulated in SARS-CoV-2 datasets. Additionally, we catalogued a set of genes that were conserved or differentially regulated across all the respiratory viruses. These finding provide foundational and genome-wide data to gauge the markers of respiratory viral infections individually and under co-infection.

sentences: Differences in nanoscale organization of DNase I hypersensitive and insensitive chromatin in single human cells Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. By using a combination of high throughput super-resolution microscopy and coarse-grained modelling we investigated properties of active and inactive chromatin in interphase nuclei. Using DNase I hypersensitivity as a criterion, we have selected prototypic active and inactive regions from ENCODE data that are representative for K-562 and more than 150 other cell types. By using oligoFISH and automated STED microscopy we systematically measured physical distances of the endpoints of 5kb DNA segments in these regions. These measurements result in high-resolution distance distributions which are right-tailed and range from very compact to almost elongated configurations of more than 200 nm length for both the active and inactive regions. Coarse-grained modeling of the respective DNA segments suggests that in regions with high DNase I hypersensitivity cell-to-cell differences in nucleosome occupancy determine the histogram shape. Simulations of the inactive region cannot sufficiently describe the compaction measured by microscopy, although internucleosomal interactions were elevated and the linker histone H1 was included in the model. These findings hint at further organizational mechanisms while the microscopy-based distance distribution indicates high cell-to-cell differences also in inactive chromatin regions. The analysis of the distance distributions suggests that direct enhancer-promoter contacts, which most models of enhancer action assume, happen for proximal regulatory elements in a probabilistic manner due to chromatin flexibility.

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Linked machine learning classifiers improve species classification of fungi when using error-prone long-reads on extendedmetabarcodes BackgroundThe increased usage of error-prone long-read sequencing for metabarcoding of fungi has not been matched with adequate public databases and concomitant analysis approaches. We address this gap and present a proof-of-concept study for classifying fungal taxa using linked machine learning classifiers. We demonstrate the capability of linked machine learning classifiers to accurately classify species and strains using real-world and simulated fungal ribosomal DNA datasets, including plant and human pathogens. We benchmark our new approach in comparison to current alignment and k-mer based methods based on synthetic mock communities. We also assess real world applications of species identification in complex unlabelled datasets.

ResultsOur machine learning approach assigned individual nanopore long-read amplicon sequences to fungal species with high recall rates and low false positive rates. Importantly, our approach successfully distinguished between closely-related species and strains when individual read errors were higher than the genetic distance between individual taxa, which the alignment and k-mer methods could not do. The machine learning approach showed an ability to identify key species with high recall rates, even in complex samples of unknown species composition.

ConclusionsA proof of concept machine learning approach using a tree-descent approach on a decision tree of classifiers can identify known taxa with high accuracy, and precisely detect known target species from complex samples with high recall rates. We propose this approach is suitable for detecting the known knowns of pathogens or invasive species in any environment of mostly unknown composition, including agriculture and wild ecosystems.

sentences: ZBTB18 inhibits SREBP-dependent fatty acid synthesis by counteracting CTBPs and KDM1A/LSD1 activity in glioblastoma Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. Our study points at CTBP1/2 and LSD1 as co-activators of SREBP genes whose complex functional activity is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3 active marks leading to increased di-methylation of lysine 4 (H3K4me2). Concomitantly, we observed increased di-methylation of lysine 9 (H3K9me2), and decrease of the active mark H3K4me3 with consequent repression of the SREBP genes. In line with our findings, lipidomic analysis shows a reduction of several phospholipid species upon ZBTB18 expression. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its cofactors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

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Gene exchange networks define species-like units in marine prokaryotes Post-submission note. Since the original submission of this manuscript to bioRxiv, we discovered that some of our results may be impacted by the limitations of some of the comparative genomics tools used in this study. We are working on a revised version of the manuscript.

Although horizontal gene transfer is recognized as a major evolutionary process in Bacteria and Archaea, its general patterns remain elusive, due to difficulties tracking genes at relevant resolution and scale within complex microbiomes. To circumvent these challenges, we analyzed a randomized sample of >12,000 genomes of individual cells of Bacteria and Archaea in the tropical and subtropical ocean - a well-mixed, global environment. We found that marine microorganisms form gene exchange networks (GENs) within which transfers of both flexible and core genes are frequent, including the rRNA operon that is commonly used as a conservative taxonomic marker. The data revealed efficient gene exchange among genomes with <28% nucleotide difference, indicating that GENs are much broader lineages than the nominal microbial species, which are currently delineated at 4-6% nucleotide difference. The 42 largest GENs accounted for 90% of cells in the tropical ocean microbiome. Frequent gene exchange within GENs helps explain how marine microorganisms maintain millions of rare genes and adapt to a dynamic environment despite extreme genome streamlining of their individual cells. Our study suggests that sharing of pangenomes through horizontal gene transfer is a defining feature of fundamental evolutionary units in marine planktonic microorganisms and, potentially, other microbiomes.

sentences: Nucleosome-directed replication origin licensing independent of a consensus DNA sequence The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c.. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with new analysis of genome-wide ORC binding, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

sentences: Cepo uncovers cell identity through differential stability We present Cepo, a method to generate cell-type-specific gene statistics of differentially stable genes from single-cell RNA-sequencing (scRNA-seq) data to define cell identity. Cepo outperforms current methods in assigning cell identity and enhances several cell identification applications such as cell-type characterisation, spatial mapping of single cells, and lineage inference of single cells.

sentences: Thermal adaptation and fatty acid profiles of bone marrow and muscles in mammals: implications of a study of caribou (Rangifer tarandus caribou) Mammals have evolved several physiological mechanisms to cope with changes in ambient temperature. Particularly critical among them is the process of keeping cells in a fluid phase to prevent metabolic dysfunction. In this paper, we examine variation in the fatty acid composition of bone marrow and muscle tissues in the cold-adapted caribou (Rangifer tarandus caribou) to determine whether there are systematic differences in fatty acid profiles between anatomical regions that could potentially be explained by thermal adaptation. Our results indicate that the bone marrow and muscle tissues from the appendicular skeleton are more unsaturated than the same tissues in the axial skeleton, a finding that is consistent with physiological adaptation of the appendicular regions to thermal challenges. Because mechanisms of thermal adaptation appear to be widely shared among terrestrial mammals, we suggest that the same patterns may prevail in other species, possibly including humans.

sentences: Complex Activity and Short-term Memories in Reciprocally Connected Cerebral Organoids Macroscopic axonal connections in the human brain distribute information and neuronal activity across the brain. Although brain organoid technologies have recently provided novel avenues to investigate human brain function by replicating small neuronal networks of the brain in vitro, functionality of the macroscopic connections between distant regions has not been investigated with organoids. Here, we describe the neural activity of human cerebral organoids reciprocally connected by a bundle of axons. The connected organoids produced significantly more intense and complex oscillatory activity than conventional cerebral organoids. Optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the connected organoids exhibit plasticity as a neuronal network. Our findings highlight functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. can allow researchers to develop and test compounds to modulate activity of in vitro neuronal network models in unprecedented ways.

sentences: Effect of stimulation time on the expression of human macrophage polarization markers Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1{beta}, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGF{beta}) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

sentences: A CTDNEP1-lipin 1-mTOR regulatory network restricts ER membrane biogenesis to enable chromosome motions necessary for mitotic fidelity The endoplasmic reticulum (ER) dramatically restructures in open mitosis to become excluded from the mitotic spindle; however, the significance of ER reorganization to mitotic progression is not known. Here, we demonstrate that limiting ER membrane biogenesis enables mitotic chromosome movements necessary for chromosome biorientation and prevention of micronuclei formation. Aberrantly expanded ER membranes increase the effective viscosity of the mitotic cytoplasm to physically restrict chromosome dynamics - slowed chromosome motions impede correction of mitotic errors induced by transient spindle disassembly, leading to severe micronucleation. We define the mechanistic link between regulation of ER membrane biogenesis and mitotic fidelity by demonstrating that a CTDNEP1-lipin 1-mTOR regulatory network limits ER lipid synthesis to prevent chromosome missegregation. Together, this work shows that ER membranes reorganize in mitosis to enable chromosome movements necessary for mitotic error correction and reveal dysregulated lipid metabolism as a potential source of aneuploidy in cancer cells.

sentences: Neurogenesis and angiogenesis are associated features of aggressive breast cancer The tumor microenvironment (TME) is important for cancer growth and progress. Whereas angiogenesis is an established hallmark of cancer, the field of cancer neuroscience is now emerging. Here, we asked whether neurogenesis and angiogenesis are associated features in breast cancer and related to aggressive behavior. Single cell-based spatial analysis by imaging mass cytometry (IMC) indicated significant colocalization of neural and vascular structures, suggesting the presence of neuro-vascular niches within the tumor tissue. By protein and gene expression data from human breast cancer cohorts, we found that neurogenesis and angiogenesis, together with a consolidated neuro-angiogenic signature score, were linked to high-grade breast cancer characteristics and reduced survival, also within the luminal tumor subgroup. Cases with high neuro-angiogenic score were split in two subgroups, one hormone receptor negative and one receptor positive. Our findings might be relevant for improved patient stratification and further exploration of novel treatment strategies.

sentences: Reexamining Waddington: Canalization and new mutations are not required for the evolution of genetic assimilation Over 65 years ago, Waddington demonstrated ancestrally phenotypically plastic traits can evolve to become constitutive, a process he termed genetic assimilation. Genetic assimilation evolves rapidly, assumed to be in large part due to segregating genetic variation only expressed in rare/novel environments, but otherwise phenotypically cryptic. Despite previous work suggesting a substantial role of cryptic genetic variation contributing to the evolution of genetic assimilation, some have argued for a prominent role for new mutations of large effect concurrent with selection. Interestingly, Waddington was less concerned by the relative contribution of CGV or new variants, but aimed to test the role of canalization, an evolved form of robustness. While canalization has been extensively studied, its role in the evolution of genetic assimilation is disputed, in part because explicit tests of evolved robustness are lacking. To address these questions, we recreated Waddingtons selection experiments on an environmentally sensitive change in Drosophila wing morphology (crossvein development), using many independently evolved replicate lineages. Using these, we show that 1) a polygenic CGV, but not new variants of large effect are largely responsible for the evolved response demonstrated using both genomic and genetic approaches. 2) Using both environmental manipulations and mutagenesis of the evolved lineages that there is no evidence for evolved changes in canalization contributing to genetic assimilation. Finally, we demonstrate that 3) CGV has potentially pleiotropic and fitness consequences in natural populations and may not be entirely "cryptic".

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Quantitative measurement of antibiotic resistance in Mycobacterium tuberculosis reveals genetic determinants of resistance and susceptibility in a target gene approach The World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.

This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

sentences: Simulation of foraging behavior using a decision-making agent with Bayesian and inverse Bayesian inference: Levy and Brownian walk-like search patterns derived from differences in prey distributions It has been stated that in human migratory behavior, the step length series may have temporal correlation and that there is some relationship between this time dependency and the fact that the frequency distribution of step length follows a power-law distribution. Furthermore, the frequency of occurrence of the step length in some large marine organisms has been found to switch between power-law and exponential distributions, depending on the difficulty of prey acquisition. However, to date it has not been clarified how the aforementioned three phenomena arise: the positive correlation created in the step length series, the relation between the positive correlation of the step length series and the form of an individuals step length distribution, and the switching between power-law and exponential distributions depending on the abundance of prey. This study simulated foraging behavior using the Bayesian decision-making agent simultaneously performing both knowledge learning and knowledge-based inference to analyze how the aforementioned three phenomena arise. In the agent with learning and inference, past experiences were stored as hypotheses (knowledge) and they were used in current foraging behavior; at the same time, the hypothesis continued to be updated based on new experiences. The simulation results show that the agent with both learning and inference has a mechanism that simultaneously causes all the phenomena.

sentences: The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite Malaria pathology is caused by the exponential replication of Plasmodium parasites in the blood stream. The bottleneck of the parasite life cycle is the invasion of erythrocytes immediately after parasites egress from infected red blood cells. DNA damage-inducible protein 1 (Ddi1) is a conserved eukaryotic proteasome shuttle protein containing an internal retroviral-like protease domain. Using conditional genetics, we now show that the proteolytic activity of the P. falciparum homologue, PfDdi1, is critically required for invasion of red blood cells. Furthermore, PfDdi1 disruption results in the accumulation of highly polyubiquitinated proteins that can be processed by purified PfDdi1 or distant eukaryotic homologues. We also show that PfDdi1 interacts with multiple components of the ubiquitin-proteasome system and that parasites lacking PfDdi1 are more sensitive to proteasome inhibition. Overall, this study establishes PfDdi1 as a key component of the eukaryotic ubiquitin-proteasome system and as a promising antimalarial target.

sentences: The functional connectivity of the human claustrum according to the Human Connectome Project database The claustrum is an irregular and fine sheet of grey matter in the basolateral telencephalon present in almost all mammals. The claustrum has been the object of several studies using animal models and more recently in human beings using neuroimaging. Believed to be involved in cognition and disease such as fear recognition, suppression of natural urges, multisensory integration, conceptual integration, seizures, multiple sclerosis, and Parkinsons disease. Nevertheless, the function of the claustrum still remains unclear. We present a functional connectivity study of the claustrum in order to identify its main networks. Resting state functional and anatomical MRI data from 100 healthy subjects were analyzed; taken from the Human Connectome Project (HCP, NIH Blueprint: The Human Connectome Project), with 2x2x2 mm3 voxel resolution. Positive functional connectivity was found (p<0.05, FDR corrected) between the claustrum and the insula, anterior cingulate cortex, pre-central and postcentral gyrus, superior temporal gyrus, and subcortical areas. Our findings coincide with the results previously reported in both animal models and studies with humans. Showing the claustrum as a well-connected structure not only structurally but also functionally. Evidencing the claustrum as a node participating in different neural networks.

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Ancestral contributions to contemporary European complex traits The contemporary European genetic makeup formed in the last 8000 years as the combination of three main genetic components: the local Western Hunter-Gatherers, the incoming Neolithic Farmers from Anatolia and the Bronze Age component from the Pontic Steppes. When meeting into the post-Neolithic European environment, the genetic variants accumulated during their three distinct evolutionary histories mixed and came into contact with new environmental challenges.

Here we investigate how this genetic legacy reflects on the complex trait landscape of contemporary European populations, using the Estonian Biobank as a case study.

For the first time we directly connect the phenotypic information available from biobank samples with the genetic similarity to these ancestral groups, both at a genome-wide level and focusing on genomic regions associated with each of the 27 complex traits we investigated. We also found SNPs connected to pigmentation, cholesterol, sleep, diastolic blood pressure, and body mass index (BMI) to show signals of selection following the post Neolithic admixture events. We recapitulate existing knowledge about pigmentation traits, corroborate the connection between Steppe ancestry and height and highlight novel associations. Among others, we report the contribution of Hunter Gatherer ancestry towards high BMI and low blood cholesterol levels.

Our results show that the ancient components that form the contemporary European genome were differentiated enough to contribute ancestry-specific signatures to the phenotypic variability displayed by contemporary individuals in at least 11 out of 27 of the complex traits investigated here.

sentences: Biophysical demonstration of co-packaging of glutamate and GABA in individual synaptic vesicles in the central nervous system Many mammalian neurons release multiple neurotransmitters to activate diverse classes of ionotropic receptors on their postsynaptic targets. Entopeduncular nucleus somatostatin (EP Sst+) neurons that project to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear if these are packaged into the same or segregated pools of synaptic vesicles. Here we describe a novel method combining electrophysiology, spatially-patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA corelease. We find that the properties of PSCs elicited in LHb neurons by optogenetic activation of EP Sst+ terminals are only consistent with co-packaging of glutamate and GABA into individual vesicles. Furthermore, serotonin, which acts presynaptically to weaken EP Sst+ to LHb synapses, does so by altering the release probability of vesicles containing both transmitters. Our approach is broadly applicable to the study of multi-transmitter neurons throughout the brain and our results constrain mechanisms of neuromodulation in LHb.

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rbioacc: an R-package to analyse toxicokinetic data O_LI rbioacc is an  R-package dedicated to the analysis of experimental data collected from bioaccumulation tests during which organisms are exposed to a chemical (exposure phase) and then put into a clean media (depuration phase). Internal concentrations are regularly measured over time all along the experiment.
C_LIO_LI rbioacc provides ready-to-use functions to visualize and fully analyze such data. Under a Bayesian framework, this package fits a generic one-compartment toxicokinetic (TK) model automatically built from the data. It provides TK parameter estimates (appropriate uptake and elimination rates) and bioaccumulation metrics (e.g., BCF, BSAF, BMF). All parameter estimates, bioaccumulation metrics as well as predictions of internal concentrations into organisms are delivered with their uncertainty.
C_LIO_LIBioaccumulation metrics are primarily provided in support of environmental risk assessment, in full compliance with regulatory requirements required to approve marketing applications of chemical substances.
C_LIO_LIThis paper gives brief worked examples of the use of  rbioacc from data collected through standard bioaccumulation tests, and publicly available within the scientific literature. These examples constitute step-by-step user-guides to analyze any new data set, uploaded in the right format.
C_LI

sentences: Light sets the brain's daily clock by regional quickening and slowing of the molecular clockworks at dawn and dusk How daily clocks in the brain are set by light to local environmental time and encode the seasons is not fully understood. The suprachiasmatic nucleus (SCN) is a central circadian clock that orchestrates physiology and behavior in tune with daily and seasonal light cycles. Here we have found that optogenetically simulated light input to explanted SCN changes the waveform of the molecular clockworks from sinusoidal in free-running conditions to highly asymmetrical shapes with accelerated synthetic (rising) phases and extended degradative (falling) phases marking clock advances and delays at simulated dawn and dusk. Daily waveform changes are sufficient to entrain to simulated winter and summer photoperiods, and to non-24h periods. SCN imaging further reveals that acute waveform shifts are greatest in the ventrolateral SCN, while period effects are greatest in the dorsomedial SCN. Thus, circadian entrainment is encoded by SCN clock gene waveform changes that arise from spatiotemporally distinct intrinsic responses within the SCN neural network.

sentences: Single-cell resolution of MET and EMT programs during zebrafish fin regeneration Zebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA-sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1, a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.

sentences: Chemical characterization; Antimicrobial and Larvicidal activity of essential oil from Callistemon citrinus (Bottle brush) leaves Mosquito-borne diseases are prevalent in more than 100 countries across the world. They are the major vectors for transmission of Malaria, dengue, yellow fever, filariasis, schistosomiasis, Japanese encephalitis etc. Many of the formerly employed insecticides in mosquito control have harmful effects on human health and other non-target populations, their non-biodegradable nature, the higher rate of bio-magnification in our ecosystem and increasing insecticide resistance on global scale are raising serious concerns. . Therefore, search for natural, eco-friendly alternatives such as bio-insecticides is imperative. In this study, Larvicidal activity of the essential oil obtained from the leaves of Callistemon citrinus was tested on Dengue vector mosquito Aedes aegypti & Chikungunya vector mosquito Culex sp. Also, the chemical composition of the essential oil was recorded using GC-MS analysis. The anti-microbial activity of the essential oil was checked against a few common bacteria and fungi. Callistemon citrinus comes out to be one of such bio-insecticides with many therapeutic active constituents, showing appreciable anti-microbial activity and 80-100% larvicidal activity.

sentences: A Self-attention Graph Convolutional Network for Precision Multi-tumour Early Diagnostics with DNA Methylation Data DNA methylation data-based precision tumour early diagnostics is emerging as the state of the art for molecular tumour recognition, which could capture the signals of cancer occurrence 3~5 years in advance and clinically more homogenous groups. However, the sensitive of early detection for many tumors is about 30%, which needs to be greatly improved. Nevertheless, on the basis of the whole genome bisulfite sequencing methylation data, a comprehensive characterisation of the entire molecular genetic landscape of the tumor as well as the subtle differences between different tumours could be identified. With the accumulation of methylation data, high performance deep learning models that considering and modeling more unbiased information need to be developed. According to the above analysis, we designed a pipeline to investigate genome-wide DNA methylation patterns for precision multi-tumour early diagnostics. We proposed a graph convolutional network considering the attention mechanism to dissect DNA methylation heterogeneity of different cancer types. The attention mechanism in the graph convolutional network architecture could detect the cancer signals from genome-wide methylated molecular interactions, improving the robustness and sensitivity of multi-tumor classification. Experimental results demonstrates the feasibility of precision multi-tumour early diagnostics with the DNA methylation data. The workflow presented here is very useful for tumor classification, which highly relevant for the future blood diagnosis and treatment of the tumour.

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Host-symbiont population genomics provide insights into partner fidelity, transmission mode and habitat adaptation in deep-sea hydrothermal vent snails Symbiont specificity, both at the phylotype and strain level, can have profound consequences for host ecology and evolution. However, except for insights from a few model symbiosis systems, the degree of partner fidelity and the influence of host versus environmental factors on symbiont composition are still poorly understood. Nutritional symbioses between invertebrate animals and chemosynthetic bacteria at deep-sea hydrothermal vents are examples of relatively selective associations, where hosts affiliate only with particular, environmentally acquired phylotypes of gammaproteobacterial or campylobacterial symbionts. In hydrothermal vent snails of the sister genera Alviniconcha and Ifremeria this phylotype specificity has been shown to play a role in habitat distribution and partitioning among different holobiont species. However, it is currently unknown if fidelity goes beyond species level associations that might influence genetic structuring, connectivity and habitat adaptation of holobiont populations. We used metagenomic analyses to assess sequence variation in hosts and symbionts and identify correlations with geographic and environmental factors. Our analyses indicate that host populations are not differentiated across a ~800 km gradient, while symbiont populations are clearly structured between vent locations due to a combination of neutral and selective processes. Overall, these results suggest that host individuals flexibly associate with locally adapted strains of their specific symbiont phylotypes, which supports a long-standing but untested paradigm of the benefits of horizontal transmission. Strain flexibility in these snails likely enables host populations to exploit a range of habitat conditions, which might favor wide-spread genetic connectivity and ecological resilience unless physical dispersal barriers are present.

Significance StatementSymbiont composition in horizontally transmitted symbioses is influenced by a combination of host genetics, environmental conditions and geographic barriers. Yet the relative importance of these factors and the effects of adaptive versus neutral evolutionary forces on symbiont population structure remain unknown in the majority of marine symbioses. To address these questions, we applied population genomic approaches in four species of deep-sea hydrothermal vent snails that live in obligate association with chemosynthetic bacteria. Our analyses show that host genetics plays a minor role compared to environment for symbiont strain composition despite specificity to symbiont species and corroborate a long-standing hypothesis that vent invertebrates affiliate with locally adapted symbiont strains to cope with the variable habitat conditions characterizing hydrothermal vents.

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Fine-scale spatial patterns of wildlife disease are common and understudied O_LIAll parasites are heterogeneous in space, yet little is known about the prevalence and scale of this spatial variation, particularly in wild animal systems. To address this question, we sought to identify and examine spatial dependence of wildlife disease across a wide range of systems.
C_LIO_LIConducting a broad literature search, we collated 31 such datasets featuring 89 replicates and 71 unique host-parasite combinations, only 51% of which had previously been used to test spatial hypotheses. We analysed these datasets for spatial dependence within a standardised modelling framework using Bayesian linear models, and we then meta-analysed the results to identify generalised determinants of the scale and magnitude of spatial autocorrelation.
C_LIO_LIWe detected spatial autocorrelation in 48/89 model replicates (54%) across 21/31 datasets (68%), spread across parasites of all groups. Even some very small study areas (under 0.01km2) exhibited substantial spatial variation.
C_LIO_LIDespite the common manifestation of spatial variation, our meta-analysis was unable to identify host-, parasite-, or sampling-level determinants of this heterogeneity across systems. Parasites of all transmission modes had easily detectable spatial patterns, implying that structured contact networks and susceptibility effects are potentially as important in spatially structuring disease as are environmental drivers of transmission efficiency.
C_LIO_LIOur findings demonstrate that fine-scale spatial patterns of infection manifest frequently and across a range of wild animal systems, and many studies are able to investigate them whether or not the original aim of the study was to examine spatially varying processes. Given the widespread nature of these findings, studies should more frequently record and analyse spatial data, facilitating development and testing of spatial hypotheses in disease ecology. Ultimately, this may pave the way for an a priori predictive framework for spatial variation in novel host-parasite systems.
C_LI

sentences: Gene family evolution underlies cell type diversification in the hypothalamus of teleosts Hundreds of cell types form the vertebrate brain, but it is largely unknown how similar these cellular repertoires are between or within species, or how cell type diversity evolves. To examine cell type diversity across and within species, we performed single-cell RNA sequencing of [~]130,000 hypothalamic cells from zebrafish (Danio rerio) and surface- and cave-morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which last shared a common ancestor over 150 million years ago. Orthologous cell types displayed differential paralogue expression that was generated by sub-functionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes, and characterized by the neo-functionalization of members of recently expanded or contracted gene families. Within species comparisons revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences between surface- and cave-morphs. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types, and reveal how gene family evolution and the neo- and sub-functionalization of paralogs contribute to cellular diversity.

sentences: AlphaFold2 transmembrane protein structure prediction shines Transmembrane (TM) proteins are major drug targets, indicated by the high percentage of prescription drugs acting on them. For a rational drug design and an understanding of mutational effects on protein function, structural data at atomic resolution are required. However, hydrophobic TM proteins often resist experimental structure determination and in spite of the increasing number of cryo-EM structures, the available TM folds are still limited in the Protein Data Bank. Recently, the DeepMinds AlphaFold2 machine learning method greatly expanded the structural coverage of sequences, with high accuracy. Since the employed algorithm did not take specific properties of TM proteins into account, the validity of the generated TM structures should be assessed. Therefore, we investigated the quality of structures at genome scales, at the level of ABC protein superfamily folds, and also in specific individual cases. We tested template-free structure prediction also with a new TM fold, dimer modeling, and stability in molecular dynamics simulations. Our results strongly suggest that AlphaFold2 performs astoundingly well in the case of TM proteins and that its neural network is not overfitted. We conclude that a careful application of its structural models will advance TM protein associated studies at an unexpected level.

sentences: DNA methylation Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-cell Heterogeneity The molecular and functional heterogeneity of pancreatic {beta}-cells is well recognized. Pancreatic islets harbor a small subset of {beta}-cells that co-express Tyrosine Hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of this sub-population within islets is essential for appropriate insulin secretion. However, the distinguishing characteristics of this subpopulation and the mechanisms that restrict TH expression in {beta}-cells are not known. Here, we define the specific molecular and metabolic characteristics of the TH+ {beta}-cells and show that TH expression in {beta}-cells is restricted by DNA methylation patterning during {beta}-cell lineage specification. Ablation of de novo DNA methyltransferase Dnmt3a in the pancreatic- and endocrine-progenitor lineages results in a dramatic increase in the proportion of TH+ {beta}-cells, while {beta}-cell specific ablation of Dnmt3a has no effect on this sub-population. We demonstrate that maintenance of Th promoter DNA methylation patterns is essential for its continued restriction in postnatal {beta}-cells, and that loss of DNA methylation dysregulates TH expression in {beta}-cells in response to chronic overnutrition, contributing to impairment of {beta}-cell identity. These data highlight the essential requirement of DNA methylation patterning in regulating endocrine cell fates, and reveal a novel role of DNA methylation in {beta}-cell heterogeneity.

sentences: Chigno/CG11180 and SUMO are Chinmo-Interacting Proteins with a Role in Drosophila Testes Stem Cells Maintenance of sexual identity on the cellular level ensures the proper function of sexually dimorphic genes expressed in the brain and gonads. Disruption of genes that regulate sex maintenance alters the cellular structure of these tissues and leads to infertility and diseases, such as diabetes, obesity, and gonadal cancers. Sex maintenance in the testis of Drosophila melanogaster depends on the previously identified gene chinmo (Chronologically inappropriate morphogenesis). Chinmos effect on testis differentiation has been investigated in detail, but there is still much to be elucidated about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (Ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmos interaction with CG11180 is SUMOdependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work indicates that SUMO functionally links Chinmo and CG11180 in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Childless Gambino (Chigno) for CG11180.

sentences: LysM receptors in Coffea arabica: identification, characterization, and gene expression in response to Hemileia vastatrix Pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) localized on the host plant cell wall. These receptors activate a broad-spectrum and durable defense, which are desired characteristics for disease resistance in plant breeding programs. In this study, candidate sequences for PRRs with lysin motifs (LysM) were investigated in the Coffea arabica genome. For this, approaches based on the principle of sequence similarity, conservation of motifs and domains, phylogenetic analysis, and modulation of gene expression in response to Hemileia vastatrix were used. The candidate sequences for PRRs in C. arabica (Ca1-LYP, Ca2-LYP, Ca1-CERK1, Ca2-CERK1, Ca-LYK4, Ca1-LYK5 and Ca2-LYK5) showed high similarity with the reference PRRs used: Os-CEBiP, At-CERK1, At-LYK4 and At-LYK5. Moreover, the ectodomains of these sequences showed high identity or similarity with the reference sequences, indicating structural and functional conservation. The studied sequences are also phylogenetically related to the reference PRRs described in Arabidopsis, rice, and other plant species. All candidates for receptors had their expression induced after the inoculation with H. vastatrix, since the first time of sampling at 6 hours post-inoculation (hpi). At 24 hpi, there was a significant increase in expression, for most of the receptors evaluated, and at 48 hpi, a suppression. The results showed that the candidate sequences for PRRs in the C. arabica genome display high homology with fungal PRRs already described in the literature. Besides, they respond to pathogen inoculation and seem to be involved in the perception or signaling of fungal chitin, acting as receptors or coreceptors of this molecule. These findings represent an advance in the understanding of the basal immunity of this species.

sentences: Population differentiation and structural variation in the genome of Manduca sexta across the United States Many species that are extensively studied in the laboratory are less well characterized in their natural habitat, and laboratory strains represent only a small fraction of the variation in a species genome. Here we investigate genomic variation in three natural North American populations of an agricultural pest and a model insect for many scientific disciplines, the tobacco hornworm (Manduca sexta). We show that hornworms from Arizona, Kansas, and North Carolina are genetically distinct, with Arizona being particularly differentiated from the other two populations using Illumina whole-genome resequencing. Peaks of differentiation exist across the genome, but here we focus in on the most striking regions. In particular, we identify two likely segregating inversions found in the Arizona population. One inversion on the Z chromosome may enhance adaptive evolution of the sex chromosome. The larger, autosomal inversion contains a pseudogene may be involved in the exploitation of a novel hostplant in Arizona, but functional genetic assays will be required to support this hypothesis. Nevertheless, our results reveal undiscovered natural variation and provide useful genomic data for both pest management and evolutionary genetics of this insect species.

sentences: Feasibility of Using Wearable EMG Armbands combined with Unsupervised Transfer Learning for Seamless Myoelectric Control Myoelectric control uses electromyography (EMG) signals as human-originated input to enable intuitive interfaces with machines. As such, recent rehabilitation robotics employs myoelectric control to autonomously classify user intent or operation mode using machine learning. However, performance in such applications inherently suffers from the non-stationarity of EMG signals across measurement conditions. Current laboratory-based solutions rely on careful, time-consuming control of the recordings or periodic recalibration, impeding real-world deployment. We propose that robust yet seamless myoelectric control can be achieved using a low-end, easy-to-"don" and "doff" wearable EMG sensor combined with unsupervised transfer learning. Here, we test the feasibility of one such application using a consumer-grade sensor (Myo armband, 8 EMG channels @ 200 Hz) for gesture classification across measurement conditions using an existing dataset: 5 users x 10 days x 3 sensor locations. Specifically, we first train a deep neural network using Temporal-Spatial Descriptors (TSD) with labeled source data from any particular user, day, or location. We then apply the Self-Calibrating Asynchronous Domain Adversarial Neural Network (SCADANN), which automatically adjusts the trained TSD to improve classification performance for unlabeled target data from a different user, day, or sensor location. Compared to the original TSD, SCADANN improves accuracy by 12{+/-}5.2% (avg{+/-}sd), 9.6{+/-}5.0%, and 8.6{+/-}3.3% across all possible user-to-user, day-to-day, and location-to-location cases, respectively. In one best-case scenario, accuracy improves by 26% (from 67% to 93%), whereas sometimes the gain is modest (e.g., from 76% to 78%). We also show that the performance of transfer learning can be improved by using a "better" model trained with "good" (e.g., incremental) source data. We postulate that the proposed approach is feasible and promising and can be further tailored for seamless myoelectric control of powered prosthetics or exoskeletons.

sentences: Context-dependent transcriptional regulation of microglial proliferation Microglia proliferation occurs during brain development and brain lesions, but how this is coordinated at the transcriptional level is not well understood. Here, we investigated transcriptional mechanisms underlying proliferation of mouse microglia during postnatal development and in adults in models of induced microglial depletion-repopulation and brain demyelination. While each proliferative subset displayed globally a distinct signature of gene expression, they also co-expressed a subgroup of 1,370 genes at higher levels than quiescent microglia. Furthermore, expression of these may be coordinated by one of two modes of regulation. A first mode augments expression of genes already expressed in quiescent microglia and is subject to regulation by Klf/Sp, Nfy, and Ets transcription factors. Alternatively, a second mode enables de novo transcription of cell cycle genes and requires additional regulatory input from Lin54 and E2f factors. Overall, proliferating microglia integrate regulation of cell cycle gene expression with their broader, context-dependent, transcriptional landscape.

sentences: Fuzzy RNA-recognition by the Trypanosoma brucei editosome The recognition of RNA-molecules by proteins and protein complexes is a critical step on all levels of gene expression. Typically, the generated ribonucleoprotein complexes rely on the binary interaction of defined RNA-sequences or precisely folded RNA-motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA-molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome we identified multiple similar but nonidentical RNA-motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.523 ({lambda}2). We establish that a synthetic 4V(3E)-RNA is sufficient to compete for the editosomal pre-mRNA binding site and that it is able to inhibit RNA-editing in vitro. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA-folding space by recognizing a fuzzy continuum of RNA-folds that fit a consensus graph-descriptor. This provides a mechanism on how the protein complex is able to bind the structurally pleomorphic pool of pre- and partially edited mRNAs. We speculate that other fuzzy RNA-recognition motifs exist especially for proteins that interact with multiple RNA-species.

sentences: Phenotype bias determines how RNA structures occupy the morphospace of all possible shapes Morphospaces representations of phenotypic characteristics are often populated unevenly, leaving large parts unoccupied. Such patterns are typically ascribed to contingency, or else to natural selection disfavouring certain parts of the morphospace. The extent to which developmental bias, the tendency of certain phenotypes to preferentially appear as potential variation, also explains these patterns is hotly debated. Here we demonstrate quantitatively that developmental bias is the primary explanation for the occupation of the morphospace of RNA secondary structure (SS) shapes. Upon random mutations, some RNA SS shapes (the frequent ones) are much more likely to appear than others. By using the RNAshapes method to define coarse-grained SS classes, we can directly compare the frequencies that non-coding RNA SS shapes appear in the RNAcentral database to frequencies obtained upon random sampling of sequences. We show that: a) Only the most frequent structures appear in nature; the vast majority of possible structures in the morphospace have not yet been explored. b) Remarkably small numbers of random sequences are needed to produce all the RNA SS shapes found in nature so far. c) Perhaps most surprisingly, the natural frequencies are accurately predicted, over several orders of magnitude in variation, by the likelihood that structures appear upon uniform random sampling of sequences. The ultimate cause of these patterns is not natural selection, but rather strong phenotype bias in the RNA genotype-phenotype map, a type of developmental bias or "findability constraint", which limits evolutionary dynamics to a hugely reduced subset of structures that are easy to "find".

sentences: Choice-selective sequences dominate in cortical relative to thalamic inputs to the nucleus accumbens, providing a potential substrate for credit assignment How are actions linked with subsequent outcomes to guide choices? The nucleus accumbens, which is implicated in this process, receives glutamatergic inputs from the prelimbic cortex and midline regions of the thalamus. However, little is known about whether and how representations differ across these input pathways. By comparing these inputs during a reinforcement learning task in mice, we discovered that prelimbic cortical inputs preferentially represent actions and choices, whereas midline thalamic inputs preferentially represent cues. Choice-selective activity in the prelimbic cortical inputs is organized in sequences that persist beyond the outcome. Through computational modeling, we demonstrate that these sequences can support the neural implementation of reinforcement learning algorithms, both in a circuit model based on synaptic plasticity, and one based on neural dynamics. Finally, we test and confirm predictions of our circuit models by direct manipulation of nucleus accumbens input neurons. Thus, we integrate experiment and modeling to suggest neural solutions for credit assignment.

sentences: Full-likelihood analysis of genomic data clarifies a complex history of species divergence and introgression in the erato-sara group of Heliconius butterflies AO_SCPLOWBSTRACTC_SCPLOWIntrogressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species makes estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood analysis methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ full-likelihood methods under the multispecies coalescent (MSC) model with and without gene flow to analyze the genomic data from six members of the erato-sara clade of Heliconius butterflies and infer the species phylogeny and cross-species introgression events. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of H. telesiphe, a likely hybrid speciation origin for H. hecalesia, and gene flow between sister species H. erato and H. himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the multispecies coalescent makes it possible to extract rich historical information of species divergence and gene flow from genomic data.

sentences: A muscle-related contractile tissue specified by MRTF-activity in Porifera Muscle-based movement is a hallmark of animal biology, but the evolutionary origins of myocytes - the cells that comprise muscle tissues - are unknown. Sponges (Porifera) provide an opportunity to reconstruct the earliest periods of myocyte evolution. Although sponges are believed to lack muscle, they are capable of coordinated whole-body contractions that purge debris from internal water canals. This behavior has been observed for decades, but their contractile tissues remain uncharacterized; it is an open question whether they have affinity to muscle or non-muscle contractile tissues in other animals. Here, we characterize the endothelial-like lining of water canals (the endopinacoderm) as a primary contractile tissue in the sponge Ephydatia muelleri. We find tissue-wide organization of contractile actin-bundles that contain striated-muscle myosin II and transgelin, and that contractions are regulated by the release of internal Ca2+ stores upstream of the myosin-light-chain-kinase (MLCK) pathway. Further, we show that the endopinacoderm is developmentally specified by myocardin-related transcription factor (MRTF) as part of an environmentally-inducible transcriptional complex that otherwise is known to function in muscle development, plasticity, and regeneration in other animals. We conclude that both muscle tissues and the endopinacoderm evolved from a common, multifunctional contractile tissue in the animal stem-lineage. Furthermore, as an actin-regulated force-sensor, MRTF-activity offers a mechanism for how water canals dynamically remodel in response to flow and can re-form normally from stem-cells in the absence of the intrinsic positional cues characteristic of embryogenesis in other animals.

sentences: Staying in touch: how highly specialised moth pollinators track host plant phenology in unpredictable climates For pollinating insects that visit just a single flowering species, the co-occurrence of flowers and insects in time is likely to have critical implications for both plant and pollinator. Insects often utilise diapause to persist through periods in which resources are unavailable, timing their re-emergence by responding to the same environmental cues as their host plants. The obligate pollination mutualisms (OPMs) between Epicephala moths (Gracillariidae) and their leaf flower host plants are some of the most specialised interactions between plants and insects. However, to date there have been very few studies of Epicephala moth lifecycles and none of how they synchronise their activity with the flowering of their host plants. Breynia oblongifolia (Phyllanthaceae) is known to be exclusively pollinated by two highly specific species of Epicephala moth (Gracillariidae). We surveyed populations of both the host plant and its pollinators over multiple years to determine their annual phenology and then modelled the climatic factors that drive their activity. Using our newly gained knowledge of moth and host plant phenology, we then looked for evidence of diapause at both the egg and pre-pupal stages. Our phenology surveys showed that although female flowers were present throughout the entire year, the abundance of flowers and fruits was highly variable between sites and strongly associated with local rainfall and photoperiod. Fruit abundance, but not flower abundance, was a significant predictor of adult Epicephala activity, suggesting that eggs or early instar larvae diapause within dormant flowers and emerge as fruits mature. Searches of overwintering flowers confirmed this, with many containing evidence of pollen and diapausing pollinators. We also observed the behaviour of adult Epicephala prior to pupation and found that ~10% of the Autumn emerging Epicephala enter diapause, eclosing to adulthood after 38-56 weeks. The remaining 90% of autumn emerging adults pupate directly with no diapause, suggesting a bet hedging strategy for adult emergence. As such, Epicephala moths appear to utilise diapause at multiple stages in their lifecycle, and possibly bet hedging, in order to deal with variable flowering phenology and climatic unpredictability.

sentences: Cooperative protein allosteric transition mediated by a fluctuating transmission network Allosteric communication between distant protein sites represents a key mechanism of biomolecular regulation and signal transduction. Compared to other processes such as protein folding, however, the dynamical evolution of allosteric transitions is still not well understood. As example of allosteric coupling between distant protein regions, we consider the global open-closed motion of the two domains of T4 lysozyme, which is triggered by local motion in the hinge region. Combining extensive molecular dynamics simulations with a correlation analysis of interresidue contacts, we identify a network of interresidue distances that move in a concerted manner. The cooperative process originates from a cogwheel-like motion of the hydrophobic core in the hinge region, which constitutes a flexible transmission network. Through rigid contacts and the protein backbone, the small local changes of the hydrophobic core are passed on to the distant terminal domains and lead to the emergence of a rare global conformational transition. As in an Ising-type model, the cooperativity of the allosteric transition can be explained via the interaction of local fluctuations.

sentences: Evolutionary transition from a single RNA replicator to a multiple replicator network In prebiotic evolution, self-replicating molecules are believed to have evolved into complex living systems by expanding their information and functions open-endedly. Theoretically, such evolutionary complexification could occur through successive appearance of novel replicators that interact with one another to form replication networks. Here we performed long-term evolution experiments using an RNA that replicates by a self-encoded RNA replicase. The RNA diversified into multiple coexisting host and parasite lineages, whose frequencies in the population initially fluctuated and gradually stabilized. The final population, comprising five RNA lineages, forms a replicator network with diverse interactions, including cooperation to help the replication of all other members. These results support the capability of molecular replicators to spontaneously develop complexity through Darwinian evolution, a critical step for the emergence of life.

sentences: Effects of early life adversity on immediate early gene expression: systematic review and 3-level meta-analysis of rodent studies Early-life adversity (ELA) causes long-lasting structural and functional changes to the brain, rendering affected individuals vulnerable to the development of psychopathologies later in life. Immediate-early genes (IEGs) provide a potential marker for the observed alterations, bridging the gap between activity-regulated transcription and long-lasting effects on brain structure and function. Several heterogeneous studies have used IEGs to identify differences in cellular activity after ELA; systematically investigating the literature is therefore crucial for comprehensive conclusions. Here, we performed a systematic review on 39 pre-clinical studies in rodents to study the effects of ELA on IEG expression. Females and IEGs other than cFos were investigated in only a handful of publications. We meta-analyzed publications investigating specifically cFos expression. ELA increased cFos expression after an acute stressor only if the animals (control and ELA) had experienced multiple negative life events. At rest, ELA increased cFos expression irrespective of other life events, suggesting that ELA creates a phenotype similar to naive, acutely stressed animals. We present a conceptual theoretical framework to interpret the unexpected results. Overall, ELA likely alters IEG expression across the brain, especially in interaction with other negative life events. The present review highlights current knowledge gaps and provides guidance to aid the design of future studies.

sentences: Long-term evolution of quantitative traits in the Drosophila melanogaster species subgroup Quantitative genetics aims at untangling the genetic and environmental effects on phenotypic variation. Trait heritability, which summarizes the relative importance of genetic effects, is estimated at the intraspecific level, but theory predicts that heritability could influence long-term evolution of quantitative traits. The phylogenetic signal concept bears resemblance to heritability and it has often been called species-level heritability. Under certain conditions, such as trait neutrality or contribution to phylogenesis, within-species heritability and between-species phylogenetic signal should be correlated. Here, we investigate the potential relationship between these two concepts by examining the evolution of multiple morphological traits for which heritability has been estimated in Drosophila melanogaster. Specifically, we analysed 42 morphological traits in both sexes on a phylogeny inferred from 22 nuclear genes for nine species of the melanogaster subgroup. We used Pagels {lambda} as a measurement of phylogenetic signal because it is the least influenced by the number of analysed taxa. Pigmentation traits showed the strongest concordance with the phylogeny, but no correlation was found between phylogenetic signal and heritability estimates mined from the literature. We obtained data for multiple climatic variables inferred from the geographical distribution of each species. Phylogenetic regression of quantitative traits on climatic variables showed a significantly positive correlation with heritability. Convergent selection, the response to which depends on the trait heritability, may have led to the null association between phylogenetic signal and heritability for morphological traits in Drosophila. We discuss the possible causes of discrepancy between both statistics and caution against their confusion in evolutionary biology.

sentences: Enhancer-promoter competition between homologous alleles leads to reduced transcription in early Drosophila embryos The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. Here, we ask if inter-allelic interactions between two homologous alleles can affect gene regulation. Using MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcription of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the hemizygous allele, suggesting the possibility of allelic competition in homozygotes. Moreover, the MS2-yellow reporter gene showed reduced transcriptional activity when a partial transcription unit (enhancer or promoter only) was in the homologous position. We propose that the transcriptional machinery that binds to both the enhancer and promoter region, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. To support this idea, we showed that the homologous alleles did not interfere with each other in earlier nuclear cycles when Pol II is in excess, while the degree of interference gradually increased in nuclear cycle 14. Such allelic competition was observed for endogenous snail as well. Our study provides new insights into the role of 3D inter-allelic interactions in gene regulation.

sentences: Intranasal immunization with a vaccinia virus vaccine vector expressing pre-fusion stabilized SARS-CoV-2 spike fully protected mice against lethal challenge with the heavily mutated mouse-adapted SARS2-N501YMA30 strain of SARS-CoV-2 The Omicron SARS-CoV-2 variant has been designated a variant of concern because its spike protein is heavily mutated. In particular, Omicron spike is mutated at 5 positions (K417, N440, E484, Q493 and N501) that have been associated with escape from neutralizing antibodies induced by either infection with or immunization against the early Washington strain of SARS-CoV-2. The mouse-adapted strain of SARS-CoV-2, SARS2-N501YMA30, contains a spike that is also heavily mutated, with mutations at 4 of the 5 positions in Omicron spike associated with neutralizing antibody escape (K417, E484, Q493 and N501). In this manuscript we show that intranasal immunization with a pre-fusion stabilized Washington strain spike, expressed from a highly attenuated, replication-competent vaccinia virus construct, NYVAC-KC, fully protected mice against disease and death from SARS2-N501YMA30. Similarly, immunization by scarification on the skin fully protected against death, but not from mild disease. This data demonstrates that Washington strain spike, when expressed from a highly attenuated, replication-competent poxvirus, administered without parenteral injection can fully protect against the heavily mutated mouse-adapted SARS2-N501YMA30.

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Sustainable use of plant protection products in heroic viticulture areas.Reducing risks to the environment and winemaking. ObjectiveThe aim and objective of this article is to focus on the importance of the rational use of plant protection products to combat vine fungi and at the same time reduce their risks to human health and the environment. The integrated management of fungal diseases aims to achieve the development of healthy crops with the minimum alteration of agro-ecosystems and the promotion of natural mechanisms.

Material and methodsThe study was carried out in one vineyard in Ribeira Sacra (North-West Spain) during 2018. Souto vineyard is located at 438 m above mean sea level (42{degrees} 24 27.67" N 7{degrees} 28 20.06" W; northwest-southeast orientation) in the lower terraces of the river Sils banks, following the contour lines and with gradients of up to 80%. The variety studied was Godello, for sampling the reproductive structures in the air (spores of Botrytis and Erysiphe and sporangia of Plasmopara), a Lanzoni VPPS-2000(R) spore trap (Lanzoni s.r.l., Bologna, Italy) was used.

ResultsThe Botrytis Seasonal Spore Integral (SSIn) was markedly higher than for the other pathogens under consideration. Taking into account the maximum daily values, a clear dominance of Botrytis spores was also found, with a maximum of 397 spores/m3 at the beginning of June, while Erysiphe and Plasmopara were recorded at around 26 and 227 spores/m3, respectively, at the beginning of august and mid-July. The statistical analysis of the spore concentrations and the main meteorological variables showed for Erysiphe that the highest Spearmans r correlation coefficient corresponded to the rainfall, as for Plasmopara airbone sporangia, but with a negative sign, while for Botrytis spores, no significant correspondence was found for any meteorological parameter.

ConclusionThe use of plant protection products can be much more effective if fungicides are applied at the right time, at the precise doses and combined with agricultural techniques of management of the vineyards. There are sustainable and profitable alternatives that can improve vine yields while protecting the environment in areas of heroic viticulture where the vineyard, is a fundamental element of the wine-growing landscape, combining as it does historical, cultural and landscape characteristics.

sentences: Pipeline to detect the positional relationship between transposable elements and adjacent genes in host genome AO_SCPLOWBSTRACTC_SCPLOWUnderstanding the relationship between transposable elements (TEs) and their closest positional genes in the host genome is a key point to explore their potential role in genome evolution. Transposable elements can regulate and affect gene expression not only because of their mobility within the genome but also because of their transcriptional activity. A comprehensive knowledge of structural organization between transposable elements and neighboring genes is important to study TE functional role in gene regulation. We implemented a pipeline to display the relationship between the distribution of TEs and adjacent genes in host genomes. Our tool is freely available here: https://github.com/marieBvr/TEs_genes_relationship_pipeline.

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Aminooxadiazolyl kainic acid reveals that kainic acid receptors contribute to astrocytoma glutamate signaling The excitatory neurotransmitter glutamate triggers a Ca2+ rise and the extension of processes in astrocytes. Our results suggest that kainic acid receptors (KAR) can independently initiate glutamate signaling in astrocytoma U118-MG cells. The natural product kainic acid triggered glioexcitablity in cells and was inhibited by the KAR antagonist CNQX, but its activity was lower than glutamate on KARs. We created a new heteroaryl kainoid based on rational design: aminooxadiazolyl kainic acid 1 (AODKA). AODKA induced a larger calcium influx and a faster processes extension than kainic acid in U118-MG cells. AODKA is a new tool to study KAR activity in the nervous system.

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org.highwire.dtl.DTLVardef@1b63f72org.highwire.dtl.DTLVardef@1ff92c4org.highwire.dtl.DTLVardef@1fcb212org.highwire.dtl.DTLVardef@10ed76e_HPS_FORMAT_FIGEXP  M_FIG C_FIG

sentences: The Skeletal Muscle Circadian Clock Regulates Titin Splicing Through RBM20 Circadian rhythms are maintained by a cell autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression through both RNAseq and LC-MS. This alteration in titins spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. Finally, we identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

sentences: Modulation of RNA binding properties of the RNA helicase UPF1 by its activator UPF2 The NMD helicase UPF1 is a prototype of the superfamily 1 (SF1) of RNA helicases that bind RNA with high affinity and translocate on it in an ATP-dependent manner. Previous studies showed that UPF1 has a low basal catalytic activity that is greatly enhanced upon binding of its interaction partner, UPF2. Activation of UPF1 by UPF2 entails a large conformational change that switches the helicase from an RNA-clamping mode to an RNA-unwinding mode. The ability of UPF1 to bind RNA was expected to be unaffected by this activation mechanism. Here we show, using a combination of biochemical and biophysical methods, that binding of UPF2 to UPF1 drastically reduces the affinity of UPF1 for RNA, leading to a release of the bound RNA. Although UPF2 is capable of binding RNA in vitro, our results suggest that dissociation of the UPF1-RNA complex is not a consequence of direct competition in RNA binding but rather an allosteric effect that is likely mediated by the conformational change induced by binding of the helicase to its activator. We discuss these results in light of transient interactions forged during mRNP assembly, particularly in the UPF1-dependent mRNA decay pathways.

sentences: Low-Cost, Modular Modification to a Desktop 3D Printer for General Purpose Gel/Paste Extrusion & Direct Ink Writing We propose a design for a simple paste extruder modification that can be used for the selective deposition and patterning of gels and pastes, using a desktop 3D printer as the primary platform. This technology has found use with a variety of materials in seemingly disparate fields, including the printing of ceramics, food and biological materials, each with a variety of material-specific solutions to enhance printability. However, we focus on a syringe-pump driven system that is simple, low-cost, modular, easily assembled and highly modifiable with a low barrier of entry in order to maximise the generalisability and range of printable materials.

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Functional characterisation of banana (Musa spp.) 2-oxoglutarate-dependent dioxygenases involved in flavonoid biosynthesis Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well-known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon.

Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta.

Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

sentences: Solid optical clearing agents based through-Intact-Skull (TIS) window technique for long-term observation of cortical structure and function in mice In vivo cortical optical imaging needs to overcome the scattering of skull. Compared to the traditional transcranial surgery-based open-skull glass window and thinned-skull preparation, chemical tissue optical clearing techniques can provide a skull-remained optical access to the brain while maintaining its original environment. However, previously demonstrated skull optical clearing windows could only maintain transparency for a couple of hours and hardly capable for high-resolution monitoring of awake animals. Here, we developed a convenient and easy-handling chronic skull optical clearing technique, named "Through-Intact-Skull (TIS) window", which was compatible with long-term observation at high resolution, and yielded large imaging depth of 900 m for cortical neurovascular visualization. In addition, our TIS window could monitor neuron activity in awake mice for a long term. Therefore, our bio-compatible and non-invasive TIS window is a new promising approach for intravital brain microscopy with great potential for basic research in neuroscience.

sentences: Methods for recording and imaging spreading depolarizations and seizure-like events in mouse hippocampal slices Spreading depolarization (SD) is a sudden and synchronized depolarization of principal cells followed by depression of activity, which slowly propagates across brain regions like cortex or hippocampus. SD is considered to be mechanistically relevant to migraine, epilepsy, and traumatic brain injury. Interestingly, research into SD typically uses SD triggered immediately after a focal stimulus. Here we optimize an in vitro experimental model allowing us to record SD without focal stimulation. This method uses electrophysiological recordings and intrinsic optical imaging in slices. The method is also relatively easy and inexpensive. Acute hippocampal slices from mice or rats were prepared and used for extracellular and whole-cell recordings. Recordings were made in a submerged-style chamber with flow of artificial cerebrospinal fluid (aCSF) above and below the slices. Flow was fast (> 5ml/min), and temperature was 32{degrees}C. As soon as slices were placed in the chamber, aCSF containing 0 mM Mg2+ and 5 mM K+ (0 Mg2+/5 K+ aCSF) was used. Two major types of activity were observed: SD and seizure-like events (SLEs). Both occurred after many minutes of recording. Although both mouse and rat slices showed SLEs, only mouse slices developed SD and did so in the first hour of 0 Mg2+/5 K+ aCSF exposure. Intrinsic optical imaging showed that most SDs initiated in CA3 and could propagate into CA1 and dentate gyrus. In dentate gyrus, SD propagated in two separate waves: (1) into the hilus and (2) into granule cell and molecular layers simultaneously. This in vitro model can be used to better understand the mechanisms and relationship between SD and SLEs. It could also be useful in preclinical drug screening.

sentences: High Quality Phasing Using Linked-Read Whole Genome Sequencing of Patient Cohorts Informs Genetic Understanding of Complex Traits Phasing of heterozygous alleles is critical for interpretation of cis-effects of disease-relevant variation. For population studies, phase is often inferred from external data but read-based phasing approaches that span long genomic distances would be more accurate because they enable both genotype and phase to be obtained from a single dataset. To demonstrate how read-based phasing can provide functional insights, we sequenced 477 individuals with Cystic Fibrosis (CF) using linked-read sequencing. We benchmark read-based phasing with different short- and long-read sequencing technologies, prioritize linked-read technology as the most informative and produce a benchmark phase call set from reference sample HG002 for the community. The 477 samples display an average phase block N50 of 4.39 Mb. We use these samples to construct a graph representation of CFTR haplotypes, which facilitates understanding of complex CF alleles. Fine-mapping and phasing of the chr7q35 trypsinogen locus associated with CF meconium ileus demonstrates a 20 kb deletion and a PRSS2 missense variant p.Thr8Ile (rs62473563) independently contribute to meconium ileus risk (p=0.0028, p=0.011, respectively) and are PRSS2 pancreas eQTLs (p=9.5e-7 and p=1.4e-4, respectively), explaining the mechanism by which these polymorphisms contribute to CF. Phase enables access to haplotypes that can be used for genome graph or reference panel construction, identification of cis-effects, and for understanding disease associated loci. The phase information from linked-reads provides a causal explanation for variation at a CF-relevant locus which also has implications for the genetic basis of non-CF pancreatitis to which this locus has been reported to contribute.

sentences: Time-adaptive Unsupervised Auditory Attention Decoding Using EEG-based Stimulus Reconstruction The goal of auditory attention decoding (AAD) is to determine to which speaker out of multiple competing speakers a listener is attending based on the brain signals recorded via, e.g., electroencephalography (EEG). AAD algorithms are a fundamental building block of so-called neuro-steered hearing devices that would allow identifying the speaker that should be amplified based on the brain activity. A common approach is to train a subject-specific stimulus decoder that reconstructs the amplitude envelope of the attended speech signal. However, training this decoder requires a dedicated  ground-truth EEG recording of the subject under test, during which the attended speaker is known. Furthermore, this decoder remains fixed during operation and can thus not adapt to changing conditions and situations. Therefore, we propose an online time-adaptive unsupervised stimulus reconstruction method that continuously and automatically adapts over time when new EEG and audio data are streaming in. The adaptive decoder does not require ground-truth attention labels obtained from a training session with the end-user and instead can be initialized with a generic subject-independent decoder or even completely random values. We propose two different implementations: a sliding window and recursive implementation, which we extensively validate on three independent datasets based on multiple performance metrics. We show that the proposed time-adaptive unsupervised decoder outperforms a time-invariant supervised decoder, representing an important step toward practically applicable AAD algorithms for neuro-steered hearing devices.

sentences: Robust Inference of Bi-Directional Causal Relationships in Presence of Correlated Pleiotropy with GWAS Summary Data To infer a causal relationship between two traits, several correlation-based causal direction (CD) methods have been proposed with the use of SNPs as instrumental variables (IVs) based on GWAS summary data for the two traits; however, none of the existing CD methods can deal with SNPs with correlated pleiotropy. Alternatively, reciprocal Mendelian randomization (MR) can be applied, which however may perform poorly in the presence of (unknown) invalid IVs, especially for bi-directional causal relationships. In this paper, first, we propose a CD method that performs better than existing methods regardless of the presence of correlated pleiotropy. Second, along with a simple but yet effective IV screening rule, we propose applying a closely related and state-of-the-art MR method in reciprocal MR, showing its almost identical performance to that of the new CD method when their model assumptions hold; however, if the modeling assumptions are violated, the new CD method is expected to better control type I errors. Notably bi-directional causal relationships impose some unique challenges beyond those for uni-directional ones, and thus requiring special treatments. For example, we point out for the first time several scenarios where a bi-directional relationship, but not a uni-directional one, can unexpectedly cause the violation of some weak modeling assumptions commonly required by many robust MR methods. Finally we applied the proposed methods to 12 risk factors and 4 common diseases, confirming mostly well-known uni-directional causal relationships, while identifying some novel and plausible bi-directional ones such as between BMI and T2D, and between BMI and CAD.

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Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue -helical sequence is replaced by -helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this -helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although -helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

Statement of Significance-helices are ubiquitous components of protein structure that exhibit a degree of independent folding behavior, making them plausible structural modules within proteins. Here, we assess the effects of switching the sequence of an -helix in an essential enzyme for -helical sequences from evolutionarily unrelated proteins. The resultant chimeric proteins can still fold but enzymatic activity, stability, and cellular growth rates are negatively affected. Computational investigations reveal how residues in an -helix have been shaped by selection pressure to maintain catalytic activity and a specific, helical conformation of the protein. More broadly, we illustrate how molecular and fitness effects of switching protein segments depend on the protein and cellular context.

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Intracellular Ca2+ channels initiate physiological glucose signaling in beta cells examined in situ ObjectiveInsulin release from pancreatic beta cells is driven by cytosolic [Ca2+]c oscillations of several different time scales that are primarily attributed to plasma membrane ion channel activity. However, the majority of past studies have been performed at supraphysiological glucose concentrations above 10 mM using electrophysiologic approaches that solely measure plasma membrane ion fluxes. The role of endoplasmic reticulum (ER) Ca2+ stores in glucose-stimulated Ca2+ signaling remains poorly understood.

MethodsIn this study, we hypothesized new, brighter [Ca2+]c sensors coupled with high-resolution functional Ca2+ imaging could be used to test a previously unappreciated role for the ryanodine and IP3 intracellular Ca2+ release channels in [Ca2+]c oscillations stimulated by increases from 6 mM to 8 mM glucose.

ResultsUsing mouse pancreas tissue slices exposed to physiological glucose increments, our results show that glucose-dependent activation of IP3 and ryanodine receptors produces two kinetically distinct forms of compound events involving calcium-induced Ca2+ release. Ca2+ release mediated by IP3 and ryanodine receptors was sufficient to generate Ca2+ oscillations and necessary for the response to physiological glucose, which could be initiated in the absence of Ca2+ influx across the plasma membrane through voltage-gated Ca2+ channels.

ConclusionsIn aggregate, these data suggest that intracellular Ca2+ receptors play a key role in shaping glucose-dependent [Ca2+]c responses in pancreatic beta cells in situ. In our revised model, the primary role for plasma membrane Ca2+ influx at physiological glucose concentrations is to refill ER Ca2+ stores.

sentences: Fat2 polarizes the WAVE complex in trans to align cell protrusions for collective migration For a group of cells to migrate together, each cell must couple the polarity of its migratory machinery with that of the other cells in the cohort. Although collective cell migrations are common in animal development, little is known about how protrusions are coherently polarized among groups of migrating epithelial cells. We address this problem in the collective migration of the follicular epithelial cells in Drosophila melanogaster. In this epithelium, the cadherin Fat2 localizes to the trailing edge of each cell and promotes the formation of lamellipodia at the leading edge of the cell behind. We show that Fat2 performs this function by acting in trans to restrict WAVE complex activity to one long-lived region along each cells leading edge. Without Fat2, the WAVE complex distribution expands around the cell perimeter and fluctuates over time, resulting in reduced, unpolarized protrusive activity. We further show that Fat2s influence is very local, with sub-micron-scale puncta of Fat2 concentrating the WAVE complex in corresponding puncta just across the leading-trailing cell-cell interface. These findings demonstrate that a trans interaction between Fat2 and the WAVE complex creates stable regions of protrusive activity in each cell and aligns the cells protrusions across the epithelium for directionally persistent collective migration.

sentences: Above- and below-ground biodiversity responses to the prolonged flood pulse in central-western Amazonia, Brazil. Amazonia encompasses forests that grow in areas that are periodically inundated by overflowing rivers. The inundation depth and duration vary according to the slope of the terrain, creating a flooding gradient. This gradient directly affects the biota, but the effect on soil organisms remains elusive. Here, we use DNA metabarcoding to estimate prokaryote and eukaryote diversity from soil and litter samples in a seasonally flooded forest and its adjacent unflooded forest in central-western Amazonia using 16S and 18S gene sequences, respectively. We characterize the below-ground diversity and community composition based on Amplicon Sequence Variants (ASVs) along the flooding gradient. We test for the relationship of soil biota with the flooding gradient, soil properties and above-ground woody plant diversity. The flooding gradient did not explain below-ground biodiversity. Nor was the below-ground diversity explained by the above-ground woody plant diversity. However, we found taxonomic groups not previously reported in Amazonian seasonally flooded forests. Also, the flooding gradient and woody plant diversity did, in part, explain the community composition of soil bacteria. Although the effects of the flooding gradient, soil properties and above-ground woody plant diversity is hard to quantify, our results thus indicate that flood stress could influence below-ground bacterial community composition.

sentences: Mechanistic insights into the success of xenobiotic degraders resolved from metagenomes of microbial enrichment cultures Even though microbial communities can be more effective at degrading xenobiotics than cultured micro-organisms, yet little is known about the microbial strategies that underpin xenobiotic biodegradation by microbial communities. Here, we employ metagenomic community sequencing to explore the mechanisms that drive the development of 49 xenobiotic-degrading microbial communities, which were enriched from 7 contaminated soils or sediments with a range of xenobiotic compounds. We show that multiple microbial strategies likely drive the development of xenobiotic degrading communities, notably (i) presence of genes encoding catabolic enzymes to degrade xenobiotics; (ii) presence of genes encoding efflux pumps; (iii) auxiliary catabolic genes on plasmids; and (iv) positive interactions dominate microbial communities with efficient degradation. Overall, the integrated analyses of microbial ecological strategies advance our understanding of microbial processes driving the biodegradation of xenobiotics and promote the design of bioremediation systems.

sentences: Comparative Analysis of Sorghum EMS Mutants and Natural Populations To build a large-scale genomic resource for functional validation of sorghum genes through EMS-mutagenized BTx623 seeds, we deep sequenced (30-60X) an additional 445 phenotyped EMS mutant lines. 4.2 million EMS mutations are called with nearly 36,800 mutations that could have a disruptive effect on functions of over 15,500 genes. Combining variants carried by both the natural population and previous EMS efforts, over 69% of sorghum coding genes (23644) are now presented with one or more mutations that are, or are predicted to be, disruptive to their functions. Our results show that the EMS population carries more significant mutations but less in each sample than the natural population, which makes it more powerful in elucidating sorghum gene functions on a large scale and requiring less work in validation of candidate causal genes. We have made the data available through two ways, one is the integration with the BSAseq workflow that supports retrieving independent EMS samples carrying the same genes with significant mutation for complementary testing, and the other is a web application for directly querying genes with significant mutations on SciApps.org.

sentences: ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals. Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with {micro}M affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

sentences: The dynamics of adaptation to stress from standing genetic variation and de novo mutations Adaptation from standing genetic variation is an important process underlying evolution in natural populations but we rarely get the opportunity to observe the dynamics of fitness changes in real time. Here, we used the power of microbial experimental evolution and whole population sequencing to track the phenotypic and genomic changes of genetically diverse yeast populations in environments with different stress levels. We found that populations rapidly and in parallel increased in fitness in stressful environments. The founders genetic diversity was quickly depleted, however, not to the same degree in all populations and environments. Some populations fixed all ancestral variation in < 30 generations while others maintained diversity across hundreds of generations. We also observed parallelism at the gene and pathway level. Specifically, we detected up to seven genes harbouring multiple independent mutations in different populations, and a general enrichment for mutations affecting downstream effectors of the high-osmolarity-glycerol pathway in three out of four environments. Adaptation to the most stressful environment was characterised by the fast evolution of functional haploidy, likely driven by standing genetic variation. Almost 40% of all populations contained aneuploidies (losses or gains of chromosomes) at least once during experimental evolution. Some aneuploidies were maintained for hundreds of generations in parallel in different replicates, suggesting they were adaptive. This work shows that experimental evolution is a great tool to address the interplay between standing variation and the influx of de novo mutations, leading to a better understanding of the demographic and environmental drivers and constraints of a populations capacity to adapt to environmental change.

sentences: The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2 Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

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Antibacterial 3D-printed PMMA/ceramic composites Following the rising interested on 3D-printed technologies, this research explores the possibility to use stereo-lithography to 3D print PMMA resins reinforced with up to 15% in weight of antibacterial ceramic powders. Three different reinforcements were tested, following previous literature data: aluminum nitride, titanium oxide and barium titanate.

Between the three powders, the most uniform dispersion was achieved using aluminum nitride. Initial screenings with mixed and cured composite resins showed that between the three composite materials, only aluminum nitride or barium titanate PMMA showed a clear antibacterial effect when compared to the pristine reference, with aluminum nitride being the most effective against E. coli. When 3D printed using stereo-lithography, the composite containing aluminum nitride showed an even higher degree of dispersion and comparable antibacterial effects. Moreover, aluminum nitride reinforced PMMA resins showed good mechanical properties, comparable to the basic resin, and could be further strengthened by a standard post-curing process.

sentences: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from human induced pluripotent stem cells The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

sentences: An amyloidosis-associated polymorphism does not alter LECT2 stability in vitro Amyloid fibrils formed from leukocyte chemotactic factor 2 (LECT2), a secreted human cytokine, are associated with kidney failure in the disease amyloid LECT2 (ALECT2) amyloidosis. This rare disease was recognized in 2008 and has a variable prevalence worldwide. The mechanisms which lead to ALECT2 fibril deposition are not known and there are no treatments other than kidney transplant. The LECT2 gene harbors a single nucleotide polymorphism that leads to either a valine or isoleucine residue at position 40 of the mature protein. Most of the individuals diagnosed with ALECT2 amyloidosis are homozygous for valine at this position, which led us to hypothesize that the valine-containing variant of LECT2 protein is less stable and more prone to aggregation than the isoleucine-containing variant. Here, we investigate the structure, stability and aggregation of both variants of recombinant LECT2. Both variants have similar structures in solution; unfold in similar concentrations of urea; and aggregate at similar rates under native-like conditions, forming structures that bind to thioflavin T. Chelation of the structural zinc ion destabilizes both variants to a similar extent, and increases the rate at which they aggregate. We do not observe a consistent difference in stability or aggregation between the variants of LECT2, so we suggest that the presence of the valine residue at position 40 does not determine whether an individual is at increased risk of ALECT2 amyloidosis.

sentences: GTAGMe-seq: joint profiling of genetic variants, DNA methylation, GpC methyltransferase footprints, and 3D genome in the same DNA molecules Cis-regulatory elements (CREs) coordinate to regulate the expression of their targeted genes, up to megabases away. However, the joint measurement of CREs activities and their interactions over large genomic distances is limited by the current sequencing approaches. We report a method, GTAGMe-seq, which simultaneously captures the single nucleotide polymorphisms, DNA methylation, GpC methyltransferase footprints, and chromosome conformation changes from the same DNA molecules, together with transcriptome in the same assay. GTAGMe-seq shows high concordance with the state-of-art mono-omics assays across different molecular measurements. It reveals the coordinated GpC methyltransferase footprints at distal genomic segments in spatial proximity and different types of long-range allele-specific methyltransferase footprints.

sentences: High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans. Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.

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Visual Cortical Area MT is Required For Development of the Dorsal Stream and Associated Visuomotor Behaviours The middle temporal (MT) area of the extrastriate visual cortex has long been studied in adulthood for its distinctive physiological properties and function as a part of the dorsal stream, yet interestingly possesses a similar maturation profile as the primary visual cortex (V1). Here we examined whether an early-life lesion of MT altered the dorsal stream development and the behavioural precision of reaching to grasp sequences. We observed permanent changes in the anatomy of cortices associated with both reaching (PE and MIP) and grasping (AIP), as well as in reaching and grasping behaviours. In addition, we observed a significant impact on the anatomy of V1 and the direction sensitivity of V1 neurons in the lesion projection zone. These findings indicate that area MT is a crucial node for the development of the primate vision, impacting both V1 and areas in the dorsal visual pathway known to mediate visually guided manual behaviours.

TeaserThe early life loss of visual area MT leads to significant anatomical, physiological and behavioural changes.

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Excitatory/inhibitory imbalance in autism: the role of glutamate and GABA gene-sets in symptoms and cortical brain structure BackgroundThe excitatory/inhibitory (E/I) imbalance hypothesis posits that an imbalance between excitatory (glutamatergic) and inhibitory (GABAergic) mechanisms underlies the behavioral characteristics of autism spectrum disorder (autism). However, how E/I imbalance arises and how it may differ across autism symptomatology and brain regions is not well understood.

MethodsWe used innovative analysis methods - combining competitive gene-set analysis and gene-expression profiles in relation to cortical thickness (CT)- to investigate the relationship between genetic variance, brain structure and autism symptomatology of participants from the EU-AIMS LEAP cohort (autism=360, male/female=259/101; neurotypical control participants=279, male/female=178/101) aged 6 to 30 years. Competitive gene-set analysis investigated associations between glutamatergic and GABAergic signaling pathway gene-sets and clinical measures, and CT. Additionally, we investigated expression profiles of the genes within those sets throughout the brain and how those profiles relate to differences in CT between autistic and neurotypical control participants in the same regions.

ResultsThe glutamate gene-set was associated with all autism symptom severity scores on the Autism Diagnostic Observation Schedule-2 (ADOS-2) and the Autism Diagnostic Interview-Revised (ADI-R) within the autistic group, while the GABA set was associated with sensory processing measures (using the SSP subscales) across all participants. Brain regions with greater gene expression of both glutamate and GABA genes showed greater differences in CT between autistic and neurotypical control participants.

ConclusionsOur results suggest crucial roles for glutamate and GABA genes in autism symptomatology as well as CT, where GABA is more strongly associated with sensory processing and glutamate more with autism symptom severity.

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TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry, and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP.

HighlightsO_LITRIM25 and ZAP play a major role on type I IFN-mediated inhibition of EBOV trVLP replication
C_LIO_LITRIM25 interacts with the EBOV NP and is recruited to vRNPs in the cytoplasm after viral entry
C_LIO_LITRIM25 ubiquitinates NP and displaces it from the viral genome, facilitating ZAP interaction
C_LIO_LIZAP targets CpGs in the EBOV genome to inhibit EBOV trVLP replication
C_LI

sentences: Entropic pressure controls oligomerization of Vibrio cholerae ParD2 antitoxin ParD2 is the antitoxin component of the parDE2 toxin-antitoxin module from Vibrio cholerae and consists of an ordered DNA binding domain followed by an intrinsically disordered ParE-neutralizing domain. In absence of the C-terminal IDP domain, VcParD2 crystallizes as a doughnut-shaped hexadecamer formed by the association of eight dimers. This assembly is stabilized via hydrogen bonds and salt bridges rather than hydrophobic contacts. In solution, oligomerization of the full-length protein is restricted to a stable, open 10-mer or 12-mer, likely as a consequence of entropic pressure from the IDP tails. The relative positioning of successive VcParD2 dimers mimics the arrangement of Streptococcus agalactiae CopG dimers on their operator and allows for an extended operator to wrap around the VcParD2 oligomer.

sentences: An isogenic panel of single App knock-in mouse models of Alzheimer's disease confers differential profiles of β-secretase inhibition and endosomal abnormalities We previously developed single App knock-in mouse models of Alzheimers disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL- G-F and AppNL-F mice). These models showed the development of amyloid {beta} peptide (A{beta}) pathology, neuroinflammation and cognitive impairment with aging. We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of {beta}-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by {beta}-secretase (CTF-{beta}) affect endosomal properties as previously reported as well as other pathological events? A{beta} pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a {beta}-secretase inhibitor, verubecestat, inhibited A{beta} production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of {beta}-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-{beta} generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-{beta}, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

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Learning Divisive Normalization in Primary Visual Cortex Divisive normalization (DN) is a prominent computational building block in the brain that has been proposed as a canonical cortical operation. Numerous experimental studies have verified its importance for capturing nonlinear neural response properties to simple, artificial stimuli, and computational studies suggest that DN is also an important component for processing natural stimuli. However, we lack quantitative models of DN that are directly informed by measurements of spiking responses in the brain and applicable to arbitrary stimuli. Here, we propose a DN model that is applicable to arbitrary input images. We test its ability to predict how neurons in macaque primary visual cortex (V1) respond to natural images, with a focus on nonlinear response properties within the classical receptive field. Our model consists of one layer of subunits followed by learned orientation-specific DN. It outperforms linear-nonlinear and wavelet-based feature representations and makes a significant step towards the performance of state-of-the-art convolutional neural network (CNN) models. Unlike deep CNNs, our compact DN model offers a direct interpretation of the nature of normalization. By inspecting the learned normalization pool of our model, we gained insights into a long-standing question about the tuning properties of DN that update the current textbook description: we found that within the receptive field oriented features were normalized preferentially by features with similar orientation rather than non-specifically as currently assumed.

Author summaryDivisive normalization (DN) is a computational building block throughout sensory processing in the brain. We currently lack an understanding of what role this normalization mechanism plays when processing complex stimuli like natural images. Here, we use modern machine learning methods to build a general DN model that is directly informed by data from primary visual cortex (V1). Contrary to high-predictive deep learning models, our DN-based models parameters offer a straightforward interpretation of the nature of normalization. Within the receptive field, we found that neurons responding strongly to a specific orientation are preferentially normalized by other neurons that are highly active for similar orientations, rather than being normalized by all neurons as it is currently assumed by textbook models.

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Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3 end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5 end of dusA and the 3 end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.

Author summaryWe identified integrative elements distantly related to the Salmonella Genomic Island 1 (SGI1), a key vector of antibiotic resistance genes in Gammaproteobacteria. SGI1 and its variants reside at the 3 end of trmE, share a large, highly conserved core of genes, and carry a complex integron that confers multidrug resistance phenotypes to their hosts. Unlike members of the SGI1 group, these novel genomic islands target the 5 end dusA or the 3 end of yicC, lack multidrug resistance genes, and seem much more diverse. We showed here that, like SGI1, these elements are mobilized by conjugative plasmids of the IncC group. Based on comparative genomics and functional analyses, we propose a hypothetical model of the evolution of SGI1 and its siblings from the progenitor of IncA and IncC conjugative plasmids via an intermediate dusA-specific integrative element through gene losses and gain of alternative integration/excision modules.

sentences: Novel and highly distributed classes of hindbrain neuronal activity contribute to sensory processing and motor control in the Xenopus laevis tadpole. An animals survival depends heavily on the selection and execution of timely and well-coordinated motor responses. The brainstem controls the activity of spinal neural circuits to produce and modify movements. However, important questions remain unanswered about the origin of this descending control and how brainstem neuronal activity integrates sensory inputs and determines motor functions. Here, we record hindbrain extracellular activity in response to trunk skin stimulation, which in turn leads to fictive swimming in the hatchling Xenopus laevis tadpole. We identify four distinct classes of single unit activity, distributed along the hindbrain rostro-caudal axis, whose firing patterns correlate to distinct motor states. We observe different firing patterns in response to stimulation that leads to fictive swimming versus the application of a weak stimulus which does not evoke movement. We identify differences in the temporal activation of the four classes of hindbrain activity in relation to the initiation of fictive swimming. We propose a simple network encompassing the novel neuronal populations embedded within the currently known sensory pathway and central pattern generators of the tadpole brainstem. By identifying the contribution of the individual supraspinal neuronal populations we build a better understanding of how the brain controls and modulates movement.

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Identification of blood autosomal cis-expression quantitative trait methylation (cis-eQTMs) in children BackgroundThe identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in childrens blood, using data from 832 children of the Human Early Life Exposome (HELIX) project.

MethodsBlood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition.

ResultsWe identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at <250 kb from eGenes TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. 40.4% of eQTMs had at least one genetic variant associated with methylation and expression levels. The overlap of autosomal cis eQTMs in childrens blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants.

ConclusionsThis catalogue of autosomal cis eQTMs in childrens blood can help the biological interpretation of EWAS findings and is publicly available at https://helixomics.isglobal.org/.

Funding: The study has received funding from the European Communitys Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Unions Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the project PI17/01225, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, "A way to make Europe") and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII).

sentences: Symbiont-conferred immunity interacts with the effects of parasitoid genotype and intraguild predation to shape pea aphid immunity in a clone-specific fashion Host-parasite interactions represent complex co-evolving systems in which genetic variation within a species can significantly affect selective pressure on traits in the other (for example via inter-species indirect genetic effects). While often viewed as a two-species interaction between host and parasite species, some systems are more complex due to the involvement of symbionts in the host that influence its immunity, enemies of the host, and the parasite through intraguild predation. However, it remains unclear what the joint effects of intraguild predation, defensive endosymbiosis, within-species genetic variation and indirect genetic effects on host immunity are. We have addressed this question in an important agricultural pest system, the pea aphid Acyrthosiphon pisum, which shows significant intraspecific variability in immunity to the parasitoid wasp Aphidius ervi due to immunity conferring endosymbiotic bacteria. In a complex experiment involving a quantitative genetic design of the parasitoid, two ecologically different aphid lineages and the aphid lion Chrysoperla carnea as an intraguild predator, we demonstrate that aphid immunity is affected by intraspecific genetic variation in the parasitoid and the aphid, as well as by associated differences in the defensive endosymbiont communities. Using 16s rRNA sequencing, we identified secondary symbionts that differed between the lineages. We further show that aphid lineages differ in their altruistic behaviour once parasitised whereby infested aphids move away from the clonal colony to facilitate predation. The outcome of these complex between-species interactions not only shape important host-parasite systems but have also implications for understanding the evolution of multitrophic interactions, and aphid biocontrol.

sentences: Versatile Encapsulation and Synthesis of Potent Therapeutic Liposomes by Thermal Equilibration The wide-scale use of liposomal delivery systems is hampered by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes, but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes. Integrating a thermal equilibration technique with our unique liposome synthesis approach, co-loaded targeting liposomes can be engineered in an efficient and scalable manner with potencies 200-fold higher than typical passive encapsulation techniques. We demonstrate this capability through simultaneous co-loading of hydrophilic and hydrophobic small molecules and through targeted delivery of liposomal Doxorubicin to a metastatic breast cancer cell line MDA-MB-231. Molecular dynamic simulations are used to explain interactions between Doxorubicin and liposome membrane during thermal equilibration. By addressing the existing challenges, we have developed an unparalleled approach that will facilitate the formulation of novel theranostic and pharmaceutical strategies.

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DysRegNet: Patient-specific and confounder-aware dysregulated network inference Gene regulation is frequently altered in diseases in unique and often patient-specific ways. Hence, personalized strategies have been proposed to infer patient-specific gene-regulatory networks. However, existing methods do not focus on disease-specific dysregulation or lack assessments of statistical significance. Moreover, they do not account for clinically important confounders such as age, sex or treatment history.

To overcome these shortcomings, we present DysRegNet, a novel method for inferring patient-specific regulatory alterations (dysregulations) from gene expression profiles. We compared DysRegNet to state-of-the-art methods and demonstrated that DysRegNet produces more interpretable and biologically meaningful networks. Independent information on promoter methylation and single nucleotide variants further corroborate our results. We apply DysRegNet to eleven TCGA cancer types and illustrate how the inferred networks can be used for down-stream analysis. We show that unique as well as cancer-type-specific dysregulation patterns exist and highlight immune-related mechanisms that are not obvious when focusing on individual genes rather than their interactions.

DysRegNet is available as a Python package (https://github.com/biomedbigdata/DysRegNet_package) and analysis results for eleven TCGA cancer types are further available through an interactive web interface (https://exbio.wzw.tum.de/dysregnet).

sentences: Combination of successor representation-based appetitive learning and individual representation-based aversive learning is a good strategy and can be implemented in the cortico-basal ganglia circuits The direct and indirect pathways of the basal ganglia (BG) have been suggested to learn mainly from positive and negative feedbacks, respectively. Since these pathways unevenly receive inputs from different cortical neuron types and/or regions, they may preferentially use different state/action representations. We explored whether such combined use of different representations coupled with appetitive or aversive learning has computational benefits. We simulated reward learning tasks in dynamic environments, and examined the performance of animal modeled as an agent equipped with two learning systems, each of which used individual representation (IR) or successor representation (SR) of states. With varying the combination of IR or SR and also the learning rates from positive and negative reward prediction errors (RPEs) in each system, we found that combination of an SR-based system learning mainly from positive RPEs and an IR-based system learning mainly from negative RPEs could achieve good performance, as compared to other combinations, in many situations. The architecture of such a combination provides a novel coherent explanation for the functional significance and underlying mechanism of diverse findings about the cortico-BG circuits. These results suggest that combining different representations with appetitive and aversive learning is an effective learning strategy adopted by the brain.

sentences: An epigenetic memory of inflammation controls context-dependent lineage plasticity in the pancreas Inflammation is essential to the disruption of tissue homeostasis, and, in the pancreas, can destabilize the identity of terminally differentiated acinar cells. Herein we employ lineage-traced mouse models to delineate the chromatin dynamics that accompany the cycle of metaplasia and regeneration following pancreatitis, and unveil the presence of an epigenetic memory of inflammation in the pancreatic acinar cell compartment. We observe that despite histologic resolution of pancreatitis, acinar cells fail to return to their molecular baseline after several months, representing an incomplete cell fate decision. In vivo, this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second inflammatory insult but increased tumorigenesis with an oncogenic Kras mutation. We demonstrate that both persistent chromatin and transcriptional changes constituting memory are recalled with oncogenic stress. Together, our findings define the dynamics and recall of an epigenetic memory of inflammation that impacts cell fate decisions in a context-dependent manner.

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Sod1 Integrates Oxygen Availability to Redox Regulate NADPH Production and the Thiol Redoxome Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major new aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is exclusively dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this new mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2- toxicity, which only requires < 1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Significance StatementCu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress, metabolic defects, and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2-) in a manner that produces an oxidant as a byproduct, hydrogen peroxide (H2O2). While much is known about the necessity of scavenging O2-, it is less clear what the physiological roles of Sod1-derived H2O2 are. Herein, we discovered that Sod1-derived H2O2 plays a very important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for ROS scavenging enzymes, as well as redox regulating a large network of enzymes.

sentences: epiAneufinder: identifying copy number variations from single-cell ATAC-seq data Single-cell open chromatin profiling via the single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) assay has become a mainstream measurement of open chromatin in single-cells. Here we present a novel algorithm, epiAneufinder, that exploits the read count information from scATAC-seq data to extract genome-wide copy number variations (CNVs) for individual cells, allowing to explore the CNV heterogeneity present in a sample at the single-cell level. Using different cancer scATAC-seq datasets, we show how epiAneufinder can identify intratumor clonal heterogeneity in populations of single cells based on their CNV profiles. These profiles are concordant with the ones inferred from single-cell whole genome sequencing data for the same samples. epiAneufinder allows the addition of single-cell CNV information to scATAC-seq data, without the need of additional experiments, unlocking a layer of genomic variation which is otherwise unexplored.

sentences: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies. NF-kB driven cellular immunity is essential for both pro- and anti-inflammatory responses to microbes, which makes it one of the most frequently targeted pathways by bacteria during pathogenesis. How NF-kB tunes the epithelial response to Streptococcus pneumoniae across the spectrum of commensal to pathogenic host phenotypic outcomes is not fully understood. In this study, we compare a commensal-like 6B ST90 strain to an invasive TIGR4 isolate and demonstrate that TIGR4 both blunts and antagonizes NF-kB activation. We identified, through comparative mass spectrometry of the p65 interactome, that the 6B ST90 isolate drives a non-canonical NF-kB RelB cascade, whereas TIGR4 induces p65 degradation though aggrephagy. Mechanistically, we show that during TIGR4 challenge a novel interaction of COMMD2 with p65 and p62 is established to mediate degradation of p65. With these results, we establish a role for COMMD2 in negative NF-kB regulation, and present a paradigm for diverging NF-kB responses to pneumococcus. Thus, our studies reveal for the first time a new bacterial pathogenesis mechanism to repress host inflammatory response though COMMD2 mediated turnover of p65.

sentences: Endocytic Trafficking Promotes Vacuolar Enlargements for Fast Cell Expansion Rates in Plants The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Lofke et al., 2015; Scheuring et al., 2016; Dunser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Counterintuitively, our data reveal that endocytic trafficking from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this "compromise" mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

sentences: Beyond BOLD: Evidence for diffusion fMRI contrast in the human brain distinct from neurovascular response Functional Magnetic Resonance Imaging (fMRI) is an essential method to measure brain activity non-invasively. While fMRI almost systematically relies on the blood oxygenation level-dependent (BOLD) contrast, there is an increasing interest in alternative methods that would not rely on neurovascular coupling. A promising but controversial such alternative is diffusion fMRI (dfMRI), which relies instead on dynamic fluctuations in apparent diffusion coefficient (ADC) due to microstructural changes underlying neuronal activity, i.e. neuromorphological coupling. However, it is unclear whether genuine dfMRI contrast, distinct from BOLD contamination, can be detected in the human brain in physiological conditions. Here, we present the first dfMRI study in humans attempting to minimize BOLD contamination sources and comparing functional responses at two field strengths (3T and 7T). Our study benefits from unprecedented high spatio-temporal resolution, harnesses novel denoising strategies and examines characteristics of not only task but also resting-state dfMRI. We report task-induced decrease in ADC with temporal and spatial features distinct from the BOLD response and yielding more specific activation maps. Furthermore, we report dfMRI resting-state functional connectivity which, compared to its BOLD counterpart, is essentially free from physiological artifacts and preserves positive correlations but preferentially suppresses anti-correlations, which are likely of vascular origin. A careful acquisition and processing design thus enable the detection of genuine dfMRI contrast on clinical MRI systems. As opposed to BOLD, diffusion functional contrast could be particularly well suited for low-field MRI.

sentences: Diversity and distribution of sediment bacteria across an ecological and trophic gradient Can we cluster bacterial sediment communities based on lake size, depth, and trophic status? Or, are bacterial microbial communities an emergent property of their geography, integrating regional physical and climatic conditions? Lakes in Minnesota are uniquely situated to address these questions because of their wide geographic range and variability in size and basin land-use. In this study, we selected twenty lakes with varying morphological and chemical properties across four ecological regions of Minnesota. Our objectives were to (i)) evaluate the diversity and spatial variation of the bacterial community at the sediment-water interface and (ii) determine how lake location and watershed land-use impact aqueous chemistry and influence community structure. Our data indicate that sediment communities from similar depth intervals are more likely to cluster by ecological region rather than any individual lake properties (e.g., trophic status, TP concentration, lake depth). However, composition is tied to a given lake, wherein samples from the same core were more alike than samples at similar depths across lakes. Our results illustrate the diversity within lake sediment microbial communities and provide insight into relationships between taxonomy, physicochemical, and geographic properties of north temperate lakes.

sentences: LIN-35 is necessary in both the soma and germline for preserving fertility in Caenorhabditis elegans under moderate temperature stress Maintenance of germline function under stress conditions is crucial for species survival. The germline across many species is especially sensitive to elevated temperature stress. We have investigated the role that the pocket protein LIN-35 plays in preserving fertility in C. elegans under moderate high temperature stress. Here we show that lin-35 mutants have a number of temperature sensitive germline defects. The decreased brood size seen at elevated temperatures is more severe in lin-35 mutants than wild type. This loss of fertility under temperature stress is primarily due to loss of zygotic but not maternal LIN-35. Additionally, we have found that expression of LIN-35 is necessary in both the germline and soma for the preserving fertility at elevated temperature. Specifically, germline expressed LIN-35 is primarily what is important for maintaining fertility in hermaphrodites, but broad somatic expression of LIN-35 is also necessary for oocyte formation and/or function at elevated temperatures. Additionally, we found that overexpression of LIN-35 in the germline only can stabilize the formation of P granules in primordial germ cells. Our data adds to the emerging critical role that LIN-35 plays in buffering tissues against stress.

sentences: Closed-loop automated reaching apparatus (CLARA) for interrogating motor systems Objective: Personalized neurostimulation is a rapidly expanding category of therapeutics for a broad range of indications. Development of these innovative neurological devices requires high-throughput systems for closed-loop stimulation of model organisms, while monitoring physiological signals and complex, naturalistic behaviors. To address this need, we developed CLARA, a closed-loop automated reaching apparatus. Approach: Using breakthroughs in computer vision, CLARA integrates fully-automated, markerless kinematic tracking of multiple features we use to classify animal behavior and precisely deliver neural stimulation based on behavioral outcomes. CLARA is compatible with advanced neurophysiological tools, enabling the testing of neurostimulation devices and identification of novel neurological biomarkers. Results: The CLARA system tracks unconstrained skilled reach behavior in 3D at 150hz without physical markers. The system fully automates trial initiation and pellet delivery and is capable of accurately delivering stimulation in response to trial outcome with sub-quarter second latency. Mice perform the skilled reach task in the CLARA system at a proficiency similar to manually trained animals. Kinematic data from the CLARA system provided novel insights into the dynamics of reach consistency over the course of learning, suggesting that changes are driven entirely by unsuccessful reach accuracy. Additionally, using the closed-loop capabilities of CLARA, we demonstrate that vagus nerve stimulation (VNS) delivered on reach success improves skilled reach performance and increases reach trajectory consistency in healthy animals. Significance: The CLARA system is the first mouse behavior apparatus that uses markerless pose tracking to provide real-time closed-loop stimulation in response to the outcome of an unconstrained motor task. Additionally, we demonstrate that the CLARA system was essential for our finding that VNS given after successful completion of a motor task improves performance in healthy animals. This approach has high translational relevance for developing neurostimulation technology based on complex human behavior.

sentences: Dopamine enhanced auditory perceptual learning in humans via long-term memory consolidation Dopamine is known to modulate sensory plasticity in animal brain, but how it impacts perceptual learning in humans remains largely unknown. In a placebo-controlled, double-blinded training experiment with young healthy adults (both male and female), oral administration of Madopar, a dopamine precursor, during each of multiple training sessions was shown to enhance auditory perceptual learning, particularly in late training sessions. Madopar also enhanced learning and transfer to working memory when tested outside the time widow of drug effect, which appeared to retain for at least 20 days. To test whether such learning modulation was mediated by the dopaminergic working memory network, the same dopamine manipulation was applied to working memory training, but to little influence on learning or transfer. Further, a neural network model of auditory perceptual learning revealed distinctive behavioural modulation patterns for proposed dopaminergic functions in the auditory cortex: trial-by-trial reinforcement signals (reward/reward prediction error and expected reward) and across-session memory consolidation. Only the memory consolidation simulations matched experimental observations. The results thus demonstrate that dopamine modulates human perceptual learning, mostly likely via enhancing memory consolidation over extended time scales.

sentences: A quantitative gibberellin signalling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signalling biosensor by engineering one of the DELLA repressors, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this novel degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signalling activity in the SAM. We show that high GA signalling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organisation of internodes, thus contributing to internode specification in the SAM.

sentences: Does Pisum sativum exhibit a response to neighbour and pot volume manipulations? A bayesian hierarchical meta-analysis Plant-plant competition is ubiquitous in nature. However, studying below ground behaviour of roots has always posed certain difficulties. Pea (Pisum sativum L.) has become a sort of model species for ecological studies about how plant roots respond to neighbouring plant roots and barriers in soil. However, published results point in several different directions. This has sometimes been interpreted as pea having sophisticated context dependent responses that can change in complex ways depending on its surroundings. To explore this further, here, we combine the result of five new experiments with published results to examine 18 unique experiments from 7 different studies for a total of 254 replicates. We used a Bayesian hierarchical meta-analysis approach to estimating the likely effect size from the available data, as well as quantify heterogeneity among different experiments, studies and cultivars. The posterior distributions show that, at the coarsest possible scale of total root production, it is unlikely that P. sativum root growth is influenced by either neighbours or barriers to root growth imposed by the walls of pots that vary in volume. We suggest that further work should consider repeating experiments that reported finer scale root plasticity in pea at the rhizosphere scale, and also consider alternative model species for the study of plant root responses to external cues.

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Leveraging joint mechanics simplifies the neural control of movement Behaviors we perform every day, such as manipulating an object or walking, require precise control of interaction forces between our bodies and the environment. These forces are generated by active muscle contractions, specified by the nervous system, and by joint mechanics, determined by the intrinsic properties of the musculoskeletal system. Depending on behavioral goals, joint mechanics might simplify or complicate control of movement by the nervous system. However, whether humans can exploit joint mechanics to simplify neural control remains unclear. Here we evaluated if leveraging joint mechanics can simplify neural control by comparing performance in three tasks that required subjects to generate specified torques about the ankle during imposed sinusoidal movements; only one task required torques that could be generated by leveraging the intrinsic mechanics of the joint. We developed a novel approach that used continuous estimates of impedance, a quantitative description of joint mechanics, and measures of muscle activity to determine the mechanical and neural contributions to the net ankle torque generated in each motor task. We found that the torque resulting from changes in neural control was reduced when ankle impedance was consistent with the task being performed, resulting in a task that required less muscular effort. Subjects perceived this task to be easier than those that were not consistent with the impedance of the ankle and were able to perform it with the highest level of consistency. These results demonstrate that leveraging the mechanical properties of a joint can simplify task completion and improve performance.

KEY POINTSO_LIInteracting with our environment requires production of interaction forces, which are generated by muscle contractions, specified by the nervous system, and by joint mechanics, determined by the intrinsic properties of the musculoskeletal system.
C_LIO_LIWe assessed whether leveraging joint mechanics can simplify neural control by having subjects complete 3 tasks, only one of which could be accomplished by leveraging joint mechanics.
C_LIO_LIWe found that subjects reduced their muscular effort, perceived the task to be easier and completed the task more consistently when the mechanics of the ankle were consistent with the task.
C_LIO_LIThese results highlight the importance of considering limb mechanics when interpreting measures of neural control related to movement and may benefit the design of mechanical interfaces that optimize human performance.
C_LI

sentences: Reduced sensorimotor beta dynamics could represent a "slowed movement state" in healthy individuals Beta oscillations (~13-30 Hz) recorded from the sensorimotor cortex have canonical amplitude changes during movement. Specifically, a movement-related beta decrease (MRBD) occurs before movement, and a post-movement beta rebound (PMBR) follows. We investigated how the MRBD and PMBR vary with movement speed. Individuals performed a task with blocks that generated longer reaction times (RTs) and shorter RTs (Slow and Fast blocks, respectively) while scalp-electroencephalography (EEG) was recorded. The timing of task events before movement was also modulated to generate blocks with certain and uncertain timing (Fixed and Varied blocks, respectively). Beta modulation was reduced in Slow blocks compared to Fast blocks (i.e., a less negative MRBD and less positive PMBR). For the movement certainty manipulation, we saw mixed behavioral and EEG results. Our primary findings align with previous work which has shown reduced movement-related beta modulation in patients with Parkinsons disease. We propose that a "slowed movement state", whether it is experimentally induced or a manifestation of Parkinsons disease bradykinesia, is represented through reduced beta dynamics. Altogether, the MRBD and PMBR may represent motor speed on a continuum with Parkinsons disease as an extreme example of slowed movement.

sentences: Engineering bacterial blight-resistant plants through CRISPR/Cas9-targeted editing of the MeSWEET10a promoter in cassava Editing of the promoter in the disease-susceptibility gene MeSWEET10a of SC8 cassava confers resistance to CBB. All mutated cassava lines had normal morphological and yield-related traits as the wild type. The results lay a research foundation for breeding cassava resistant to bacterial blight.

sentences: Improved prediction of protein-protein interactions using AlphaFold2 Predicting the structure of interacting protein chains is a fundamental step towards understanding protein function. Unfortunately, no computational method can produce accurate structures of protein complexes. AlphaFold2, has shown unprecedented levels of accuracy in modelling single chain protein structures. Here, we apply AlphaFold2 for the prediction of heterodimeric protein complexes. We find that the AlphaFold2 protocol together with optimized multiple sequence alignments, generate models with acceptable quality (DockQ[≥]0.23) for 63% of the dimers. From the predicted interfaces we create a simple function to predict the DockQ score which distinguishes acceptable from incorrect models as well as interacting from non-interacting proteins with state-of-art accuracy. We find that, using the predicted DockQ scores, we can identify 51% of all interacting pairs at 1% FPR. The protocol can be found at: https://gitlab.com/ElofssonLab/FoldDock.

sentences: Adjusting for Common Variant Polygenic Scores Improves Yield in Rare Variant Association Analyses With the emergence of large-scale sequencing data, methods for improving power in rare variant analyses (RVAT) are needed. Here, we show that adjusting for common variant polygenic scores improves the yield in gene-based RVAT across 65 quantitative traits in the UK Biobank (up to 20% increase at =2.6x10-6), without a marked increase in false-positive rates or genomic inflation. Our results illustrate how adjusting for common variant effects can aid in rare variant association discovery.

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PHIST: fast and accurate prediction of prokaryotichosts from metagenomic viral sequences SummaryPHIST (Phage-Host Interaction Search Tool) predicts prokaryotic hosts of viruses from their genomic sequences. It improves host prediction accuracy at species level over current alignment-based tools (on average by 3 percentage points) as well as alignment-free and CRISPR-based tools (by 14-20 percentage points). PHIST is also two orders of magnitude faster than alignment-based tools making it suitable for metagenomics studies.

Availability and implementationGNU-licensed C++ code wrapped in Python API available at: https://github.com/refresh-bio/phist

Contactandrzej.zielezinski@amu.edu.pl, adam.gudys@polsl.pl

Supplementary informationSupplementary data are available at publisher Web site.

sentences: Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex Lactate oxidation with NAD+ as electron acceptor is a highly endergonic reaction and some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron transferring proteins EtfA and EtfB. The electron cryo-microscopically (cryo-EM) characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 [A] resolution consists of a mobile EtfAB shuttle located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle contact each other thereby forming the D (dehydrogenase conducting) state. The intermediary Asp37 and Asp139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. A plausible novel B (bifurcation conducting) state with the EtfAB base and shuttle FADs in a productive electron transfer distance was derived by integrating Alphafold2 calculations. Kinetic analysis of enzyme variants shed light on the connection between NAD binding/release and D-to-B state transition. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is based on the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was outlined.

sentences: Relationships between biomechanical parameters, neurological recovery, and neuropathology following concussion in swine Mild traumatic brain injury (mTBI) affects millions of individuals annually primarily through falls, traffic collisions, or blunt trauma and can generate symptoms that persist for years. Closed-head rotational injury is the most common form of mTBI and is defined by a rapid change in acceleration within an intact skull. Injury kinematics - the mechanical descriptors of injury-inducing motion - explain movement of the head, energy transfer to the brain, and, therefore, determine injury severity. However, the relationship between closed-head rotational injury kinematics - such as angular velocity, angular acceleration, and injury duration - and outcome after mTBI is currently unknown. To address this gap in knowledge, we analyzed archived surgical records of 24 swine experiencing a diffuse closed-head rotational acceleration mTBI against 12 sham animals. Kinematics were contrasted against acute recovery outcomes, specifically apnea, extubation time, standing time, and recovery duration. Compared to controls, animals with mTBI were far more likely to have apnea (p<0.001) along with shorter time to extubation (p=0.023), and longer time from extubation to recovery (p=0.006). Using regression analyses with variable selection, we generated simplified linear models relating kinematics to apnea (R2=0.27), standing time (R2=0.39) and recovery duration (R2=0.42). Neuropathology was correlated with multiple kinematics, with maximum acceleration exhibiting the strongest correlation (R2=0.66). Together, these data suggest the interplay between multiple injury kinematics, including minimum velocity and middle to minimum acceleration time, best explain acute recovery parameters and neuropathology after mTBI in swine. Future experiments that independently manipulate individual kinematics could be instrumental in developing translational diagnostics for clinical mTBI.

sentences: Characterization of late structural maturation with a neuroanatomical marker that considers both cortical thickness and intracortical myelination The cortical ribbon changes throughout a persons lifespan, with the most significant changes occurring during crucial development and aging periods. Changes during adulthood are rarely investigated due to the scarcity of neuroimaging data during this period. After childhood, the brain loses gray matter, which is evidenced by an apparent reduction in cortical thickness (CT); one factor of this thinning process is intense ongoing intracortical myelination (MYEL). Here, we report age-related changes in CT, MYEL, and their ratio in 447 participants aged 18 to 57 years (BIL&GIN cohort). We propose the CT/MYEL ratio to be a multimodal cortical maturation index (MATUR) capable of reflecting 1) stages during which CT and MYEL patterns diverge and 2) the regional differences in cortical maturation that occur in adulthood. Age mainly decreased CT in all cortical regions, with larger reductions occurring in the bilateral insular lobes, temporal and frontal poles, and cingulate cortices. Age led to a linear increase in MYEL in the entire cortex and larger increases in the primary motor, auditory, and visual cortices. The effects of age on the MATUR index were characterized by both linear and quadratic components. The linear component mimicked the pattern found in CT, with 1) a robust amplification of the global and regional effects of age on CT and 2) evidence of new bilateral linear decreases in the frontal and cortical cortices. Most importantly, age exhibited additional large quadratic effects on the MATUR index in the bilateral frontal (more prominent in the right hemisphere), parietal, temporal, and cingulate regions that were not highlighted by the CT metric. Thus, the MATUR index was more sensitive to age-related cortical structural changes during adulthood than was either CT or MYEL alone. As evidenced by the large quadratic component of the effect of age, the newly proposed maturation index dramatically improved the characterization of the regional cortical territories, uncovering the latest brain maturation steps that occur before stabilization and deterioration occur in mid- and late adulthood.

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Aspergillus fumigatus drives tissue damage via iterative assaults upon mucosal integrity and immune homeostasis The human lung is constantly exposed to Aspergillus fumigatus spores, the most prevalent worldwide cause of fungal respiratory disease. Pulmonary tissue damage is a unifying feature of Aspergillus-related diseases; however, the mechanistic basis of damage is not understood. In the lungs of susceptible hosts A. fumigatus undergoes an obligatory morphological switch involving spore germination and hyphal growth. We modelled A. fumigatus infection in cultured A549 human pneumocytes, capturing phosphoactivation status of five host signalling pathways, nuclear translocation & DNA binding of eight host transcription factors, and expression of nine host response proteins over six time points encompassing exposures to live fungus and the secretome thereof. The resulting dataset, comprised of more than 1000 data points, reveals that pneumocytes mount differential responses to A. fumigatus spores, hyphae and soluble secreted products via the NF-kB, JNK, and JNK + p38 pathways respectively. Importantly, via selective degradation of host pro-inflammatory (IL-6 and IL-8) cytokines and growth factors (FGF-2), fungal secreted products reorchestrate the host response to fungal challenge as well as driving multiparametric epithelial damage, culminating in cytolysis. Dysregulation of NF-kB signalling, involving iterative stimulation of canonical and non-canonical signalling, was identified as a significant feature of host damage both in vitro and in a mouse model of invasive aspergillosis. Our data demonstrate that composite tissue damage results from iterative exposures to different fungal morphotypes and secreted products and suggest that modulation of host responses to fungal challenge might represent a unified strategy for therapeutic control of pathologically distinct types of Aspergillus-related disease.

IMPORTANCEPulmonary aspergillosis is a spectrum of diseases caused primarily by Aspergillus fumigatus. This fungus is ubiquitous in the environment and grows as a mold, which harbors and disperses spores into the environment. Like other airborne pathogens, the lung mucosa is the first point of contact with the fungus post inhalation. The outcome and severity of disease depends on the host-fungal interaction at the lung interface. We studied how the human lung interacts with spore, germ tube and hyphae growth forms to understand the sequence and dynamics of the early events, which are critical drivers of disease development and progression. Our work is significant in identifying, in response to fungal secreted products, non-canonical NF-kB activation via RelB as being a driving factor in fungus-mediated lung damage. This process could be modulated therapeutically to protect the integrity of infected lung mucosae.

sentences: The P600, but not N400, is modulated by sustained attention The functional significance of the two prominent language-related ERP components N400 and P600 is still under debate. It has recently been suggested that one important dimension along which the two vary, is in terms of automaticity versus attentional control, with N400 amplitudes reflecting more automatic and P600 amplitudes reflecting more controlled aspects of sentence comprehension. The availability of executive resources necessary for controlled processes depends on sustained attention, which fluctuates over time. Here, we thus tested whether P600 and N400 amplitudes depend on the level of sustained attention. We re-analyzed EEG and behavioral data from a sentence processing task by Sassenhagen & Bornkessel-Schlesewsky (2015, Cortex), which included sentences with morphosyntactic and semantic violations. Participants read sentences phrase by phrase and indicated whether a sentence contained any type of anomaly as soon as they had the relevant information. To quantify periods of high versus low sustained attention, we extracted a moving reaction time coefficient of variation over the entire course of the task. We found that the P600 amplitude was significantly larger during periods of low reaction time variability (high sustained attention) than in periods of high reaction time variability (low sustained attention). In contrast, the amplitude of the N400 was not affected by reaction time variability. These results thus suggest that the P600 component is sensitive to sustained attention while the N400 component is not, which provides independent evidence for accounts suggesting that P600 amplitudes reflect more controlled and N400 amplitudes more automatic aspects of sentence comprehension.

sentences: The DEAD-box RNA helicases RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis The RhlE DEAD-box RNA helicase protein family is widespread among Proteobacteria, but it is the least understood due to the lack of a clear biological function. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. RhlE1 and RhlE2 diverged during P. aeruginosa evolution; our data indicate that this resulted in a non-redundant biological role, a distinct molecular action and an enzymatic activity differentially stimulated by RNA. Whereas RhlE1 is specifically necessary for bacteria growth in cold, we show that RhlE2 acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts and multiple functionalities indispensable not only for P. aeruginosa environmental adaptation, but also for its virulence. The global action of RhlE2 relies on a unique C-terminal extension, which establishes an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery.

sentences: Greater neural pattern dissimilarity in retrieval stage of spaced learning is associated with higher meaningfulness and better memory The question of whether repeated studies bring more variability or less to our brain is a critical problem before scientist continue their further study about memory. In the past ten years, a series of neural pattern representation studies have found that under the condition of spaced learning, the neural pattern similarity(NPS) between two encoding stages increases, and the researchers claimed that these results support the idea that repeated studies bring more similarity along with repetition in our brain, which is conflicted with the encoding variability theory(Feng et al., 2019; Y. Lu et al., 2015; Xue et al., 2010, 2011a). However, we doubt this viewpoint because we think the difference between encoding processing cannot be used to represent the difference between memory states. In current experiments, we used a new experimental paradigm with a longer lag and elaboration learning task to test the encoding variability theory. By comparing the difference between neural pattern dissimilarity(NPDS1)(spaced learning - one-time learning) and NPDS2(massed learning - one-time learning) in the final test (retrieval) stage, we get the result that the NPDS1 was significantly greater than NPDS2 in the parietal lobe of 400ms and the right frontal lobe of 600ms, which is more fitting to the encoding variability mechanism. However, we believe that there is no contradiction between these two experimental evidences. On the contrary, we think they reflect different aspects of the process of spaced learning. We propose that the deficient processing in encoding stage exactly lead to less encoding variability in our memory. More importantly, this result gives us reason to double the paper published ten years ago in Science, which claimed repetition brings greater neural pattern similarity.

sentences: Non-target effects of ten essential oils on the egg-parasitoid Trichogramma evanescens Essential oils (EOs) are increasingly used as biopesticides due to their insecticidal potential. This study addresses their non-target effects on a biological control agent: the egg parasitoid Trichogramma evanescens. In particular, we tested whether EOs affected parasitoid fitness either directly, by decreasing pre-imaginal survival, or indirectly, by disrupting parasitoids orientation abilities. The effect of Anise, Fennel, Sweet orange, Basil, Coriander, Oregano, Peppermint, Mugwort, Rosemary and Thyme EOs were studied on five strains of T. evanescens. Specific experimental setups were developed, and data obtained from image analysis were interpreted with phenomenological models fitted with Bayesian inference. Results highlight the fumigant toxicity of EOs on parasitoid development. Anise, Fennel, Basil, Coriander, Oregano, Peppermint and Thyme EOs are particularly toxic and drastically reduce the emergence rate of T. evanescens. Most EOs also affect parasitoid behavior: (i) Basil, Coriander, Oregano, Peppermint, Mugwort and Thyme EOs are highly repellent for naive female parasitoids; (ii) Anise and Fennel EOs can have repellent to attractive effects depending on strains; and (iii) Sweet orange, Oregano and Rosemary EOs have no detectable impact on orientation behavior. This study shows that EOs fumigation have non-target effects on egg parasitoids. This highlights the need to cautiously precise the deployment framework of biopesticides in an agroecological perspective.

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MetaMutationalSigs: Comparison of mutational signature refitting results made easy SummaryThe analysis of mutational signatures is becoming increasingly common in cancer genetics, with emerging implications in cancer evolution, classification, treatment decision and prognosis. Recently, several packages have been developed for mutational signature analysis, with each using different methodology and yielding significantly different results. Because of the nontrivial differences in tools refitting results, researchers may desire to survey and compare the available tools, in order to objectively evaluate the results for their specific research question, such as which mutational signatures are prevalent in different cancer types. There is a need for a software that can aggregate results from different refitting packages and present them in a user-friendly way to facilitate effective comparison of mutational signatures.

Availability and implementationMetaMutationalSigs is implemented using R and python and is available for installation using Docker and available at: https://github.com/EESI/MetaMutationalSigs

ContactGail Rosen (glr26@drexel.edu).

Supplementary informationMore information about the package including test data and results are available at https://github.com/EESI/MetaMutationalSigs

sentences: Visual and auditory brain areas share a representational structure that supports emotion perception Emotionally expressive music and dance occur together across the world. This may be because features shared across the senses are represented the same way even in different sensory brain areas, putting music and movement in directly comparable terms. These shared representations may arise from a general need to identify environmentally relevant combinations of sensory features, particularly those that communicate emotion. To test the hypothesis that visual and auditory brain areas share a representational structure, we created music and animation stimuli with crossmodally matched features expressing a range of emotions. Participants confirmed that each emotion corresponded to a set of features shared across music and movement. A subset of participants viewed both music and animation during brain scanning, revealing that representations in auditory and visual brain areas were similar to one another. This shared representation captured not only simple stimulus features, but also combinations of features associated with emotion judgments. The posterior superior temporal cortex represented both music and movement using this same structure, suggesting supramodal abstraction of sensory content. Further exploratory analysis revealed that early visual cortex used this shared representational structure even when stimuli were presented auditorily. We propose that crossmodally shared representations support mutually reinforcing dynamics across auditory and visual brain areas, facilitating crossmodal comparison. These shared representations may help explain why emotions are so readily perceived and why some dynamic emotional expressions can generalize across cultural contexts.

sentences: Antibiotic action revealed by real-time imaging of the mycobacterial membrane The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

sentences: Sparse latent factor regression models for genome-wide and epigenome-wide association studies Association of phenotypes or exposures with genomic and epigenomic data faces important statistical challenges. One of these challenges is to account for variation due to unobserved confounding factors, such as individual ancestry or cell-type composition in tissues. This issue can be addressed with penalized latent factor regression models, where penalties are introduced to cope with high dimension in the data. If a relatively small proportion of genomic or epigenomic markers correlate with the variable of interest, sparsity penalties may help to capture the relevant associations, but the improvement over non-sparse approaches has not been fully evaluated yet. Here, we present least-squares algorithms that jointly estimate effect sizes and confounding factors in sparse latent factor regression models. In simulated data, sparse latent factor regression models generally achieved higher statistical performance than other sparse methods, including the least absolute shrinkage and selection operator (LASSO) and a Bayesian sparse linear mixed model (BSLMM). In generative model simulations, statistical performance was slightly lower (while being comparable) to non-sparse methods, but in simulations based on empirical data, sparse latent factor regression models were more robust to departure from the model than the non-sparse approaches. We applied sparse latent factor regression models to a genome-wide association study of a flowering trait for the plant Arabidopsis thaliana and to an epigenome-wide association study of smoking status in pregnant women. For both applications, sparse latent factor regression models facilitated the estimation of non-null effect sizes while overcoming multiple testing issues. The results were not only consistent with previous discoveries, but they also pinpointed new genes with functional annotations relevant to each application.

sentences: Decisive Role of Polymer-BSA Interactions in Biofilm Substrates on Philicity and EPS Composition Formation of extracellular polymeric substances (EPS) is a crucial step for bacterial biofilm growth. Dependence of EPS composition on the growth substrate and the conditioning of the latter is thus of primary importance. Here, we present results of studies on the growth of biofilms of two different strains each, of the Gram negative bacteria Escherichia coli and Klebsiella pneumoniae, on four polymers used commonly in indwelling medical devices - Polyethene, Polypropylene, Polycarbonate, and Polytetrafluoroethylene immersed in Bovine Serum Albumin (BSA) for 24 hrs. The polymer substrates are studied before and after immersing in BSA for 9 hrs and 24 hrs, using contact angle measurement (CAM) and Field Emission Scanning Electron Microscopy (FE-SEM) to extract, respectively, the  philicity (defined as{phi} {equiv} sin ({theta}-90{degrees}), where{theta} is contact angle of the liquid on the solid at a particular temperature and ambient pressure) and spatial Hirsch parameter H (defined from the relation, F(r) ~ r2H, where F(r) is the mean squared density fluctuation at the sample surface). H =, <0.5 or >0.5 signifies no correlation, anti-correlation, and correlation, respectively. The substrates are seen to transform from large hydrophobicity to near amphiphilicity with the formation of BSA conditioning surface layer, and the H-values distinguish the length scales of ~ 100 nm, 500 nm, and 2000 nm, with the anti-correlation increasing with length scale. Biofilms grown on the BSA-covered surfaces are studied with CAM, FE-SEM, Fourier Transform Infrared (FTIR) and Surface Enhanced Raman Spectroscopy (SERS). Most notably, the{phi} -values are independent of the bacterial species and strain but dependent on the polymer, as is also shown strikingly by both types of spectra, while H-values show some bacterial variation. Thus, the EPS composition and consequently the wetting properties of the corresponding bacterial biofilms seems to be decided by the interaction of the conditioning BSA layer with a specific polymer used as the growth substrate.

sentences: The anthropometric and physical qualities of womens rugby league Super League and international players; identifying differences in playing position and standard Participation in womens rugby league has been growing since the foundation of the English womens rugby league Super League in 2017. However, the evidence base to inform womens rugby league remains sparse. This study provides the largest quantification of anthropometric and physical qualities of womens rugby league players to date, identifying differences between positions (forwards & backs) and playing standard (Womens Super League [WSL] vs. International). The height, weight, body composition, lower body strength, jump height, speed and aerobic capacity of 207 players were quantified during the pre-season period. Linear mixed models and effects sizes were used to determine differences between positions and standards. Forwards were significantly (p < 0.05) heavier (forwards: 82.5 {+/-} 14.8kg; backs: 67.7 {+/-} 9.2kg) and have a greater body fat % (forwards: 37.7 {+/-} 6.9%; backs: 30.4 {+/-} 6.3%) than backs. Backs had significantly greater lower body power measured via jump height (forwards: 23.5 {+/-} 4.4cm; backs: 27.6 {+/-} 4.9cm), speed over 10m (forwards: 2.12 {+/-} 0.14s; backs: 1.98 {+/-} 0.11s), 20m (forwards: 3.71 {+/-} 0.27s; backs: 3.46 {+/-} 0.20s), 30m (forwards: 5.29 {+/-} 0.41s; backs: 4.90 {+/-} 0.33s), 40m (forwards: 6.91 {+/-} 0.61s; backs: 6.33 {+/-} 0.46s) and aerobic capacity (forwards: 453.4 {+/-} 258.8m; backs: 665.0 {+/-} 298.2m) than forwards. Additionally, international players were found to have greater anthropometric and physical qualities in comparison to their WSL counterparts. This study adds to the limited evidence base surrounding the anthropometric and physical qualities of elite womens rugby league players. Comparative values for anthropometric and physical qualities are provided which practitioners may use to evaluate the strengths and weaknesses of players, informing training programs to prepare players for the demands of womens rugby league.

sentences: Herbicidal activity of fluoroquinolone derivatives Development of herbicides with novel modes of action are crucial for weed control and to hinder herbicide resistance. An attractive novel herbicidal target is plant DNA gyrase, which has been demonstrated to be effectively inhibited by the known antimicrobial ciprofloxacin. Despite this good herbicidal activity ciprofloxacin is not suitable as a herbicide due to its antimicrobial activity, therefore, a diverse library of analogues was analysed to gain insight into the aspects required for herbicidal activity. This analysis revealed significant structural modifications were tolerated and that the fluoride at C-6 and a cyclic amino group at C-7 were not crucial for herbicidal activity. The analysis also revealed that these modifications also affected the antibacterial activity with one compound demonstrating good herbicidal activity and weak antibacterial activity, against both Gram-positive and Gram-negative bacteria.

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Chromosome-encoded IpaH ubiquitin ligases indicate non-human pathogenic Escherichia Until recently, Shigella and enteroinvasive Escherichia coli were thought to be primate-restricted pathogens. The base of their pathogenicity is the type 3 secretion system (T3SS) encoded by the pINV virulence plasmid, which facilitates host cell invasion and subsequent proliferation. A large family of T3SS effectors, E3 ubiquitin-ligases encoded by the ipaH genes, have a key role in the Shigella pathogenicity through the modulation of cellular ubiquitination that degrades host proteins. However, recent genomic studies identified ipaH genes in the genomes of Escherichia marmotae, a potential marmot pathogen, and an E. coli extracted from fecal samples of bovine calves, suggesting that non-human hosts may also be infected by these strains, potentially pathogenic to humans.

We performed a comparative genomic study of the functional repertoires in the ipaH gene family in Shigella and enteroinvasive Escherichia from human and predicted non-human hosts. We found that fewer than half of Shigella genomes had a complete set of ipaH genes, with frequent gene losses and duplications that were not consistent with the species tree and nomenclature. Non-human host IpaH proteins had a diverse set of substrate-binding domains and, in contrast to the Shigella proteins, two different types of the NEL C-terminal domain. Only the ipaH9.8 gene was found in Escherichia derived from both human and non-human hosts. These results provide a framework for understanding of ipaH-mediated host-pathogens interactions and suggest a need for a genomic study of fecal samples from diseased animals.

sentences: Fishing for biodiversity Fisheries are damaging, and seemingly incompatible with the conservation of marine ecosystems. Yet fish are an important source of food, and support the lives of many people in coastal communities. This paper considers an idea that a moderate intensity of fishing, appropriately scaled across species, could help in maintaining biodiversity, rather than reducing it. The scaling comes from an intuition that rates of fishing mortality of species should be kept in line with production rates of the species, a notion known as balanced harvesting. This places species conservation and exploitation on an equal footing in a single equation, showing quantitatively the relative levels of fishing mortality that species of different abundance can support. Using a dynamic model of a marine ecosystem, we give numerical evidence showing for the first time that fishing, if scaled in this way, can protect rarer species, while allowing some exploitation of species with greater production. This works because fishing mortality rates, when scaled by production, are density-dependent. Such fishing, operating adaptively to follow species production rates over time, contains a feedback that would help to protect species from overfishing in the presence of uncertainty about how marine ecosystems work.

sentences: The potential of dynamic physiological traits in young tomato plants to predict field-yield performance To address the challenge of predicting tomato yields in the field, we used whole-plant functional phenotyping to evaluate water relations under well-irrigated and drought conditions. The genotypes tested are known to exhibit variability in their yields in wet and dry fields. The examined lines included two lines with recessive mutations that affect carotenoid biosynthesis, zeta z2083 and tangerine t3406, both isogenic to the processing tomato variety M82. The two mutant lines were reciprocally grafted onto M82, and multiple physiological characteristics were measured continuously, before, during and after drought treatment in the greenhouse. A comparative analysis of greenhouse and field yields showed that the whole-canopy stomatal conductance (gsc) in the morning and cumulative transpiration (CT) were strongly correlated with field measurements of total yield (TY: r2 = 0.9 and 0.77, respectively) and plant vegetative weight (PW: r2 = 0.6 and 0.94, respectively). Furthermore, the minimum CT during drought and the rate of recovery when irrigation was resumed were both found to predict resilience.

sentences: Sparse Epistatic Regularization of Deep Neural Networks for Inferring Fitness Functions Despite recent advances in high-throughput combinatorial mutagenesis assays, the number of labeled sequences available to predict molecular functions has remained small for the vastness of the sequence space combined with the ruggedness of many fitness functions. Expressive models in machine learning (ML), such as deep neural networks (DNNs), can model the nonlinearities in rugged fitness functions, which manifest as high-order epistatic interactions among the mutational sites. However, in the absence of an inductive bias, DNNs overfit to the small number of labeled sequences available for training. Herein, we exploit the recent biological evidence that epistatic interactions in many fitness functions are sparse; this knowledge can be used as an inductive bias to regularize DNNs. We have developed a method for sparse epistatic regularization of DNNs, called the epistatic net (EN), which constrains the number of non-zero coefficients in the spectral representation of DNNs. For larger sequences, where finding the spectral transform becomes computationally intractable, we have developed a scalable extension of EN, which subsamples the combinatorial sequence space uniformly inducing a sparse-graph-code structure, and regularizes DNNs using the resulting greedy optimization method. Results on several biological landscapes, from bacterial to protein fitness functions, show that EN consistently improves the prediction accuracy of DNNs and enables them to outperform competing models which assume other forms of inductive biases. EN estimates all the higher-order epistatic interactions of DNNs trained on massive sequence spaces--a computational problem that takes years to solve without leveraging the epistatic sparsity in the fitness functions.

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Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant ({Delta}csp1). We observed decreases in cellular aggregation and surface attachment with the {Delta}csp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that {Delta}csp1 had reduced pili compared to the wild type and complemented strains. The {Delta}csp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and {Delta}csp1 revealed changes in expression of several genes important for attachment and biofilm formation in {Delta}csp1. One gene of intertest, pilA1, encodes a type IV pili subunit protein and was up regulated in {Delta}csp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines. X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.

ImportanceXylella fastidiosa is a major threat to the worldwide agriculture industry (1, 2). Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood. There are currently few effective solutions to suppress X. fastidiosa disease development or eliminate bacteria from infected plants(3). Recently, disease epidemics due to X. fastidiosa have greatly expanded(2, 4, 5), exacerbating the need for better disease prevention and control strategies. Our studies show that Csp1 is involved in X. fastidiosa virulence and stress tolerance. Understanding how Csp1 influences pathogenesis and bacteria survival can aide in developing novel pathogen and disease control strategies. We also streamlined a bioinformatics protocol to process and analyze long read Nanopore bacterial RNA-Seq data, which has previously not been reported for X. fastidiosa.

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Parkinson' disease medication alters rat small intestinal motility and microbiota composition Parkinsons disease (PD) is known to be associated with altered gastrointestinal function and microbiota composition. Altered gastrointestinal function is key in the development of small intestinal bacterial overgrowth, which is a comorbidity often observed in PD patients. Although PD medication could be an important confounder in the reported alterations, its effect per se on the microbiota composition or the gastrointestinal function at the site of drug absorption has not been studied. To this end, healthy (i.e., not PD model) wild-type Groningen rats were employed and treated with dopamine, pramipexole (in combination with levodopa/carbidopa), or ropinirole (in combination with levodopa/carbidopa) for 14 sequential days. Rats treated with dopamine agonists showed a significant reduction in the small intestinal motility and an increase in bacterial overgrowth in the distal small intestine. Importantly, significant alterations in microbial taxa were observed between the treated and vehicle groups, analogous to the changes previously reported in human PD vs HC microbiota studies. These microbial changes included an increase in Lactobacillus and Bifidobacterium, and decrease in Lachnospiraceae and Prevotellaceae. Importantly, certain Lactobacillus species correlated negatively with the plasma levels of levodopa. Overall, the results highlight the significant effect of PD medication per se on the gut microbiota, and the disease-associated comorbidities, including gastrointestinal dysfunction and small intestinal bacterial overgrowth.

Research in contextO_ST_ABSEvidence before this studyC_ST_ABSIn the last years many studies focused on the faecal microbiota profiles of Parkinsons disease (PD) patients and compared them to healthy control (HC) subjects and reported a difference between the microbiota profiles. Although some bacterial taxa have been reported to be differential abundant across studies there is a low consensus between the overall changes observed. It remains to be elucidated whether and how the disease status itself, the PD medication or the gastrointestinal dysfunction, an often reported comorbidity, plays a role in the altered microbiota profiles of Parkinsons disease patients.

Added value of this studyTwo major confounding factors potentially affecting the microbiota profiles differentiating PD patients from HC subjects are the PD medication and gastrointestinal dysfunction. We showed that PD medication elicits an decreasing effect on the small intestinal gut motility in a healthy (i.e. non Parkinson) rat model which was a contributing factor to the altered microbiota profile observed. We found that taxa reported previously to be differentially abundant were mirrored in our healthy rat model showing the impact of PD medication on microbiota profiles.

Implications of all the available evidenceThis study showed that PD medication and gastrointestinal motility are important factors in the microbiota profiles and could explain some of the differential abundant taxa reported in the cross-sectional PD microbiota studies. Future studies should take the potential side effects of medication that could elicit alterations of the microbiota composition into account when seeking for microbial biomarkers of disease status.

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The Arabidopsis thaliana PeptideAtlas; harnessing world-wide proteomics data for a comprehensive community proteomics resource We developed a new resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis proteome, such as the significance of protein splice forms, post-translational modifications (PTMs), or simply obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) analyses collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical and biological metadata. Nearly 40 million out of [~]143 million MSMS spectra were matched to the reference genome Araport11, identifying [~]0.5 million unique peptides and 17858 uniquely identified proteins (only isoform per gene) at the highest confidence level (FDR 0.0004; 2 non-nested peptides [≥] 9 aa each), assigned canonical proteins, and 3543 lower confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified, generated from pseudogenes, alternative start, stops and/or splice variants and sORFs; these features should be considered for updates to the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. This new PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS data.

One sentence summaryA new web resource providing the global community with mass spectrometry-based Arabidopsis proteome information and its spectral, technical and biological metadata integrated with TAIR and JBrowse

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Regulatory interplay between RNase III and asRNAs in E. coli; the case of AsflhD and the master regulator of motility, flhDC In order to respond to ever-changing environmental cues, bacteria have evolved resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases) and RNA chaperones, and regulatory RNAs including antisense RNAs (asRNAs). AsRNAs bound to their complementary mRNA are primary targets for the double-strand-specific endoribonuclease, RNase III. By comparing primary and processed transcripts in an rnc strain, mutated for RNase III, and its isogenic wild type strain, we detected several asRNAs. We confirmed the existence of RNase III-sensitive asRNA for crp, ompR, phoP and flhD genes, encoding master regulators of gene expression. AsflhD, the asRNA to the master regulator of motility flhDC, is slightly induced under heat-shock conditions in a sigma24 (RpoE)-dependent manner. We demonstrate that expression of AsflhD asRNA is involved in the transcriptional attenuation of flhD and thus participates in the control of the whole motility cascade. This study demonstrates that AsflhD and RNase III are additional players in the complex regulation ensuring a tight control of flagella synthesis and motility.

ImportanceThe importance of asRNAs in the regulation of gene expression has long been underestimated. Here, we confirm that asRNAs can be part of layered regulatory networks since some are found opposite to genes encoding global regulators. In particular, we show how an antisense RNA (AsflhD) to the gene expressing a transcription factor serving as the primary regulator of bacterial swimming motility (FlhD4C2) is involved in the transcriptional attenuation of flhD, which in turn impacts the expression of other genes of the motility cascade. The role of AsflhD highlights the importance of discrete fine-tuning mechanisms in the control of complex regulatory networks.

sentences: Dispersed Sleep Microstates and Associated Structural Changes in GBA1 Mouse: Relevance to Rapid Eye Movement Behavior Disorder Rapid eye movement (REM) sleep behaviour disorder (RBD) is a rare parasomnia that may predict the later occurrence of alpha-synucleinopathies. Variants in the gene encoding for the lysosomal enzyme glucocerebrosidase, GBA, strongly increase the risk of RBD. In a GBA1-mouse model recently shown to mimic prodromal stages of -synucleinopathy, we now demonstrate striking REM and NREM sleep abnormalities accompanied by distinct structural changes in the more widespread sleep neurocircuitry.

sentences: Hierarchical architecture of dopaminergic circuits enables second-order conditioning in Drosophila Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. Here, we identify a feedforward circuit formed between dopamine subsystems and show that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. We find that a slow and stable memory compartment can serve as an effective "teacher" by instructing other faster and transient memory compartments via a single key interneuron, which we identify by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the "student" compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists.

sentences: Multimodal Thrombectomy Device for Treatment of Acute Deep Venous Thrombosis Deep vein thrombosis (DVT) is a potentially deadly medical condition that is costly to treat and impacts thousands of Americans every year. DVT is characterized by the formation of blood clots within the deep venous system of the body. If a DVT dislodges it can lead to venous thromboembolism (VTE) and pulmonary embolism (PE), both of which can lead to significant morbidity or death. Current treatment options for DVT are limited in both effectiveness and safety, in part because the treatment of the DVT cannot be confined to a defined sequestered treatment zone. We therefore developed and tested a thrombectomy device that enables the sequesteration of a DVT to a defined treatment zone during fragmentation and evacuation. We observed that, compared to a predicate thrombectomy device, the sequestered approach reduced distal DVT embolization during ex vivo thrombectomy. The sequestered approach also facilitated isovolumetric infusion and suction that enabled clearance of the sequestered treatment zone without significantly impacting vein wall diameter. Results suggest that our novel device using sequestered therapy holds promise for the treatment of high risk large-volume DVTs.

sentences: Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2 The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we applied single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrated widespread tropism for nasal epithelial cell types. The host response was dominated by type I and III IFNs and interferon-stimulated gene products. This response was notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response began to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN{beta} or IFN{lambda}1 induced an efficient antiviral state that potently restricted SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data suggest that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

sentences: Secondary structure of subgenomic RNA M of SARS-CoV-2 SARS-CoV-2 belongs the Coronavirinae family. As other coronaviruses, SARS-CoV-2 is enveloped and possesses positive-sense, single-stranded RNA genome of [~] 30 kb. Genome RNA is used as the template for replication and transcription. During these processes, positive-sense genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) are created. Several studies showed importance of genomic RNA secondary structure in SARS-CoV-2 replication. However, the structure of sgRNAs have remained largely unsolved so far. In this study, we performed probing of sgRNA M of SARS-CoV-2 in vitro. This is the first experimentally informed secondary structure model of sgRNA M, which presents features likely to be important in sgRNA M function. The knowledge about sgRNA M provides insights to better understand virus biology and could be used for designing new therapeutics.

sentences: Spatial Transcriptomics-based Identification of Molecular Markers for Nanomedicine Distribution in Tumor Tissue The intratumoral accumulation of nanomedicine has been considered a passive process, referred to as the enhanced permeability and retention (EPR) effect. Recent studies have suggested that the tumor uptake of nanomedicines follows an energy-dependent pathway rather than being a passive process. Herein, to explore the factor candidates that are associated with nanomedicine tumor uptake, we developed a molecular marker identification platform by integrating microscopic fluorescence images of a nanomedicine distribution with spatial transcriptomics (ST) information. When this approach is applied to PEGylated liposomes, molecular markers related to hypoxia, glucose metabolism and apoptosis can be identified as being related to the intratumoral distribution of the nanomedicine. We expect that our method can be applied to explain the distribution of a wide range of nanomedicines and that the data obtained from this analysis can enhance the precise utilization of nanomedicines.

sentences: Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282{+/-}199.6 vs 838.1{+/-}91.33; p=0.0757), PGN (34.64{+/-}3.178 vs 17.53{+/-}2.12; p<0.0001), and LPS (405.5{+/-}48.37 vs 249.6{+/-}17.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

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Genomic estimation of quantitative genetic parameters in wild admixed populations O_LIHeritable genetic variation among free-living animals or plants is essential for populations to respond to selection and adapt. It is therefore important to be able to estimate additive genetic variance VA, which can be obtained using a generalized linear mixed model known as the animal model. An underlying assumption of the standard animal model is that the study population is genetically unstructured, which is often unrealistic. In fact, admixture might be the norm rather than the exception in the wild, like in geographically structured populations, in the presence of (im)migration, or in re-introduction and conservation contexts. Unfortunately, animal model estimators may be biased in such cases. So-called genetic group animal models that account for genetically differentiated subpopulations have recently become popular, but methodology is currently only available for cases where relatedness among individuals can be estimated from pedigrees.
C_LIO_LITo ensure that genetic group animal models with heterogeneous VA remain applicable to populations with genomic data but no pedigrees, there is a clear need to generalize these models to the case when exclusively genomic data is available. We therefore introduce such methodology for wild admixed systems by extending methods that were recently suggested in the context of plant breeding. Our extension relaxes the limiting assumptions that currently restrict their use to artificial breeding setups.
C_LIO_LIWe illustrate the usefulness of the extended genomic genetic groups animal model on a wild admixed population of house sparrows resident in an island system in Northern Norway, where genome-wide data on more than 180 000 single nucleotide polymorphisms (SNPs) is available to derive genomic relatedness. We compare our estimates of quantitative genetic parameters to those derived from a corresponding pedigree-based genetic groups animal model. The satisfactory agreement indicates that the new method works as expected.
C_LIO_LIOur extension of the very popular animal model ensures that the upcoming challenges with increasing availability of genomic data for quantitative genetic studies of wild admixed populations can be handled. To make the method widely available to the scientific community, we offer guidance in the form of a tutorial including step-by-step instructions to facilitate implementation.
C_LI

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Strain dependent structural effects and in vivo efficacy of enterovirus-D68 inhibitors Acute flaccid myelitis (AFM) leads to loss of limb control in young children and is likely due to Enterovirus-D68 (EV-D68), for which there is no current treatment. We have developed a lead isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of the pleconaril-resistant CVB3-Woodruff (IC50 6-20 nM), EV-D68 (IC50 58 nM), and other enteroviruses. A mouse respiratory model of EV-D68 infection, in which pleconaril is inactive, showed decreased viremia of 3 log units as well as statistically significant 1 log reduction in lung titer reduction at day 5 after treatment with 11526092. A cryo-electron microscopy (cryo-EM) structure of EV-D68 in complex with 11526092 suggests that the increased potency may be due to additional hydrophobic interactions. Cryo-EM structures of 11526092 and pleconaril demonstrate destabilization of EV-D68 (MO strain) compared to the previously described stabilization of EV-D68 (Fermon strain) with pleconaril, illustrating clear strain dependent mechanisms of this molecule. 11526092 represents a more potent inhibitor in vitro with in vivo efficacy providing a potential future treatment for EV-D68 and AFM, suggesting an improvement over pleconaril for further optimization.

One-Sentence Summary11526092 demonstrates protein destabilization, improved in vitro potency and in vivo efficacy when compared with pleconaril against EV-D68.

sentences: Putting the theory into 'burstlet theory': A biophysical model of bursts and burstlets in the respiratory preBötzinger complex Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBotzinger complex (preBotC). In in vitro slice preparations containing the preBotC, extracellular potassium must be elevated above physiological levels (to 7 - 9 mM) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBotC projects. Reexamination of how extracellular K+ affects preBotC neuronal activity has revealed that low amplitude oscillations persist at physiological levels. These oscillatory events are sub-threshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBotC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBotC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBotC population requires intracellular Ca2+ dynamics and activation of a calcium-activated non-selective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca2+ influx as well as Ca2+ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated non-selective cationic current can explain all of the key observations underlying the burstlet theory of respiratory rhythm generation. Thus, we provide a mechanistic basis to unify the experimental findings on rhythm generation and motor output recruitment in the preBotC.

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Adeno-associated virus type 2 (AAV2) uncoating is a stepwise process and is linked to structural reorganization of the nucleolus Nucleoli are membrane-less structures located within the nucleus and are known to be involved in many cellular functions, including stress response and cell cycle regulation. Besides, many viruses can employ the nucleolus or nucleolar proteins to promote different steps of their life cycle such as replication, transcription and assembly. While adeno-associated virus type 2 (AAV2) capsids have previously been reported to enter the host cell nucleus and accumulate in the nucleolus, both the role of the nucleolus in AAV2 infection, and the viral uncoating mechanism remain elusive. In all prior studies on AAV uncoating, viral capsids and viral genomes were not directly correlated on the single cell level, at least not in absence of a helper virus. To elucidate the properties of the nucleolus during AAV2 infection and to assess viral uncoating on a single cell level, we combined immunofluorescence analysis for detection of intact AAV2 capsids and capsid proteins with fluorescence in situ hybridization for detection of AAV2 genomes. The results of our experiments provide evidence that uncoating of AAV2 particles occurs in a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar structure.

Author SummaryAdeno-associated virus (AAV) capsids have been reported to enter the host cell nucleus and accumulate in the nucleolus. However, both the role of the nucleolus in AAV2 infection as well as the viral uncoating mechanism remain unknown. Here, we provide evidence that uncoating of the AAV2 particle is a stepwise process that is completed in the nucleolus and supported by alteration of the nucleolar morphology.

sentences: The META tool optimizes metagenomic analyses across sequencing platforms and classifiers. A major challenge in the field of metagenomics is the selection of the correct combination of sequencing platform and downstream metagenomic analysis algorithm, or  classifier. Here, we present the Metagenomic Evaluation Tool Analyzer (META), which produces simulated data and facilitates platform and algorithm selection for any given metagenomic use case. META-generated in silico read data are modular, scalable, and reflect user-defined community profiles, while the downstream analysis is done using a variety of metagenomic classifiers. Reported results include information on resource utilization, time-to-answer, and performance. Real-world data can also be analyzed using selected classifiers and results benchmarked against simulations. To test the utility of the META software, simulated data was compared to real-world viral and bacterial metagenomic samples run on four different sequencers and analyzed using 12 metagenomic classifiers. Lastly, we introduce  META Score: a unified, quantitative value which rates an analytic classifiers ability to both identify and count taxa in a representative sample.

sentences: Liver-secreted fluorescent blood plasma markers enable chronic imaging of microcirculation Studying blood microcirculation is vital for gaining insights into vascular diseases. Acute administration of fluorescent tracers is currently used for deep tissue blood flow imaging. This is invasive, and the plasma fluorescence decreases within an hour of administration. We report a novel approach for the longitudinal study of vasculature. Using a single systemic administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to label the blood in mice. All segments of the vasculature in brain and peripheral tissue are observable by two-photon microscopy within two weeks of vector administration. This approach allows for observation of circulation without the need for repeated administration for several months. We demonstrate the chronic assessment of vascular functions at micro-and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease.

sentences: Deactivation of SARS-CoV-2 surrogate porcine epidemic diarrhea virus with electron beam irradiation under the cold chain transportation condition Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prevailed all over the world and emerged as a significant public health emergency. The rapid outbreak of SARS-CoV-2 is largely due to its high transmission capacity. Studies implied that the cold chain logistics would be a potential route for the spread of SARS-CoV-2. The low temperature condition of the cold chain is conducive to survival and transmission of virus. Thus, the virus disinfection in cold chain should not be neglected for controlling COVID-19. However, due to the low temperature feature of the cold-chain, the virus disinfecting methods suitable in cold chain are limited. Here the high-energy electron beam irradiation is proposed to disinfect the SARS-CoV-2 in cold chain logistics. We evaluated the impact of high-energy electron beam irradiation on porcine epidemic diarrhea virus (PEDV), an enveloped virus surrogate for SARS-CoV-2, and explored the possible mechanism of the action of high-energy electron beam irradiation on PEDV. The irradiation dose of 10 kGy inactivated 98.1 % PEDV on the both top and bottom surfaces of various packaging materials under cold chain frozen condition. High-energy electron beam inactivated PEDV by inducing damages on viral genome or even capsid.

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Transposable elements in the genome of the lichen-forming fungus Umbilicaria pustulata, and their distribution in different climate zones along elevation BackgroundTransposable elements (TEs) are an important source of genome plasticity across the tree of life. Accumulating evidence suggests that TEs may not be randomly distributed in the genome. Drift and natural selection are important forces shaping TE distribution and accumulation, acting directly on the TE element or indirectly on the host species. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the hosts ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold-temperate. We trace the occurrence of the newly identified TEs in populations along three replicated elevation gradients using a Pool-Seq approach, to identify TE insertions of potential adaptive significance.

ResultsWe found that TEs cover 21.26 % of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. Out of a total of 182 TE copies we identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function.

ConclusionsThis pioneering study into the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution. Particularly, it may serve as a foundation for assessing the impact of TE dynamics on fungal adaptation to the abiotic environment, and the impact of TE activity on the evolution and maintenance of a symbiotic lifestyle.

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Common breast cancer risk loci predispose to distinct tumor subtypes BackgroundGenome-wide association studies (GWAS) have identified multiple common breast cancer susceptibility variants. Many of these variants have differential associations by estrogen receptor (ER), but how these variants relate with other tumor features and intrinsic molecular subtypes is unclear.

MethodsAmong 106,571 invasive breast cancer cases and 95,762 controls of European ancestry with data on 173 breast cancer variants identified in previous GWAS, we used novel two-stage polytomous logistic regression models to evaluate variants in relation to multiple tumor features (ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and grade) adjusting for each other, and to intrinsic-like subtypes.

ResultsEighty-five of 173 variants were associated with at least one tumor feature (false discovery rate <5%), most commonly ER and grade, followed by PR and HER2. Models for intrinsic-like subtypes found nearly all of these variants (83 of 85) associated at P<0.05 with risk for at least one luminal-like subtype, and approximately half (41 of 85) of the variants were associated with risk of at least one non-luminal subtype, including 32 variants associated with triple-negative (TN) disease. Ten variants were associated with risk of all subtypes in different magnitude. Five variants were associated with risk of luminal A-like and TN subtypes in opposite directions.

ConclusionThis report demonstrates a high level of complexity in the etiology heterogeneity of breast cancer susceptibility variants and can inform investigations of subtype-specific risk prediction.

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Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration Diatoms are unicellular algae, characterized by silica cell walls. The silica elements are formed intracellularly in a membrane-bound silica deposition vesicle (SDV), and are exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements is a long-standing enigma. We studied membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during the formation of the mineral phase it is in tight association with the SDV membranes, which are forming a precise mold of the delicate geometrical patterns. During exocytosis, the distal SDV membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. Within the cell, there is no evidence for the formation of a new plasma membrane, thus the proximal SDV membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.

Significance StatementExocytosis is a fundamental process for cell metabolism, communication, and growth. During exocytosis, an intracellular vesicle fuses with the plasma membrane to release its contents. In classical exocytosis, where the exocytosed vesicles are much smaller than the cell, membrane homeostasis is maintained by recycling excess membranes back into the cell. However, an extreme case of exocytosis is the extrusion of large and rigid cell wall elements by unicellular marine algae. During this process, the cell needs to deal with a potential doubling of its plasma membrane. This study reports on a unique exocytosis mechanism used by these organisms, in which the cells cope with the geometrical and physical challenges of exocytosis by releasing a significant amount of membranes to the extracellular space.

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

sentences: Immune phase transition under steroid treatment The steroid hormone, Glucocorticoid (GC) is a well-known immunosuppressant that controls T cell-mediated adaptive immune response. In this work, we have developed a minimal kinetic network model of T-cell regulation connecting relevant experimental and clinical studies to quantitatively understand the long-term effects of GC on pro-inflammatory T-cell (Tpro) and anti-inflammatory T-cell (Tanti) dynamics. Due to the antagonistic relation between these two types of T-cells, their long-term steady-state population ratio helps us to characterize three classified immune-regulations: (i) weak ([Tpro]>[Tanti]); (ii) strong ([Tpro]<[Tanti]), and (iii) moderate ([Tpro] [~] [Tanti]); holding the characteristic bistability). In addition to the differences in their long-term steady-state outcome, each immune-regulation shows distinct dynamical phases. In the pre-steady, a characteristic intermediate stationary phase is observed to develop only in the moderate regulation regime. In the medicinal field, the resting time in this stationary phase is distinguished as a clinical latent period. GC dose-dependent steady-state analysis shows an optimal level of GC to drive a phase-transition from the weak/auto-immune prone to the moderate regulation regime. Subsequently, the pre-steady state clinical latent period tends to diverge near that optimal GC level where [Tpro]: [Tanti] is highly balanced. The GC-optimized elongated stationary phase explains the rationale behind the requirement of long-term immune diagnostics, especially when long-term GC-based chemotherapeutics and other immunosuppressive drugs are administrated. Moreover, our study reveals GC sensitivity of clinical latent period which might serve as an early warning signal in the diagnosis of different immune phases and determining immune phase-wise steroid treatment.

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Black chromatin is indispensable for accurate simulations of Drosophila melanogaster chromatin structure. The interphase chromatin structure is extremely complex, precise and dynamic. Experimental methods can only show the frequency of interaction of the various parts of the chromatin. Therefore, it is extremely important to develop theoretical methods to predict the chromatin structure. In this publication, we describe the necessary factors for the effective modeling of the chromatin structure in Drosophila melanogaster. We also compared Monte Carlo with Molecular Dynamic methods. We showed that incorporating black, non-reactive chromatin is necessary for successfully prediction of chromatin structure, while the loop extrusion model or using Hi-C data as input are not essential for the basic structure reconstruction.

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Cerebellar neuronal dysfunction accompanies early motor symptoms in Spinocerebellar Ataxia Type 3 and is partially alleviated upon chronic citalopram treatment Spinocerebellar Ataxia Type 3 (SCA3) is an adult-onset, progressive ataxia. SCA3 presents with ataxia before any gross neuropathology. A feature of many cerebellar ataxias is aberrant cerebellar output that contributes to motor dysfunction. We examined whether abnormal cerebellar output was present in the CMVMJD135 SCA3 mouse model and, if so, whether it correlated with the disease onset and progression. In vivo recordings showed that the activity of deep cerebellar nuclei neurons, the main output of the cerebellum, was altered. The aberrant activity correlated with the onset of ataxia. However, although the severity of ataxia increased with age, the severity of the aberrant cerebellar output was not progressive. The abnormal cerebellar output, however, was accompanied with non-progressive abnormal activity of their upstream synaptic inputs, the Purkinje cells. In vitro recordings indicated that alterations in both intrinsic Purkinje cell pacemaking and in their synaptic inputs contributed to abnormal Purkinje cell activity. These findings implicate abnormal cerebellar physiology as an early, consistent contributor to pathophysiology in SCA3, and suggest that the aberrant cerebellar output could be an appropriate therapeutic target in SCA3.

Summary StatementIn a mouse model of Spinocerebellar ataxia type 3 aberrant cerebellar physiology is apparent early in disease, before overt neuronal pathology or neuronal death.

sentences: Transcriptome Analysis of the Nematode Caenorhabditis elegans and Litoditis marina in a Chemically Defined Food Environment Diets regulate animal development, reproduction, and lifespan. However, the underlying molecular mechanisms remain elusive. A chemically defined CeMM diet attenuates development and promotes longevity of C. elegans, but whether it impact on other nematodes is unknown. Here, we studied the effect of the CeMM diet on the development and longevity of the marine nematode Litoditis marina, which belongs to the same family as C. elegans. We further investigated genome-wide transcriptional responses to CeMM and OP50 diet for both nematodes, respectively. We observed that the CeMM diet attenuated L. marina development but did not extend its lifespan. We found that many of the FOXO DAF-16 target genes, lysosome and xenobiotic metabolism related genes were significantly increased on the CeMM, which might contribute to the lifespan extension of C. elegans. Notably, we found that the expression of lysosome and xenobiotic metabolism pathway genes was significantly down regulated in L. marina on CeMM, which might explain why the CeMM diet could not promote the lifespan of L. marina compared to bacterial feeding. Additionally, down-regulation of several RNA transcription and protein generation and related processes genes might not only be involved in extending longevity but also contribute to attenuating development of C. elegans on CeMM, while down-regulation of unsaturated fatty acids synthesis genes might contribute to slow down the growth of L. marina on CeMM. Further genetic analysis of candidate gene(s) of longevity and development in C. elegans and L. marina will provide the molecular mechanisms underlying how diets regulate animal physiology and health in the context of global climate change with variable nutritional environments.

sentences: Impaired spatial learning and suppression of sharp wave ripples by cholinergic activation at the goal location The hippocampus plays a central role in long-term memory formation, and different hippocampal network states are thought to have different functions in this process. These network states are controlled by neuromodulatory inputs, including the cholinergic input from the medial septum. Here, we used optogenetic stimulation of septal cholinergic neurons to understand how cholinergic activity affects different stages of spatial memory formation in a reward-based navigation task in mice. We found that optogenetic stimulation of septal cholinergic neurons (1) impaired memory formation when activated at goal location but not during navigation; (2) reduced sharp wave-ripple (SWR) incidence at goal location; and (3) reduced SWR incidence and enhanced the-ta-gamma oscillations during sleep. These results underscore the importance of appropriate timing of cholinergic input in long-term memory formation, which might help explain the limited success of cholinesterase inhibitor drugs in treating memory impairment in Alzheimers disease.

sentences: A Fatty Acid Anabolic Pathway in Specialized-Cells Remotely Controls Oocyte Activation in Drosophila Pheromone-mediated partner recognition is crucial for maintenance of animal species. Here, we discover a metabolic link between pheromone and gamete physiology. In female genital tract, oocyte maturation is arrested at a specific meiotic-phase. Release of this arrest, called oocyte-activation, is triggered by a species-dependent signal. We show in Drosophila melanogaster that oenocytes, which produce the fatty acids (FAs) used as precursors of cuticular hydrocarbons (CHCs), including pheromones, are also essential for oocyte activation. We identified a set of FA-anabolic enzymes required within oenocytes for the synthesis of a particular FA that is not a CHC precursor but controls oocyte activation. Our study thus reveals that two tightly linked FA-anabolic pathways act in parallel, one to produce sexual pheromones, the other to initiate embryonic development. Given that pheromone-deficient Drosophila melanogaster females are highly attractive for males irrespective of their species, this oenocyte function might have evolved to prevent hybrid development.

sentences: PhycoMine: A Microalgae Data Warehouse PhycoMine is data warehouse system created to fostering the analysis of complex and integrated data from microalgae species in a single computational environment. The PhycoMine was developed on top of the InterMine software system, and it has implemented an extended database model, containing a series of tools that help the users in the analysis and mining of individual data and group data. The platform has widgets created to facilitate simultaneous data mining of different datasets. Among the widgets implemented in PhycoMine, there are options for mining chromosome distribution, gene expression variation via transcriptomics, proteomics sets, Gene Onthology enrichment, KEGG enrichment, publication enrichment, EggNOG, Transcription factors and transcriptional regulators enrichment and phenotypical data. These widgets were created to facilitate data visualization of the gene expression levels in different experimental setups, for which RNA-seq experimental data is available in data repositories. For comparative purposes, we have reanalyzed 200 RNA-seq datasets from Chlamydomonas reinhardtii, a model unicellular microalga, for optimizing the performance and accuracy of data comparisons. We have also implemented widgets for metabolic pathway analysis of selected genes and proteins and options for biological network analysis. The option for analysis of orthologue genes was also included. With this platform, the users can perform data mining for a list of genes or proteins of interest in an integrated way through accessing the data from different sources and visualizing them in graphics and by exporting the data into table formats. The PhycoMine platform is freely available and can be visited through the URL https://PhycoMine.iq.usp.br.

sentences: Wikidata and the bibliography of life Biological taxonomy rests on a long tail of publications spanning nearly three centuries. Not only is this literature vital to resolving disputes about taxonomy and nomenclature, for many species it represents a key source - indeed sometimes the only source - of information about that species. Unlike other disciplines such as biomedicine, the taxonomic community lacks a centralised, curated literature database (the "bibliography of life"). This paper argues that Wikidata can be that database as it has flexible and sophisticated models of bibliographic information, and an active community of people and programs ("bots") adding, editing, and curating that information. The paper also describes a tool to visualise and explore bibliography information in Wikidata and how it links to both taxa and taxonomists.

sentences: Dynamic persistence of intracellular bacterial communities of uropathogenic Escherichia coli in a human bladder-chip model of urinary tract infections Uropathogenic Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbours of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.

sentences: Modeling the C. elegans Germline Stem Cell Genetic Network using Automated Reasoning Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible model system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. This work lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.

sentences: Type I IFN promotes pathogenic inflammatory monocyte maturation during H5N1 infection Ly6Chi inflammatory monocytes show high IFN responses, and contribute to both protective and pathogenic functions following influenza virus infection. In order to understand the significance of IFN responses in this subset, we examined monocytes during infection with a lethal H5N1 virus that induces high levels of IFN and a low-pathogenicity H1N1 virus that induces low levels of IFN. We show that H5N1 infection results in early recruitment of high numbers of Ly6Chi monocytes and induction of chemokines and Ifnb1. Using unbiased transcriptomic and proteomic approaches, we also find that monocytes are significantly enriched during H5N1 infection and are associated with chemokine and IFN signatures in mice, and with severity of symptoms after influenza virus infection in humans. Recruited Ly6Chi monocytes subsequently become infected in the lung, produce IFN-{beta}, and mature into FasL+ monocyte-derived cells (FasL+MCs) expressing dendritic cell markers. Both Ccr2-/- and Faslgld mice are protected from lethal infection, indicating that monocytes contribute to pathogenesis. Global loss of type I and type III IFN signaling in Stat2-/- mice results in loss of monocyte recruitment, likely reflecting a requirement for IFN-dependent chemokine induction. Here we show that IFN is not directly required for monocyte recruitment on an IFN-sufficient background, but is required for maturation to FasL+MCs. Loss of IFN signaling skews to a Ly6Clo phenotype associated with tissue repair, suggesting that IFN signaling in monocytes is a critical determinant of influenza virus pathogenesis.

sentences: Nucleolin loss-of-function leads to aberrant FGF signaling and craniofacial anomalies rRNA transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies, however the cellular and molecular reasons for this are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. We discovered that Nucleolin is dynamically expressed during embryonic development with high enrichment in the craniofacial tissues. Consistent with this pattern of expression, ncl homozygous mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observe that ncl-/- mutants exhibit decreased rRNA synthesis and p53-dependent neuroepithelial cell death. In addition, the half-life of fgf8a mRNA is reduced in ncl-/- mutants, which perturbs Fgf signaling, resulting in misregulation of Sox9a mediated chondrogenesis and Runx2 mediated osteogenesis. Exogenous addition of human recombinant FGF8 to the mutant zebrafish significantly rescues the cranioskeletal phenotype, suggesting that Nucleolin regulates osteochondroprogenitor differentiation during craniofacial development by post-transcriptionally regulating Fgf signaling. Our work has therefore uncovered a novel tissue-specific function for Nucleolin in rRNA transcription and growth factor signaling during embryonic craniofacial development.

sentences: A brainstem integrator for self-localization and positional homeostasis To accurately track self-location, animals need to integrate their movements through space. In amniotes, representations of self-location have been found in regions such as the hippocampus. It is unknown whether more ancient brain regions contain such representations and by which pathways they may drive locomotion. Fish displaced by water currents must prevent uncontrolled drift to potentially dangerous areas. We found that larval zebrafish track such movements and can later swim back to their earlier location. Whole-brain functional imaging revealed the circuit enabling this process of positional homeostasis. Position-encoding brainstem neurons integrate optic flow, then bias future swimming to correct for past displacements by modulating inferior olive and cerebellar activity. Manipulation of position-encoding or olivary neurons abolished positional homeostasis or evoked behavior as if animals had experienced positional shifts. These results reveal a multiregional hindbrain circuit in vertebrates for optic flow integration, memory of self-location, and its neural pathway to behavior.

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MORPHOLOGICAL DETERMINANTS OF CELL-TO-CELL VARIATIONS IN ACTION POTENTIAL DYNAMICS IN SUBSTANTIA NIGRA DOPAMINERGIC NEURONS Action potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. AP shape is also used to distinguish neuronal populations, as it greatly varies between neuronal types. For instance, AP duration ranges from hundreds of microseconds in cerebellar granule cells to 2-3 milliseconds in substantia nigra pars compacta (SNc) dopaminergic (DA) neurons. While most of this variation seems to arise from differences in the subtypes of voltage- and calcium-gated ion channels expressed, a few studies suggested that dendritic morphology may also affect AP shape. However, AP duration also displays significant variability in a same neuronal type, while the determinants of these variations are poorly known. Using electrophysiological recordings, morphological reconstructions and realistic Hodgkin-Huxley modeling, we investigated the relationships between dendritic morphology and AP shape in SNc DA neurons. In this neuronal type where the axon arises from an axon-bearing dendrite (ABD), the duration of the somatic AP could be predicted from a linear combination of the complexities of the ABD and the non-ABDs. Dendrotomy simulation and experiments showed that these correlations arise from the causal influence of dendritic topology on AP duration, due in particular to a high density of sodium channels in the somato-dendritic compartment. In addition, dendritic morphology also modulated AP back-propagation efficiency in response to barrages of EPSCs in the ABD. In line with previous findings, these results demonstrate that dendritic morphology plays a major role in defining the electrophysiological properties of SNc DA neurons and their cell-to-cell variations.

SIGNIFICANCE STATEMENTAction potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. AP shape (e.g. duration) greatly varies between neuronal types but also within a same neuronal type. While differences in ion channel expression seem to explain most of AP shape variation across cell types, the determinants of cell-to-cell variations in a same neuronal type are mostly unknown. We used electrophysiological recordings, neuronal reconstruction and modeling to show that, due to the presence of sodium channels in the somato-dendritic compartment, a large part of cell-to-cell variations in somatic AP duration in substantia nigra pars compacta dopaminergic neurons is explained by variations in dendritic topology.

sentences: Large Bi-ethnic Study of Plasma Proteome Leads to Comprehensive Mapping of cis-pQTL and Models for Proteome-wide Association Studies Improved understanding of genetic regulation of proteome can facilitate the identification of causal mechanisms for complex traits. We analyzed data on 4,657 plasma proteins from 7,213 European American (EA) and 1,871 African American (AA) individuals from the ARIC study, and further replicated findings on 467 AA individuals from the AASK study. Here we identified 2,004 proteins in EA and 1,618 in AA, with majority overlapping, which showed associations with common variants in cis-regions. Availability of AA samples led to smaller credible sets and significant number of population-specific cis-pQTLs. Elastic-net produced powerful models for protein prediction in both populations. An application of proteome-wide association studies (PWAS) to serum urate and gout, implicated several proteins, including IL1RN, revealing the promise of the drug anakinra to treat acute gout flares. Our study demonstrates the value of large and diverse ancestry study for genetic mechanisms of molecular phenotypes and their relationship with complex traits.

sentences: Molecular Dynamics Reveals the Effects of Temperature on Critical SARS-CoV-2 Proteins Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly identified RNA virus that causes the serious infection Coronavirus Disease 2019 (COVID-19). The incidence of COVID-19 is still increasing worldwide despite the summer heat and cool winter. However, little is known about seasonal stability of SARS-CoV-2. Herein, we employ Molecular Dynamics (MD) simulations to explore the effect of temperature on four critical SARS-CoV-2 proteins. Our work demonstrates that the spike Receptor Binding Domain (RBD), Main protease (Mpro), and nonstructural protein 3 (macro X) possesses extreme thermos-stability when subjected to temperature variations rendering them attractive drug targets. Furthermore, our findings suggest that these four proteins are well adapted to habitable temperatures on earth and are largely insensitive to cold and warm climates. Furthermore, we report that the critical residues in SARS-CoV-2 RBD were less responsive to temperature variations as compared to the critical residues in SARS-CoV. As such, extreme summer and winter climates, and the transition between the two seasons, are expected to have a negligible effect on the stability of SARS-CoV-2 which will marginally suppress transmission rates until effective therapeutics are available world-wide.

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Synthetic dysmobility screen unveils an integrated STK40-YAP-MAPK system driving cell migration Integrating signals is essential for cell survival, leading to the concept of synthetic lethality. However, how signaling is integrated to control cell migration remains unclear. By conducting a "two-hit" screen, we revealed the synergistic reduction of cell migration when serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) were simultaneously suppressed. Single-cell analyses showed that STK40 knockdown reduced cell motility and coordination by strengthening focal adhesion (FA) complexes. Furthermore, STK40 knockdown reduced translocation of yes-associated protein (YAP) into the nucleus, while MAPK inhibition further weakened YAP activities in the nucleus to disturb FA remodeling. Altogether, we unveiled an integrated STK40-YAP-MAPK system regulating cell migration, and introduced "synthetic dysmobility" as a novel strategy to collaboratively control cell migration.

One Sentence SummaryBlocking collaborative pathways within the integrated signaling network synergistically disrupts the migration of cells.

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Characterizing the middle-age neurophysiology using EEG/MEG Middle adulthood - the period of life between 40 and 60 years of age - is accompanied by important physical and emotional changes, as well as cognitive and neuronal ones. Nevertheless, middle age is often overlooked in neuroscience under the assumption that this is a time of relative stability, although cognitive decline, as well as changes in brain structure and function are well-established by the age of 60.

Here we characterized the middle-aged brain in the context of healthy younger and older adults by assessing resting-state electrophysiological and neuromagnetic activity in two different samples (N = 179, 631). Alpha and beta oscillations - two key ageing signatures - were analyzed in terms of spectral power and burst events. While posterior alpha power and burst rate features changed linearly with age, similarly to behavioral measures, sensorimotor beta power and burst rate properties varied non-linearly, with inflection points during middle age. The findings suggest that ageing is characterized by distinct spatial and temporal brain dynamics, some critically arising in middle age.

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Principles of Dopamine Binding to Carbon Surfaces Fast Scan Cyclic Voltammetry (FSCV) combined with carbon electrodes is considered as the gold standard method for real-time detection of oxidizable neurotransmitters. The bioinert nature, rapid electron transfer kinetics and long-term stability make carbon an attractive material for probing brain electrochemistry. Herein, we first demonstrate a rapid fabrication process of carbonized nanopipettes and subsequently perform experimental measurements and theoretical simulations to study mechanisms of dopamine binding on carbonized surfaces. To explain the kinetics of dopamine oxidation on carbonized electrodes we adapted the electron-proton transfer model originally developed by Compton and found that the electron-proton transfer model best explains the experimental observations. We further investigated the electron-proton transfer theory by constructing a Density Function Theory (DFT) for visualization of dopamine binding to graphite-like surfaces consisting of heteroatoms. For graphite surfaces that are capped with hydrogen alone, we found that dopamine is oxidized, whereas, on graphite surfaces doped with heteroatoms such as nitrogen and oxygen, we found deprotonation of dopamine along with oxidation thus validating our experimental and theoretical data. These observations provide mechanistic insights into multistep electron transfer during dopamine oxidation on graphite surfaces.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/457508v1_ufig1.gif" ALT="Figure 1">
View larger version (29K):
org.highwire.dtl.DTLVardef@8bee9corg.highwire.dtl.DTLVardef@de66ecorg.highwire.dtl.DTLVardef@1373032org.highwire.dtl.DTLVardef@3d61d0_HPS_FORMAT_FIGEXP  M_FIG A: Pictorial view of the experimental setup of carbonized electrodes. The application of waveform causes the oxidation of dopamine. B. Background subtracted voltammogram of dopamine, wherein the waveform applied is -0.4V to 1.3V and cycled back at -0.4V at 200 V s-1 at 10 Hz. C: A hotspot showing the oxidation and reduction of dopamine, wherein two distinct redox spots can be seen. The first redox spot can be seen at 0.0V and the second one at 0.5V. Thus showing a multistep electron transfer for dopamine. D: A DFT model for dopamines interaction with graphite surfaces doped with nitrogen atoms. Oxidation of oxygen (red) can be seen with loss of protons.

C_FIG

sentences: Transactive response DNA-binding Protein (TDP-43) regulates early HIV-1 entry and infection. The transactive response DNA-binding protein (TDP-43) is an important regulator of mRNA, being reported to stabilize the anti-HIV factor, histone deacetylase 6 (HDAC6). However, little is known about the role of TDP-43 in HIV infection. In this work, we seek for the TDP-43 function on regulating CD4+ T cell permissibility to HIV infection. We observed that over-expression of wt-TDP-43 in CD4+ T cells stabilized HDAC6, increasing mRNA and the protein levels of this antiviral enzyme. Under this experimental condition, HIV-1 infection was impaired, independently of the viral envelope glycoprotein (Env) complex tropism. The results obtained by using an HIV-1 Env-mediated cell-to-cell fusion model, under the same experimental conditions, suggest that the increase in TDP-43 levels negatively affects the viral Env fusion capacity. Moreover, the specific siRNA silencing of endogenous TDP-43 in target cells lead to a significant decrease in the levels of HDAC6 which consistently induces an increase in the fusogenic and infection activities of the HIV-1 Env. These observations were confirmed by using primary viral Envs from HIV+ individuals with different clinical phenotypes. An increase in the level of expression of wt-TDP-43 strongly reduced the Envs infection activity of viremic non-progressors (VNP) and rapid progressors (RP) HIV+ individuals down to the levels of the inefficient HIV-1 Envs from long-term non-progressor elite controllers (LTNP-EC) individuals. On the contrary, low levels of endogenous TDP-43, obtained after specific siRNA-TDP-43 knocking-down, significantly favors the infection activity of primary HIV-1 Envs of VNP and RP individuals, leading to an increase in the infection ability of the primary HIV-1/LTNP-EC Envs. Based on this evidence, we interpret that TDP-43 conditions cell permissibility to HIV infection by affecting viral Env fusion and infection capacities, at least by altering the cellular levels of the antiviral enzyme HDAC6.

sentences: Novel measures of Morris water maze performance that use vector field maps to assess accuracy, uncertainty, and intention of navigational searches Most commonly used behavioural measures for testing learning and memory in the Morris water maze (MWM) involve comparisons of an animals residence time in different quadrants of the pool. Such measures are limited in their ability to test different aspects of the animals performance. Here, we describe novel measures of performance in the MWM that use vector fields to capture the motion of mice as well as their search pattern in the maze. Using these vector fields, we develop quantitative measures of performance that are intuitive and more sensitive than classical measures. First, we describe search patterns in terms of vector field properties and use these properties to define three metrics of spatial memory namely Spatial Accuracy, Uncertainty and, Intensity of Search. We demonstrate the usefulness of these measures using four different data sets including comparisons between different strains of mice, an analysis of two mouse models of Noonan syndrome (Ptpn11 D61G and Ptpn11 N308D/+), and a study of goal reversal training. Importantly, besides highlighting novel aspects of performance in this widely used spatial task, our measures were able to uncover previously undetected differences, including in an animal model of Noonan syndrome, which we rescued with the mitogen activated protein kinase kinase (MEK) inhibitor SL327. Thus, our results show that our approach breaks down performance in the Morris water maze into sensitive measurable independent components that highlight differences in spatial learning and memory in the MWM that were undetected by conventional measures.

sentences: Multiple paths lead to salt tolerance - pre-adaptation vs dynamic responses from two closely related extremophytes Salt tolerance is a complex trait with much of the underlying genetic variation and integrated response strategies yet to be discovered from stress adapted plants. Schrenkiella parvula and Eutrema salsugineum are salt-tolerant extremophytes related to Arabidopsis thaliana in Brassicaceae. We investigated their response strategies contrasted against the salt-sensitive model, A. thaliana to cope with salt stresses via transcriptomic, metabolomic, and ionomic adjustments. The extremophytes exemplified divergent routes to achieve nutrient balance, build osmotolerance, boost antioxidant capacity, and extend transcriptomic support for modified ion transport and stress signaling. Those led to similar molecular phenotypes adapted to salt stress in the extremophytes, absent in A. thaliana. The predominant transcriptomic signals in all three species were associated with salt stress. However, root architecture modulation mediated by negative regulation of auxin and ABA signaling supported minimally-affected root growth unique to each extremophyte during salt treatments. Overall, E. salsugineum exhibited pre-adapted responses at the metabolome level, whereas S. parvula showed dynamic metabolomic responses coupled to a pre-adapted transcriptome to survive salt stress. Our work shows that the two extremophytes share common salt tolerance features, but differ substantially in pathways leading to the convergent, adaptive traits.

sentences: The 'squalene route' to carotenoid biosynthesis is widespread in Bacteria. Squalene is mostly associated with the biosynthesis of polycyclic triterpenes. Although there have been suggestions that squalene could be involved in the biosynthesis of carotenoids, functionally and evolutionarily related to polycyclic triterpenes, evidence of this  squalene route in nature was lacking. We demonstrate that planctomycetes synthesize C30 carotenoids via squalene and that this  squalene route is widely distributed in Bacteria. We also investigated the functional roles of hopanoids and carotenoids in Planctomycetes and show that their protective functions under stress conditions are complementary. Our evolutionary analyses suggest that the C30 carotenoid biosynthetic pathway is the most ancestral, with a potential origin in Firmicutes or Planctomycetes. In addition, we propose an evolutionary scenario to explain the diversification of the different carotenoid and squalene pathways. Together, these results improve the evolutionary contextualization of these molecules. Likewise, the widespread occurrence of the squalene route in bacteria increases the functional repertoire of squalene.

sentences: Vaccination with SARS-CoV-2 Spike Protein and AS03 Adjuvant Induces Rapid Anamnestic Antibodies in the Lung and Protects Against Virus Challenge in Nonhuman Primates Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FC receptors mediating effector functions in vitro. Pseudovirus and live virus neutralizing IC50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3x106 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

sentences: Necroptosis inhibition counteracts axonal degeneration, cognitive decline and key hallmarks of aging, promoting brain rejuvenation. Age is the main risk factor for the development of neurodegenerative diseases. In the aged brain, axonal degeneration is an early pathological event, preceding neuronal dysfunction, and cognitive disabilities. Necroptosis activation mediates degeneration of injured axons, but whether necroptosis triggers neurodegeneration and cognitive impairment along aging is unknown. Here we show necroptosis activation in hippocampal axons during aging. Loss of the necroptotic effector Mlkl was sufficient to delay age-associated axonal degeneration, protecting against decreased synaptic transmission and memory decline in aged mice. Moreover, short-term pharmacologic inhibition of necroptosis in aged mice reverted structural and functional hippocampal impairment. Finally, a quantitative proteomic analysis revealed that necroptosis inhibition leads to an overall improvement of the aged hippocampal proteome, including molecular biofunctions associated with brain rejuvenation. Our results demonstrate that necroptosis contributes to the functional decline of the aged brain, and necroptosis inhibition constitute a potential geroprotective strategy to treat age-related disabilities.

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WAT3R: Recovery of T-Cell Receptor Variable Regions From 3' Single-Cell RNA-Sequencing SummaryDiversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of  and {beta} chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3-biased single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3 scRNA-seq is not available. Here we present an analysis pipeline to process TCR variable regions enriched from 3 scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells (PBMCs) from a healthy donor and detect TCR sequences in a high proportion of single T-cells. Detection of TCR sequences is negligible in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T-cells than other T-cell types, indicating an association between T-cell clonotypes and differentiation states.

Availability and implementationThe Workflow for Association of T-cell receptors from 3 single-cell RNA-seq (WAT3R), including test data, is available on GitHub (https://github.com/mainciburu/WAT3R), Docker Hub (https://hub.docker.com/r/mainciburu/wat3r), and a workflow on the Terra platform (https://app.terra.bio). The test dataset is available on GEO (accession number pending).

sentences: Organoid-based human stomach micro-physiological system to recapitulate the dynamic mucosal defense mechanism Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host-microbe interactions are limited due to the lack of physiological stimuli. Herein, we describe the first human stomach micro-physiological system (hsMPS) with physiologically relevant gastric mucosal defense system based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial-mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh-like mucus layer containing high level of mucus protective peptides and well-developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori are successfully demonstrated in our system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies.

sentences: Biphasic roles of hedgehog signaling in the production and self-renewal of outerradial glia in the ferret cerebral cortex The neocortex, the center for higher brain function, emerged in mammals and expanded in the course of evolution. The expansion of outer radial glia (oRGs) and intermediate progenitor cells (IPCs) plays key roles in the expansion and consequential folding of the neocortex. Therefore, understanding the mechanisms of oRG and IPC expansion is important for understanding neocortical development and evolution. By using mice and human cerebral organoids, we previously revealed that hedgehog (HH) signaling expands oRGs and IPCs. Nevertheless, it remained to be determined whether HH signaling expanded oRGs and IPCs in vivo in gyrencephalic species, in which oRGs and IPCs are naturally expanded. Here, we show that HH signaling is necessary and sufficient to expand oRGs and IPCs in ferrets, a gyrencephalic species, through conserved cellular mechanisms. HH signaling increases oRG-producing division modes of ventricular radial glia (vRGs), oRG self-renewal, and IPC proliferation. Notably, HH signaling affects vRG division modes only in an early restricted phase before superficial-layer neuron production peaks. Beyond this restricted phase, HH signaling promotes oRG self-renewal. Thus, HH signaling expands oRGs and IPCs in two distinct but continuous phases during cortical development.

sentences: From Diversity to Complexity: Microbial Networks in Soils Network analysis has been used for many years in ecological research to analyze organismal associations, for example in food webs, plant-plant or plant-animal interactions. Although network analysis is widely applied in microbial ecology, only recently has it entered the realms of soil microbial ecology, shown by a rapid rise in studies applying co-occurrence analysis to soil microbial communities. While this application offers great potential for deeper insights into the ecological structure of soil microbial ecosystems, it also brings new challenges related to the specific characteristics of soil datasets and the type of ecological questions that can be addressed. In this Perspectives Paper we assess the challenges of applying network analysis to soil microbial ecology due to the small-scale heterogeneity of the soil environment and the nature of soil microbial datasets. We review the different approaches of network construction that are commonly applied to soil microbial datasets and discuss their features and limitations. Using a test dataset of microbial communities from two depths of a forest soil, we demonstrate how different experimental designs and network constructing algorithms affect the structure of the resulting networks, and how this in turn may influence ecological conclusions. We will also reveal how assumptions of the construction method, methods of preparing the dataset, and definitions of thresholds affect the network structure. Finally, we discuss the particular questions in soil microbial ecology that can be approached by analyzing and interpreting specific network properties. Targeting these network properties in a meaningful way will allow applying this technique not in merely descriptive, but in hypothesis-driven research.

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Mountain radiations are not only rapid and recent: ancient diversification of South American frog and lizard families related to Paleogene Andean orogeny and Cenozoic climate variations Mountainous areas host a disproportionately large fraction of Earths biodiversity, suggesting a causal relationship between mountain building and biological diversification. Mountain clade radiations are generally associated with changes in environment, climate, and the increase in heterogeneity therein during mountain building. However, examining the causal relationship between mountain building and diversification is a complex challenge, because isolating the effects of surface uplift from other abiotic (climate) or biotic variables is not straightforward. In this study, we investigate the relative contributions of abiotic climate-driven (temperature) and geology-driven (elevation) drivers on evolutionary rates of ancient groups of organisms in the South American Andes. We present regional curves of Andean elevation based on a recent compilation of paleo-elevational data back to the Late Cretaceous, and analyse the diversification history of six Andean frog and lizard families that originated equally far back in time. For two clades (Aromobatidae and Leptodactylidae), we find that they diversified most rapidly during the early phase of mountain building (Late Cretaceous - Paleogene), when the first high-elevation habitats emerged in South America. The diversification of one clade (Centrolenidae) is correlated with Cenozoic temperature variations, with higher speciation rates during warm periods. The last three clades (Dendrobatidae, Hemiphractidae and Liolaemidae) are best explained by environment-independent diversification, although for Liolaemidae, an almost equally strong positive correlation was found between speciation and Andean elevation since the late Eocene. Our findings imply that throughout the long-lived history of surface uplift in the Andes, mountain building drove the diversification of different clades at different times, while not directly affecting other clades. Our study illustrates the importance of paleogeographic reconstructions that capture the complexity and heterogeneity of mountain building in our understanding of the effects that a changing environment plays in shaping biodiversity patterns observed today.

HighlightsO_LIWe provide novel regional paleoelevation curves for the Andes back to the Late Cretaceous
C_LIO_LIThe diversification history of six Andean-centered clades is studied
C_LIO_LIWe find clade-specific responses to environmental changes
C_LIO_LIThe impact of Andean uplift could reach further back in time than previously thought
C_LI

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Nicotinic modulation insecticides act on diverse receptor subtypes with distinct subunit compositions Insect nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of nicotinic modulation insecticides including neonicotinoids, the most widely used insecticides in the world. However, the resistance development from pests and the negative impacts on the pollinators affect their applications and create demand for the alternatives. Thus, it is very important to understand the mode of action of these insecticides at the molecular level, which is actually unclear for more than 30 years. In this study, we systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunits mutants against eleven nicotinic modulation insecticides. Our results showed that there are several subtypes of nAChRs with distinct subunits compositions that are responsible for the toxicity of different insecticides, respectively. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. On the other hand, the spinosyns may exclusively act on the 6 homomeric nAChR but not any other heteromeric pentamers. Behavioral assays using thermogenetic tools further confirmed the bioassay results and support the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for resistance management and the development of novel insecticides targeting this important ion channels.

Author SummaryThe neonicotinoids and spinosyns make up about 27% of the insecticides by world market value. Novel insecticides like sulfoxaflor, flupyradifurone and triflumezopyrim are developed as alternatives due to the negative effects of neonicotinoids on pollinators. Although all act via insect nicotinic acetylcholine receptors, the mode of action is unclear. Our work shows that these insecticides act on diverse receptor subtypes with distinct subunit compositions. This finding could lead to the development of more selective insecticides to control pests with minimal effects on beneficial insects.

sentences: Human primary visual cortex shows larger population receptive fields for binocular disparity-defined stimuli 1The visual perception of 3D depth is underpinned by the brains ability to combine signals from the left and right eyes to produce a neural representation of binocular disparity for perception and behavior. Electrophysiological studies of binocular disparity over the past two decades have investigated the computational role of neurons in area V1 for binocular combination, while more recent neuroimaging investigations have focused on identifying specific roles for different extrastriate visual areas in depth perception. Here we investigate the population receptive field properties of neural responses to binocular information in striate and extrastriate cortical visual areas using ultra-high field fMRI. We measured BOLD fMRI responses while participants viewed retinotopic-mapping stimuli defined by different visual properties: contrast, luminance, motion, correlated and anti-correlated stereoscopic disparity. By fitting each condition with a population receptive field model, we compared quantitatively the size of the population receptive field for disparity-specific stimulation. We found larger population receptive fields for disparity compared with contrast and luminance in area V1, the first stage of binocular combination, which likely reflects the binocular integration zone, an interpretation supported by modelling of the binocular energy model. A similar pattern was found in region LOC, where it may reflect the role of disparity as a cue for 3D shape. These findings provide insight into the binocular receptive field properties underlying processing for human stereoscopic vision.

sentences: Evolution of recombination landscapes in diverging populations of bread wheat. Reciprocal exchanges of DNA (crossovers) that occur during meiosis are mandatory to ensure the production of fertile gametes in sexually reproducing species. They also contribute to shuffle parental alleles into new combinations thereby fuelling genetic variation and evolution. However, due to biological constraints, the recombination landscape is highly heterogenous along the genome which limits the range of allelic combinations and the adaptability of populations. An approach to better understand the constraints on the recombination process is to study how it evolved in the past. In this work we tackled this question by constructing recombination profiles in four diverging bread wheat (Triticum aestivum L.) populations established from 371 landraces genotyped at 200,062 SNPs. We used linkage disequilibrium (LD) patterns to estimate in each population the past distribution of recombination along the genome and characterize its fine-scale heterogeneity. At the megabase scale, recombination rates derived from LD patterns were consistent with family-based estimates obtained from a population of 406 recombinant inbred lines. Among the four populations, recombination landscapes were significantly positively correlated between each other and shared a statistically significant proportion of highly recombinant intervals. However, this comparison also highlighted that the similarity in recombination landscapes between populations was significantly decreasing with their genetic differentiation in most regions of the genome. This observation was found to be robust to SNP ascertainment and demography and suggests a relatively rapid evolution of factors determining the fine-scale localization of recombination in bread wheat.

sentences: Chromosome-level Thlaspi arvense genome provides new tools for translational research and for a newly domesticated cash cover crop of the cooler climates Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.

sentences: Mycobacterium tuberculosis transcriptional regulator Rv1019 is upregulated in hypoxia and negatively regulates Rv3230c-Rv3229c operon encoding enzymes in the oleic acid biosynthetic pathway The main obstacle in eradicating tuberculosis is the ability of Mycobacterium tuberculosis to remain dormant in the host, and then to get reactivated even years later under immuno-compromised conditions. Transcriptional regulation in intracellular pathogens plays an important role in adapting to the challenging environment inside the host cells. Previously, we demonstrated that Rv1019, a putative transcriptional regulator of M. tuberculosis H37Rv, is an autorepressor. We showed that, Rv1019 is cotranscribed with Rv1020 (mfd) and Rv1021 (mazG) encoding DNA repair proteins and negatively regulates the expression of these genes. In the present study, we show that Rv1019 also regulates the expression of the genes Rv3230c and Rv3229c (desA3) which form a two-gene operon in M. tuberculosis. Constitutive expression of Rv1019 in M. tuberculosis significantly downregulated the expression of these genes. Employing Waynes hypoxia-induced dormancy model of M. tuberculosis, we show that Rv1019 is upregulated (3-fold) under hypoxia. Finally, by reporter assay, using M. smegmatis as a model, we validate that Rv1019 is recruited to the promoter of Rv3230c-Rv3229c during hypoxia and negatively regulates this operon which is involved in the biosynthesis of oleic acid.

sentences: Wnt-responsive enhancer regulation in mammals by a CDX/TCF transcription factor collective and a novel DNA sequence motif Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of {beta}-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal-related homeodomain (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.

sentences: Optogenetic stimulation of BLA terminals in the BNST elicits long term synaptic plasticity and restores synaptic alterations induced by adolescent Shank3 downregulation Anxiety disorders are the most prevalent co-morbidity factor associated with the core domains of Autism Spectrum Disorders (ASD). Investigations on potential common neuronal mechanisms that may explain the co-occurrence of ASD and anxiety disorders are still poorly unexplored. One of the key questions which remained unsolved is the role of Shank3 protein in anxiety behaviors. Here we used shRNA strategy to model Shank3 insufficiency in the bed nucleus of the stria terminalis (BNST). We found that Shank3 downregulation in the BNST induced anxiogenic effects. Associated with these behavioural defects, we showed alteration of glutamatergic synaptic functions in the BNST induced by Shank3 insufficiency during adolescence. In addition, we pointed that adolescence represents a crucial time window to interfere with BNST maturation, a key hub for anxiety control. Together, these results unravelled the crucial role of Shank3 expression in the BNST during adolescence. Our study provided a new insight in the neuronal mechanisms underlying anxiety disorders. We proposed to screen a novel molecular target essential for BNST integrity during adolescence. This result may further help for the diagnosis and the development of therapeutic strategy for anxiety disorders and anxiety disorders implicated in some forms of ASD.

sentences: Quantifying and cataloguing unknown sequences within human microbiomes Advances in genome sequencing technologies and lower costs have enabled the exploration of a multitude of known and novel environments and microbiomes. This has led to an exponential growth in the raw sequence data that is deposited in online repositories. Metagenomic and metatranscriptomic data sets are typically analysed with regards to a specific biological question. However, it is widely acknowledged that these data sets are comprised of a proportion of sequences that bear no similarity to any currently known biological sequence, and this so-called  dark matter is often excluded from downstream analyses. In this study, a systematic framework was developed to assemble, identify, and measure the proportion of unknown sequences present in distinct human microbiomes. This framework was applied to forty distinct studies, comprising 963 samples, and covering ten different human microbiomes including fecal, oral, lung, skin and circulatory system microbiomes. The framework was used to determine the proportion of taxonomically unknown sequences present within samples, and to compare such sequences both within and across assembled metagenomes. We found that whilst the human microbiome is one of the most extensively studied, on average 2% of assembled sequences have not yet been taxonomically defined. However, this proportion varied extensively among different microbiomes and was as high as 25% for skin and oral microbiomes that have more interactions with the environment. The publicly available data sets used have not previously been systematically mined to quantify and compare such dark matter. Typically, these unknown sequences are found in several microbiomes and potentially belong to unidentified novel microbes that we interact with on a daily basis. A cross-study comparison led to the identification of similar unknown sequences in different samples and/or microbiomes. A rate of taxonomic characterisation of 1.64% of unknown sequences being characterised per month was calculated from these taxonomically unknown sequences discovered in this study. Additionally, the approach led to the discovery of several potentially novel viral genomes that bear no similarity to sequences in the public databases. Both our computational framework and the novel unknown sequences produced are publicly available for future cross-referencing.

sentences: Conserved meiotic mechanisms in the cnidarian Clytia hemisphaerica revealed by Spo11 knockout During meiosis, each duplicated chromosome pairs and recombines with its unique homolog to ensure the shuffling of genetic information across generations. Functional studies in classical model organisms have revealed a surprising diversity in the chronology and interdependency of the earliest meiotic steps such as chromosome movements, pairing, association via Synaptonemal Complex formation (synapsis), recombination and the formation of chiasmata. A key player is Spo11, an evolutionarily conserved topoisomerase-related transesterase that initiates meiotic recombination via the catalysis of programmed DNA double stranded breaks (DSBs). While DSBs are required for pairing and synapsis in budding yeast and mouse, alternative pathways are employed during female meiosis of the fruit fly and nematode Caenorhabditis elegans. Here, to provide a comparative perspective on meiotic regulation from a distinct animal clade, we chart gametogenesis in Clytia hemisphaerica jellyfish and examine the role of Spo11 using CRISPR-Cas9 mutants, generated clonally from F0 polyp colonies. Spo11 mutant females fail to assemble synaptonemal complexes and chiasmata, such that homologous chromosome pairs disperse during oocyte growth. Subsequent meiotic divisions are abnormal but produce viable progeny. Clytia thus shares an ancient eukaryotic dependence of synapsis and chromosome segregation on Spo11-generated DSBs. It provides a valuable additional experimental model for dissecting meiotic mechanisms during animal gametogenesis, and for building a comparative framework for distinguishing evolutionarily conserved versus flexible features of meiosis.

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CanIsoNet: A Database to Study the Functional Impact of Isoform Switching Events in Cancer MotivationAlternative splicing, as an essential regulatory mechanism in normal mammalian cells, is frequently disturbed in cancer. Switches in the expression of alternative isoforms can alter protein interaction networks of associated genes giving rise to cancer progression and metastases. We have recently analyzed the pathogenic impact of switching events in 1209 cancer samples covering 24 different cancer types. Here, we are presenting CanIsoNet (Cancer Isoform specific interaction Network), a database to view, browse and search these isoform switching events. CanIsoNet is the first webserver that incorporates isoform expression data with STRING interaction networks and COSMIC annotations to predict the pathogenic impact of isoform switching events in various cancer types.

ResultsData in CanIsoNet can be browsed by cancer types or searched by genes or isoforms in annotation rich data tables. Various annotations for 11,041 isoforms and 31,748 unique isoform switching events are provided across 24 cancer types, including proximity information to COSMIC cancer census genes, network density data for each cancer-specific isoform, PFAM domain IDs of disrupted interactions, domain structure visualization of transcripts and expression data of switched isoforms for each sample.

AvailabilityCanIsoNet is freely available at https://caniso.net under a Creative Common License. The source codes can be found at https://github.com/KarakulakTulay/CanIsoNet_Web

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Transcriptomics provides a genetic signature of vineyard site with insight into vintage-independent regional wine characteristics In wine fermentations, the metabolic activity of both Saccharomyces cerevisiae and non-Saccharomyces organisms impact wine chemistry. Ribosomal DNA amplicon sequencing of grape musts has demonstrated that microorganisms occur non-randomly and are associated with the vineyard of origin, suggesting a role for the vineyard, grape, and wine microbiome in shaping wine fermentation outcomes. We used ribosomal DNA amplicon sequencing of grape must and RNA sequencing of primary fermentations to profile fermentations from 15 vineyards in California and Oregon across two vintages. We find that the relative abundance of fungal organisms detected by ribosomal DNA amplicon sequencing did not correlate with transcript abundance from those organisms within the RNA sequencing data, suggesting that the majority of the fungi detected in must by ribosomal DNA amplicon sequencing are not active during these inoculated fermentations. Additionally, we detect genetic signatures of vineyard site and region during fermentation that are predictive for each vineyard site, identifying nitrogen, sulfur, and thiamine metabolism as important factors for distinguishing vineyard site and region.

ImportanceThe wine industry generates billions of dollars of revenue annually, and economic productivity is in part associated with regional distinctiveness of wine sensory attributes. Microorganisms associated with grapes and wineries are influenced by region of origin, and given that some microorganisms play a role in fermentation, it is thought that microbes may contribute to the regional distinctiveness of wine. We show that while the presence of microbial DNA is associated with wine region and vineyard site, the presence of microbial DNA is not associated with gene expression of those microorganisms during fermentation. We further show that detected gene expression signatures associated with wine region and vineyard site provide a means to address differences in fermentations that may drive regional distinctiveness.

sentences: Organizing laboratory information to analyze the reproducibility of experimental workflows Reproducibility is the cornerstone of scientific experiments. Assessing the reproducibility of an experiment requires analyzing the contribution of different factors to the variation of the observed data. Suitable data structures need to be defined prior to the data collection effort so that data associated with these factors can be recorded and associated with observations of the variable of interest. The resulting datasets can be analyzed statistically to estimate the effect of experimental factors on the observed data using ANOVA models. Custom data structures to document the execution of experimental workflows are defined in a research data management system. The data produced by multiple repetitions of a plasmid purification process and a cell culture process are analyzed using the Kruskal-Wallis H-test to identify factors contributing to their variation. Repetitions of the plasmid purification process do not lead to significant differences in extraction yields. Statistically significant differences in plasmid solution purity are identified but the differences are small enough that are not biologically relevant. The maintenance of two cell lines over many generations leads to similar datasets. However, different media preparations appear to influence the variation of cell viability and harvested cell counts in unexpected ways that may be the indirect expression of hidden effects not captured in the data structure.

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Differential impact of rhamnolipids and polysorbate80 on composition and functionality of the gut microbiota in the SHIME system. Dietary emulsifiers have been shown to affect the composition and function of the gut microbial community, both in vivo and in vitro. Yet, several knowledge gaps remain to be addressed: the impact from a longer timeframe exposure on the gut microbiota, interindividual variability in microbiome response and the putative impact from novel clean label alternatives for current food emulsifiers.

In the present study, the impact of one conventional dietary emulsifier, TWEEN80, and one potential novel alternative, rhamnolipids, on the human gut microbiota was investigated using the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). The faecal microbiota from two human donors, with high and low responsiveness to the emulsifiers, were exposed to 0,05 m% and 0,5 m% of the emulsifiers for 7 days.

The results confirmed previous observations that the effects on the composition and functionality are both emulsifier- and donor dependent. The effects reached an equilibrium after about 3 days of exposure. Overall, TWEEN80 and rhamnolipids displayed opposite effects: TWEEN80 increased cell counts, reduced propionate concentration, increased butyrate levels, increased a.o. Faecalibacterium, Blautia and Hungatella abundance, while rhamnolipids did the opposite. Rhamnolipids also sharply increased the abundance of unclassified Lachnospiraceae. On the other hand, both emulsifiers increased the relative abundance of unclassified Enterobacteriaceae. Both emulsifiers also altered the microbial metabolome in different ways and a pathway enrichment analysis tool revealed that the metabolome alterations could be reminiscent of gut issues and obesity.

Overall, the impact from the rhamnolipids was larger than that of TWEEN80 at similar concentrations, indicating that the former may not necessarily be a safer alternative for the latter. The microbiotas response also depended on its original composition and the sensitivity status for which the faecal donors were selected, was preserved. Whether the same donor-diversity and longitudinal impact can be expected in the human colon as well and what impact this has on the host will have to be further investigated.

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Reticular Dysgenesis-associated Adenylate Kinase 2 deficiency causes failure of myelopoiesis through disordered purine metabolism Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect.

This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.

sentences: Integrative small and long RNA-omics analysis of human healing and non-healing wounds discovers cooperating microRNAs as therapeutic targets MicroRNAs (miR) are important posttranscriptional regulators and exhibit a high potential to be utilized in diagnosis and therapy. However, our insufficient knowledge of the miR-mediated gene regulation in human skin wound healing severely hinders the identification of clinically relevant miRs. Here, we performed paired small RNA and long RNA sequencing in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic non-healing venous ulcers (VU). With integrative small and long RNA-omics analysis, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte growth while promoting inflammatory response. Collectively, our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

sentences: Visual Field Analysis: a reliable method to score left- and right eye-use using automated tracking Brain and behavioural asymmetries have been documented in various taxa. Many of these asymmetries involve preferential left- and right-eye use. However, measuring eye use through manual frame-by-frame analyses from video recordings is laborious and may lead to biases. Recent progress in technology allowed the development of accurate tracking techniques for measuring animal behaviour. Amongst these techniques, DeepLabCut, a python-based tracking toolbox using transfer learning with deep neural networks, offers the possibility to track different body parts with unprecedented accuracy. Exploiting the potentialities of DeepLabCut, we developed  Visual Field Analysis, an additional open-source application for extracting eye-use data. To our knowledge, this is the first application that can automatically quantify left-right preferences in eye use. Here we test the performance of our application in measuring preferential eye-use in young domestic chicks. The comparison with manual scoring methods revealed a perfect correlation in the measures of eye-use obtained by  Visual Field Analysis. With our application, eye-use can be analysed reliably, objectively and at a fine scale in different experimental paradigms.

sentences: Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here we developed a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions (16304 reactions in total). The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data revealed that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation - known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimization, providing a framework to understand metabolic adaptation in eukaryal cells.

sentences: Electrochemical Detection of Adrenaline and Hydrogen Peroxide on Carbon Nanotube Electrodes Adrenaline and hydrogen peroxide have neuromodulatory functions in the brain. Considerable interest exists in developing electrochemical sensors that can detect their levels in vivo due to their important biochemical roles. Challenges associated with electrochemical detection of hydrogen peroxide and adrenaline are that the oxidation of these molecules usually requires highly oxidising potentials (beyond 1.4 V vs Ag/AgCl) where electrode damage and biofouling are likely and the signals of adrenaline, hydrogen peroide and adenosine overlap. To address these issues we fabricated pyrolysed carbon electrodes coated with oxidised carbon nanotubes (CNTs). Using these electrodes for fast-scan cyclic voltammetric (FSCV) measurements showed that the electrode offers reduced overpotentials compared with graphite and improved resistance to biofouling. The Adrenaline peak is reached at 0.75({+/-}0.1) V and reduced back at -0.2({+/-}0.1) V while hydrogen peroxide is detected at 0.85({+/-}0.1) V on this electrode. The electrodes are highly sensitive with a sensitivity of 16nA {micro}M-1 for Adrenaline and 11nA {micro}M-1 for hydrogen peroxide on an 80 {micro}m2 electrode. They are also suitable to distinguish between adrenaline, hydrogen peroxide and adenosine thus these probes can be used for multimodal detection of analytes.

sentences: Endothelial integration of mechanosensory signals by the spectrin cytoskeleton Physiological blood flow induces the secretion of vasoactive compounds, notably NO, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. We demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrin modulates the cell surface density of hyaluronan, which senses and translates shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive Piezo1 channels are thought to reside. Accordingly, shear-induced Piezo1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced eNOS stimulation were similarly dependent on spectrin. We concluded that the apical spectrin network is not only required for shear sensing, but transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

sentences: Association of stress-related limbic activity and baseline interleukin-6 plasma levels in healthy adults Several studies suggest a link between acute changes in inflammatory parameters due to an endotoxin or (psychological) stressor and the brains stress response. The extent to which basal circulating levels of inflammatory markers are associated with the brains stress response has been hardly investigated so far. In the present study, plasma levels of the cytokine interleukin (IL)-6 were obtained as a baseline measure of peripheral inflammatory processes and linked to neural markers of psychosocial stress using a modified version of the Montreal Imaging Stress Task in a sample of N=65 healthy subjects. Of three a-priori defined regions of interest - the amygdala, anterior insula, and anterior cingulate cortex - baseline IL-6 was significantly and negatively associated with stress-related neural activation in the right amygdala and left anterior insula. Our results suggest that baseline inflammatory physiology might be related to differences in the neural stress response and that this relationship could be inverse to that previously shown by studies on acutely induced inflammation.

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Using generalized additive models to analyze biomedical non-linear longitudinal data 1In biomedical research, the outcome of longitudinal studies has been traditionally analyzed using the repeated measures analysis of variance (rm-ANOVA) or more recently, linear mixed models (LMEMs). Although LMEMs are less restrictive than rm-ANOVA as they can work with unbalanced data and non-constant correlation between observations, both methodologies assume a linear trend in the measured response. It is common in biomedical research that the true trend response is nonlinear and in these cases the linearity assumption of rm-ANOVA and LMEMs can lead to biased estimates and unreliable inference.

In contrast, GAMs relax the linearity assumption of rm-ANOVA and LMEMs and allow the data to determine the fit of the model while also permitting incomplete observations and different correlation structures. Therefore, GAMs present an excellent choice to analyze longitudinal data with non-linear trends in the context of biomedical research. This paper summarizes the limitations of rm-ANOVA and LMEMs and uses simulated data to visually show how both methods produce biased estimates when used on data with non-linear trends. We present the basic theory of GAMs and using reported trends of oxygen saturation in tumors, we simulate example longitudinal data (2 treatment groups, 10 subjects per group, 5 repeated measures for each group) to demonstrate their implementation in R. We also show that GAMs are able to produce estimates with non-linear trends even when incomplete observations exist (with 40% of the simulated observations missing). To make this work reproducible, the code and data used in this paper are available at: https://github.com/aimundo/GAMs-biomedical-research.

sentences: On a Reef Far, Far Away: Anthropogenic Impacts Following Extreme Storms Affect Sponge Health and Bacterial Communities Terrestrial runoff can negatively impact marine ecosystems through stressors including excess nutrients, freshwater, sediments, and contaminants. Severe storms, which are increasing with global climate change, generate massive inputs of runoff over short timescales (hours to days); such runoff impacted offshore reefs in the northwest Gulf of Mexico (NW GoM) following severe storms in 2016 and 2017. Several weeks after coastal flooding from these events, NW GoM reef corals, sponges, and other benthic invertebrates -185 km offshore experienced mortality (2016 only) and/or sub-lethal stress (both years). To assess the impact of storm-derived runoff on reef filter feeders, we characterized the bacterial communities of two sponges, Agelas clathrodes and Xestospongia muta, from offshore reefs during periods of sub-lethal stress and no stress over a three-year period (2016-2018). Sponge-associated bacterial communities were altered during both flood years. Additionally, we found evidence of wastewater contamination (based on 16S rRNA gene libraries and quantitative PCR) in offshore sponges following these flood years, but not during the no-flood year. We show that flood events from severe storms have the capacity to reach offshore reef ecosystems and impact resident benthic organisms. Such impacts are most readily detected if baseline data on organismal physiology and associated microbiome composition are available. This highlights the need for molecular and microbial time series of benthic organisms in near- and offshore reef ecosystems, and the continued mitigation of stormwater runoff and climate change impacts.

sentences: Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that Actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1-Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

sentences: uPIC-M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost. Here, we present a scalable, rapid, and inexpensive approach for creating User-designed Physically Isolated Clonal-Mutant (uPIC-M) libraries that utilizes recent advances in oligo synthesis, high-throughput sample preparation, and next-generation sequencing. To demonstrate uPIC-M, we created a scanning mutant library of SpAP, a 541 amino acid alkaline phosphatase, and recovered 94% of desired mutants in a single iteration. uPIC-M uses commonly available equipment and freely downloadable custom software and can produce a 5000 mutant library at 1/3 the cost and 1/5 the time of traditional techniques.

sentences: Privacy-preserving harmonization via distributed ComBat Challenges in clinical data sharing and the need to protect data privacy have led to the development and popularization of methods that do not require directly transferring patient data. In neuroimaging, integration of data across multiple institutions also introduces unwanted biases driven by scanner differences. These scanner effects have been shown by several research groups to severely affect downstream analyses. To facilitate the need of removing scanner effects in a distributed data setting, we introduce distributed ComBat, an adaptation of a popular harmonization method for multivariate data that borrows information across features. We present our fast and simple distributed algorithm and show that it yields equivalent results using data from the Alzheimers Disease Neuroimaging Initiative. Our method enables harmonization while ensuring maximal privacy protection, thus facilitating a broad range of downstream analyses in functional and structural imaging studies.

sentences: Amyloid fibrils in frontotemporal lobar degeneration with TDP-43 inclusions are composed of TMEM106B, rather than TDP-43 FTLD is the third most common neurodegenerative condition, following only Alzheimers and Parkinsons diseases. FTLD typically presents in 45-64-year-olds with behavioral changes or progressive decline of language skills. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions detected by TDP-43 immunoreactivity. Here, we extracted amyloid fibrils from brains of four patients, representing four out of five FTLD-TDP subclasses and determined their near-atomic resolution structures by cryo-EM. Unexpectedly, all amyloid fibrils examined are composed of a 135-residue C-terminal fragment of TMEM106B, a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP. In addition to TMEM106B fibrils, abundant non-fibrillar aggregated TDP-43 is present, as revealed by immunogold labeling. Our observations confirm that FTLD-TDP is an amyloid-involved disease and suggest that amyloid involvement in FTLD-TDP is of protein TMEM106B, rather than of TDP-43.

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Spiking recurrent neural networks represent task-relevant neural sequences in rule-dependent computation Prefrontal cortical neurons play in important roles in performing rule-dependent tasks and working memory-based decision making. Motivated by experimental data, we develop an excitatory-inhibitory spiking recurrent neural network (SRNN) to perform a rule-dependent two-alternative forced choice (2AFC) task. We imposed several important biological constraints onto the SRNN, and adapted the spike frequency adaptation (SFA) and SuperSpike gradient methods to update the network parameters. These proposed strategies enabled us to train the SRNN efficiently and overcome the vanishing gradient problem during error back propagation through time. The trained SRNN produced rule-specific tuning in single-unit representations, showing rule-dependent population dynamics that strongly resemble experimentally observed data in rodent and monkey. Under varying test conditions, we further manipulated the parameters or configuration in computer simulation setups and investigated the impacts of rule-coding error, delay duration, weight connectivity and sparsity, and excitation/inhibition (E/I) balance on both task performance and neural representations. Overall, our modeling study provides a computational framework to understand neuronal representations at a fine timescale during working memory and cognitive control.

Author SummaryWorking memory and decision making are fundamental cognitive functions of the brain, but the circuit mechanisms of these brain functions remain incompletely understood. Neuroscientists have trained animals (rodents or monkeys) to perform various cognitive tasks while simultaneously recording the neural activity from specific neural circuits. To complement the experimental investigations, computational modeling may provide an alternative way to examine the neural representations of neuronal assemblies during task behaviors. Here we develop and train a spiking recurrent neural network (SRNN) consisting of balanced excitatory and inhibitory neurons to perform the rule-dependent working memory tasks Our computer simulations produce qualitatively similar results as the experimental findings. Moreover, the imposed biological constraints on the trained network provide additional channel to investigate cell type-specific population responses, cortical connectivity and robustness. Our work provides a computational platform to investigate neural representations and dynamics of cortical circuits a fine timescale during complex cognitive tasks.

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ECCsplorer: a pipeline to detect extrachromosomal circular DNA (eccDNA) from next-generation sequencing data MotivationExtrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. Apart from carrying tandemly repeated DNA, eccDNAs may also harbor extra copies of genes or recently activated transposable elements. As eccDNAs occur in all eukaryotes investigated so far and likely play roles in stress, cancer, and aging, they have been prime targets in recent research - with their investigation limited by the scarcity of computational tools.

ResultsHere, we present the ECCsplorer, a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing techniques. Following Illumina-sequencing of amplified circular DNA (circSeq), the ECCsplorer enables an easy and automated discovery of eccDNA candidates. The data analysis encompasses two major procedures: First, read mapping to the reference genome allows the detection of informative read distributions including high coverage, discordant mapping, and split reads. Second, reference-free comparison of read clusters from amplified eccDNA against control sample data reveals specifically enriched DNA circles. Both software parts can be run separately or jointly, depending on the individual aim or data availability. To illustrate the wide applicability of our approach, we analyzed semiartificial and published circSeq data from the model organisms H. sapiens and A. thaliana, and generated circSeq reads from the non-model crop B. vulgaris. We clearly identified eccDNA candidates from all datasets, with and without reference genomes. The ECCsplorer pipeline specifically detected mitochondrial mini-circles and retrotransposon activation, showcasing the ECCsplorers sensitivity and specificity. The derived eccDNA targets are valuable for a wide range of downstream investigations - from analysis of cancer-related eccDNAs over organelle genomics to identification of active transposable elements.

Availability and implementationThe ECCsplorer pipeline is available on GitHub at https://github.com/crimBubble/ECCsplorer under the GNU license.

ContactTony Heitkam (tony.heitkam@tu-dresden.de)

Supplementary informationSupplementary data are available online.

sentences: Strain dynamics of specific contaminant bacteria modulate the performance of ethanol biorefineries Bioethanol is a viable alternative for fossil fuels, and its use has lowered CO2 emissions by over 500 million tonnes in Brazil alone by replacing more than 40% of the national gasoline consumption. However, contaminant bacteria reduce yields during fermentation. Our understanding of these contaminants is limited to targeted studies, and the interplay of the microbial community and its impact on fermentation efficiency remains poorly understood. Comprehensive surveying and longitudinal analysis using shotgun metagenomics of two major biorefineries over a production season revealed similar patterns in microbial community structure and dynamics throughout the entire fermentation system. Strain resolution metagenomics identified specific Lactobacillus fermentum strains as strongly associated with poor industrial performance and laboratory-scale fermentations revealed yield reductions of up to 4.63{+/-}1.35% depending on the specific contaminating strains. Selective removal of these strains could reduce emissions from the bioethanol industry by more than 2x106 tonnes per year. Using the large-scale Brazilian ethanol fermentations as a model system for studying microbiome-phenotype relationships this study further demonstrates how high-resolution metagenomics can identify culprits of large scale industrial biomanufacturing.

sentences: Lichen fungi do not depend on the alga for ATP production Lichen fungi live in a symbiotic association with unicellular phototrophs and have no known aposymbiotic stage. A recent study postulated that some of them have lost mitochondrial oxidative phosphorylation and rely on their algal partners for ATP. This claim originated from an apparent lack of ATP9, a gene encoding one subunit of ATP synthase, from a few mitochondrial genomes. Here we show that while these fungi indeed have lost the mitochondrial ATP9, each retain a nuclear copy of this gene. Our analysis reaffirms that lichen fungi produce their own ATP.

sentences: Preparation of cationized albumin nanoparticles loaded indirubin by high pressure hemogenizer Indirubin can be applied as an anti-cancer drug for inhibition of brain tumors. However, its performance is reduced due to hydrophobicity. In this study, we synthesized cationic human serum albumin (CHSA) nanoparticle by a new hybrid approach for improvement the surface chemistry of albumin and investigate the amount of indirubin loaded CHSA nanoparticle. In this study, the generated mechanical force from a high-pressure homogenizer (HPH) was used to make nanoparticles with a certain size with narrow polydispersity. The results indicated that the size of indirubin loaded CHSA nanoparticles were 130 nm and their zeta potential were +9. Besides, the encapsulation efficiency and drug loading capacity were found to be 85% and 5.8 %, respectively. To the best to our knowledge, this is the first time that indirubin has been used in albumin nanoparticles. In this study, indirubin loaded CHSA nanoparticles was shown can be a potential candidate for drug delivery in the treatment of glioblastoma. Moreover, the cationized form allows the chemical agent to be transmitted to the brain.

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Variable Selection via Grace-AKO with Applications in Genomics MotivationVariable selection is a common statistical approach to detecting genes associated with clinical outcomes of scientific interest. There are hundreds of thousands of genes in genomic studies, while only a limited number of individual samples are available. Therefore, it is important to develop a method to identify genes associated with outcomes of interest that can control finite-sample false discovery rate (FDR) in high-dimensional data settings.

ResultsThis article proposes a novel method named Grace-AKO for graph-constrained estimation (Grace), which incorporates aggregation of multiple knockoffs (AKO) with the network-constrained penalty. Grace-AKO can control FDR in finite sample settings and improve stability. Simulation studies show that Grace-AKO has better performance in FDR control in finite sample settings than the original Grace model. We apply Grace-AKO to the prostate cancer data in the The Cancer Genome Atlas (TCGA) program by incorporating prostate-specific antigen (PSA) pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) as the prior information. Grace-AKO finally identifies 47 candidate genes associated with PSA level, and more than 75% of the detected genes can be validated.

Availability and implementationWe developed an R package for Grace-AKO available at: https://github.com/mxxptian/GraceAKO

Contactdoraz@hku.hk

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Trajectory Recording and Analysis System for Cockroach Robot In this study, We design a trajectory recording and analysis system to record and analysis the changes in the movement behavior of the cockroach robot after stimulation. The external hardware of this system is an infrared touchpad as the experimental platform for the cockroach robot to crawl freely, and the infrared matrices densely distributed in the X and Y directions of the infrared touchpad are used to detect and locate the position of the cockroach robot. The cockroach robots movement trajectory is displayed visually through the projectors interface projection on the infrared touchpad. The system software consists of three main parts: the electrical signal parameter setting module, the movement trajectory recording module, and the data analysis module. The electrical signal parameter setting module sets the stimulation parameters and configures the corresponding serial port to independently stimulate the left and right antenna and cercus of the cockroach; the trajectory recording module is used to record the trajectory of the cockroach robot through the coordinate positioning method. The data analysis module explores the change of motion behavior of the cockroach robot with time after receiving the stimulus by using the stage analysis method, and explores the change of motion of the cockroach robot with different voltage stimulus by using the module analysis method. The system is tested in experiments and the results demonstrated its applicability to the recording and analysis of the cockroach robots trajectories.

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The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that impact energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here we demonstrate how a physiologically relevant ribosome population arises during high Na+ and pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes re-incorporation of the protein, thereby repairing the subunit after the stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input, and without bypass of quality control mechanisms.

Graphical Abstract

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C_LIO_LITsr2 stores released Rps26 and allows for ribosome repair after stress
C_LIO_LIRps26 exploits a Mg binding site linked to Diamond Blackfan Anemia (DBA)
C_LIO_LIChaperone-mediated ribosome remodeling might be common for chaperoned RPs
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sentences: Loss of the cleaved-protamine 2 domain leads to incomplete histone-to-protamine exchange and infertility in mice Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete PRM2 incorporation, resulting in a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

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HYBRID REACTION-DIFFUSION AND CLOCK-AND-WAVEFRONT MODEL FOR THE ARREST OF OSCILLATIONS IN THE SOMITOGENESIS SEGMENTATION CLOCK AO_SCPLOWBSTRACTC_SCPLOWThe clock and wavefront paradigm is arguably the most widely accepted model for explaining the embryonic process of somitogenesis. According to this model, somitogenesis is based upon the interaction between a genetic oscillator, known as segmentation clock, and a differentiation wavefront, which provides the positional information indicating where each pair of somites is formed. Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation. Cotterell et al. called it a progressive oscillatory reaction-diffusion (PORD) model. In the Meinhardt-PORD model, somitogenesis is driven by short-range interactions and the posterior movement of the front is a local, emergent phenomenon, which is not controlled by global positional information. With this model, it is possible to explain some experimental observations that are incompatible with the clock and wavefront model. However the Meinhardt-PORD model has some important disadvantages of its own. Namely, it is quite sensitive to fluctuations and depends on very specific initial conditions (which are not biologically realistic). In this work, we propose an equivalent Meinhardt-PORD model, and then amend it to couple it with a wavefront consisting of a receding morphogen gradient. By doing so, we get a hybrid model between the Meinhardt-PORD and the clock-and-wavefront ones, which overcomes most of the deficiencies of the two originating models.

Somitogenesis, the process by which somites are formed, is an essential developmental stage in many vertebrates. This process occurs with a strikingly regular periodicity, that is preserved among embryos of a single species. The clock and wavefront paradigm is arguably the most widely accepted model for explaining somitogenesis. However, it is incapable of explaining some experimental facts, like the appearance of somites in the absence of an external wavefront (i.e. a receding morphogen gradient). Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation, and called it a progressive oscillatory reaction-diffusion (PORD) model. The Meinhardt-PORD model tackles some of the deficiencies of the clock and wave-front models, but it has some serious issues of its own. In the present work, we introduce an equivalent Meinhardt-PORD model, and then amend it to couple it with a receding morphogen gradient. By doing so, we get a hybrid model that incorporates characteristics of the Meinhardt-PORD and clock-and-wavefront models. We show that this hybrid model under-goes a bifurcation, from a stable to an unstable limit cycle, as the value of the parameter accounting for a background regulatory input (associated to the receding morphogen gradient) decreases. This bifurcation allows the model to explain why somites can form in the absence of an external wavefront, reassesses the role of the receding morphogen gradient as a conductor for somitogenesis, and makes the model behavior robust to random fluctuations, as well as independent from specific initial conditions (the latter, are two of the weak points of the Meinhardt-PORD model). We argue that this findings provide convincing evidence that reaction-diffusion and positional information (receding morphogen gradient) mechanisms could work together in somitogenesis.

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Impact of Glycosylation on SARS-CoV-2 Infection and Broadly Protective Vaccine Design A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (Smg) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the Smg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development.

One-Sentence SummaryRemoving glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.

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The transcriptomic landscape of the photoperiodic stress response in Arabidopsis thaliana resembles the response to pathogen infection. Plants are exposed to regular diurnal rhythms of light and dark. Changes in the photoperiod by the prolongation of the light period cause photoperiod stress in short day-adapted Arabidopsis thaliana. Here we report on the transcriptional response to photoperiod stress of wild-type A. thaliana and photoperiod stress-sensitive cytokinin signalling and clock mutants. Transcriptomic changes induced by photoperiod stress included numerous changes in reactive oxygen (ROS)-related transcripts and showed a strong overlap with changes occurring in response to ozone stress and pathogen attack, which have in common the induction of an apoplastic oxidative burst. A core set of photoperiod stress-responsive genes has been identified, including salicylic acid (SA) biosynthesis and signalling genes. Genetic analysis revealed a central role for NPR1 in the photoperiod stress response as npr1-1 mutants were stress-insensitive. Photoperiod stress treatment led to a strong increase in camalexin levels which is consistent with shared photoperiod stress and pathogen response pathways. Photoperiod stress induced resistance of Arabidopsis plants to a subsequent infection by Pseudomonas syringae cv. tomato DC3000 indicating priming of the defence response. Together, photoperiod stress causes transcriptional reprogramming resembling plant pathogen defence responses and induces systemic acquired resistance in the absence of a pathogen.

One sentence summaryPhotoperiod stress results in significant dynamic transcriptomic changes related to oxidative stress similar to those caused by pathogen attack and primes the defence response against a subsequent pathogen infection.

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Comprehensive analysis of meiosis-derived cDNA libraries reveals gene isoforms and mitochondrial proteins important for competitive fitness Gametogenesis is a highly regulated and dynamic developmental program where a diploid progenitor cell differentiates into haploid gametes, the precursors for sexual reproduction. During meiosis, several pathways converge to initiate ploidy reduction and organelle remodelling to render gametes competent for zygote formation and subsequent organismal development. Additionally, meiosis inherently rejuvenates the newly formed gametes resulting in lifespan resetting. Here, we construct five stage-specific, inducible meiotic cDNA libraries that represent over 84% of the yeast genome. We employ computational strategies to detect stage-specific meiotic transcript isoforms in each library and develop a robust screening pipeline to test the effect of each cDNA on competitive fitness. Our multi-day proof-of-principle time course reveals gene isoforms that are important for competitive fitness as well as mitochondrial proteins that cause dose-dependent disruption of respiration. Together, these novel meiotic cDNA libraries provide an important resource for systematically studying meiotic genes and gene isoforms in future studies.

HIGHLIGHTSO_LIConstruction of five stage-specific, inducible meiotic cDNA libraries in budding yeast that collectively represent 5563 genes, which is over 84% of the genome
C_LIO_LIAnalysis of the cDNA libraries reveal the presence of meiosis-specific transcript isoforms that are largely uncharacterized
C_LIO_LIDevelopment of a robust gain-of-function screening pipeline identifies previously characterized genes and novel gene isoforms important for competitive fitness
C_LIO_LIMulti-day proof-of-principle screen reveals mitochondrial proteins that cause dosage-specific respiration defects
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sentences: A cell atlas of the fly kidney Like humans, insects rely on precise regulation of their internal environments to survive. The insect renal system consists of Malpighian tubules and nephrocytes that share similarities to the mammalian kidney. Studies of the Drosophila Malpighian tubules and nephrocytes have provided many insights into our understanding of the excretion of waste products, stem cell regeneration, protein reabsorption, and as human kidney disease models. Here, we analyzed single-nucleus RNA sequencing (snRNA-seq) data sets to characterize the cell types of the adult fly kidney. We identified 11 distinct clusters representing renal stem cells (RSCs), stellate cells (SCs), regionally specific principal cells (PCs), garland nephrocyte cells (GCs) and pericardial nephrocytes (PNs). Analyses of these clusters revealed many new interesting features. For example, we found a new, previously unrecognized cell cluster: lower segment PCs that express Esyt2. In addition, we find that the SC marker genes RhoGEF64c, Frq2, Prip and CG10939 regulate their unusual cell shape. Further, we identified transcription factors specific to each cluster and built a network of signaling pathways that are potentially involved in mediating cell-cell communication between Malpighian tubule cell types. Finally, cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia - knowledge that will help the generation of kidney disease models. To visualize this dataset, we provide a web-based resource for gene expression in single cells (https://www.flyrnai.org/scRNA/kidney/). Altogether, our study provides a comprehensive resource for addressing gene function in the fly kidney and future disease studies.

sentences: The paraventricular thalamus provides a polysynaptic brake on limbic CRF neurons to sex-dependently blunt binge alcohol drinking and avoidance behavior Bed nucleus of the stria terminalis (BNST) neurons that synthesize and release the stress neuropeptide corticotropin-releasing factor (CRF) drive binge alcohol drinking and anxiety, behaviors that are primary risk factors for alcohol use disorder (AUD) and comorbid neuropsychiatric diseases more common in women than men. Here, we show that female C57BL/6J mice binge drink more than males and have greater basal BNSTCRF neuron excitability and synaptic excitation. We identified a dense VGLUT2+ glutamatergic synaptic input from the paraventricular thalamus (PVT) that is anatomically similar in males and females. These PVTBNST neurons release glutamate directly onto BNSTCRF neurons but also engage a large BNST interneuron population to ultimately provide a net inhibition of BNSTCRF neurons, and both components of this polysynaptic PVTVGLUT2-BNSTCRF circuit are more robust in females than males. Chemogenetic inhibition of the PVTBNST projection promoted binge alcohol drinking in females without affecting males, and chemogenetic activation of the pathway was sufficient to reduce avoidance behavior in both sexes in anxiogenic contexts. Lastly, we show that withdrawal from repeated binge drinking produces a female-like phenotype in the male PVT-BNSTCRF excitatory synapse without altering the function of PVTBNST neurons per se. Our data describe a complex feedforward inhibitory PVTVGLUT2-BNSTCRF glutamatergic circuit that is more robust in females, plays sex-dependent roles in alcohol drinking and avoidance behavior, and undergoes sex-dependent alcohol-induced plasticity.

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Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesnt affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cell of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.

Authors summaryBiomineralization is a widespread, fundamental process by which organisms use minerals to harden their tissues. Mineral-bearing vesicles were observed in biomineralizing cells and play an essential role in biomineralization, yet little is known about their three-dimensional (3D) dynamics. Here we quantify 3D-vesicle-dynamics during calcite skeleton formation in sea urchin larvae, using lattice-light-sheet microscopy. We discover that calcium vesicles perform a diffusive motion in both calcifying and non-calcifying cells of the embryo. The diffusion coefficient and vesicle speed are higher in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. This difference is possibly due to the higher rigidity of the ectodermal cells as demonstrated by the enhanced signal of f-actin and myosinII activity in these cells compared to the skeletogenic cells. The motion of the vesicles in the skeletogenic cells, is not directed toward the biomineralization compartment but the vesicles slow down near it, possibly to deposit their content. Blocking skeletogenesis through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR), increases vesicle volume but doesnt change the diffusion mode and the cytoskeleton markers in the cells. Our studies reveal the active diffusive motion of mineral bearing vesicles that is apparently defined by the mechanical properties of the cells.

Calcium-vesicle diffusion in biomineralization

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A network information theoretic framework to characterise muscle synergies in space and time ObjectiveCurrent approaches to muscle synergy extraction rely on linear dimensionality reduction algorithms that make specific assumptions on the underlying signals. However, to capture nonlinear time varying, large-scale but also muscle-specific interactions, a more generalised approach is required.

ApproachHere we developed a novel framework for muscle synergy extraction that relaxes model assumptions by using a combination of information- and network theory and dimensionality reduction. We first quantify informational dynamics between muscles, time-samples or muscle-time pairings using a novel mutual information formulation. We then model these pairwise interactions as multiplex networks and identify modules representing the network architecture. We employ this modularity criterion as the input parameter for dimensionality reduction, which verifiably extracts the identified modules, and also to characterise salient structures within each module.

Main resultsThis novel framework captures spatial, temporal and spatiotemporal interactions across two benchmark datasets of reaching movements, producing distinct spatial groupings and both tonic and phasic temporal patterns. Readily interpretable muscle synergies spanning multiple spatial and temporal scales were identified, demonstrating significant task dependence, ability to capture trial-to-trial fluctuations and concordance across participants. Furthermore, our framework identifies submodular structures that represent the distributed networks of co-occurring signal interactions across scales.

SignificanceThe capabilities of this framework are illustrated through the concomitant continuity with previous research and novelty of the insights gained. Several previous limitations are circumvented including the extraction of functionally meaningful and multiplexed pairwise muscle couplings under relaxed model assumptions. The extracted synergies provide a holistic view of the movement while important details of task performance are readily interpretable. The identified muscle groupings transcend biomechanical constraints and the temporal patterns reveal characteristics of fundamental motor control mechanisms. We conclude that this framework opens new opportunities for muscle synergy research and can constitute a bridge between existing models and recent network-theoretic endeavours.

sentences: Comparative whole-genome approach to identify bacterial traits for microbial interactions Microbial interactions shape the structure and function of microbial communities with profound consequences for biogeochemical cycles and ecosystem health. Yet, most interaction mechanisms are studied only in model systems and their prevalence is unknown. To systematically explore the functional and interaction potential of sequenced marine bacteria, we developed a new trait-based approach, and applied it to 473 complete genomes (248 genera), representing a significant fraction of marine microbial communities. We identified genome functional clusters (GFCs) which condense bacterial diversity into groups with potentially common ecology and life history. Most GFCs revealed unique combinations of interaction traits, some widely distributed (e.g., antimicrobial activity), others less common (e.g., production of siderophores and phytohormones, biosynthesis and export of specific B vitamins). Specific GFCs, comprising Alpha- and Gammaproteobacteria, are predicted to preferentially interact synergistically and/or antagonistically with bacteria and phytoplankton. Moreover, linked trait clusters (LTCs) identify traits that may have evolved together and point to specific modes of interactions. Our approach translates multidimensional genomic information into an atlas of marine bacteria and their ecosystem functions, relevant for understanding the fundamental rules that govern community assembly and dynamics.

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Early lock-in of structured and specialised information flows during neural development The brains of many organisms are capable of complicated distributed computation underpinned by a highly advanced information processing capacity. Although substantial progress has been made towards characterising the information flow component of this capacity in mature brains, there is a distinct lack of work characterising its emergence during neural development. This lack of progress has been largely driven by the lack of effective estimators of information processing operations for the spiking data available for developing neural networks. Here, we leverage recent advances in this estimation task in order to quantify the changes in information flow during development. We do so by studying the changes in the intrinsic dynamics of the spontaneous activity of developing dissociated neural cell cultures. We find that the quantity of information flowing across these networks undergoes a dramatic increase across development. Moreover, the spatial structure of these flows is locked-in during early development, after which there is a substantial temporal correlation in the information flows across recording days. We analyse the flow of information during the crucial periods of population bursts. We find that, during these bursts, nodes undertake specialised computational roles as either transmitters, mediators or receivers of information, with these roles tending to align with their spike ordering -- either early, mid or late in the bursts. Further, we find that the specialised computational roles occupied by nodes during bursts tend to be locked-in early. Finally, we briefly compare these results to information flows in a model network developing according to an STDP learning rule from a state of independent firing to synchronous bursting. The phenomena of large increases in information flow, early lock-in of information flow spatial structure and computational roles based on burst position were also observed in this model, hinting at the broader generality of these phenomena.

AUTHOR SUMMARYThis paper studies the development of computation in biological systems by analysing changes in the flow of information in developing neural cell cultures. Although there have been a number of previous studies of information flows in neural cell cultures, this work represents the first study which compares information flows in the intrinsic dynamics across development time. Moreover, we make use of a recently proposed continuous-time transfer entropy estimator for spike trains, which, in comparison to the discrete-time estimator used previously, is able to capture important effects occurring on both small and large timescales simultaneously. We find that information flows begin to emerge after 5-10 days of activity, and crucially, the spatial structure of information flows remains significantly temporally correlated over the first month of recording. Furthermore, the magnitude of information flows across the culture are strongly related to burst position, and the roles of regions as information flow sources, sinks and mediators are found to remain consistent across development. Finally, we confirm that these early lock-ins also occur in a simple model network developing under an STDP update rule, suggesting a plausible mechanism undergirding this phenomenon.

sentences: SpatialCorr: Identifying Gene Sets with Spatially Varying Correlation Structure Recent advances in spatially resolved transcriptomics technologies enable both the measurement of genome-wide gene expression profiles and their mapping to spatial locations within a tissue. A first step in spatial transcriptomics data analysis is identifying genes with expression that varies spatially, and robust statistical methods exist to address this challenge. While useful, these methods do not detect spatial changes in the coordinated expression within a group of genes. To this end, we present SpatialCorr, a method for identifying sets of genes with spatially varying correlation structure. Given a collection of gene sets pre-defined by a user, SpatialCorr tests for spatially induced differences in the correlation of each gene set within tissue regions, as well as between and among regions. An application to cutaneous squamous cell carcinoma demonstrates the power of the approach for revealing biological insights not identified using existing methods.

sentences: Gametophyte genome activation occurs at pollen mitosis I in maize Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase1-3. Pollen actively transcribes its haploid genome4, providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

sentences: Genome-wide mediation analysis: bridging the dividebetween genotype and phenotype via transcriptomicdata in maize Mapping genotype to phenotype is an essential topic in genetics and genomics research. As the Omics data become increasingly available, genome-wide association study (GWAS) has been widely applied to establish the relationship between genotype and phenotype. However, signals detected by GWAS usually span broad genomic regions with many underneath candidate genes, making it challenging to interpret and validate the molecular functions of the candidate genes. Under the context of genetics research, we hypothesized a causal chain from genotype to phenotype partially mediated by intermediate molecular processes, i.e., gene expression. To test this hypothesis, we applied the high dimensional mediation analysis, a class of causal inference method with an assumed causal chain from the exposure to the mediator to the outcome, and implemented it to the maize diversity panel (N=280 lines). Using 40 publicly available agronomic traits, 66 newly generated metabolic traits, and published RNA-seq data from seven different tissues, we detected N=736 unique mediating genes, explaining an average of 12.7% phenotypic variance due to mediation. Noticeably, 83/736 (11%) genes were identified in mediating more than one trait, suggesting the prevalence of pleiotropic mediating effects. Among those pleiotropic mediators, benzox-azinone synthesis 13 (Bx13), a well-characterized gene encoding a 2-oxoglutarate-dependent dioxygenase, was identified mediating 40 agronomic and metabolic traits in different tissues. Further genetic and genomic analyses of the Bx13 and adjacent mediating genes suggested a 3D co-regulation modulation likely affect their expression levels and eventually lead to phenotypic consequences. Our results suggested the genome-wide mediation analysis is a powerful tool to integrate Omics data in providing causal inference to connect genotype to phenotype.

sentences: A Novel Mice Model of Catecholaminergic Polymorphic Ventricular Tachycardia Generated by CRISPR/Cas9 Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of childrens sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT.

sentences: Improving Small Molecule pKa Prediction Using Transfer Learning with Graph Neural Networks Enumerating protonation states and calculating micro-state pKa values of small molecules is an important yet challenging task for lead optimization and molecular modeling. Commercial and non-commercial solutions have notable limitations such as restrictive and expensive licenses, high CPU/GPU hour requirements, or the need for expert knowledge to set up and use. We present a graph neural network model that is trained on 714,906 calculated mico-state pKa predictions from molecules obtained from the ChEMBL database. The model is fine-tuned on a set of 5,994 experimental pKa values significantly improving its performance on two challenging test sets. Combining the graph neural network model with Dimorphite-DL, an open-source program for enumerating ionization states, we have developed the open-source Python package pkasolver, which is able to generate and enumerate protonation states and calculate micro-state pKa values with high accuracy.

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Integration of visual and antennal mechanosensory feedback during head stabilization in hawkmoths During flight maneuvers, insects exhibit compensatory head movements which are essential for stabilizing the visual field on their retina, reducing motion blur, and supporting visual self-motion estimation. In Diptera, such head movements are mediated via visual feedback from their compound eyes that detect retinal slip, as well as rapid mechanosensory feedback from their halteres - the modified hindwings that sense the angular rates of body rotations. Because non-Dipteran insects lack halteres, it is not known if mechanosensory feedback about body rotations plays any role in their head stabilization response. Diverse non-Dipteran insects are known to rely on visual and antennal mechanosensory feedback for flight control. In hawkmoths, for instance, reduction of antennal mechanosensory feedback severely compromises their ability to control flight. Similarly, when the head movements of freely-flying moths are restricted, their flight ability is also severely impaired. The role of compensatory head movements as well as multimodal feedback in insect flight raises an interesting question: in insects that lack halteres, what sensory cues are required for head stabilization? Here, we show that in the nocturnal hawkmoth Daphnis nerii, compensatory head movements are mediated by combined visual and antennal mechanosensory feedback. We subjected tethered moths to open-loop body roll rotations under different lighting conditions, and measured their ability to maintain head angle in the presence or absence of antennal mechanosensory feedback. Our study suggests that head stabilization in moths is mediated primarily by visual feedback during roll movements at lower frequencies, whereas antennal mechanosensory feedback is required when roll occurs at higher frequency. These findings are consistent with the hypothesis that control of head angle results from a multimodal feedback loop that integrates both visual and antennal mechanosensory feedback, albeit at different latencies. At adequate light levels, visual feedback is sufficient for head stabilization. However, under dark conditions, antennal mechanosensory feedback is essential for the control of head movements.

HighlightsO_LIVisual feedback contributes to head stabilization primarily during slower body rotation in hawkmoths.
C_LIO_LIAntennal mechanosensors contribute to head stabilization primarily during faster body roll.
C_LIO_LIAntennal mechanosensory feedback in head stabilization is mediated via Johnstons organ.
C_LIO_LIRestricting head movements affects flight control.
C_LI

sentences: Adduction Induces Large Optic Nerve Head Deformations in Subjects with Normal Tension Glaucoma Purpose: To assess optic nerve head (ONH) deformations and strains during adduction, abduction, and intraocular pressure (IOP) elevation in subjects with high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). Design: Clinic-based cross-sectional study. Participants: 114 HTG subjects and 114 NTG subjects. Methods. We recruited 228 subjects (114 subjects with HTG [pre-treatment IOP > 21mmHg] and 114 with NTG [pre-treatment IOP < 21mmHg]). For each subject, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze, (2) 20 degree adduction, (3) 20 degree abduction, and (4) primary gaze with acute IOP elevation (to approximately 33 mmHg) achieved through ophthalmodynamometry. For each OCT volume, we automatically segmented the prelaminar tissue (PLT), the choroid, the sclera and the lamina cribrosa (LC) using a deep learning algorithm. We also digitally aligned the OCT volumes obtained from (2)-(4) to the primary gaze volume (1) before performing digital volume correlation (DVC) analysis to quantify IOP- and gaze-induced ONH tissues three-dimensional displacements and effective strain (a local measure of tissue deformation) for all scenarios. Main Outcome Measures: Three-dimensional ONH displacements and strains. Results: Across all subjects, adduction generated high effective strain (4.2 {+/-} 1.4%) in the ONH tissues with no significant difference (p>0.05) with those induced by IOP elevation (4.5 {+/-} 1.5%); while abduction generated significantly lower (p = 0.014) effective strain (3.8 {+/-} 1.1%). Interestingly, the LC of HTG subjects exhibited significantly higher effective strain than those of NTG subjects under IOP elevation (HTG:4.6 {+/-} 1.7% vs NTG:4.1 {+/-} 1.5%, p = 0.047). Conversely, the LC tissue of NTG subjects exhibited significantly higher effective strain than those of HTG subjects under adduction (NTG: 4.9 {+/-} 1.9% vs HTG: 4.0 {+/-} 1.4%, p = 0.041). Conclusion: We found that adduction produced comparable strains and displacements as IOP elevation. We also found that NTG subjects experienced higher strains due to adduction than HTG subjects, while HTG subjects experienced higher strain due to IOP elevation than NTG subjects - and that these differences were most pronounced in the LC tissue.

sentences: Sequence-space expansion is driven by the emergence of new signaling pathways This report presents multi-genomes evidence that sequence-space expansion occurs when pathways with incompatible functions emerge. It is speculated that as pathways evolve within the sequence-space of highly paralogous signaling systems, they insulate against unwanted interactions, and can expand into new sequence-space. To prove that sequence-space expansion is driven by pathway insulation against emergent pathways, we develop a comprehensive, holistic (whole genomes) new pathway discovery pipeline. The holistic pipeline, which uses t-distributed stochastic neighbor embedding algorithms and phylogeny to map the extant sequence-space of thousands of bacterial two-component systems, reveals a new signaling pathway, resultant from domain swapping. By employing functional genomics screens in our model system Pseudomonas putida KT2440, we provide empirical evidence that descendents of the functionally discordant domain swapped pathway and its nearest neighbors in sequence-space are paralogous. We conclude that the emergence of this new pathway necessitated pathway insulation and drove sequence-space expansion.

sentences: GlycoSHIELD: a versatile pipeline to assess glycan impact on protein structures More than 75% of surface and secreted proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, influencing protein dynamics and interactions with other molecules. Glycan conformational freedom impairs complete structural elucidation of glycoproteins. Computer simulations may be used to model glycan structure and dynamics. However, such simulations typically require thousands of computing hours on specialized supercomputers, thus limiting routine use. Here, we describe a reductionist method that can be implemented on personal computers to graft ensembles of realistic glycan conformers onto static protein structures in a matter of minutes. Using this open-source pipeline, we reconstructed the full glycan cover of SARS-CoV-2 Spike protein (S-protein) and a human GABAA receptor. Focusing on S-protein, we show that GlycoSHIELD recapitulates key features of extended simulations of the glycosylated protein, including epitope masking, and provides new mechanistic insights on N-glycan impact on protein structural dynamics.

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

sentences: Redox-active secondary metabolites act as interspecies modulators of antibiotic resilience Bacterial opportunistic pathogens make a wide range of secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of two redox-active secondary metabolites, pyocyanin and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin is made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. Both molecules alter the susceptibility profile of pathogenic species within the "Burkholderia cepacia complex" to different antibiotics, either antagonizing or potentiating their effects, depending on the drugs class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

sentences: Integration of Odor-Induced Activity of Kenyon Cells in an Electrotonically Compact Drosophila Mushroom Body Output Neuron (MBON) The formation of an ecologically useful lasting memory requires that the brain has an accurate internal representation of the surrounding environment. In addition, it must have the ability to integrate a variety of different sensory stimuli and associate them with rewarding and aversive behavioral outcomes. Over the previous years, a number of studies have dissected the anatomy and elucidated some of the working principles of the Drosophila mushroom body (MB), the flys center for learning and memory. As a consequence, we now have a functional understanding of where and how in the MB sensory stimuli converge and are associated. However, the molecular and cellular dynamics at the critical synaptic intersection for this process, the Kenyon cell-mushroom body output neuron (KC-MBON) synapse, are largely unknown. Here, we introduce a first approach to understand this integration process and the physiological changes occurring at the KC-MBON synapse during Kenyon cell (KC) activation. We use the published connectome of the Drosophila MB to construct a functional computational model of the MBON-3 dendritic structure. We simulate synaptic input by individual KC-MBON synapses by current injections into precisely (m) identified local dendritic sections, and the input from a model population of KCs representing an odor by a spatially distributed cluster of current injections. By recording the effect of the simulated current injections on the membrane potential of the neuron, we show that the MBON-3 is electrotonically compact. This suggests that odor-induced MBON activity is likely governed by input strength while the positions of KC input synapses are largely irrelevant.

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Widespread Utilization of Diverse Organophosphate Pollutants by Marine Bacteria Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

One sentence summaryWidespread utilization of diverse organophosphate pollutants by over 20 marine bacterial strains represents a new hope for ocean bioremediation.

sentences: The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDAs Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methylcytosine modifications. Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), one enzymatic deamination method (EMseq), targeted methylation sequencing (Illumina Methyl Capture EPIC), and single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, BWAMeth, and GemBS), we found overall high concordance between assays (R=0.87-R0.93), differences in efficency of read mapping and CpG capture and coverage, and platform performance. The data provided herein can guide continued used of these reference materials in epigenomics assays, as well as provide best practices for epigenomics research and experimental design in future studies.

sentences: Escaping through the predator's gill cleft: A defensive tactic of juvenile eels after capture by predatory fish Some prey animals can actively escape from predators even after being captured, but knowledge of such behaviors is still limited, especially in vertebrates. Here, we report the unique active escape behavior of Japanese eel Anguilla japonica juveniles through the gill cleft of the predatory fish Odontobutis obscura. Of the 54 A. japonica juveniles captured by the predator, 28 escaped via the predators gill, and most escaped individuals survived afterwards. In all escaped individuals, their tails emerged first from the gill cleft of the predator, and then their whole bodies slipped out in a backward direction from the cleft. These findings indicate that A. japonica juveniles have the specialized antipredator tactic, which provides the basis for further investigation from behavioral, ecological, and evolutionary perspectives.

sentences: A general hidden Markov model for multi-parent populations A common step in the analysis of multi-parent populations is genotype reconstruction: identifying the founder origin of haplotypes from dense marker data. This process often makes use of a probability model for the pattern of founder alleles along chromosomes, including the relative frequency of founder alleles and the probability of exchanges among them, which depend on a model for meiotic recombination and on the mating design for the population. While the precise experimental design used to generate the population may be used to derive a precise characterization of the model for exchanges among founder alleles, this can be tedious, particularly given the great variety of experimental designs that have been proposed. We describe an approximate model that can be applied for a variety of multi-parent populations. We have implemented the approach in the R/qtl2 software, and we illustrate its use in applications to publicly-available data on Diversity Outbred and Collaborative Cross mice

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De novo transcriptome of Taverniera cuneifolia (Roth) Ali. Taverniera cuneifolia has been described as a potent substitute of Licorice in India. It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Glycyrrhizin is one of the most useful bioactive sesquiterpenoid present in this plant. The present study aim to carry out transcriptome analysis in root tissue of Taverniera cuneifolia to identify specific functional genes involved in the biosynthesis of secondary metabolites. The root transcriptome sequencing of Taverniera cuneifolia resulted in a total of ~7.29 Gb of raw data and generated 55,991,233 raw reads. The high quality reads were de novo assembled by Trinity assembler followed through CD-HIT resulted into 35,590 "Unigene" transcripts with an average size of 419 bp. The unigenes were analyzed using BLAST2GO resulted in 27,884 (78.35%) transcript with blast hits, 22,510 (63.25%) transcript with mapping and 21,066 (59.19%) transcript with annotation. Functional annotation was carried out using NCBIs non-redundant and Uniprot databases resulted in the identification of 21,066 (59.19%) annotated transcripts and GO assigned to 24751 (69.54%) transcripts. The gene ontology result shows maximum sequences match with Biological Processes (48%), Molecular Function (27%) and Cellular components (23%). A total of 289 metabolic enriched pathways were identified, which included pathways like Sesquiterpenoid and triterpenoid pathway which were involved in synthesis of secondary metabolite Glycyrrhizin biosynthesis. The enzymes, squalene monooxygenase, farnesyl-diphosphate farnesyltransferase, beta amyrin synthase, beta-amyrin 24-hydroxylase, were identified by functional annotation of transcriptome data. There were several other pathways like terpenoid backbone biosynthesis, steroid biosynthesis, Carotenoid biosynthesis, Flavonoids biosynthesis etc. which have been reported first time from this plant. Transcription factors were predicted by comparison with Plant Transcription Factor Database, and 1557 trancripts belonging to 85 trancription factor families were identified. This transcriptome analysis provided an important resource for future genomic studies in Taverniera cuneifolia, therefore representing basis in further investigation of the plant.

SignificanceLicorice (Glycyrrhiza glabra roots) is used as traditional Chinese herbal medicines in majority of formulations. Licorice is also used in Industries like food, herbal and cosmetics etc. due to its high demand in the market it is imported from foreign countries and is not available locally of superior quality (Liu et al., 2015). In India, Taverniera cuneifolia has been described as a potent substitute of Licorice, it has been quoted in ancient books like Charak Samhita during the Nigandu period (Kamboj, 2000) and Barda dungar ni Vanaspati ane upyog (Thaker 1910). It has been used as an expectorant, anti-inflammatory, anti-ulcer, wound healing, blood purifier etc. Transcriptomic studies will assist in understanding the basic molecular structure, function and organization of information within the genome of Taverniera cuniefolia. This study will help us to identify the key metabolites their expressions and genes responsible for their production.

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Navigating the amino acid sequence space between functional proteins using a deep learning framework MotivationShedding light on the relationships between protein se-quences and functions is a challenging task with many implications in protein evolution, diseases understanding, and protein design. Protein sequence / function space is however hard to comprehend due to its com-plexity. Generative models help to decipher complex systems thanks to their abilities to learn and recreate data specificity. Applied to protein sequences, they can point out relationships between protein positions and functions capture the sequence patterns associated with functions or navigate through uncharted area of molecular evolution.

ResultsIn this study, an unsupervised generative approach based on adversarial auto-encoder (AAE) is proposed to generate and explore new sequences with respect to their functions thanks to the prior distribution allowing a continuous exploration of the latent space. AAEs are tested on three protein families known for their multiple functions. Clustering re-sults on the encoded sequences from the latent space computed by AAEs display high level of homogeneity regarding the protein sequence func-tions. The study also reports and analyzes for the first time two sampling strategies based on latent space interpolation and latent space arithmetic to generate intermediate protein sequences sharing sequential and functional properties of original sequences issued from different families and functions. Generated sequences by interpolation between latent space data points demonstrate the ability of the AAE to generalize and to pro-duce meaningful biological sequences from an evolutionary uncharted area of the biological sequence space. Finally, 3D structure models generated by comparative modelling between different combinations of structures of different sub-families and of generated sequences from latent space or sub-family sequences point out to the ability of the latent space arithmetic to successfully transfer functional properties between sub-families. All in all this study confirms the ability of deep learning frameworks to model biological complexity and bring new tools to explore amino acid sequence and functional spaces.

AvailabilityCode and data used for this study are freely available at https://github.com/T-B-F/aae4seq.

Contacttristan@bitardfeildel.fr

Supplementary informationSupplementary data are available at online.

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A novel PNGase Rc for improved protein N-deglycosylation in bioanalytics and HDX-MS epitope mapping under challenging conditions N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry (LC-MS) and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS).

Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quench conditions (pH 2.5, 0 {degrees}C) and in the presence of 0.4 M Tris(2-carboxyethyl)phosphine (TCEP), 4 M urea and 1 M guanidinium chloride (GdmCl). Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRP).

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Sequence dependencies and mutation rates of localized mutational processes in cancer BackgroundCancer mutations accumulate through replication errors and DNA damage coupled with incomplete repair. Individual mutational processes often show strong sequence and regional preferences. As a result, some sequence contexts mutate at much higher rates than others. Mutational hotspots, with recurrent mutations across cancer samples, represent genomic positions with elevated mutation rates, often caused by highly localized mutational processes.

ResultsWe analyze the mutation rates of all 11-mer genomic sequence contexts using the PCAWG set of 2,583 pan-cancer whole genomes. We further associate individual mutations and contexts to mutational signatures and estimate their relative mutation rates. We show that hotspots generally identify highly mutable sequence contexts. Using these, we show that some mutational signatures are enriched in hotspot sequence contexts, corresponding to well-defined sequence preferences for the underlying localized mutational processes. This includes signature 17b (of unknown etiology) and signatures 62 (POLE), 7a (UV), and 72 (linked to lymphomas). In some cases, the mutation rate increases further when focusing on certain genomic regions, such as signature 62 in poised promoters, where the mutation is increased several thousand folds over the overall data set average.

ConclusionWe summarize our findings in a catalog of localized mutational processes, their sequence preferences, and their estimated mutation rates.

sentences: Neuropsychological assessment and virtual reality training of social prediction in patients with cerebellar malformation It has been proposed that impairments of the predictive function exerted by the cerebellum may account for social cognition deficits. Here, we integrated cerebellar functions in a predictive coding framework to elucidate how cerebellar alterations could affect the predictive processing of others behavior. Experiment 1 demonstrated that cerebellar patients were impaired in relying on contextual information during action prediction, and this impairment was significantly associated with social cognition abilities. Experiment 2 indicated that patients with cerebellar malformation showed a domain-general deficit in using contextual information to predict both social and physical events. Experiment 3 provided first evidence that a social-prediction training in virtual reality could boost the ability to use context-based predictions to understand others intentions. These findings shed new light on the predictive role of the cerebellum and its contribution to social cognition, paving the way for new approaches to the rehabilitation of the Cerebellar Cognitive Affective Syndrome.

sentences: Lymphatics constitute a novel component of the intestinal stem cell niche Intestinal stem cells (ISCs) depend on niche factors for their proper function. However, the source of these ISC niche factors and how they support ISCs remain controversial due to their redundant expression patterns. Here, we report that the maintenance of ISCs depends on both lymphatic endothelial cells (LECs) and Rspo3+Grem1+ fibroblasts (RGFs). We found that LECs are surrounded by RGFs and located in close proximity to Lgr5+ ISCs. RSPO3 production is restricted to LECs and RGFs and they can partially compensate for each other; however, RSPO3 loss in both of LECs and RGFs drastically compromises ISC numbers, villi length, and repair after irradiation-induced injury. Mechanistically, irradiation-induced damage expands LEC and RGF numbers and enhances the latters generation of RSPO3 through IL-1 receptor activation. We propose that LECs represent a novel component of the ISC niche, which together with RGFs, provide essential RSPO3 to sustain ISCs in homeostasis and regeneration.

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Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci Repetitive DNA represents an important driver of sex chromosome differentiation. Yet, repetitive sequences tend to be misrepresented or overlooked in genomic studies. We analysed repetitive DNA landscape of sex chromosomes in several populations of a turquoise killifish Nothobranchius furzeri and its sister species N. kadleci (Teleostei: Nothobranchiidae), representatives of African annual killifishes with high rate of karyotype and sex chromosome evolution. We combined bioinformatic analyses of repeatome with molecular cytogenetic techniques such as comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, genes for ribosomal RNAs (rDNA) and bacterial artificial chromosomes (BACs) and immunostaining of SYCP3 and MLH1 proteins, which marked lateral elements of synaptonemal complexes and recombination sites, respectively. We revealed that N. furzeri and N. kadleci share the XY sex chromosome system, which is thus much older than previously assumed. Sex chromosomes are mostly heteromorphic as evidenced by distinct distribution of satellite DNAs and major rDNA. Yet, the heteromorphic XY sex chromosomes pair almost exclusively regularly in meiosis, which implies synaptic adjustment. Physical mapping of BACs identified inversions on Y chromosomes of the N. kadleci populations, akin to the pattern previously reported in N. furzeri. Yet, repetitive DNA landscape of X and Y sex chromosomes either diverged in parallel in populations of both species or it evolved in their common ancestor and thus predates the inversions. The observed differentiation via repeat repatterning thus cannot be explained by the classical sexually antagonistic model. Rather, we hypothesized that relaxed meiotic drive and recombination reduced by neutral processes could drive changes in repeatome and secondary inversions could be maintained by sexually antagonistic regulatory effects resulting from early evolution of dosage compensation.

Author summaryEarly differentiation of sex chromosomes is not yet satisfactorily understood despite intensive research effort. Homomorphic sex chromosomes and their rapid turnover are common in teleost fishes, which makes them excellent models for studying evolution of nascent sex chromosomes. We investigated sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, particularly their repetitive landscape, which was misrepresented in previous genomic studies. Combination of cytogenetic and genomic approaches revealed that both species share heteromorphic XY sex chromosome system. The N. furzeri XY sex chromosomes thus evolved earlier than previously expected. In N. kadleci, Y-linked inversions analogous to those reported in N. furzeri were detected. Changes in repetitive DNA distribution on sex chromosomes are either convergent or occurred in a common ancestor of both species, prior to the inversion events. The observed sex chromosome differentiation on repetitive DNA level thus cannot be reconciled with the classical theoretical model of sex chromosome evolution driven by sexually antagonistic selection. We invoke alternative explanations such as relaxed meiotic drive and recombination reduced by neutral processes, and we hypothesize that secondary inversions could be maintained by early evolution of dosage compensation resulting in sexually antagonistic regulatory effects.

sentences: Drivers and Determinants of Strain Dynamics Following Faecal Microbiota Transplantation Faecal microbiota transplantation (FMT) is an efficacious therapeutic intervention, but its clinical mode of action and underlying microbiome dynamics remain poorly understood. Here, we analysed the metagenomes associated with 142 FMTs, in a time series-based meta-study across five disease indications. We quantified strain-level dynamics of 1,089 microbial species based on their pangenome, complemented with 47,548 newly constructed metagenome-assembled genomes. Using subsets of procedural-, host- and microbiome-based variables, LASSO-regularised regression models accurately predicted the colonisation and resilience of donor and recipient microbes, as well as turnover of individual species. Linking this to putative ecological mechanisms, we found these sets of variables to be informative of the underlying processes that shape the post-FMT gut microbiome. Recipient factors and complementarity of donor and recipient microbiomes, encompassing entire communities to individual strains, were the main determinants of individual strain population dynamics, and mostly independent of clinical outcomes. Recipient community state and the degree of residual strain depletion provided a neutral baseline for donor strain colonisation success, in addition to inhibitive priority effects between species and conspecific strains, as well as putatively adaptive processes. Our results suggest promising tunable parameters to enhance donor flora colonisation or recipient flora displacement in clinical practice, towards the development of more targeted and personalised therapies.

sentences: Epitopedia: identifying molecular mimicry of known immune epitopes Upon infection, foreign antigenic proteins stimulate the hosts immune system to produce antibodies targeting the pathogen. These antibodies bind to regions on the antibody called epitopes. Structural similarity (molecular mimicry) of epitopes between an infecting pathogen and host proteins or other pathogenic proteins the host has previously encountered can impact the host immune response to the pathogen and may lead to cross-reactive antibodies. The ability to identify potential regions of molecular mimicry in a pathogen can illuminate immune effects which are especially important to pathogen treatment and vaccine design. Here we present Epitopedia, a software pipeline that facilitates the identification of regions that may exhibit potential three-dimensional molecular mimicry between an antigenic pathogen protein and known immune epitopes as catalogued by the immune epitope database (IEDB). Epitopedia is open-source software released under the MIT license and is freely available on GitHub, including a Docker container with all other software dependencies preinstalled. We performed an analysis describing how various secondary structure states, identity between pentapeptide pairs, and identity between the parent sequences of pentapeptide pairs affects RMSD. We found that pentapeptides pairs in a helical conformation had considerably lower RMSD values than those in Extended or Coil conformations. We also found that RMSD is significantly increased when pentapeptide pairs are from non-homologous sequences.

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The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against coronavirus disease in 2019 (COVID-19). Clinical trials and ongoing vaccinations present with very high protection levels and varying degrees of side effects. However, the nature of the reported side effects remains poorly defined. Here we present evidence that LNPs used in many preclinical studies are highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate.

In summary, here we show that the LNPs used for many preclinical studies are highly inflammatory. Thus, their potent adjuvant activity and reported superiority comparing to other adjuvants in supporting the induction of adaptive immune responses likely stem from their inflammatory nature. Furthermore, the preclinical LNPs are similar to the ones used for human vaccines, which could also explain the observed side effects in humans using this platform.

sentences: BonZeb: Open-source, modular software tools for high-resolution zebrafish tracking and analysis We present BonZeb - a suite of modular Bonsai packages which allow high-resolution zebrafish tracking with dynamic visual feedback. Bonsai is an increasingly popular software platform that is accelerating the standardization of experimental protocols within the neurosciences due to its speed, flexibility, and minimal programming overhead. BonZeb can be implemented into novel and existing Bonsai workflows for online behavioral tracking and offline tracking with batch processing. We demonstrate that BonZeb can run a variety of experimental configurations used for gaining insights into the neural mechanisms of zebrafish behavior. BonZeb supports head-fixed closed-loop and free-swimming virtual open-loop assays as well as multi-animal tracking, optogenetic stimulation, and calcium imaging during behavior. The combined performance, ease of use and versatility of BonZeb opens new experimental avenues for researchers seeking high-resolution behavioral tracking of larval zebrafish.

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Direct Energy Transfer from Allophycocyanin-Free Rod-Type CpcL-Phycobilisome to Photosystem I Phycobilisomes (PBSs) are photosynthetic antenna megacomplexes comprised of pigment-binding proteins (cores and rods) joined with linker proteins. A rod-type PBS that does not have a core is connected to photosystem I (PSI) by a pigment-free CpcL linker protein, which induces a red-shift of the absorption band of phycocyanobilin (PCB) in the rod (red-PCB). Herein, the isolated supercomplex of the rod-type PBS and the PSI tetramer from Anabaena sp. PCC 7120 were probed by picosecond laser spectroscopy at 77 K and by decay-associated spectral analysis to show that red-PCB mediates the fast (time constant = 90 ps) and efficient (efficiency = 95%) transfer of excitation energy from PCB in rod to chlorophyll a (Chl a) in PSI. According to the Forster energy transfer mechanism, this high efficiency corresponds to a 4-nm distance between red-PCB and Chl a, suggesting that {beta}-84 PCB in rod acts as red-PCB.

TOC GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Pharmacologic and genetic inhibition of cholesterol esterification reduces tumour burden: a pan-cancer systematic review and meta-analysis of preclinical models Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, and Web of Science for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p<0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p[≤]0.002). SOAT inhibition increased tumour apoptosis (p=0.007), CD8+ lymphocyte infiltration and cytotoxicity (p[≤]0.05), and reduced proliferation (p=0.0003) and metastasis (p<0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size was overestimated. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.

sentences: Fracture healing is delayed in the absence of gasdermin signaling Amino-terminal fragments from proteolytically cleaved gasdermins (GSDMs) form plasma membrane pores that enable the secretion of interleukin-1{beta} (IL-1{beta}) and IL-18. Excessive GSDM- mediated pore formation can compromise the integrity of the plasma membrane thereby causing the lytic inflammatory cell death, pyroptosis. We found that GSDMD and GSDME were the only GSDMs that were readily expressed in bone microenvironment. Therefore, we tested the hypothesis that GSDMD and GSDME are implicated in fracture healing owing to their role in the obligatory inflammatory response following injury. We found that bone callus volume and biomechanical properties of injured bones were significantly reduced in mice lacking either GSDM compared with wild-type (WT) mice, indicating that fracture healing was compromised in mutant mice. However, compound loss of GSDMD and GSDME did not exacerbate the outcomes, suggesting shared actions of both GSDMs in fracture healing. Mechanistically, bone injury induced IL-1{beta} and IL-18 secretion in vivo, a response that was mimicked in vitro by bone debris and ATP, which function as inflammatory danger signals. Importantly, the secretion of these cytokines was attenuated in conditions of GSDMD deficiency. Finally, deletion of IL-1 receptor reproduced the phenotype of Gsdmd or Gsdme deficient mice, implying that inflammatory responses induced by the GSDM-IL-1 axis promote bone healing after fracture.

sentences: Miniaturizing wet scrubbers for aerosolized droplet capture Aerosols dispersed and transmitted through the air (e.g., particulate matter pollution, bioaerosols) are ubiquitous and one of the leading causes of adverse health effects and disease transmission. A variety of sampling methods (e.g., filters, cyclones, impactors) have been developed to assess personal exposures. However, a gap still remains in the accessibility and ease-of-use of these technologies for people without experience or training in collecting airborne samples. Additionally, wet scrubbers (large non-portable industrial systems) utilize liquid sprays to remove aerosols from the air; the goal is to "scrub" (i.e., clean) the exhaust of industrial smokestacks, not collect the aerosols for analysis. Inspired by wet scrubbers, we developed a device fundamentally different from existing portable air samplers by using aerosolized microdroplets to capture aerosols in personal spaces (e.g., homes, offices, schools). Our aerosol-sampling device is the size of a small teapot, can be operated without specialized training, and features a winding flow path in a supersaturated relative humidity environment enabling droplet growth. The integrated open mesofluidic channels shuttle coalesced droplets to a collection chamber for subsequent sample analysis. Here, we present the experimental demonstration of aerosol capture into water droplets. Iterative study optimized the non-linear flow manipulating baffles and enabled an 83% retention of the aerosolized microdroplets in the confined volume of our device. As a proof-of-concept for aerosol capture into a liquid medium, 0.5-3 {micro}m model particles were used to evaluate aerosol capture efficiency. Finally, we demonstrate the device can capture and keep a bioaerosol (bacteriophage MS2) viable for downstream analysis.

sentences: Tolerance of Wildlife in Protected Area Borderlands Increases in human-wildlife conflict globally threaten human wellbeing and biodiversity conservation. Sustainable solutions that promote coexistence of people and wildlife are needed, especially in human-dominated landscapes surrounding protected areas. Peoples attitudes toward wildlife influence their behaviours, including tolerance for human-wildlife interactions, poaching, and habitat degradation. Better understanding of how to improve peoples attitudes toward wildlife is instrumental to promoting coexistence between people and wildlife in shared spaces. Efforts to promote coexistence often fail because they are based on the inaccurate assumption that peoples attitudes towards wildlife are directly and proportionally related to wildlife-based financial incentives and costs. In reality, peoples attitudes towards wildlife are far more complex. We analysed surveys (n=237) from Mozambique to examine peoples attitudes toward wildlife in the buffer zones surrounding protected areas using logistic regression and Getis-Ord hot-spot analysis (GI*). Mozambique, which is under-represented in the wildlife-based research literature, is characterized by extreme poverty and rewilding efforts. We found that most respondents were tolerant of wildlife and tolerance was positively correlated with peoples age, gender, and agreement with rules governing wildlife conservation. Peoples tolerance for wildlife was also reinforced if they receive benefits from wildlife and are situated further from the park fence. Predation, human harm, and crop loss were not significant predictors of tolerance. We found no evidence of spatial patterns in tolerance for wildlife. Our results suggest that wildlife conservation programs are more likely to be successful if benefits are distributed equitably and community members are actively involved in decision making.

sentences: Characterizing circadian connectome of O. tenuiflorum using an integrated network theoretic framework Ocimum tenuiflorum (Tulsi) is among the most valuable medicinal plants as almost every part of this herb and the essential oil it produces possess pharmaceutical properties that have been utilized since ancient times to cure a large number of diseases. Like in other plants, circadian clock in Tulsi regulate important physiological processes like growth, development, defence etc. by anticipating environmental cues. In the present work, identification and characterization of core circadian clock and clock associated proteins in Tulsi genome is reported. We mined 24 core clock (CC) proteins in 56 template plant genomes to build their hidden Markov models (HMMs). These HMMs were used to identify 24 core clock proteins in O. tenuiflorum which were further subjected to functional annotation. A hybrid network theoretic methodology comprising of random walk with restart (RWR) and graphlet degree vectors (GDV) was implemented to explore the local topology of the interologous, Tulsi protein interaction network (TulsiPIN) and mine CC associated raw candidate proteins. Statistical and biological significance of the raw candidates was determined using permutation and enrichment tests. A total of 70 putative CC associated proteins were identified which were further subjected to functional annotation.

sentences: Rat sensitivity to multipoint statistics is predicted by efficient coding of natural scenes Efficient processing of sensory data requires adapting the neuronal encoding strategy to the statistics of natural stimuli. Previously, in Hermundstad et al. 2014, we showed that local multipoint correlation patterns that are most variable in natural images are also the most perceptually salient for human observers, in a way that is compatible with the efficient coding principle. Understanding the neuronal mechanisms underlying such adaptation to image statistics will require performing invasive experiments that are impossible in humans. Therefore, it is important to understand whether a similar phenomenon can be detected in animal species that allow for powerful experimental manipulations, such as rodents. Here we selected four image statistics (from single- to four-point correlations) and trained four groups of rats to discriminate between white noise patterns and binary textures containing variable intensity levels of one of such statistics. We interpreted the resulting psychometric data with an ideal observer model, finding a sharp decrease in sensitivity from 2- to 4-point correlations and a further decrease from 4- to 3-point. This ranking fully reproduces the trend we previously observed in humans, thus extending a direct demonstration of efficient coding to a species where neuronal and developmental processes can be interrogated and causally manipulated.

sentences: Testicular Dysgenesis Syndrome-like morphology and gene expression, and activation of Hypoxia Inducible Factor 1 Alpha in juvenile lamb testes following developmental exposure to low-level environmental chemical mixture. Current declines in male reproductive health may, in part, be driven by anthropogenic environmental chemical exposure. Using the biosolid treated pasture (BTP) sheep model, this study examined the effects of gestational exposure to a translationally relevant mixture of environmental chemicals. At 8 weeks of age, ram lambs gestationally exposed to BTP were lighter than control, and their testes contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. Effects of biosolid exposure on the testicular transcriptome correlated with human testicular dysgenesis syndrome (TDS) patient data. Common differentially expressed genes indicated changes in apoptotic and mTOR signalling, which concurs with previous gene expression data from neonatal BTP lambs. Gene expression data and immunohistochemistry indicates increased HIF1 activation and nuclear localisation in BTP exposed animals, known to disrupt testosterone synthesis. Together, these results provide a potential mechanism for the pathogenesis of this phenotype, and TDS in humans.

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TRPA1: A Potential Prognostic Indicator for Oral Squamous Cell Carcinoma IntroductionTransient receptors are related to oral cancer pain. Previously capsaicin (TRPV1 agonist) was shown to induce cell death in oral cancer cells. We hypothesised that these receptors are present in oral cancer.

MethodWe examined the presence of cannabinoid receptors (CB1 and CB2) and targets (TRPV1, TRPA1, CaV 3.1, CaV 3.2, CaV 3.3) via quantitative polymerase chain reaction (qPCR) in oral cancer cells SCC4, SCC9, SCC25, Cal27, and normal oral cell line OKF6.

ResultCannabinoid receptors are absent in all the cell lines, while TRPA1 is only present in normal cells, but absent in all the oral cancer cell lines. Voltage-gated calcium channels are present in all the cell lines.

Conclusion and Future AspectsTRPA1 could be the possible future prognostic indicator of oral squamous cell carcinoma. Future functionality assays could use precancerous cell lines to follow the loss of TRPA1.

sentences: Meta-analysis reveals the global picture of antibiotic resistance gene prevalence across environments The environment is an important component in the emergence and transmission of antimicrobial resistance (AMR). Despite that, current AMR monitoring initiatives lack comprehensive reference data for the vast majority of environments. To enable monitoring to detect deviations from the normal background resistance levels in the environment, it is necessary to establish the typical baseline of AMR in a variety of settings. In an attempt to establish this baseline level, we here performed a comprehensive literature survey across 2516 scientific papers, 150 of which contained relevant qPCR data on antibiotic resistance genes (ARGs) in environments associated with potential routes of AMR dissemination. The collected data include 1487 samples distributed across 30 different countries and 17 environmental types, in a time span from 2005 to 2018. More than 330 different genes were identified from the collected studies. Most surveyed environments contained a diverse set of ARGs, but generally at low abundances. We used linear mixed models and overrepresentation analysis to identify time trends and associations between ARGs and specific environments. Altogether these data represent a comprehensive overview of the occurrence and levels of ARGs in different environments, providing background data for risk assessment models within current and future AMR monitoring frameworks.

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SAMase of bacteriophage T3 inactivates E. coli methionine S-adenosyltransferase by forming hetero-polymers S-adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host E. coli cells, thus inactivating a crucial metabolite involved in plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy -- it allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle Cryo-EM and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM, but also by interacting with and efficiently inhibiting the hosts methionine S-adenosyltransferase (MAT) -- the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined together by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency high-amplitude active site-deforming modes. The amplification of uncorrelated motions between active site residues weakens MATs ability to withhold substrates, explaining the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools.

IMPORTANCESelf-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy -- silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes might be a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

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Refining conformational ensembles of flexible proteins against small-angle X-ray scattering data Intrinsically disordered proteins and flexible regions in multi-domain proteins display substantial conformational heterogeneity. Characterizing the conformational ensembles of these proteins in solution typically requires combining one or more biophysical techniques with computational modelling or simulations. Experimental data can either be used to assess the accuracy of a computational model or to refine the computational model to get a better agreement with the experimental data. In both cases, one generally needs a so-called forward model, i.e. an algorithm to calculate experimental observables from individual conformations or ensembles. In many cases, this involve one or more parameters that need to be set, and it is not always trivial to determine the optimal values or to understand the impact on the choice of parameters. For example, in the case of small-angle X-ray scattering (SAXS) experiments, many forward models include parameters that describe the contribution of the hydration layer and displaced solvent to the background-subtracted experimental data. Often, one also needs to fit a scale factor and a constant background for the SAXS data, but across the entire ensemble. Here, we present a protocol to dissect the effect of free-parameters on the calculated SAXS intensities, and to identify a reliable set of values. We have implemented this procedure in our Bayesian/Maximum Entropy framework for ensemble refinement, and demonstrate the results on four intrinsically disordered proteins and a three-domain protein connected by flexible linkers. Our results show that the resulting ensembles can depend on the parameters used for solvent effects, and suggests that these should be chosen carefully. We also find a set of parameters that work robustly across all proteins.

SIGNIFICANCEThe flexibility of a protein is often key to its biological function, yet understanding and characterizing its conformational heterogeneity is difficult. We here describe a robust protocol for combining small-angle X-ray scattering experiments with computational modelling to obtain a conformational ensemble. In particular, we focus on the contribution of protein hydration to the experiments and how this is included in modelling the data. Our resulting algorithm and software should make modelling intrinsically disordered proteins and multi-domain proteins more robust, thus aiding in understanding the relationship between protein dynamics and biological function.

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On optimal temozolomide scheduling for slowly growing gliomas BackgroundTemozolomide (TMZ) is an oral alkylating agent active against gliomas with a favorable toxicity profile. It is part of the standard of care in the management of glioblastoma, and is commonly used in low-grade gliomas. In-silico mathematical models can potentially be used to personalize treatments and to accelerate the discovery of optimal drug delivery schemes.

MethodsAgent-based mathematical models fed with either mouse or patient data were developed for the in-silico studies. The experimental test beds used to confirm the results were: mouse glioma models obtained by retroviral expression of EGFR wt or EGFR vIII in primary progenitors from p16/p19 ko mice and grown in vitro and in vivo in orthotopic allografts, and human glioblastoma U251 cells immobilized in alginate microfibers. The patient data used to parametrize the model were obtained from the TCGA/TCIA databases and the TOG clinical study.

ResultsSlow growth  virtual murine gliomas benefited from increasing TMZ dose separation in silico. In line with the simulation results, improved survival, reduced toxicity, lower expression of resistance factors and reduction of the tumor mesenchymal component were observed in experimental models subject to long-cycle treatment, particularly in slowly-growing tumors. Tissue analysis after long-cycle TMZ treatments revealed epigenetically-driven changes in tumor phenotype, which could explain the reduction in glioma growth speed. In-silico trials provided support for methods of implementation in human patients.

ConclusionsIn-silico simulations, and in-vitro and in-vivo studies show that TMZ administration schedules with increased time between doses may reduce toxicity, delay the appearance of resistances and lead to survival benefits mediated by changes in the tumor phenotype in gliomas.

IMPORTANCE OF THE STUDYIn-vivo evidence is provided of improvements in survival, resistance, and toxicity from TMZ schemes with long rest periods between doses in slowly-growing GBM mouse models. The results match hypotheses generated in silico using a mathematical model incorporating the main biological features and fed with real patient data. An epigenetically-driven change in tumor phenotype was also revealed experimentally, which could explain the reduction in glioma growth speed under the  long cycle scheme. To determine the extent to which our results hold for human patients, large sets of simulations were performed on virtual patients. These in-silico trials suggest different ways to bring the benefits observed in experimental models into clinical practice.

sentences: In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for potential chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the leading target candidates therein. In order to successfully validate NP-GPCRs as targets for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identifying receptor activating ligand(s) in a process known as receptor deorphanisation. Such efforts first require the identification of all neuropeptide ligands within parasites. Here we comb the genomes of nine therapeutically relevant pathogenic nematode species to comprehensively characterise the nematode parasite neuropeptide-like protein (NLP) complements, and details the discovery of several previously unreported, yet conserved, neuropeptides and neuropeptide-encoding genes. We identify the neuropeptides that are most highly conserved in all parasites examined, and characterise their physiological activity on the reproductive musculature of the parasite, Ascaris suum. These data suggest conserved neuropeptide functions in both free living and parasitic nematodes, and support the potential for exploitation of the neuropeptide signalling system as an anthelmintic target.

sentences: Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells Cardiac directed differentiation of human induced pluripotent stem cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types even when using very well-characterized protocols. We wondered whether differentiated cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. By associating individual differentiated cells that share a common hiPS cell precursor, we were able to test whether expression variability in differentiated cells was pre-determined from the hiPS cell state. Although within a single experiment, differentiated cells that share an hiPS cell progenitor were more transcriptionally similar to each other than to other cells in the differentiated population, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurred during differentiation in a manner that suggested that all cells were equally likely to survive or die, suggesting that there was no intrinsic selection bias for cells descended from particular hiPS cell progenitors. These results led us to wonder about how cells grow out spatially during the directed differentiation process. Labeling cells by their expression of a few canonical cell type marker genes, we showed that cells expressing the same marker tended to occur in patches observable by visual inspection, suggesting that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. Altogether, our results show that while there is substantial heterogeneity in the initial hiPS cell population, that heterogeneity is not responsible for heterogeneous outcomes, and that the window during which cell type specification occurs is likely to begin shortly after the seeding of hiPS cells for differentiation.

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In vivo Evaluation and in silico Prediction of the Toxicity of Drepanoalpha Hard capsules GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/411124v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@12dbf1aorg.highwire.dtl.DTLVardef@5d6e9forg.highwire.dtl.DTLVardef@9ba26org.highwire.dtl.DTLVardef@9c2017_HPS_FORMAT_FIGEXP  M_FIG C_FIG ABSTRACTO_ST_ABSEthnopharmacological relevanceC_ST_ABSDrepanoalpha(R) hard capsules, a dry ethanolic extract (drug-extract ratio, 100/11) of a mixture of Justicia secunda Vahl and Moringa oleifera Lam dried leaves (1: 1, w/w) are used for the management of sickle cell disease in the Democratic Republic of Congo.

Aim of the studyThis phytomedicine safety was investigated by acute and sub-acute administration in Guinea pigs.

Materials and methodsHealthy, male and nulliparous and non-pregnant female Guinea pigs were obtained from "Laboratory of Animal Experimentation and Toxicology" of the Department of Biology, Faculty of Sciences, University of Kinshasa. The animals were randomly selected, marked and divided into 2 groups of 5 animals each (3 males and 2 females) and 4 groups of 3 animals each for acute and sub-acute toxicity studies, respectively. The contents of hard capsules were dissolved in normal saline solution (NaCl 9 {per thousand}). Animals received by gavage a single dose of 5000 mg/ kg of body weight (B.W.) of Drepanoalpha(R) hard capsules (acute toxicity) and 125 mg/ kg, 250 mg/ kg and 500 mg/ kg of B.W. twice daily for 28 days (sub-acute toxicity). Normal saline solution was used as control. Hematological, biochemical and histopathological analyses were performed and the behavior of the animals was observed after treatment.

ResultsThe median lethal dose (LD50) is higher than 5000 mg/ kg of B.W., and the relative weights of vital organs (kidneys, liver and heart) collected from Guinea pigs at the end of treatment on D14 (acute toxicity) and D28 (sub-acute toxicity) has not undergone significant changes (p > 0.05). The results of haematological (red and white blood cells counts, haemoglobin, haematocrit) and biochemical (ALT, AST, albumin, total protein) tests did not show significant differences between control and test groups (=0.05 for acute toxicity), while the histopathological study revealed no damage to the various organs excised.

ConclusionThe results indicate the safety of Drepanoalpha(R) hard capsules, confirming previous studies, in rats and Guinea pigs, based on aqueous decoction of its raw herbs mixtures and the corresponding lyophilizate.

sentences: Highly efficient healing of critical sized articular cartilage defect in situ using a chemically nucleoside-modified mRNA-enhanced cell therapy Critical sized cartilage defects heal poorly and MSC-based therapies holds promise functional cartilage regeneration either used alone or in combination with growth factors. However, Recombinant protein growth factors were proven to have minimal benefits while to have adverse side effects and high cost. Nonviral mRNA delivery provides a promising, alternative approach to delivering therapeutic proteins within defect lesion for an extended period of time. Despite successful therapeutic outcome in bone and other vascularized tissues, the therapeutic application of mRNA in poorly vascularized tissues such as cartilage is still facing many challenges and rarely studied. We report here using chemically modified messenger RNA encoding TGF-{beta}3(TGF-{beta}3 cmRNA) to enhance the therapeutic efficacy of BMSCs to efficient repair of cartilage defect. Local administration of TGF-{beta}3 cmRNA enhanced BMSCs therapy restored critical-sized cartilage defects in situ in a rat model within 6 weeks with structural and molecular markers similar to its nature counterparts. In addition, the development of osteoarthritis caused by cartilage damage was prevented by this mRNA-enhanced BMSCs therapy evidenced by minimal late-stage OA pharmacological presentations. This novel mRNA enhanced-MSC technology extend the development of new therapeutic approaches for treating functional cartilage repair.

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In vivo analysis of LEA-1 in C. elegans desiccation tolerance BackgroundCells and organisms typically cannot survive in the absence of water. However, there are some notable exceptions, including animals such as nematodes, tardigrades, rotifers, and some arthropods. One class of proteins known to play a role in desiccation resistance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear.

Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection.

ResultsTo study the endogenous function of an LEA protein in an animal, we created a true null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutant animals were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress. We identified minimal motifs within C. elegans LEA-1 that are sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1s in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans survived mild desiccation and osmotic stress at the same levels as worms with the full-length protein.

ConclusionsOur results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection.

sentences: The mutational landscape of single neurons and oligodendrocytes reveals evidence of inflammation-associated DNA damage in multiple sclerosis Neuroinflammation has been linked to DNA damage in multiple sclerosis (MS), but its impact on neural cell genomes at nucleotide resolution is unknown. To address this question, we performed single nucleus whole genome sequencing to determine the landscape of somatic mutation in 172 neurons and oligodendrocytes (OLs) extracted from post-mortem brain tissue from 5 MS cases and three controls. We identified two cases with a significant excess of somatic single nucleotide variants (sSNV) in neurons and OLs from MS inflammatory demyelinated lesions. For a case with primary progressive MS, this translated to a 68% increase in sSNV frequency and 32-year equivalent increase in biological age of lesion-resident cells. Mutational signature analysis conducted on all cells revealed that defective DNA repair and transcription-associated DNA damage are important mutagenic mechanism in both neurons and OLs in MS. Our findings provide the first evidence that inflammation in the brains of people with MS is associated with DNA damage, which may have implications for other neurodegenerative diseases and future drug development.

sentences: Temporal perturbations cause movement-context independent but modality-specific sensorimotor adaptation Complex, goal-directed and time-critical movements require the processing of temporal features in sensory information as well as the fine-tuned temporal interplay of several effectors. Temporal estimates used to produce such behavior may thus be obtained through perceptual or motor processes. To disentangle the two options, we tested whether adaptation to a temporal perturbation in an interval reproduction task transfers to interval reproduction tasks with varying sensory information (visual appearance of targets, modality, virtual reality (VR) environment or real-world) or varying movement types (continuous arm movements or brief clicking movements). Halfway through the experiments we introduced a temporal perturbation, such that continuous pointing movements were artificially slowed down in VR, causing participants to adapt their behavior to sustain performance. In four experiments, we found that sensorimotor adaptation to temporal perturbations is independent of environment context and movement type, but modality specific. Our findings suggest that motor errors induced by temporal sensorimotor adaptation affect the modality specific perceptual processing of temporal estimates.

sentences: Sensory cortical dynamics during optical microstimulation training Primary sensory cortex is a key locus of plasticity during learning. Exposure to novel stimuli often alters cortical activity, but isolating cortex-specific dynamics is challenging due to extensive pre-cortical processing. Here, we employ optical microstimulation of pyramidal neurons in layer (L) 2/3 of mouse primary vibrissal somatosensory cortex (vS1) to study cortical dynamics as mice learn to discriminate microstimulation intensity. Tracking activity over weeks using two-photon calcium imaging, we observe a rapid sparsification of the photoresponsive population, with the most responsive neurons exhibiting the largest declines in responsiveness. Following sparsification, the photoresponsive population attains a stable rate of neuronal turnover. At the same time, the photoresponsive population increasingly overlaps with populations encoding whisker movement and touch. Finally, we find that mice with larger declines in responsiveness learn the task more slowly than mice with smaller declines. Our results reveal that microstimulation-evoked cortical activity undergoes extensive reorganization during task learning and that the dynamics of this reorganization impact perception.

sentences: Quantification of alternative 3'UTR isoforms from single cell RNA-seq data with scUTRquant Although half of human genes use alternative polyadenylation (APA) to generate mRNA isoforms that encode the same protein but differ in their 3'UTRs, most single cell RNA-sequencing (scRNA-seq) pipelines only measure gene expression. Here, we describe an open-access pipeline, called scUTRquant (https://github.com/Mayrlab/scUTRquant), that measures gene and 3'UTR isoform expression from scRNA-seq data obtained from known cell types in any species. scUTRquant-derived gene and 3'UTR transcript counts were validated against standard methods which demonstrated their accuracy. 3'UTR isoform quantification was substantially more reproducible than previous methods. scUTRquant provides an atlas of high-confidence 3' end cleavage sites at single-nucleotide resolution to allow APA comparison across mouse datasets. Analysis of 120 mouse cell types revealed that during differentiation genes either change their expression or they change their 3'UTR isoform usage. Therefore, we identified thousands of genes with 3'UTR isoform changes that have previously not been implicated in specific biological processes.

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MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as beta-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.

HighlightsO_LIActivation of MYCN attenuates proliferation in RPE1 cells
C_LIO_LIGrowth arrest is associated with an early senescence-like transcriptional signature
C_LIO_LITranscriptional and phenotypic evidence of nucleolar stress
C_LIO_LICDKN1A upregulation in G2 phase primes cells for faltering in subsequent G1
C_LI

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Behavioral and Neurostructural changes associated with Chronic Amygdala Hyperactivation BackgroundThe amygdala (AMY) is a key brain region of the limbic system that plays a critical role in emotion processing and stress response. Functional magnetic resonance imaging (fMRI) studies identified abnormal AMY activation in psychiatric illnesses including major depressive disorder (MDD). Stress exposure is a major precipitating factor of MDD episodes which are associated with AMY hyperactivity. Preclinical studies using of pharmacologic, opto- and chemogenetic approaches to activate AMY neurons have consistently demonstrated that acute AMY hyperactivation induces anxiety-like behaviors in mice. However, it remains unknown if chronic hyperactivation of the amygdala (cHOA) is sufficient to induce chronic stress-like deficits or is a susceptibility factor for chronic stress-induced behavioral, volumetric and synaptic deficits.

MethodsUsing designer receptor exclusively activated by designer drug (DREADD) approach, basolateral amygdala (BLA) neurons of Camk2a-cre mice infected with a virus driving the expression of the Gq-coupled DREADD were activated with clozapine-N-oxide (in drink water for 5 weeks). Mice were then exposed to chronic restraint stress (CRS; 1X/day for 1hr) for 2 weeks. All mice were behaviorally assessed in the Phenotyper (PT), and sucrose consumption tests (SCT) each week and in the novelty supressed feeding (NSF, once at the end of the experiment). Animals were then perfused for ex vivo-MRI and puncta density analysis.

ResultsWe found that mice with cHOA displayed a progressive increase in baseline anxiety-like deficits in the PT test and slightly more marked deficits following CRS compared to controls, but not statistically different from animals subjected to CRS alone. Also, cHOA did not exacerbate CRS effect in the NSF. No significant cAH effect was found in the SCT before or after CRS. MRI analysis revealed no statistical charges between groups, while increased synaptic puncta density was found in cHOA mice subjected to CRS compared to cHOA or CRS alone.

ConclusionWe demonstrate that cAH is sufficient to induce anxiety and may exacerbate CRS effects on anxiety and synaptic measures. Results also suggest that cHOA was not sufficient to induce depressive-like behavior and was not a vulnerability factor for stress-induced depressive-like behavior in mice. Altogether, our findings imply that a strong causal link between AMY hyperactivity and elevated anxiety, but not depressive-like behaviors and provide critical information to clinical research focused on using AMY activity level as a biomarker in stress-related illnesses.

sentences: Transfer of weight information depends differently on used hand and handedness for perception and action When lifting an object sequentially with the two hands, information about object weight can be transferred from one hand to the other. This information can be used to predictively scale fingertip forces and to form a perceptual estimation about the objects weight. This study investigated how weight information can be transferred between the two hands and how this depends on the used hand and handedness of the participant. Right- and left-handed participants lifted light and heavy objects with the right or left hand in a pseudorandomized order and estimated the objects weight. Results showed that predictive force scaling depended on the previously lifted object, with higher forces rates if a previous object was heavy. This known effect of sensorimotor memory was mostly independent of the used hand and handedness, indicating that weight information could be transferred for fingertip force scaling. Furthermore, a perceptual bias depending on the previous lifted object was found, with lower weight estimations when the previous object was heavy compared to light. In contrast to the results on force scaling, a transfer of this perceptual bias was only found in left-handed participants. In addition, right-handers only showed a bias when they used their dominant hand. These findings indicate that the transfer of weight information depends differently on the used hand and handedness for perceptual estimations and predictive force scaling.

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Spatially Patterned Bi-electrode Epiretinal Stimulation for Axon Avoidance at Cellular Resolution ObjectiveEpiretinal prostheses are designed to restore vision to people blinded by photoreceptor degenerative diseases by stimulating surviving retinal ganglion cells (RGCs), which carry visual signals to the brain. However, inadvertent stimulation of RGCs at their axons can result in non-focal visual percepts, limiting the quality of artificial vision. Theoretical work has suggested that axon activation can be avoided with current stimulation designed to minimize the second spatial derivative of the induced extracellular voltage along the axon. However, this approach has not been verified experimentally at the resolution of single cells. Approach. In this work, a custom multi-electrode array (512 electrodes, 10 m diameter, 60 m pitch) was used to stimulate and record RGCs in macaque retina ex vivo at single-cell, single-spike resolution. RGC activation thresholds resulting from bi-electrode stimulation, which consisted of bipolar currents simultaneously delivered through two electrodes straddling an axon, were compared to activation thresholds from traditional single-electrode stimulation.

ResultsOn average, across three retinal preparations, the bi-electrode stimulation strategy reduced somatic activation thresholds ([~]21%) while increasing axonal activation thresholds ([~]14%), thus favoring selective somatic activation. Furthermore, individual examples revealed rescued selective activation of somas that was not possible with any individual electrode. Significance. This work suggests that a bi-electrode epiretinal stimulation strategy can reduce inadvertent axonal activation at cellular resolution, for high-fidelity artificial vision.

Novelty & SignificanceThe effectiveness of bi-electrode stimulation for enhancing the electrical activation of retinal neurons was tested using high-density multi-electrode recording and stimulation in isolated macaque retina. The results suggest that spatially patterned bi-electrode stimuli reduce unwanted axon activation and thus improve the selectivity of stimulation at cellular resolution. Similar patterns could be implemented in a future high-resolution prosthesis to permit a more faithful replication of normal retinal activity, at the resolution of single-cells and single-spikes, for the treatment of incurable blindness.

sentences: Binocular visual experience and sleep promote visual cortex plasticity and restore binocular vision in a mouse model of amblyopia Amblyopia arises from an altered balance of input from the two eyes to the binocular zone of primary visual cortex (bV1) during childhood, causing long-lasting visual impairment. Amblyopia is commonly treated by patching the dominant eye, however, the relative impacts of monocular vs. binocular visual experiences on restoration of bV1 function remains unclear. Moreover, while sleep has been implicated in V1 plasticity in response to vision loss, its role in recovery from amblyopia is unknown. We used monocular deprivation (MD) in juvenile mice to model amblyopia in bV1. We compared recovery of visual responses for the two eyes among bV1 regular spiking (RS, putative principal) neurons and fast-spiking (FS, putative parvalbumin-expressing [PV+]) interneurons after identical-duration, identical-quality binocular recovery (BR) or monocular, reverse occlusion (RO) experiences. We find that BR is quantitatively superior to RO with respect to renormalizing both bV1 populations visual responses. However, this recovery was seen only in freely-sleeping mice; post-BR sleep deprivation prevented functional recovery. Thus, both binocular visual experience and subsequent sleep are required to optimally renormalize bV1 responses in a mouse model of amblyopia.

sentences: FETCH: A platform for high-throughput quantification of gap junction hemichannel docking Gap junctions are membrane spanning channels that connect the cytoplasm of apposed cells, allowing for the passage of small molecules and ions. They are formed by the connexin (Cx) family of proteins which assemble into hexameric hemichannels on each cell and dock to create gap junctional channels between two cells. Despite importance of various Cx isoforms in human physiology and disease, available tools for screening and discriminating their interactions such as hemichannel compatibility, docking and permeability are limited. Here, we developed FETCH (flow enabled tracking of connexosomes in HEK cells), a method which utilizes the generation of annular gap junctions (connexosomes) as downstream indicators of hemichannel compatibility for intercellular docking. First, we show that fluorescent connexosomes create a cellular phenotype that is detectable by flow cytometry analysis. We then show that FETCH identifies homotypic and heterotypic docking of many single isoform connexin hemichannels. Finally, we demonstrate that FETCH captures the impact of disease-relevant connexin protein mutations on gap junction formation. Thus, we establish a new flow cytometry-based method that is amenable to the high-throughput classification of gap junction hemichannel docking.

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Regulus, a transcriptional regulatory networks inference tool based on Semantic Web technologies MotivationTranscriptional regulation -a major field of investigation in life science-is performed by binding of specialized proteins called transcription factors (TF) to DNA in specific, context-dependent regulatory regions, leading to either activation or inhibition of gene expression. Relations between TF, regions and genes can be described as regulatory networks, which are basically knowledge graphs containing the relationships between the different entities. Current methods of transcriptional regulatory networks inference rarely use information about TF binding or regulatory regions, often require a large number of samples and most of time do not indicate if the TF-gene relation is an activation or an inhibition. The resulting networks may then contain inconsistent relations and the methods are not applicable for common experimental or clinical settings, where the number of samples is limited. Therefore, based on our previous experience of formalizing the Regulatory Circuits data-sets with Semantic Web Technologies, we decided to create a new tool for transcriptional networks inference, that could solve these issues. Results: Our tool, Regulus, provides candidate signed TF-gene relations computed from gene expressions, regulatory region activities and TF binding sites data, together with the genomic location of all entities. After creating expressions and activities patterns, data are integrated into a RDF endpoint. A dedicated SPARQL query retrieves all potential TF-region relations for a given gene expression pattern. These ternary TF-region-gene pattern relations are then filtered and signed using a logical consistency check translated from biological knowledge. Regulus compares favorably to its closest network inference method, provides signs which are consistent with public databases and, when applied to real biological data, identifies both known and potential new regulators. We also provide several means to more stringently filter the output regulators. Altogether, we propose a new tool devoted to transcriptional network inference in settings where samples are scarce and cell populations may be closely related.

AvailabilityThe Regulus package is available at https://gitlab.com/gcollet/regulus

sentences: High-quality complete genome resource of plant pathogenic bacterium Dickeya solani strain IPO 2019 isolated from Hyacinthus orientalis Dickeya solani is an emerging plant pathogenic bacterium, causing disease symptoms in a variety of agriculturally relevant crop species worldwide. To date a number of D. solani genomes have been sequenced and characterized, the great majority of these genomes have however come from D. solani strains isolated from potato (Solanum tuberosum L.) and not from other plant hosts. Herewith, we present the first complete, high-quality genome of D. solani strain IPO 2019 (LMG 25990) isolated from ornamental plant Hyacinthus orientalis. The genome of D. solani strain IPO 2019 consists of one chromosome of 4,919,542 bp., with a GC content of 56.2% and no plasmids. The genome contains 4502 annotated features, 22 rRNA genes, 73 tRNA genes and 1 CRISPRS. We believe that the information of this high-quality, complete, closed genome of D. solani strain isolated from host plant different than potato (i.e. hyacinth) will provide resources for comparative genomic studies as well as for analyses targeting adaptation and ecological fitness mechanisms present in Dickeya solani species.

sentences: Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry. Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with two drug/protein combinations and apply our workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.

sentences: Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor ({beta}2AR). The assay involves temporally-resolved imaging of the binding of individual {beta}2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of {beta}2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The set-up combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

sentences: The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

sentences: Integument transcriptome profile of the European sea cucumber Holothuria forskali (Holothuroidea, Echinodermata) In non-model organisms, Next Generation Sequencing (NGS) technology improve our ability to analyze gene expression and identify new genes or transcripts of interest. In this research, paired-end Illumina HiSeq sequencing has been used to describe a composite transcriptome based on two libraries generated from dorsal and ventral integuments of the European sea cucumber Holothuria forskali (Holothuroidea, Echinodermata). A total of 43,044,977 million HQ reads were initially generated. After de novo assembly, a total of 111,194 unigenes were predicted. On all predicted unigenes, 32,569 show significant matches with genes/proteins present in the reference databases. Around 50% of annotated unigenes were significantly similar to sequences from the purple sea urchin Strongylocentrotus purpuratus genome. Annotation analyses were performed on predicted unigenes using public reference databases. These RNA-seq data provide an interesting resource for researchers with a broad interest in sea cucumber biology.

sentences: Genomically hardwired regulation of gene activity orchestrates cellular iron homeostasis in Arabidopsis Iron (Fe) is an essential micronutrient that plays pivotal roles as electron donor and catalyst across organisms. In plants, variable, often insufficient Fe supply necessitates mechanisms that constantly attune Fe uptake rates and recalibrate cellular Fe homeostasis. Here, we show that short-term (0.5, 6, and 12 h) exposure of Arabidopsis thaliana plants to Fe deficiency triggered massive changes in gene activity governed by transcription and alternative splicing (AS), regulatory layers that were to a large extent mutually exclusive. Such preclusion was not observed for genes that are directly involved in the acquisition of Fe, which appears to be concordantly regulated by both expression and AS. Generally, genes with lower splice site strengths and higher intron numbers were more likely to be regulated by AS, no dependence was on gene architecture was observed for transcriptionally controlled genes. Conspicuously, specific processes were associated with particular genomic features and biased towards either regulatory mode, suggesting that genomic hardwiring is functionally biased. Early changes in splicing patterns were, in many cases, congruent with later changes in transcript or protein abundance, thus contributing to the pronounced transcriptome-proteome discordance observed in plants.

sentences: SYNAPTIC RELEASE POTENTIATION AT AGING AUDITORY RIBBON SYNAPSES Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50 % loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.

sentences: Metagenomic screening for lipolytic genes reveals an ecology-clustered distribution pattern Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique genes encoding lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integral classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.

sentences: Glycan reactive anti-HIV-1 antibodies bind the SARS-CoV-2 spike protein but do not block viral entry The SARS-CoV-2 spike glycoprotein is a focal point for vaccine immunogen and therapeutic antibody design, and also serves as a critical antigen in the evaluation of immune responses to COVID-19. A common feature amongst enveloped viruses such as SARS-CoV-2 and HIV-1 is the propensity for displaying host-derived glycans on entry spike proteins. Similarly displayed glycosylation motifs can serve as the basis for glyco-epitope mediated cross-reactivity by antibodies, which can have important implications on virus neutralization, antibody-dependent enhancement (ADE) of infection, and the interpretation of antibody titers in serological assays. From a panel of nine anti-HIV-1 gp120 reactive antibodies, we selected two (PGT126 and PGT128) that displayed high levels of cross-reactivity with the SARS-CoV-2 spike. We report that these antibodies are incapable of neutralizing pseudoviruses expressing SARS-CoV-2 spike proteins and are unlikely to mediate ADE via Fc{gamma}RII receptor engagement. Nevertheless, ELISA and other immunoreactivity experiments demonstrate these antibodies are capable of binding the SARS-CoV-2 spike in a glycan-dependent manner. These results contribute to the growing literature surrounding SARS-CoV-2 S cross-reactivity, as we demonstrate the ability for cross-reactive antibodies to interfere in immunoassays.

sentences: Immune function of the serosa in hemimetabolous insect eggs Insects comprise more than a million species and many authors have attempted to explain this success by evolutionary innovations. A much overlooked evolutionary novelty of insects is the serosa, an extraembryonic epithelium around the yolk and embryo. We have shown previously that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, whether this immune competence evolved in the Tribolium lineage or is ancestral to all insects. Here, we expand our studies to two hemimetabolous insects, the bug Oncopeltus fasciatus and the swarming grasshopper Locusta migratoria. For Oncopeltus, RNA sequencing reveals an extensive response upon infection, including the massive upregulation of antimicrobial peptides (AMPs). We demonstrate antimicrobial activity of these peptides using in vitro bacterial growth assays, and describe two novel AMP families called Serosins and Ovicins. For both insects, qPCRs show immune competence of the eggs when the serosa is present, and in situ hybridizations demonstrate that immune gene expression is localized in the serosa. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have facilitated the spectacular radiation of the insects.

sentences: Binding of cardiolipin to the KcsA channel at the membrane outer leaflet allosterically opens the inner gate Membrane proteins embedded in the membrane undergo changes in their actions under the influence of membrane lipids. Here, we present a novel type of lipid action on the potassium channel KcsA from Streptomyces lividans. Cardiolipin is present in various cellular membranes, including the host membrane of KcsA. Although the M0 domain, a nontransmembrane helix, is known to sense anionic lipids in the inner leaflet, we found that divalent anionic cardiolipin in the outer leaflet of the membrane interacts with positively charged residues, Arg64 and Arg89, on the extracellular side of the transmembrane domain. This binding propagates its action across the membrane toward the intracellular region of KcsA, thus, opening the inner gate. Such a long-range allosteric effect has not been found for channel-lipid interactions.

sentences: The Tabula Sapiens: a single cell transcriptomic atlas of multiple organs from individual human donors Molecular characterization of cell types using single cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues and tissue specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, the tissue specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type specific RNA splicing was discovered and analyzed across tissues within an individual.

sentences: Acoustic camera system for measuring ultrasound communication in mice To investigate molecular, genetic, and neural mechanisms underlying social behaviors and their deficits in neuropsychiatric disorders, social communication via ultrasonic vocalizations (USVs) in mice has received considerable attention as a powerful experimental model. The advances in sound localization technology have facilitated the analysis of vocal interactions between multiple mice. However, existing sound localization systems are built around distributed-microphone arrays, which require a special recording arena and long processing time. Here we report a novel acoustic camera system, USVCAM, which enables simpler and faster USV localization and assignment. The system comprises recently developed USV segmentation algorithms with a modification for overlapping vocalizations that results in high accuracy. Using USVCAM, we analyzed USV communications in a conventional home cage, and demonstrated novel vocal interactions in female ICR mice under a resident-intruder paradigm. The extended applicability and usability of USVCAM may facilitate future studies investigating normal and abnormal vocal communication and social behaviors, as well as the underlying physiological mechanisms.

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Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution. Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Cell replacement therapy holds promise as a therapeutic approach to restore vision to those who have lost PRs through damage or disease. While PR transplant research is ongoing in animal models, success is hindered by our limited understanding of PR axon growth during development and regeneration. Using a human pluripotent stem cell (hPSC) reporter line that labels PRs (WA09 CRX+/tdTomato), we generated retinal organoids in order to study mechanisms of PR process extension. We found that the earliest born PRs exhibit autonomous axon extension from dynamic terminals that appear similar to projection neuron growth cones. However, as hPSC-derived PRs age from 40 to 80 days of differentiation, they lose dynamic terminals in 2D plated cultures and within 3D retinal organoids, which does not correlate with cell birth date. Using a rod-specific hPSC reporter line (WA09 NRL+/eGFP), we further determined that rod PRs never form motile growth cones. Interestingly, PRs without motile terminals are still capable of extending axons, but neurites are generated from process stretching via their attachment to motile non-PR cells, which underlies the observed differences in PR neurite lengths on different substrata. While immobile PR terminals express actin, it is less polymerized and less organized than actin present in motile terminals. However, immobile PRs do localize synaptic proteins to their terminals, suggesting a normal developmental progression. These findings help inform the development of PR transplant therapies to treat blinding diseases and provide a platform to test treatments that restore autonomous PR axon extension.

Significance StatementLoss of photoreceptors (PRs) in the retina through damage or disease causes irreversible vision loss and blindness. One treatment approach is to replace lost cells with transplanted human stem cell-derived PRs, but this requires PR axons to integrate into the host retina to restore the required neural connections. For this strategy to succeed, we need to understand how PRs extend processes to their targets during development in situ, and whether dissociated human stem cell (hPSC)-derived PRs behave in a similar fashion. In this paper, we show that hPSC-PRs have only a short window during which they are capable of autonomous axon extension, which has implications for PR transplant efforts and for our basic understanding of human retinal development.

sentences: HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver Hepatocyte Nuclear Factor 4 (HNF4), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2). P1-HNF4 is the major isoform in the adult liver while P2-HNF4 is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2- HNF4 is expressed at ZT9 and ZT21 in the normal adult liver and orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions. We demonstrate that while P1-HNF4 drives gluconeogenesis, P2-HNF4 drives ketogenesis and is required for elevated levels of ketone bodies in females. Exon swap mice expressing only P2- HNF4 exhibit subtle differences in circadian gene regulation and disruption of the clock increases expression of P2-HNF4. Taken together, we propose that the highly conserved two-promoter structure of the Hnfa gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

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Independent population coding of the past and the present in prefrontal cortex during learning Medial prefrontal cortex (mPfC) activity represents information about the state of the world, including present behaviour, such as decisions, and the immediate past, such as short-term memory. Unknown is whether information about different states of the world are represented in the same mPfC neural population and, if so, how they are kept distinct. To address this, we analyse here mPfC population activity of rats learning rules in a Y-maze, with self-initiated choice trials to an arm-end followed by a self-paced return during the inter-trial interval (ITI). We find that trial and ITI population activity from the same population fall into different low-dimensional subspaces. These subspaces encode different states of the world: multiple features of the task can be decoded from both trial and ITI activity, but the decoding axes for the same feature are roughly orthogonal between the two task phases, and the decodings are predominantly of features of the present during the trial but features of the preceding trial during the ITI. These subspace distinctions are carried forward into sleep, where population activity is preferentially reactivated in post-training sleep, but differently for activity from the trial and ITI subspaces. Our results suggest that the problem of interference when representing different states of the world is solved in mPfC by population activity occupying different subspaces for the world states, which can be independently decoded by downstream targets and independently addressed by upstream inputs.

Significance statementActivity in the medial prefrontal cortex plays a roles in representing the current and past states of the world. We show that during a maze task the activity of a single population in medial prefrontal cortex represents at least two different states of the world. These representations were sequential and sufficiently distinct that a downstream population could separately read out either state from that activity. Moreover, the activity representing different states is differently reactivated in sleep. Different world states can thus be represented in the same medial prefrontal cortex population, but in such a way that prevents potentially catastrophic interference between them.

sentences: Modulation of the renin-angiotensin system inhibits memory advantage for negative emotional material via decreasing hippocampus activation and its coupling with the amygdala Exaggerated arousal and dysregulated emotion-memory interactions are key pathological dysregulations that accompany the development of post-traumatic stress disorder (PTSD). Current treatments for PTSD are of moderate efficacy and preventing the dysregulations already during exposure to threatening events may attenuate the development of PTSD-symptomatology. In a preregistered double-blind, between-subject, placebo-controlled pharmaco-fMRI design, the present proof-of-concept study examined the potential of a single dose of angiotensin II type 1 receptor (AT1R) antagonist losartan (LT) to attenuate the mnemonic advantage of threatening stimuli and the underlying neural mechanism via combining an emotional subsequent memory paradigm with LT (n=29) or placebo treatment (n=30) and a surprise memory test after 24h washout. LT generally improved memory performance and abolished emotional memory enhancement for negative yet not positive material while emotional experience during encoding remained intact. LT further suppressed hippocampus activity during encoding of subsequently remembered negative stimuli. On the network level LT reduced coupling between hippocampus and basolateral amygdala during successful memory formation of negative stimuli. Our findings suggest that LT may have the potential to attenuate memory formation for negative yet not positive information by decreasing hippocampus activity and its functional coupling strength with amygdala. These findings suggest a promising potential of LT to prevent preferential encoding and remembering of negative events, a mechanism that could prevent the emotion-memory dysregulations underlying the development of PTSD-symptomatology.

sentences: Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory elements position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting  naive genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

sentences: Pushing the limits of HiFi assemblies reveals centromere diversity between two Arabidopsis thaliana genomes Although long-read sequencing can often enable chromosome-level reconstruction of genomes, it is still unclear how one can routinely obtain gapless assemblies. In the model plant Arabidopsis thaliana, other than the reference accession Col-0, all other accessions de novo assembled with long-reads until now have used PacBio continuous long reads (CLR). Although these assemblies sometimes achieved chromosome-arm level contigs, they inevitably broke near the centromeres, excluding megabases of DNA from analysis in pan-genome projects. Since PacBio high-fidelity (HiFi) reads circumvent the high error rate of CLR technologies, albeit at the expense of read length, we compared a CLR assembly of accession Ey15-2 to HiFi assemblies of the same sample performed by five different assemblers starting from subsampled data sets, allowing us to evaluate the impact of coverage and read length. We found that centromeres and rDNA clusters are responsible for 71% of contig breaks in the CLR scaffolds, while relatively short stretches of GA/TC repeats are at the core of >85% of the unfilled gaps in our best HiFi assemblies. Since the HiFi technology consistently enabled us to reconstruct gapless centromeres and 5S rDNA clusters, we demonstrate the value of the approach by comparing these previously inaccessible regions of the genome between two A. thaliana accessions.

sentences: Cellular And Molecular Effects Of Understudied Kinase Pregnancy Upregulated Non-Ubiquitous Calcium-Calmodulin Dependent Kinase (PNCK) In Renal Cell Carcinoma. Renal Cell Carcinoma (RCC) is a uniformly fatal disease when advanced. While immunotherapy and tyrosine kinase inhibitor-based combinations are associated with improved progression-free and overall survival, the majority of patients eventually develop treatment resistance and succumb to progressive, refractory disease. This underscores the urgent need to identify novel, non-canonical RCC targets for drug development. Through a comprehensive pan-cancer, pan-kinome analysis of the Cancer Genome Atlas (TCGA), the understudied kinase, pregnancy upregulated non-ubiquitous calcium-calmodulin dependent kinase (PNCK) was identified as the most differentially overexpressed kinase in RCC. PNCK mRNA was significantly overexpressed in RCC tissues compared to adjacent normal tissue, and its overexpression correlated with tumor T-stage grade and poor disease specific survival in both clear cell and papillary RCCs. PNCK overexpression in VHL mutant and VHL wild type RCC cell lines was associated with increased CREB phosphorylation, as well as increased cell proliferation and cell cycle progression. PNCK down-regulation, conversely, was associated with inhibition of CREB phosphorylation, decreased cell proliferation, cell cycle arrest and increased apoptosis, with differential effects observed between VHL mutant and VHL wild type cell lines. Pathway analyses in PNCK knockdown cells showed significant down regulation of hypoxia and angiogenesis pathways, as well as modulation of pathways promoting cell cycle arrest and apoptosis. The above results demonstrate for the first time the biological role of PNCK, an understudied kinase, in renal cell carcinoma and validate PNCK as a potential novel target for drug development in this fatal disease.

sentences: Metamodal Coupling of Vibrotactile and Auditory Speech Processing Systems Through Matched Stimulus Representations It has been postulated that the brain is organized by "metamodal", sensory-independent cortical modules implementing particular computations, leading to the intriguing hypothesis that brain areas can perform tasks (such as word recognition) not just in "standard" sensory modalities but also in novel sensory modalities. Yet, evidence for this theory, especially in neurotypical subjects, has been variable. We hypothesized that effective metamodal engagement of a brain area requires congruence between the novel and standard sensory modalities not only at the task level (e.g., "word recognition") but critically also a match at the algorithmic level (in Marrs terminology), i.e., at the level of neural representation of the information of interest. To test this hypothesis, we trained participants to recognize vibrotactile versions of auditory words using two encoding schemes. The vocoded approach preserved the dynamics and representational similarities of auditory speech while the token-based approach used an abstract phoneme-based code. Although both groups learned the vibrotactile word recognition task, only in the vocoded group did trained vibrotactile stimuli recruit the auditory speech network and lead to increased coupling between somatosensory and auditory speech areas. In contrast, the token-based encoding appeared to rely on paired-associate learning. Thus, matching neural input representations is a critical factor for assessing and leveraging the metamodal potential of cortical modules.

sentences: Ultra-high-throughput microbial single-cell whole genome sequencing for genome-resolved metagenomics Microbes exist widely in nature. However, less than 1% of the species of microorganisms have been discovered so far, and more than 99% of microorganisms still remain unknown, which is called microbial dark matter. Unravelling microbial dark matter is not only helpful for exploring the unknown microbial world, but also of great significance for human health research. Ever since a long time ago, due to technical limitations, the microbiome, to a great extent, is still a black box. The traditional population-based metagenomic analysis tools are without single microbial resolution and they are stretched when studying rare and unknown microbial species. Here, we use high-throughput microdroplet technology to achieve the whole genome amplification of microbial single-cell genomes in pico-litter sized microdroplets, and develop subsequent library preparation steps such as indexing of the genome, to achieve the development of ultra-high-throughput microbial single-cell whole genome sequencing for genome-resolved metagenomics, aiming to provide a powerful new weapon for exploring microbial dark matter and other microbiological studies.

sentences: PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation The inter-cellular prion-like propagation of -synuclein aggregation is emerging as an important mechanism driving the progression of neurodegenerative diseases including Parkinsons disease and multiple system atrophy (MSA). To discover therapeutic strategies reducing the spread of -synuclein aggregation, we performed a genome-wide CRISPR interference screen in a human cell-based model. We discovered that inhibiting PIKfyve dramatically reduced -synuclein aggregation induced with both recombinant -synuclein fibrils and fibrils isolated from MSA patient brain. While PIKfyve inhibition did not affect fibril uptake or -synuclein clearance or secretion, it reduced -synuclein trafficking from the early endosome to the lysosome, thereby limiting fibril escape from the lysosome and reducing the amount of fibrils that reach cytosolic -synuclein to induce aggregation. These findings point to the endolysosomal transport of fibrils as a critical step in the propagation of -synuclein aggregation and a potential therapeutic target.

sentences: PMD Uncovers Widespread Cell-State Erasure by scRNAseq Batch Correction Methods Single cell RNAseq (scRNAseq) batches range from technical replicates to multi-tissue atlases, thus requiring robust batch correction methods that operate effectively across this similarity spectrum. Currently, no metrics allow for full benchmarking across this spectrum, resulting in benchmarks that quantify removal of batch effects without quantifying preservation of real batch differences. Here, we address these gaps with a new statistical metric [Percent Maximum Difference (PMD)] that linearly quantifies batch similarity, and simulations generating cells from mixtures of distinct gene expression programs (cell-lineages/-types/-states). Using 690 real-world and 672 simulated integrations (7.2e6 cells total) we compared 7 batch integration approaches across the spectrum of similarity with batch-confounded gene expression. Count downsampling appeared the most robust, while others left residual batch effects or produced over-merged datasets. We further released open-source PMD and downsampling packages, with the latter capable of downsampling an organism atlas (245,389 cells) in tens of minutes on a standard computer.

sentences: HIF-dependent expression of creatine kinase brain isoform (CKB) promotes breast cancer metastasis, and blocking creatine kinase activity with cyclocreatine impairs invasion and improves the efficacy of conventional chemotherapies The oxygen-responsive Hypoxia Inducible Factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple negative breast cancers (TNBCs) and correlates with poor patient survival. Since inhibiting the HIF transcription factors with small molecules is challenging, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss and gain of function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, formation of invadopodia, and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

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Social selection and the evolution of maladaptation Evolution by natural selection is often viewed as a process that inevitably leads to adaptation, or an increase in population fitness over time. However, maladaptation, an evolved decrease in fitness, may also occur in response to natural selection under some conditions. Social selection, which arises from the effects of social partners on fitness, has been identified as a potential cause of maladaptation, but we lack a general rule identifying when social selection should lead to a decrease in population mean fitness. Here we use a quantitative genetic model to develop such a rule. We show that maladaptation is most likely to occur when social selection is strong relative to nonsocial selection and acts in an opposing direction. In this scenario, the evolution of traits that impose fitness costs on others may outweigh evolved gains in fitness for the individual, leading to a net decrease in population mean fitness. Further, we find that maladaptation may also sometimes occur when phenotypes of interacting individuals negatively covary. We outline the biological situations where maladaptation in response to social selection can be expected, provide both quantitative genetic and phenotypic versions of our derived result, and suggest what empirical work would be needed to test it. We also consider the effect of social selection on inclusive fitness and support previous work showing that inclusive fitness cannot suffer an evolutionary decrease. Taken together, our results show that social selection may decrease population mean fitness when it opposes individual-level selection, even as inclusive fitness increases.

Subject areaQuantitative genetics and Mendelian inheritance

sentences: M1 dynamics share similar inputs for initiating and correcting movement Motor cortex is integral to generating voluntary movement commands. However, as a dynamical system, it is unclear how motor cortical movement commands are informed by either new or sensory-driven corrective instructions. Here, we examine population activity in the primary motor cortex of macaques during a continuous, sequential arm movement task in which the movement instruction is updated several times over the course of a trial. We use Latent Factor Analysis via Dynamical Systems (LFADS) to decompose population activity into a portion explainable via dynamics, and a stream of inferred inputs required to instruct that dynamical system. The time series of inferred inputs had several surprising properties. First, input timing was more strongly locked to target appearance than to movement onset, suggesting that variable reaction times may be a function of how inputs interact with ongoing dynamics rather than variability in instruction timing. Second, inferred inputs were tuned nearly identically for both initial and corrective movements, suggesting a commonality in the structure of inputs across visually-instructed and corrective movements that was previously obscured by the complexity of the dynamical system that is M1.

sentences: Parallel and non-parallel divergence within polymorphic populations of brook stickleback, Culaea inconstans (Actinopterygii: Gasterosteidae) Studying parallel evolution allows us to draw conclusions about the repeatability of adaptive evolution. Whereas populations likely experience similar selective pressures in similar environments, it is not clear if this will always result in parallel divergence of ecologically relevant traits. Our study investigates the extent of parallelism associated with the evolution of pelvic spine reduction in brook stickleback populations. We find that populations with parallel divergence in pelvic spine morphology do not exhibit parallel divergence in head and body morphology but do exhibit parallel divergence in diet. In addition, we compare these patterns associated with pelvic reduction in brook stickleback to well-studied patterns of divergence between spined and unspined threespine stickleback. Whereas spine reduction is associated with littoral habitats and a benthic diet in threespine stickleback, spine reduction in brook stickleback is associated with a planktonic diet. Hence, we find that pelvic spine divergence is associated with largely non-parallel ecological consequences across species.

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DcPAR1 Is Required For Development And Carotenoid Synthesis In The Dark-Grown Carrot Taproot Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange Daucus carota (carrot) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes such as DcPSY1 and DcPSY2 reducing carotenoid accumulation. By means of an RNA-seq, in previous analysis we observed that carrot PHYTOCHROME RAPIDLY REGULATED 1 (DcPAR1) is more expressed in the underground grown taproot respect to those grown in light. PAR1 is a transcriptional cofactor with a negative role in the shade avoidance syndrome regulation in Arabidopsis thaliana through the dimerization with PHYTOCHROME INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here we show that overexpressing AtPAR1 in carrot produces an increment of carotenoids in taproots grown underground as well as higher DcPSY1 expression. The high identity of AtPAR1 and DcPAR1 let us to suggest a functional role of DcPAR1 that was verified through the in vivo binding to AtPIF7 and the overexpression in Arabidopsis, where it increments AtPSY expression and carotenoid accumulation together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoids levels and in the relative expression of key carotenogenic genes as well as impairment in taproot development. These results let us to propose that DcPAR1 is a key factor for secondary root development, plastid differentiation and carotenoid synthesis in carrot taproot grown underground.

One-sentence summaryDcPAR1 is a key factor for secondary root development, plastid differentiation and carotenoid synthesis in carrot taproot grown underground.

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

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

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

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

sentences: Homogenization of a Reaction Diffusion Equation can Explain Influenza A Virus Load Data We study the influence of spatial heterogeneity on the antiviral activity of mouse embryonic fibroblasts (MEF) infected with influenza A. MEF of type Ube1L-/- are composed of two distinct sub-populations, the strong type that sustains a strong viral infection and the weak type, sustaining a weak viral load. We present new data on the virus load infection of Ube1L-/-, which have been micro-printed in a checker board pattern of different sizes of the inner squares. Surprisingly, the total viral load at one day after inoculation significantly depends on the sizes of the inner squares. We explain this observation by using a reaction diffusion model and we show that mathematical homogenization can explain the observed inhomogeneities. If the individual patches are large, then the growth rate and the carrying capacity will be the arithmetic means of the patches. For finer and finer patches the average growth rate is still the arithmetic mean, however, the carrying capacity uses the harmonic mean. While fitting the PDE to the experimental data, we also predict that a discrepancy in virus load would be unobservable after only half a day. Furthermore, we predict the viral load in different inner squares that had not been measured in our experiment and the travelling distance the virions can reach after one day.

sentences: VEGF-A, PDGF-BB and HB-EGF engineered for promiscuous super affinity to the extracellular matrix improve wound healing in a model of type 1 diabetes Chronic non-healing wounds, frequently caused by diabetes, lead to lower quality of life, infection, and amputation. These wounds have limited treatment options. We have previously engineered growth factors to bind to exposed extracellular matrix (ECM) in the wound environment using the heparin-binding domain of placental growth factor-2 (PlGF-2123-144), which binds promiscuously to ECM proteins. Here, in the type 1 diabetic (T1D) NOD mouse model, engineered growth factors improved both re-epithelialization and granulation tissue formation. Engineered growth factors were even more potent in combination, and the "triple therapy" of vascular endothelial growth factor-A (VEGF-PlGF-2123-144), platelet-derived growth factor-BB (PDGF-BB-PlGF-2123-144), and heparin-binding epidermal-growth factor (EGF-PlGF-2123-144) both improved wound healing and remained at the site of administration for significantly longer than wild-type growth factors. In addition, we also found that changes in the cellular milieu of a wound, including changing amounts of M1 macrophages, M2 macrophages and effector T cells, are most predictive of wound healing success in the NOD mouse model. These results suggest that the triple therapy of VEGF-PlGF-2123-144, PDGF-BB-PlGF-2123-144, and EGF-PlGF-2123-144 may be an effective therapy for chronic non-healing wounds in that occur as a complication of diabetes.

sentences: Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

sentences: Joint effect of changing selection and demography on the site frequency spectrum The site frequency spectrum (SFS) is an important statistic that summarizes the molecular variation in a population, and used to estimate population-genetic parameters and detect natural selection. While the equilibrium SFS in a constant environment is quite well studied, recent research has focused on nonequilibrium SFS to elucidate the role of demography when selection is constant in time and of fluctuating selection in a population of constant size. However, the joint effect of time-dependent selection and population size has not been investigated so far. Here, we study the SFS in a randomly mating, diploid population in which both the population size and selection coefficient vary periodically with time using a diffusion theory approach, and derive simple analytical expressions for the time-averaged SFS in slowly and rapidly changing environments. We show that for strong selection and in slowly changing environments, the time-averaged SFS differs significantly from the equilibrium SFS when the population experiences both positive and negative cycles of the selection coefficient. The deviation depends on the time spent by the population in the deleterious part of the selection cycle and the phase difference between the selection coefficient and population size. In particular, we find that the time-averaged SFS in slowly to moderately fast varying, on-average neutral environment has the same qualitative shape as the equilibrium SFS for positively selected mutant but differs quantitatively from it which can be captured by an effective population size.

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BIO-LGCA: a cellular automaton modelling class for analysing collective cell migration 1Collective dynamics in multicellular systems such as biological organs and tissues plays a key role in biological development, regeneration, and pathological conditions. Collective tissue dynamics - understood as population behaviour arising from the interplay of the constituting discrete cells - can be studied with on- and off-lattice agent-based models. However, classical on-lattice agent-based models, also known as cellular automata, fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems.

To overcome drawbacks of classical on-lattice models, we introduce an on-lattice, agent-based modelling class for collective cell migration, which we call biological lattice-gas cellular automaton (BIO-LGCA). The BIO-LGCA is characterised by synchronous time updates, and the explicit consideration of individual cell velocities. While rules in classical cellular automata are typically chosen ad hoc, rules for cell-cell and cell-environment interactions in the BIO-LGCA can also be derived from experimental cell migration data or biophysical laws for individual cell migration. We introduce elementary BIO-LGCA models of fundamental cell interactions, which may be combined in a modular fashion to model complex multicellular phenomena. We exemplify the mathematical mean-field analysis of specific BIO-LGCA models, which allows to explain collective behaviour. The first example predicts the formation of clusters in adhesively interacting cells. The second example is based on a novel BIO-LGCA combining adhesive interactions and alignment. For this model, our analysis clarifies the nature of the recently discovered invasion plasticity of breast cancer cells in heterogeneous environments. A Python package which implements various interaction rules and visualisations of BIO-LGCA model simulations we have developed is available at https://github.com/sisyga/BIO-LGCA.

2 Author summaryPattern formation during embryonic development and pathological tissue dynamics, such as cancer invasion, emerge from individual inter-cellular interactions. In order to study the impact of single cell dynamics and cell-cell interactions on tissue behaviour, one needs to develop space-time-dependent on- or off-lattice agent-based models (ABMs), which consider the behaviour of individual cells. However, classical on-lattice agent-based models also known as cellular automata fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems. Here, we present the rule- and lattice-based BIO-LGCA modelling class which allows for (i) rigorous derivation of rules from biophysical laws and/or experimental data, (ii) mathematical analysis of collective migration, and (iii) computationally efficient simulations.

sentences: Lineage Recording Reveals the Phylodynamics, Plasticity and Paths of Tumor Evolution Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth, expansion to neighboring and distal tissues, and therapeutic resistance. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma which enabled us to track tumor evolution from single transformed cells to metastatic tumors at unprecedented resolution. We found that loss of the initial, stable alveolar-type2-like state was accompanied by transient increase in plasticity. This was followed by adoption of distinct fitness-associated transcriptional programs which enable rapid expansion and ultimately clonal sweep of rare, stable subclones capable of metastasizing to distant sites. Finally, we showed that tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates tumor progression by creating novel evolutionary paths. Overall, our study elucidates the hierarchical nature of tumor evolution, and more broadly enables the in-depth study of tumor progression.

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Perceptual and Semantic Representations at Encoding Contribute to True and False Recognition of Objects When encoding new episodic memories, visual and semantic processing are proposed to make distinct contributions to accurate memory and memory distortions. Here, we used functional magnetic resonance imaging (fMRI) and representational similarity analysis to uncover the representations that predict true and false recognition of unfamiliar objects. Two semantic models captured coarse-grained taxonomic categories and specific object features, respectively, while two perceptual models embodied low-level visual properties. Twenty-eight female and male participants encoded images of objects during fMRI scanning, and later had to discriminate studied objects from similar lures and novel objects in a recognition memory test. Both perceptual and semantic models predicted true memory. When studied objects were later identified correctly, neural patterns corresponded to low-level visual representations of these object images in the early visual cortex, lingual, and fusiform gyri. In a similar fashion, alignment of neural patterns with fine-grained semantic feature representations in the fusiform gyrus also predicted true recognition. However, emphasis on coarser taxonomic representations predicted forgetting more anteriorly in ventral anterior temporal lobe, left perirhinal cortex, and left inferior frontal gyrus. In contrast, false recognition of similar lure objects was associated with weaker visual analysis posteriorly in early visual and left occipitotemporal cortex. The results implicate multiple perceptual and semantic representations in successful memory encoding and suggest that fine-grained semantic as well as visual analysis contributes to accurate later recognition, while processing visual image detail is critical for avoiding false recognition errors.

Significance StatementPeople are able to store detailed memories of many similar objects. We offer new insights into the encoding of these specific memories by combining fMRI with explicit models of how image properties and object knowledge are represented in the brain. When people processed fine-grained visual properties in occipital and inferior temporal cortex, they were more likely to be recognize the objects later, and less likely to falsely recognize similar objects. In contrast, while object-specific feature representations in fusiform predicted accurate memory, coarse-grained categorical representations in frontal and temporal regions predicted forgetting. The data provide the first direct tests of theoretical assumptions about encoding true and false memories, suggesting that semantic representations contribute to specific memories as well as errors.

sentences: Analytical solution of linearized equations of the Morris-Lecar neuron model at large constant stimulation The classical biophysical Morris-Lecar model of neuronal excitability predicts that upon stimulation of the neuron with a sufficiently large constant depolarizing current there exists a finite interval of the current values where periodic spike generation occurs. Above the upper boundary of this interval, there is four-stage damping of the spike amplitude: 1) minor primary damping, which reflects a typical transient to stationary dynamic state, 2) plateau of nearly undamped periodic oscillations, 3) strong damping, and 4) reaching a constant asymptotic value of the neuron potential. We have shown that in the vicinity of the asymptote the Morris-Lecar equations can be reduced to the standard equation for exponentially damped harmonic oscillations. Importantly, all coefficients of this equation can be explicitly expressed through parameters of the original Morris-Lecar model, enabling direct comparison of the numerical and analytical solutions for the neuron potential dynamics at later stages of the spike amplitude damping.

sentences: Experimental characterization and automatic identification of stridulatory sounds inside wood The propagation of animal vocalizations in water and in air is a well-studied phenomenon, but sound produced by bark and wood boring insects, which feed and reproduce inside trees, is poorly understood. Often being confined to the dark and chemically-saturated habitat of wood, many bark- and woodborers have developed stridulatory mechanisms to communicate acoustically. Despite their ecological and economic importance and the unusual medium used for acoustic communication, very little is known about sound production in these insects, or their acoustic interactions inside trees. Here, we use bark beetles (Scolytinae) as a model system to study the effects of wooden tissue on the propagation of insect stridulations and propose algorithms for their automatic identification. We characterize distance-dependence of the spectral parameters of stridulatory sounds, propose data-based models for the power decay of the stridulations in both outer and inner bark, provide optimal spectral ranges for stridulation detectability, and develop automatic methods for their detection and identification. We also discuss the acoustic discernibility of species cohabitating the same log. The species tested can be acoustically identified with 99% of accuracy at distances up to 20 cm and detected to the greatest extent in the 2-6 kHz frequency band. Phloem was a better medium for sound transmission than bark.

sentences: Cell atlas of the regenerating human liver after portal vein embolization The liver has the remarkable capacity to regenerate. In the clinic, this capacity can be induced by portal vein embolization (PVE), which redirects portal blood flow resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the liver during regeneration. We first establish an atlas of cell subtypes from the healthy human liver using fresh and frozen tissues, and then compare post-PVE samples with their reference counterparts. We find that PVE alters portal-central zonation of hepatocytes and endothelial cells. Embolization upregulates expression programs associated with development, cellular adhesion and inflammation across cell types. Analysis of interlineage crosstalk revealed key roles for immune cells in modulating regenerating tissue responses. Altogether, our data provides a rich resource for understanding homeostatic mechanisms arising during human liver regeneration and degeneration.

sentences: Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus. Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the CNS, were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, viral genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.

sentences: Comparative analysis of gene prediction tools for viral genome annotation The number of newly available viral genomes and metagenomes has increased exponentially since the development of high throughput sequencing platforms and genome analysis tools. Bioinformatic annotation pipelines are largely based on open reading frame (ORF) calling software, which identifies genes independently of the sequence taxonomical background. Although ORF-calling programs provide a rapid genome annotation, they can misidentify ORFs and start codons; errors that might be perpetuated and propagated over time. This study evaluated the performance of multiple ORF-calling programs for viral genome annotation against the complete RefSeq viral database. Programs outputs varied when considering the viral nucleic acid type versus the viral host. According to the number of ORFs, Prodigal and Metaprodigal were the most accurate programs for DNA viruses, while FragGeneScan and Prodigal generated the most accurate outputs for RNA viruses. Similarly, Prodigal outperformed the benchmark for viruses infecting prokaryotes, and GLIMMER and GeneMarkS produced the most accurate annotations for viruses infecting eukaryotes. When the coordinates of the ORFs were considered, Prodigal scored high for all scenarios except for RNA viruses, where GeneMarkS generated the most reliable results. Overall, the quality of the coordinates predicted for RNA viruses was poorer than for DNA viruses, suggesting the need for improved ORF-calling programs to deal with RNA viruses. Moreover, none of the ORF-calling programs reached 90% accuracy for annotation of DNA viruses. Any automatic annotation can still be improved by manual curation, especially when the presence of ORFs is validated with wet-lab experiments. However, our evaluation of the current ORF-calling programs is expected to be useful for the improvement of viral genome annotation pipelines and highlights the need for more expression data to improve the rigor of reference genomes.

sentences: Modulation masking and fine structure shape neural envelope coding to predict speech intelligibility across diverse listening conditions A fundamental question in the neuroscience of everyday communication is how scene acoustics shape the neural processing of attended speech sounds and in turn impact speech intelligibility. While it is well known that the temporal envelopes in target speech are important for intelligibility, how the neural encoding of target-speech envelopes is influenced by background sounds or other acoustic features of the scene is unknown. Here, we combine human electroencephalography with simultaneous intelligibility measurements to address this key gap. We find that the neural envelope-domain signal-to-noise ratio in target-speech encoding, which is shaped by masker modulations, predicts intelligibility over a range of strategically chosen realistic listening conditions unseen by the predictive model. This provides neurophysiological evidence for modulation masking. Moreover, using high-resolution vocoding to carefully control peripheral envelopes, we show that target-envelope coding fidelity in the brain depends not only on envelopes conveyed by the cochlea, but also on the temporal fine structure (TFS), which supports scene segregation. Our results are consistent with the notion that temporal coherence of sound elements across envelopes and/or TFS influences scene analysis and attentive selection of a target sound. Our findings also inform speech-intelligibility models and technologies attempting to improve real-world speech communication.

sentences: Signal processing basics applied to Ecoacoustics Ecoacoustics is a research field that has attracted attention of researchers from areas as diverse as ecology, biology, engineering, and human sciences, to cite a few. Ecoacoustics studies the sounds that emanates from the environments, by gaining insights of landscape dynamics from acoustic patterns and particularities at different places. With recent advance in technology, it is common to obtain sound datasets recorded 24-h a day for several months. The analysis of these long-term sound recordings represents several challenges. Investigators already involved in acoustic are familiar with signal processing methods, which are essential tools for sound analysis. However, in general beginners interested in Ecoacoustics do not share this background formation. The present work illustrates basic topics of digital signal processing in a comprehensible style, and an effective pipeline for long-term sound explorative data analysis (EDA) is presented. Finally, the described signal processing fundamentals are applied for EDA of 1-month underwater acoustic data recorded in a Brazilian marine protected area. The Matlab codes used for the analysis will be available as supplementary material.

sentences: Characterizing Genetic Mechanisms Involved in Measuring Day-length in Drosophila melanogaster Many organisms are known to regulate seasonal behaviors and physiological processes in response to day length changes through photoperiodism. Extreme changes in photoperiods have detrimental effects on human health, which can impair development and serve as the origin of adult diseases. Since the seminal work by Bunning in 1936, there are studies supporting the view that organisms can measure the day length through an endogenous 24-hour cellular circadian clock. However, the mechanisms involved in measuring seasonal or day-length changes are not understood. In the current study, we performed a genome-wide association study (GWAS) on photoperiodism using the Drosophila Genetic Reference Panel. The GWAS identified 32 candidate genes responsible for photoperiodic regulations. The knockout mutants of the top four candidate genes (Protein Kinase C delta (Pkcdelta), Glucuronyltransferase-P (GlcAT-P), Brain-specific homeobox (Bsh), and Diuretic hormone 31 Receptor (Dh31-R1)) were analyzed for their photoperiod and circadian period phenotypes. PKCdelta and GlcAT-P mutants show a significantly different photoperiod response compared to that of the wild type strain, and also had an altered circadian period phenotype. Further molecular characterization revealed that the mutant two independent mutant alleles of PKCdelta with a defective catalytic domain had distinct photoperiod responses. Taken these data together, we concluded that there is overlap between the circadian clock and photoperiodic regulations in Drosophila, and PKCdelta is a component that is involved in both circadian and photoperiodic regulations. By identifying novel molecular components of photoperiod, the current study provides new insights into the genetic mechanisms of determining the seasonal changes.

sentences: Temporal continuity shapes visual responses of macaque face patch neurons Macaque inferior temporal cortex neurons respond selectively to complex visual images, with recent work showing that they are also entrained reliably by the evolving content of natural movies. To what extent does visual continuity itself shape the responses of high-level visual neurons? We addressed this question by measuring how cells in face-selective regions of the macaque temporal cortex were affected by the manipulation of a movies temporal structure. Sampling the movie at 1s intervals, we measured neural responses to randomized, brief stimuli of different lengths, ranging from 800 ms dynamic movie snippets to 100 ms static frames. We found that the disruption of temporal continuity strongly altered neural response profiles, particularly in the early onset response period of the randomized stimulus. The results suggest that models of visual system function based on discrete and randomized visual presentations may not translate well to the brains natural modes of operation.

sentences: Functional ultrasound imaging of the spreading activity following optogenetic stimulation of the rat visual cortex Optogenetic stimulation of the primary visual cortex (V1) is a promising therapy for sight restoration, but it remains unclear what total cerebral volume is activated after surface stimulation. In this study, we expressed the red-shifted opsin ChrimsonR in excitatory neurons within V1 in rats, and used the fine spatial resolution provided by functional ultrasound imaging (fUS) over the whole depth of the brain to investigate the brain response to focal surface stimulation. We observed optogenetic activation of a high proportion of the volume of V1. Extracellular recordings confirmed the neuronal origin of this activation. Moreover, neuronal responses were even located in deep layers under conditions of low irradiance, spreading to the LGN and V2, consistent with a normal visual information process. This study paves the way for the use of optogenetics for cortical therapies, and highlights the value of coupling fUS with optogenetics.

sentences: GANscan: continuous scanning microscopy using deep learning deblurring Most whole slide imaging (WSI) systems today rely on the "stop-and-stare" approach, where, at each field of view, the scanning stage is brought to a complete stop before the camera snaps a picture. This procedure ensures that each image is free of motion blur, which comes at the expense of long acquisition times. In order to speed up the acquisition process, especially for large scanning areas, such as pathology slides, we developed an acquisition method in which the data is acquired continuously while the stage is moving at high speeds. Using generative adversarial networks (GANs), we demonstrate this ultra-fast imaging approach, referred to as GANscan, which restores sharp images from motion blurred videos. GANscan allows us to complete image acquisitions at 30x the throughput of stop-and-stare systems. This method is implemented on a Zeiss Axio Observer Z1 microscope in brightfield mode, requires no specialized hardware, and accomplishes successful reconstructions at stage speeds of up to 5,000 m/s. We validate the proposed method by imaging H&E stained tissue sections. Our method not only retrieves crisp images from fast, continuous scans, but also corrects any defocusing that occurs during scanning. Using a consumer GPU, the inference runs at <20ms/ image.

sentences: Multifunctional role of GPCR signaling in epithelial tube formation Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the apicomedial region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data supports a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

sentences: Ultra-sensitive nanoLC-MS using second generation micro pillar array LC technology with Orbitrap Exploris 480 and FAIMS PRO to enable single cell proteomics In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition and data analysis. To demonstrate the potential for single cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar array columns with state-of-the-art Orbitrap MS/MS detection and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micro pillar dimensions that have been further downscaled (inter pillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5-0.05 ng/L) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.

sentences: Modulation of glucose metabolism by 2-Deoxy-D-Glucose (2DG) promotes IL-17 producing human T cell subsets Activation and differentiation of T cells are closely linked to their cellular metabolic programs. Glycolysis and mitochondrial metabolism are thought to be critical in modulating T cell function. Here we asked to what extent inhibition of glycolysis, using 2-Deoxy-D-Glucose (2DG), regulate activation, effector function, or differentiation of human T cell subsets. We found that glycolysis is required for T cell receptor (TCR) -mediated activation and proliferation of human naive CD4+ T cells but had less of an impact on memory subsets. CD4+ T cells cultured in the presence of 2DG displayed higher level of IL-17-secreting cells (Th17) from memory or in vitro differentiated naive regulatory T cell (Tregs) subsets. Moreover, the mucosal associated invariant T (MAIT) cell subset survived or expanded better and secreted higher IL-17 in the presence of 2DG. Remarkably, we found that the 2DG effect was reversed by mannose but not by glucose. Collectively, these findings suggest that 2DG could enrich IL-17 secreting human effector T cell subsets and their cellular functions. Our finding provides a framework to manipulate glycolytic pathways in human T cells in infectious diseases such as COVID19 and in enhancing cancer immunotherapy.

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Emergent myxobacterial behaviors arise from reversal suppression induced by kin contacts A wide range of biological systems - from microbial swarms to bird flocks, display emergent behaviors driven by coordinated movement of individuals. To this end, individual organisms interact by recognizing their kin and adjusting their motility based on others around them. However, even in the best-studied systems, the mechanistic basis of the interplay between kin recognition and motility coordination is not understood. Here, using a combination of experiments and mathematical modeling, we uncover the mechanism of an emergent social behavior in Myxococcus xanthus. By overexpressing cell surface adhesins, TraA and TraB, involved in kin recognition, large numbers of cells adhere to one another and form organized macroscopic circular aggregates that spin clockwise or counterclockwise. Mechanistically, TraAB adhesion results in sustained cell-cell contacts that trigger cells to suppress cell reversals, and circular aggregates form as the result of cells ability to follow their own cellular slime trails. Furthermore, our in-silico simulations demonstrate a remarkable ability to predict self-organization patterns when phenotypically distinct strains are mixed. For example, defying naive expectations, both models and experiments found that strains engineered to overexpress different and incompatible traAB allelles nevertheless form mixed circular aggregates. Therefore, this work provides key mechanistic insights into M. xanthus social interactions and demonstrates how local cell contacts induce emergent collective behaviors by millions of cells.

ImportanceIn many species, large populations exhibit emergent behaviors whereby all related individuals move in unison. For example, fish in schools can all dart in one direction simultaneously to avoid a predator. Currently, it is impossible to explain how such animals recognize kin through brain cognition and elicit such behaviors at a molecular level. However, microbes also recognize kin and exhibit emergent collective behaviors that are experimentally tractable. Here, using a model social bacterium, we engineer dispersed individuals to organize into synchronized collectives that create emergent patterns. With experimental and mathematical approaches we explain how this occurs at both molecular and population levels. The results demonstrate how the combination of local physical interactions triggers intracellular signaling, which in turn leads to emergent behavior on a population scale.

sentences: Hemodynamic responses link individual differences in informational masking to the vicinity of superior temporal gyrus Suppressing unwanted background sound is crucial for aural communication. A particularly disruptive type of background sound, informational masking (IM), often interferes in social settings. However, IM mechanisms are incompletely understood. At present, IM is identified operationally: when a target should be audible, based on suprathreshold target/masker energy ratios, yet cannot be heard because perceptually similar background sound interferes. Here, functional near infrared spectroscopy recordings show that task-evoked blood oxygenation changes near the superior temporal gyrus (STG) covary with behavioral speech detection performance for high-IM but not low-IM background sound, suggesting that the STG is part of an IM-dependent network. Moreover, listeners who are more vulnerable to IM show increased hemodynamic recruitment near STG. In contrast, task-evoked responses near another auditory region of cortex, the caudal inferior frontal sulcus (cIFS), do not predict behavioral sensitivity, suggesting that the cIFS belongs to an IM-independent network. Results are consistent with the idea that cortical gating shapes individual vulnerability to IM.

sentences: DeepLIIF: Deep Learning-Inferred Multiplex ImmunoFluorescence for IHC Quantification Reporting biomarkers assessed by routine immunohistochemical (IHC) staining of tissue is broadly used in diagnostic pathology laboratories for patient care. To date, clinical reporting is predominantly qualitative or semi-quantitative. By creating a multitask deep learning framework referred to as DeepLIIF, we present a single-step solution to stain deconvolution/separation, cell segmentation, and quantitative single-cell IHC scoring. Leveraging a unique de novo dataset of co-registered IHC and multiplex immunofluorescence (mpIF) staining of the same slides, we segment and translate low-cost and prevalent IHC slides to more expensive-yet-informative mpIF images, while simultaneously providing the essential ground truth for the superimposed brightfield IHC channels. Moreover, a new nuclear-envelop stain, LAP2beta, with high (>95%) cell coverage is introduced to improve cell delineation/segmentation and protein expression quantification on IHC slides. By simultaneously translating input IHC images to clean/separated mpIF channels and performing cell segmentation/classification, we show that our model trained on clean IHC Ki67 data can generalize to more noisy and artifact-ridden images as well as other nuclear and non-nuclear markers such as CD3, CD8, BCL2, BCL6, MYC, MUM1, CD10, and TP53. We thoroughly evaluate our method on publicly available benchmark datasets as well as against pathologists semi-quantitative scoring. The code, the pre-trained models, along with easy-to-run containerized docker files as well as Google CoLab project are available at https://github.com/nadeemlab/deepliif.

sentences: Temporal Dynamics of HCMV Gene Expression in Lytic and Latent Infections Primary infection with Human cytomegalovirus (HCMV) results in a persistent lifelong infection due to its ability to establish latent infection. During productive HCMV infection, viral genes are expressed in a coordinated cascade that is characteristic of all herpesviruses and traditionally relies on the dependencies of viral genes on protein synthesis and viral DNA replication. In contrast, the transcriptional landscape associated with HCMV latency is still disputed and poorly understood. Here, we examine viral transcriptomic dynamics during the establishment of both productive and latent HCMV infections. Our temporal measurements reveal that viral gene expression dynamics along productive infection and their dependencies on protein synthesis and viral DNA replication, do not fully align. This illustrates that the regulation of herpesvirus genes does not represent a simple sequential transcriptional cascade, and surprisingly, many viral genes are regulated by multiple independent modules. Using our improved classification of viral gene expression kinetics in conjunction with transcriptome-wide measurements of the effects of a wide array of chromatin modifiers, we unbiasedly show that a defining characteristic of latent cells is the unique repression of immediate early (IE) genes. Altogether, our findings provide an elaborate definition of HCMV gene expression patterns and reveal novel principles that govern viral gene expression in lytic and latent infection states.

sentences: Scrutiny of human lung infection by SARS-CoV-2 and associated human immune responses in humanized mice There is an urgent need for animal models of COVID-19 to study immunopathogenesis and test therapeutic intervenes. In this study we showed that NSG mice engrafted with human lung (HL) tissue (NSG-L mice) could be infected efficiently by SARS-CoV-2, and that live virus capable of infecting Vero cells was found in the HL grafts and multiple organs from infected NSG-L mice. RNA-seq examination identified a series of differentially expressed genes, which are enriched in viral defense responses, chemotaxis, interferon stimulation, and pulmonary fibrosis between HL grafts from infected and control NSG-L mice. Furthermore, when infecting humanized mice with human immune system (HIS) and autologous HL grafts (HISL mice), the mice had bodyweight loss and hemorrhage and immune cell infiltration in HL grafts, which were not observed in immunodeficient NSG-L mice, indicating the development of anti-viral immune responses in these mice. In support of this possibility, the infected HISL mice showed bodyweight recovery and lack of detectable live virus at the later time. These results demonstrate that NSG-L and HISL mice are susceptible to SARS-CoV-2 infection, offering a useful in vivo model for studying SARS-CoV-2 infection and the associated immune response and immunopathology, and testing anti-SARS-CoV-2 therapies.

sentences: Using machine learning and big data to explore the drug resistance landscape in HIV The authors have withdrawn this manuscript due to a duplicate posting of manuscript number 435621. Therefore, the author does not wish this work to be cited as reference for the project. Please cite and refer to the preprint 435621 (DOI: https://doi.org/10.1101/2021.03.16.435621). If you have any questions, please contact the corresponding authors.

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Systemic IgG repertoire as a biomarker for translocating gut microbiota members While the microbiota has been associated with diseases states, how specific alterations in composition, function, or localization contribute to pathologies remains unclear. The ability of defined microbes to translocate has been linked to diseases including inflammatory bowel disease (IBD) and was shown to promote responses to immune checkpoint therapy. However, scalable and unbiased tools to uncover microbes with enhanced ability to translocate are limited. Herein, we developed an approach to utilize systemic IgG in an unbiased, culture-independent, and high-throughput fashion as a biomarker to identify gut microbiota members that are capable of translocation across the gastrointestinal barrier. We validate these findings in a cohort of human subjects, and highlight a number of microbial taxa against which elevated IgG responses are unique to subjects with IBD including Collinsella, Bifidobacterium, Faecalibacterium, and Blautia spp. Collinsella and Bifidobacterium taxa identified as translocators and targets of immunity in IBD also exhibited heightened bacterial activity and growth rates in Crohns disease subjects. Our approach may represent a complementary tool to illuminate privileged interactions between host and its microbiota, and may provide an additional lens by which to uncover microbes linked to disease processes.

One Sentence SummaryCirculating microbiota-specific IgG can identify gut microbiota constituents capable of crossing the gut barrier.

sentences: Transcriptome signature of cell viability predicts drug response and drug interaction for Tuberculosis The treatment of tuberculosis (TB), which kills 1.8 million each year, remains difficult, especially with the emergence of multidrug resistant strains of Mycobacterium tuberculosis (Mtb). While there is an urgent need for new drug regimens to treat TB, the process of drug evaluation is slow and inefficient owing to the slow growth rate of the pathogen, the complexity of performing bacteriologic assays in a high-containment facility, and the context-dependent variability in drug sensitivity of the pathogen. Here, we report the development of "DRonA" and "MLSynergy", algorithms to perform rapid drug response assays and predict response of Mtb to novel drug combinations. Using a novel transcriptome signature for cell viability, DRonA accurately detects bacterial killing by diverse mechanisms in broth culture, macrophage infection and patient sputum, providing an efficient, and more sensitive alternative to time- and resource-intensive bacteriologic assays. Further, MLSynergy builds on DRonA to predict novel synergistic and antagonistic multi-drug combinations using transcriptomes of Mtb treated with single drugs. Together DRonA and MLSynergy represent a generalizable framework for rapid monitoring of drug effects in host-relevant contexts and accelerate the discovery of efficacious high-order drug combinations.

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Conformational Ensemble of Monomeric α-Synuclein in Aqueous and Crowded Environments as revealed by Markov State Model 140-residue intrinsically disordered protein -synuclein (S) is known to be susceptible to environmental cues/crowders and adopts conformations that are vastly variable in the extent of secondary structure and tertiary interactions. Depending upon the nature of these interactions, some of the conformations may be suitable for its physiological functions while some may be predisposed to aggregate with other partners into higher ordered species or to phase separate. However, the inherently heterogenous and dynamic nature of S has precluded a clear demarcation of its monomeric precursor between aggregation-prone and functionally relevant aggregation-resistant states. Here, we optimally characterise a set of metastable conformations of S by developing a comprehensive Markov state model (MSM) using cumulative 108 {micro}s-long all-atom MD simulation trajectories of monomeric S. Notably, the dimension of the most populated metastable (85%) state (Rg [~] 2.59 ({+/-}0.45) nm) corroborates PRENMR studies of S monomer and undergoes kinetic transition at 0.1-150 {micro}s time-scale with weakly populated (0.06%) random-coil like ensemble (Rg [~] 5.85 ({+/-}0.43) nm) and globular protein-like state (14%) (Rg [~] 1.95 ({+/-}0.08) nm). The inter-residue contact maps identify a set of mutually interconverting aggregation-prone {beta}-sheet networks in the NAC region and aggregation-resistant long-range interactions between N- and C-terminus or helical conformations. The presence of crowding agents compacts the MSM-derived metastable conformations in a non-monotonic fashion and skews the ensemble by either introducing new tertiary contacts or reinforcing the innate contacts to adjust to the excluded-volume effects of such environments. These observations of crucial monomeric states would serve as important steps towards rationalising routes that trigger S-associated pathologies.

Significance statement-synuclein, a neuronal protein, is often associated with neurogenerative diseases due to its tendency to self-assemble into higher ordered aggregates. While the monomeric precursor of this protein is intrinsically disordered, it is also known to be susceptible to biological environmental cues and adopts a wide range of conformations that are either primed for aggregation or remain in auto-inhibitory states. However, the inherently heterogenous nature of the monomeric form has prevented a clear dissection of aggregation-prone and functionally relevant aggregation-resistant states. Here, we resolve this via an atomistic characterisation of an optimal set of crucial metastable monomeric conformations via statistical modelling of computer simulated data. The investigation also sheds light on crowding-induced modulation of the ensemble and eventual fibrillation pathways.

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Limited consequences for loss of RNA-directed DNA methylation in Setaria viridis domains rearranged methyltransferase (DRM) mutants The Domains Rearranged Methyltransferases (DRMs) are crucial for RNA-directed DNA methylation (RdDM) in plant species. Setaria viridis is a model monocot species with a relatively compact genome that has limited transposable element content. CRISPR-based genome editing approaches were used to create loss-of-function alleles for the two putative functional DRM genes in S. viridis to probe the role of RdDM. The analysis of drm1ab double mutant plants revealed limited morphological consequences for the loss of RdDM. Whole-genome methylation profiling provided evidence for wide-spread loss of methylation in CHH sequence contexts, particularly in regions with high CHH methylation in wild-type plants. Evidence was also found for locus-specific loss of CG and CHG methylation, even in some regions that lack CHH methylation. Transcriptome profiling identified a limited number of genes with altered expression in the drm1ab mutants. The majority of genes with elevated CHH methylation directly surrounding the transcription start site or in nearby promoter regions do not have altered expression in the drm1ab mutant even when this methylation is lost, suggesting limited regulation of gene expression by RdDM. Detailed analysis of the expression of transposable elements identified several transposons that are transcriptionally activated in drm1ab mutants. These transposons likely require active RdDM for maintenance of transcriptional repression.

Significance statementMethylation profiling of Setaria viridis plants that lack functional Domains Rearranged Methyltransferase genes reveal widespread loss of DNA methylation in the CHH sequence context. Transcriptome analysis reveals a small set of genes and transposons that are silenced by RNA-directed DNA methylation.

sentences: Reducing disease and producing food: Effects of 13 agrochemicals on snail biomass and human schistosomes Agrochemical use is predicted to increase 2-5 fold by 2050 to meet food demand. Evidence suggests that agrochemical pollution could increase snails that transmit the disease schistosomiasis to 250 million people, but most agrochemicals remain unexamined. Here we quantify the relative effects of fertilizer, six insecticides, and six herbicides on snail genera responsible for 90% of global schistosomiasis cases. We identified fertilizers and 4 of 6 insecticides as high risk for increasing snail biomass by increasing snail resources (vegetative habitat and periphytic food) and reducing snail predators, respectively. Herbicides generally had negative effects on snails by reducing vegetative habitat, with two herbicides increasing snails in the absence of aquatic vegetation. Parasite production, which reflects human infection risk, scaled positively to snail biomass. Our findings suggest that fertilizers and insecticides are more likely to increase human schistosomiasis than herbicides and revealed several low risk agrochemicals that might increase crop production without increasing schistosomiasis.

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Comparative analysis of Bacillus cereus group isolates' resistance using disk diffusion and broth microdilution and the correlation between resistance phenotypes and genotypes A collection of 85 Bacillus cereus group isolates were screened for phenotypic resistance to nine antibiotics using disk diffusion and broth microdilution. The broth microdilution antimicrobial results were interpreted using the CLSI M45 breakpoints for Bacillus spp. Due to the lack of Bacillus spp. disk diffusion breakpoints, the results obtained with the disk diffusion assay were interpreted using the CLSI M100 breakpoints for Staphylococcus spp. We identified significant (p < 0.05) discrepancies in resistance interpretation between the two methods for ampicillin, gentamicin, rifampicin, tetracycline, and trimethoprim/sulfamethoxazole. Antimicrobial resistance genes were detected using unassembled and assembled whole-genome sequences with Ariba and Abricate, respectively, to assess the sensitivity and specificity for predicting phenotypic resistance based on the presence of antimicrobial resistance genes. We found antimicrobial resistance gene presence to be a poor indicator for phenotypic resistance, calling for further investigation of mechanisms underlying antimicrobial resistance in the B. cereus group. Genes with poor sensitivity and/or specificity, as determined based on broth microdilution results included rph (rifampicin, 0%, 95%), mph genes (erythromycin, 0%, 96%), and all van genes (vancomycin, 100%, 35%). However, Bc (ampicillin, 64%, 100%) and tet genes (tetracycline, 67%, 100%) were highly specific, albeit moderately sensitive indicators of phenotypic resistance based on broth microdilution results. Only beta-lactam resistance genes (Bc, BcII, and blaTEM) were highly sensitive (94%) and specific (100%) markers of resistance to ceftriaxone based on the disk diffusion results, providing further evidence of these beta-lactams role in nonsusceptibility of Bacillus cereus group isolates to ceftriaxone.

IMPORTANCEBacillus cereus group includes human pathogens that can cause severe infections requiring antibiotic treatment. Screening of environmental and food isolates for antimicrobial resistance can provide insight into what antibiotics may be more effective therapeutic options based on the lower prevalence of resistance. Currently, interpretation of antimicrobial susceptibility testing results using the disk diffusion method is complicated by the fact that there are no standard disk diffusion resistance breakpoints defined for Bacillus spp. Hence, the breakpoints for Staphylococcus are often used in research studies. By comparing the results of disk diffusion interpreted using the Staphylococcus spp. breakpoints against broth microdilution interpreted using Bacillus spp. breakpoints, this study demonstrated that disk diffusion results interpretation with Staphylococcus spp. breakpoints are inconsistent. This study also provides new insight into the poor associations between antimicrobial resistance genotypes and phenotypes for the B. cereus group.

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HIV-1 Vpu promotes phagocytosis of infected CD4+ T cells by macrophages through downregulation of CD47 Human immunodeficiency virus (HIV) remodels the cell surface of infected cells to facilitate viral dissemination and promote immune evasion. The membrane-associated Vpu accessory protein encoded by HIV-1 plays a key role in this process by altering cell surface levels of multiple host proteins. Using an unbiased quantitative plasma membrane profiling approach, we previously identified CD47 as a putative host target downregulated by Vpu. CD47 is a ubiquitously-expressed cell surface protein that interacts with the myeloid cell inhibitory receptor SIRP to deliver a "dont-eat-me" signal, thus protecting cells from phagocytosis. In this study, we investigate whether CD47 modulation by HIV-1 Vpu might promote the susceptibility of macrophages to viral infection via phagocytosis of infected CD4+ T cells. Indeed, we find that Vpu downregulates CD47 expression on infected CD4+ T cells leading to an enhanced capture and phagocytosis by macrophages. Interestingly, it is through this process that a CCR5-tropic transmitted/founder (T/F) virus, which otherwise poorly infects macrophages in its cell-free form, becomes infectious in macrophages. Importantly, we show that HIV-1-infected cells expressing a Vpu-resistant CD47 mutant are less prone to infect macrophages through phagocytosis. Mechanistically, Vpu forms a physical complex with CD47 through its transmembrane domain and targets the latter for lysosomal degradation. These results reveal a novel role of Vpu in modulating macrophage infection, which has important implications for HIV-1 transmission in early stages of infection and the establishment of viral reservoir.

IMPORTANCEMacrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus. Selective capture and engulfment of HIV-1 infected T cells was shown to drive efficient macrophage infection suggesting that this mechanism represents an important mode of infection notably for weakly macrophage-tropic T/F viruses. In this study, we provide insight into the signals that regulate this process. We show that the HIV-1 accessory protein Vpu downregulates cell surface levels of CD47, a host protein that interacts with the inhibitory receptor SIRP to deliver a "dont-eat-me" signal to macrophages. This allows for enhanced capture and phagocytosis of infected T cells by macrophages, ultimately leading to their productive infection even with T/F virus. These findings provide new insights into the mechanisms governing the intercellular transmission of HIV-1 to macrophages with implications for the establishment of the macrophage reservoir and early HIV-1 dissemination in vivo.

sentences: Epigenetic changes with age primes mammary luminal epithelia for cancer initiation Aging causes molecular changes that manifest as stereotypical phenotypes yet aging-associated diseases progress only in certain individuals. At lineage-specific resolution, we show how stereotyped and variant responses are integrated in mammary epithelia. Age-dependent directional changes in gene expression and DNA methylation (DNAm) occurred almost exclusively in luminal cells and implicated genome organizers SATB1 and CTCF. DNAm changes were robust indicators of aging luminal cells, and were either directly (anti-)correlated with expression changes or served as priming events for subsequent dysregulation, such as demethylation of ESR1-binding regions in DNAm-regulatory CXXC5 in older luminal cells and luminal-subtype cancers. Variance-driven changes in the transcriptome of both luminal and myoepithelial lineages further contributed to age-dependent loss of lineage fidelity. The pathways affected by transcriptomic and DNAm changes during aging are commonly linked with breast cancer, and together with the differential variability found across individuals, influence aging-associated cancer susceptibility in a subtype-specific manner.

sentences: Act Natural: Functional Connectivity from Naturalistic Stimuli fMRI Outperforms Resting-State in Predicting Brain Activity The search for an  ideal approach to investigate the functional connections in the human brain is an ongoing challenge for the neuroscience community. While resting-state functional magnetic resonance imaging (fMRI) has been widely used to study individual functional connectivity patterns, recent work has highlighted the benefits of collecting functional connectivity data while participants are exposed to naturalistic stimuli, such as watching a movie or listening to a story. For example, functional connectivity data collected during movie-watching were shown to predict cognitive and emotional scores more accurately than resting-state-derived functional connectivity. We have previously reported a tight link between resting-state functional connectivity and task-derived neural activity, such that the former successfully predicts the latter. In the current work we use data from the Human Connectome Project to demonstrate that naturalistic-stimulus-derived functional connectivity predicts task-induced brain activation maps more accurately than resting-state-derived functional connectivity. We then show that activation maps predicted using naturalistic stimuli are better predictors of individual intelligence scores than activation maps predicted using resting-state. We additionally examine the influence of naturalistic-stimulus type on prediction accuracy. Our findings emphasize the potential of naturalistic stimuli as a promising alternative to resting-state fMRI for connectome-based predictive modelling of individual brain activity and cognitive traits.

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Escaping from multiple visual threats: Modulation of escape responses in Pacific staghorn sculpin (Leptocottus armatus). Fish perform rapid escape responses to avoid sudden predatory attacks. During escape responses, fish bend their bodies into a C-shape and quickly turn away from the predator and accelerate. The escape trajectory is determined by the initial turn (Stage 1) and a contralateral bend (Stage 2). Previous studies have used a single threat or model predator as a stimulus. In nature, however, multiple predators may attack from different directions simultaneously or in close succession. It is unknown whether fish are able to change the course of their escape response when startled by multiple stimuli at various time intervals. Pacific staghorn sculpin (Leptocottus armatus) were startled with a left and right visual stimulus in close succession. By varying the timing of the second stimulus, we were able to determine when and how a second stimulus could affect the escape response direction. Four treatments were used: a single visual stimulus (control); or two stimuli coming from opposite sides separated by a 0 ms (simultaneous treatment); a 33 ms; or a 83 ms time interval. The 33 ms and 83 ms time intervals were chosen to occur shortly before and after a predicted 60 ms visual escape latency (i.e. during Stage 1). The 0 ms and 33 ms treatments influenced both the escape trajectory and the Stage 1 turning angle, compared to a single stimulation, whereas the 83 ms treatment had no effect on the escape response. We conclude that Pacific staghorn sculpin can modulate their escape response only between stimulation and the onset of the response, but that escape responses are ballistic after the body motion has started.

SUMMARY STATEMENTUsing double stimulation from opposite sides at different time intervals to simulate coordinated predatory attacks, Pacific staghorn sculpin escape away from the first stimulus, but were unable to turn away from the second stimulus while the escape response was in progress.

sentences: Impact of blindness onset on the representation of sound categories in occipital and temporal cortices How does blindness onset impact on the organization of cortical regions coding for the deprived and the remaining senses? We show that the coding of sound categories in the occipital cortex is enhanced and more stable within and across blind individuals when compared to sighted controls, while a reverse group difference is found in the temporal cortex. Importantly, occipital and temporal regions share a more similar representational structure in blind people, suggesting an interplay between the reorganization of occipital and temporal regions following visual deprivation. We suggest that early, and to some extent late blindness, induces network-level reorganization of the neurobiology of auditory categories by concomitantly increasing/decreasing the respective computational load of occipital/temporal regions. These results highlight the interactive nature of regional brain development in case of sensory deprivation.

sentences: Planning the potential future during multi-item visual working memory Working memory allows us to retain visual information to guide upcoming future behavior. In line with this future-oriented purpose of working memory, recent studies have shown that action planning occurs during encoding and retention of a single visual item, for which the upcoming action is certain. We asked whether and how this extends to multi-item visual working memory, when visual representations serve the potential future. Human participants performed a visual working memory task with a memory-load manipulation (one/two/four items), and a delayed orientation-reproduction report (of one item). We measured EEG to track 15-25 Hz beta activity in electrodes contralateral to the required response hand - a canonical marker of action planning. We show an attenuation of beta activity, not only in load one (with one certain future action), but also in load two (with two potential future actions), compared to load four (with low prospective-action certainty). Moreover, in load two, potential action planning occurs regardless whether both visual items afford similar or dissimilar manual responses; and it predicts the speed of ensuing memory-guided behavior. This shows that potential action planning occurs during multi-item visual working memory, and brings the perspective that working memory helps us prepare for the potential future.

sentences: Optimal spatial monitoring of populations described by reaction-diffusion models Using spatialised population measurements and related geographic habitat data, it is quite feasible nowadays to derive parsimonious spatially explicit population models and to carry on their parameter estimation. To achieve such goal, reaction-diffusion models are fairly common in conservation biology and agricultural plant health where they are used, for example, for landscape planning or epidemiological surveillance. Unfortunately, if the mathematical methods and computational power are readily available, biological measurements are not. Despite the high throughput of some habitat related remote sensors, the experimental cost of biological measurements are, in our view, one of the worst bottleneck against a widespread usage of reaction-diffusion models. Hence, in this paper, we will recall some classical methods for optimal experimental design that we deem useful to spatial ecologist. Using two case studies, one in landscape ecology and one in conservation biology, we will show how to construct a priori experimental design minimizing variance of parameter estimates, enabling optimal experimental setup with pre and post hoc filtering for accommodating additional constraints.

sentences: ASF1 activation PI3K/AKT pathway regulates sexual and asexual development in filamentous ascomycete Sexual and asexual reproduction is ubiquitous in eukaryotes. PI3K/AKT signaling pathway can modulate sexual reproduction in mammals. However, this signaling pathway modulating sexual and asexual reproduction in fungi is scarcely understood. SeASF1, a SeH4 chaperone, could manipulate sexual and asexual reproduction of Stemphylium eturmiunum. SeDJ-1, screened from Se{Delta}asf1 transcriptome, was confirmed to regulate sexual and asexual development by RNAi, of which the mechanism was demonstrated by detecting transcriptional levels and protein interactions of SeASF1, SeH4 and SeDJ-1 by qRT-PCR, and Y2H, Co-IP and Pull-down, respectively. SeASF1 coupling SeH4 bound SeDJ-1 to arouse the sexual and asexual activity. In S. eturmiunum genome, SeDJ-1 was upstream while SeGSK3 was downstream in PI3K/AKT signaling pathway. Moreover, SeDJ-1 interacted with SePI3K or SeGSK3 in vivo and in vitro. Significantly, SeDJ-1 or SePI3K could effectively stimulate sexual activity alone, but SePI3K could recover the sexual development of SiSeDJ-1. Meanwhile, SeDJ-1-M6 was a critical segment for interaction of SeDJ-1 with SePI3K. SeDJ-1-M6 played a critical role in irritating sexual reproduction in SiSePI3K, which further uncovered the regulated mechanism of SeDJ-1. Summarily, SeASF1 coupling SeH4 motivates SeDJ-1 to arouse SePI3K involved in sexual reproduction. Thus, SeASF1 can activate PI3K/AKT signaling pathway to regulate sexual and asexual development in filamentous ascomycete.

sentences: Combining evolutionary and assay-labelled data for protein fitness prediction Predictive modelling of protein properties has become increasingly important to the field of machine-learning guided protein engineering. In one of the two existing approaches, evolutionarily-related sequences to a query protein drive the modelling process, without any property measurements from the laboratory. In the other, a set of protein variants of interest are assayed, and then a supervised regression model is estimated with the assay-labelled data. Although a handful of recent methods have shown promise in combining the evolutionary and supervised approaches, this hybrid problem has not been examined in depth, leaving it unclear how practitioners should proceed, and how method developers should build on existing work. Herein, we present a systematic assessment of methods for protein fitness prediction when evolutionary and assay-labelled data are available. We find that a simple baseline approach we introduce is competitive with and often outperforms more sophisticated methods. Moreover, our simple baseline is plug-and-play with a wide variety of established methods, and does not add any substantial computational burden. Our analysis highlights the importance of systematic evaluations and sufficient baselines.

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Dynamics and self-assembly of the SARS-CoV-2 spike transmembrane domain The spike (S) protein is a trimeric, membrane-anchored fusion protein that enables coronaviruses, such as the SARS-CoV-2, to recognize and fuse with their hosts cells. While the prefusion and postfusion structures of the ectomembrane domain of the spike protein are available, the corresponding organization of its transmembrane domain is obscure. Since the transmembrane and ectomembrane domains of fusion proteins are conformationally linked, an understanding of trimerization and transmembrane conformations in the viral envelope is a prerequisite to completely understand viral fusion by the spike protein. To address this, we computationally explored the self-assembly of the SARS-CoV-2 spike transmembrane domain, starting first by determining the membrane boundaries of the spike transmembrane helix. Using atomistic molecular dynamics simulations, we found the spike protein transmembrane domain to be plastic, and the transmembrane helix to be very dynamic. The observed movements of the helix changed the membrane embedded sequence, and thereby affected the conformational ensemble of the transmembrane assembly in Martini coarse grained simulations, even flipping the super-helical handedness. Analysis of the transmembrane organization of the spike transmembrane helix provided rich insights into the interfaces utilized to self-associate. Moreover, we identified two distinct cholesterol binding regions on the transmembrane helix with different affinities for the sterol. The cholesterol binding pockets overlapped with regions involved in the initiation of transmembrane protein-protein interaction. Together, the results from our multiscale simulations not only provide insight into understudied trimeric helical interfaces in biomembranes, but also enhance our understanding of the elusive transmembrane conformational dynamics of SARS-CoV-2 spike and more generally of viral fusion proteins. These insights should enable the inclusion of the conformations of the spike protein transmembrane domain into the prevalent models of virus fusion.

SignificanceEnveloped viruses rely on fusion proteins, called spike proteins in coronaviruses, to infect cells by fusing the virus envelope with the host cell membrane. The transmembrane domain (TMD) of the coronavirus spike protein is critically involved in successful viral fusion and other aspects of the virus lifecycle, but is poorly studied. Using multiscale molecular dynamics simulations of the SARS-CoV-2 spike TMD, we explore its conformational dynamics and self-assembly in different lipid environments. The results provided here improve our understanding of transmembrane stabilization of spike trimers, which are indispensable for viral infection. Exploiting this knowledge to destabilize spike trimers should facilitate design of transmembrane domain targeted viral fusion inhibitors.

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A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva. Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on mechanisms of how the individual CPs interact, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata that survives long-term cryopreservation in liquid nitrogen. The CPs (proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine, and sarcosine) accumulate in hemolymph in a ratio of 313:108:55:26:6:4:3:0.5 mmol.L-1. Using calorimetry, we show that the artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using MALDI-MSI, we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it likely forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Summary statementThe composition of natural cryoprotectant mixture of the extremely freeze-tolerant insect is revealed. Components of the mixture work in synergy and behave differently during organismal freezing and cryopreservation.

sentences: Statistical learning of successor representations is related to on-task replay Humans automatically infer higher-order relationships between events in the environment from their statistical co-occurrence, often without conscious awareness. Neural replay of task representations, which has been described as sampling from a learned transition structure of the environment, is a candidate mechanism by which the brain could use or even learn such relational information in the service of adaptive behavior. Human participants viewed sequences of images that followed probabilistic transitions determined by ring-like graph structures. Behavioral modeling revealed that participants acquired multi-step transition knowledge through gradual updating of an internal successor representation (SR) model, although half of participants did not indicate any knowledge about the sequential task structure. To investigate neural replay, we analyzed dynamics of multivariate functional magnetic resonance imaging (fMRI) patterns during short pauses from the ongoing statistical learning task. Evidence for sequential replay consistent with the probabilistic task structure was found in occipito-temporal and sensorimotor cortices during short on-task intervals. These findings indicate that implicit learning of higher-order relationships establishes an internal SR-based map of the task, and is accompanied by cortical on-task replay.

sentences: Multitaper estimates of phase-amplitude coupling Phase-amplitude coupling (PAC) is the association of the amplitude of a high-frequency oscillation with the phase of a low-frequency oscillation. In neuroscience, this relationship provides a mechanism by which neural activity might be coordinated between distant regions. The dangers and pitfalls of assessing phase-amplitude coupling with existing statistical measures have been well-documented. The limitations of these measures include: (i) response to non-oscillatory, high-frequency, broad-band activity, (ii) response to high-frequency components of the low-frequency oscillation, (iii) adhoc selection of analysis frequency-intervals, and (iv) reliance upon data shuffling to assess statistical significance. In this work, a multitaper phase-amplitude coupling estimator is proposed that addresses issues (i)-(iv) above. Specifically, issue (i) is addressed by replacing the analytic signal envelope estimator computed using the Hilbert transform with a multitaper estimator that down-weights non-sinusoidal activity using a classical, multitaper super-resolution technique. Issue (ii) is addressed by replacing coherence between the low-frequency and high-frequency components in a standard PAC estimator with multitaper partial coherence, while issue (iii) is addressed with a physical argument regarding meaningful neural oscillation. Finally, asymptotic statistical assessment of the multitaper estimator is introduced to address issue (iv).

sentences: Angiomotin isoform 2 promotes binding of PALS1 to KIF13B at the base of primary cilia and suppresses ciliary elongation The kinesin-3 motor KIF13B functions in endocytosis, vesicle transport, and regulation of ciliary length and signaling. Direct binding of the membrane-associated guanylate kinase (MAGUK) DLG1 to KIF13Bs MAGUK-binding stalk (MBS) domain relieves motor autoinhibition and promotes microtubule plus end-directed cargo transport. Here we characterize Angiomotin isoform 2 (Ap80) as a novel KIF13B interactor that promotes binding of another MAGUK, the polarity protein and Crumbs complex component PALS1, to KIF13B. Live cell imaging analysis indicated that Ap80 is concentrated at the base of primary cilia and recruits PALS1 to this site, but is not itself a cargo of KIF13B. Consistent with a ciliary function for Ap80, its depletion led to elongated primary cilia while its overexpression caused ciliary shortening. Our results suggest that Ap80 may specifically activate KIF13B cargo binding at the base of primary cilia to regulate ciliary length.

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Fiber-associated Lachnospiraceae reduce colon tumorigenesis by modulation of the tumor-immune microenvironment Patients with colorectal cancer (CRC) harbor gut microbiomes that differ in structure and function from those of healthy individuals, suggesting this altered microbiome could contribute to tumorigenesis. Despite increasing evidence implicating the gut microbiome in CRC, the collective role of different microbial consortia in CRC carcinogenesis is unclear. We have previously described these consortia as co-abundance groups that co-exist at different abundance levels in the same patient. Here, we report that tumor biopsy tissue from patients with a "high-risk" Pathogen-type microbiome had a different immune transcriptome and immune cell infiltrate from those with a "low-risk" Lachnospiraceae-type microbiome. Transplantation from patients of the two fecal microbiome types into mice with an orthotopic tumor differentially affected tumor growth and the systemic anti-tumor immune response. The differences in tumor volume and immunophenotype between mice receiving the high-risk and the low-risk microbiome correlated with differences in the engrafted human microbial species and predicted microbiome-encoded metabolites in the two groups. Of twelve taxa whose abundance in recipient mice led to increased tumor onset, seven corresponded with differentially abundant taxa in a global dataset of 325 CRC patients versus 310 healthy controls. These data suggest that the enrichment for a Lachnospiraceae-type configuration of the gut microbiome may influence colon cancer progression and disease outcome by modulating the local and systemic anti-tumor immune response.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/432654v2_ufig1.gif" ALT="Figure 1">
View larger version (45K):
org.highwire.dtl.DTLVardef@1de9ff5org.highwire.dtl.DTLVardef@d790d5org.highwire.dtl.DTLVardef@65be8aorg.highwire.dtl.DTLVardef@1c096da_HPS_FORMAT_FIGEXP  M_FIG C_FIG Proposed model of how the high-risk Pathogen and low-risk Lachnospiraceae CAGs differentially modulate the tumor immune response.

sentences: Patterned apoptosis has an instructive role for local growth and tissue shape in a fast-growing epithelium What regulates organ size and shape remains one of the fundamental mysteries of modern biology. So far, research in this area has primarily focused on deciphering the regulation in time and space of growth and cell division, while the contribution of cell death has been much more neglected. This includes studies of the Drosophila wing imaginal disc, the prospective fly wing which undergoes massive growth during larval stage, and represents one of the best characterised systems for the study of growth and patterning. So far, it has been assumed that cell death was relatively neglectable in this tissue and as a result the pattern of growth was usually attributed to the distribution of cell division. Here, using systematic mapping and registration combined with quantitative assessment of clone size and disappearance, we show for the first time that cell death is not neglectable, and outline a persistent pattern of cell death and clone elimination in the disc. Local variation of cell death is associated with local variation of clone size, pointing to an impact of cell death on local growth which is not fully compensated by proliferation. Using morphometric analyses of adult wing shape and genetic perturbations, we provide evidence that patterned death affects locally and globally adult wing shape and size. This study describes a roadmap for accurate assessment of the contribution of cell death to tissue shape, and outlines for the first time an important instructive role of cell death in modulating quantitatively local growth and the morphogenesis of a fast-growing tissue.

sentences: Population modeling with machine learning can enhance measures of mental health - Open-Data Replication Efforts to predict trait phenotypes based on functional MRI data from large cohorts have been hampered by low prediction accuracy and/or small effect sizes. Although these findings are highly replicable, the small effect sizes are somewhat surprising given the presumed brain basis of phenotypic traits such as neuroticism and fluid intelligence. We aim to replicate previous work and additionally test multiple data manipulations that may improve prediction accuracy by addressing data pollution challenges. Specifically, we added additional fMRI features, averaged the target phenotype across multiple measurements to obtain more accurate estimates of the underlying trait, balanced the target phenotypes distribution through undersampling of majority scores, and identified data-driven subtypes to investigate the impact of between-participant heterogeneity. Our results replicated prior results from Dadi et a (2021) in a larger sample. Each data manipulation further led to small but consistent improvements in prediction accuracy, which were largely additive when combining multiple data manipulations. Combining data manipulations (i.e., extended fMRI features, averaged target phenotype, balanced target phenotype distribution) led to a three-fold increase in prediction accuracy for fluid intelligence compared to prior work. These findings highlight the benefit of several relatively easy and low-cost data manipulations, which may positively impact future work.

sentences: Structural and functional organization of visual responses in the inferior olive of larval zebrafish The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. According to a classical theory of cerebellar cortex, the inferior olive (IO) provides Purkinje cells with error information which drives motor learning in the cerebellum. Here we investigate the sensory representations in the IO of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using 2-photon imaging. We found that most IO neurons showed direction selective and binocular responses to visual stimuli and that functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between soma location, axonal projections and functional properties of IO neurons. Taken together, our results suggest that anatomically-defined classes of inferior olive neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

sentences: Evolutionary tuning of a key helix drove androgen selectivity The genetic and biophysical mechanisms by which new protein functions evolve are central concerns in evolutionary biology and molecular evolution. Despite much speculation, we know little about how protein function evolves. Here, we use ancestral proteins to trace the evolutionary history of ligand recognition in a sub-class of steroid receptors (SRs), an ancient family of ligand-regulated transcription factors that enable long-range cellular communication central to multicellular life. The most ancestral members of this family display promiscuous ligand binding due to their large ligand binding pockets, while more recently evolved SRs tend to have smaller cavities. Less obvious, however, are the forces driving the selectivity of highly similar ligands. A key example is the divergence between the progesterone and androgen receptors (PR, AR), which display a high degree of sequence similarity and yet display differential ligand preferences. This work uses the ancestral steroid receptor 2 (AncSR2), the common ancestor of all 3-ketosteroids and the ancestral androgen receptor 1 (AncAR1), the seminal androgen receptor, to explore the biophysical mechanisms that drove the evolution of androgen specificity. We determine that ligand specificity in androgen receptors is driven by changes in the conformational dynamics of the receptor as well as altered binding pocket interactions, with helix 10 (H10) playing a critical role in tuning ligand specificity.

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Long-term temporal trends in gastrointestinal parasite infection in wild Soay sheep Monitoring the prevalence and abundance of parasites over time is important for addressing their potential impact on host life-histories, immunological profiles, and their influence as a selective force. Only long-term ecological studies have the potential to shed light on both the temporal trends in infection prevalence and abundance and the drivers of such trends, because of their ability to dissect drivers that may be confounded over shorter time scales. Despite this, only a relatively small number of such studies exist. Here, we analysed changes in the prevalence and abundance of gastrointestinal parasites in the wild Soay sheep population of St Kilda across 31 years. The host population density has increased across the study, and population density is known to increase parasite transmission, but we found that density and year explained temporal variation in parasite prevalence and abundance independently. Prevalence of both strongyle nematodes and coccidian microparasites increased during the study, and this effect varied between lambs, yearlings and adults. Meanwhile, abundance of strongyles was more strongly linked to host density than to temporal (yearly) dynamics, while abundance of coccidia showed a strong temporal trend without any influence of density. Strikingly, coccidian abundance increased threefold across the course of the study in lambs, while increases in yearlings and adults were negligible. Our decades-long, intensive, individual-based study will enable the role of environmental change and selection pressures in driving these dynamics to be determined, potentially providing unparalleled insight into the drivers of temporal variation in parasite dynamics in the wild.

Key findingsO_LIWe studied temporal trends in gastrointestinal parasites of wild sheep over 31 years
C_LIO_LIYear and host population density explained temporal variation in parasites independently
C_LIO_LIPrevalence of both strongyle nematodes and coccidia varied across the study period
C_LIO_LIAbundance of strongyles was more closely linked to host density than year
C_LIO_LIAbundance of coccidia increased threefold over time in lambs, but did not vary in adults
C_LI

sentences: A mathematical framework for evo-devo dynamics Natural selection acts on phenotypes constructed over development, which raises the question of how development affects evolution. Classic evolutionary theory indicates that development affects evolution by modulating the genetic covariation upon which selection acts, thus affecting genetic constraints. However, whether genetic constraints are relative, thus diverting adaptation from the direction of steepest fitness ascent, or absolute, thus blocking adaptation in certain directions, remains uncertain. This limits understanding of long-term evolution of developmentally constructed phenotypes. Here we formulate a general tractable mathematical framework that integrates age progression, explicit development (i.e., the construction of the phenotype across life subject to developmental constraints), and evolutionary dynamics, thus describing the evolutionary developmental (evo-devo) dynamics. The framework yields simple equations that can be arranged in a layered structure that we call the evo-devo process, whereby five elementary components generate all equations including those describing genetic covariation and the evo-devo dynamics. The framework recovers evolutionary dynamic equations in gradient form and describes the evolution of genetic covariation from the evolution of gene expression, phenotype, environment, and mutational covariation. This shows that genetic and phenotypic evolution must be followed simultaneously to yield a well-defined description of long-term phenotypic evolution in gradient form, such that evolution described as the climbing of a fitness landscape occurs in geno-phenotype space. Genetic constraints in geno-phenotype space are necessarily absolute because the degrees of freedom of genetic covariation are necessarily limited by genetic space. Thus, the long-term evolutionary dynamics of developed phenotypes is strongly non-standard: (1) evolutionary equilibria are either absent or infinite in number and depend on genetic covariation and hence on development; (2) developmental constraints determine the admissible evolutionary path and hence which evolutionary equilibria are admissible; and (3) evolutionary outcomes occur at admissible evolutionary equilibria, which do not generally occur at fitness landscape peaks in geno-phenotype space, but at peaks in the admissible evolutionary path where "total genetic selection" vanishes if exogenous plastic response vanishes and mutational variation exists in all directions of gene-expression space. Development thus modulates necessarily absolute genetic constraints, and hence it affects evolutionary equilibria, the admissible evolutionary path, and which equilibria are admissible. Our framework provides an alternative method to dynamic optimization (i.e., dynamic programming or optimal control) to identify evolutionary outcomes in models with developmentally dynamic traits. These results show that development has major evolutionary effects.

sentences: Transcription factor induction of vascular blood stem cell niches in vivo The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis-regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

sentences: Costs dictate strategic investment in dominance interactions Dominance is important for access to resources. As dominance interactions are costly, individuals should be strategic in who they interact with. One hypothesis is that individuals should direct costly interactions towards those closest in rank, as they have most to gain--in terms of attaining or maintaining dominance--from winning such interactions. Here, we show that male vulturine guineafowl (Acryllium vulturinum), a gregarious species with steep dominance hierarchies, strategically express higher-cost aggressive interactions towards males occupying ranks immediately below themselves in their groups hierarchy. In contrast, lower-cost aggressive interactions are expressed towards group members further down the hierarchy. By directly evaluating differences in the strategic use of higher- and lower-cost aggressive interactions towards competitors, we show that individuals disproportionately use highest-cost interactions--such as chases--towards males found one to three ranks below themselves. Our results support the hypothesis that the costs associated with different interaction types can determine their expression in social groups with steep dominance hierarchies.

sentences: Sign inversion in selection on ploidy Ploidy - the number of homologous chromosome sets in a cell - is remarkably variable across the natural world, yet the evolutionary processes that have resulted in such diversity remain poorly understood. Here we use stochastic agent-based simulations to model ploidy evolution under the influence of indirect selection, i.e., selection mediated solely by statistical associations with fitness-affecting mutations. We find that in non-equilibrium asexual populations, the sign of selection on ploidy can change with population size - a phenomenon we have previously termed sign inversion. In large populations, ploidy dynamics are dominated by indirect effects of selection on beneficial mutations, which favors haploids over diploids. However, as population size declines, selection for beneficial mutations is neutralized by random genetic drift before drift can overwhelm selection against the cost of the deleterious mutational load. As a result, in small populations indirect selection is dominated by the cost of the deleterious load, which favors diploids over haploids. Our work adds to the growing body of evidence challenging established evolutionary theory that population size can affect only the efficiency, but not the sign, of natural selection.

sentences: Hippocampal neurons code individual memory episodes in humans The hippocampus is an essential hub for episodic memory processing. However, how human hippocampal single neurons code multi-feature associations remains unknown. Here, we provide evidence that individual neurons code discrete memory episodes, which we term Episode Specific Neurons (ESNs). These ESNs do not reflect the coding of a particular feature in the episode (i.e., concept or time). Instead, they code for the conjunction of the different elements that make up the episode.

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Effect of knee joint loading on chondrocyte mechano-vulnerability and severity of post-traumatic osteoarthritis induced by ACL-injury in mice ObjectiveThe objective of this study is to understand the role of altered in vivo mechanical environments in knee joints post anterior cruciate ligament (ACL)-injury in chondrocyte vulnerability against mechanical stimuli and in the progression of post-traumatic osteoarthritis (PT-OA).

MethodsDifferential in vivo mechanical environments were induced by unilateral ACL-injury (uni-ACL-I) and bilateral ACL-injury (bi-ACL-I) in 8-week-old female C57BL/6 mice. The gait parameters, the mechano-vulnerability of in situ chondrocytes, Youngs moduli of cartilage extracellular matrix (ECM), and the histological assessment of OA severity (OARSI score) were compared between control and experimental groups at 0[~]8-weeks post-ACL-injury.

ResultsWe found that bi-ACL-I mice experience higher joint-loading on their both injured limbs, but uni-ACL-I mice balance their joint-loading between injured and uninjured hind limbs resulting in a reduced joint-loading during gait. We also found that at 4- and 8-week post-injury the higher weight-bearing hind limbs (i.e., bi-ACL-I) had the increased area of chondrocyte death induced by impact loading and higher OARSI score than the lower weight-bearing limbs (uni-ACL-I). Additionally, we found that at 8-weeks post-injury the ECM became stiffer in bi-ACL-I joints and softer in uni-ACL-I joints.

ConclusionsOur results show that ACL-injured limbs with lower in vivo joint-loading develops PT-OA significantly slower than injured limbs with higher joint-loading during gait. Our data also indicate that articular chondrocytes in severe PT-OA are more fragile from mechanical impacts than chondrocytes in healthy or mild PT-OA. Thus, preserving physiologic joint-loads on injured joints will reduce chondrocyte death post-injury and may delay PT-OA progression.

sentences: DNA-dependent binding of nargenicin to DnaE1 inhibits replication in Mycobacterium tuberculosis Natural products provide a rich source of potential antimicrobials for use in treating infectious diseases for which drug resistance has emerged. Foremost among these is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.

sentences: CancerMIRNome: an interactive analysis and visualization database for miRNome profiles of human cancer MicroRNAs (miRNAs), which play critical roles in gene regulatory networks, have emerged as promising diagnostic and prognostic biomarkers for human cancer. In particular, circulating miRNAs that are secreted into circulation exist in remarkably stable forms, and have enormous potential to be leveraged as non-invasive biomarkers for early cancer detection. Novel and user-friendly tools are desperately needed to facilitate data mining of the vast amount of miRNA expression data from The Cancer Genome Atlas (TCGA) and large-scale circulating miRNA profiling studies. To fill this void, we developed CancerMIRNome, a comprehensive database for the interactive analysis and visualization of miRNA expression profiles based on 10,998 samples from 33 TCGA projects and 21,993 samples from 40 public circulating miRNome datasets. A series of cutting-edge bioinformatics tools and machine learning algorithms have been packaged in CancerMIRNome, allowing for the pan-cancer analysis of a miRNA of interest across multiple cancer types and the comprehensive analysis of miRNome profiles to identify dysregulated miRNAs and develop diagnostic or prognostic signatures. The data analysis and visualization modules will greatly facilitate the exploit of the valuable resources and promote translational application of miRNA biomarkers in cancer. The CancerMIRNome database is publicly available at http://bioinfo.jialab-ucr.org/CancerMIRNome.

sentences: Succession comprises a sequence of threshold-induced community assembly processes towards multidiversity Research on successions and community assembly both address the same processes such as dispersal, species sorting, and biotic interactions but lack unifying concepts. Recent theoretical advances integrated both research lines proposing a sequence of stochastic and niche-based processes along successional gradients. Shifts in assembly processes are predicted to occur abruptly once abiotic and biotic factors dominate over dispersal as main driver. Considering the multidiversity composed of five organismal groups including plants, animals, and microbes, we empirically show that stochastic dispersal-dominated community assembly is replaced by environmental filters and biotic interactions after around 60 years of succession in a glacier forefield. The niche-based character of later successional processes is further supported by a decline in multi-beta-diversity. Our results may update concepts of community assembly by considering multiple taxa, help to bridge the gap between research on successions and community assembly, and provide new insights into the emergence of multidiverse and complex ecosystems.

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Minimal phrase composition revealed by intracranial recordings The ability to comprehend phrases is an essential integrative property of the brain. Here we evaluate the neural processes that enable the transition from single word processing to a minimal compositional scheme. Previous research has reported conflicting timing effects of composition, and disagreement persists with respect to inferior frontal and posterior temporal contributions. To address these issues, 19 patients (10 male, 19 female) implanted with penetrating depth or surface subdural intracranial electrodes heard auditory recordings of adjective-noun, pseudoword-noun and adjective-pseudoword phrases and judged whether the phrase matched a picture. Stimulus-dependent alterations in broadband gamma activity, low frequency power and phase-locking values across the language-dominant left hemisphere were derived. This revealed a mosaic located in the posterior superior temporal sulcus (pSTS), in which closely neighboring cortical sites displayed exclusive sensitivity to either lexicality or phrase structure, but not both. Distinct timings were found for effects of phrase composition (210-300 ms) and pseudoword processing (approximately 300-700 ms), and these were localized to neighboring electrodes in pSTS. The pars triangularis and temporal pole encode anticipation of composition in broadband low frequencies, and both regions exhibit greater functional connectivity with pSTS during phrase composition. Our results suggest that the pSTS is a highly specialized region comprised of sparsely interwoven heterogeneous constituents that encodes both lower and higher level linguistic features. This hub in pSTS for minimal phrase processing may form the neural basis for the human-specific computational capacity for forming hierarchically organized linguistic structures.

SignificanceLinguists have claimed that the integration of multiple words into a phrase demands a computational procedure distinct from single word processing. Here, we provide intracranial recordings from a large patient cohort, with high spatiotemporal resolution, to track the cortical dynamics of phrase composition. Epileptic patients volunteered to participate in a task in which they listened to phrases ("red boat"), word-pseudoword or pseudoword-word pairs (e.g., "red fulg"). At the onset of the second word in phrases, greater broadband high gamma activity was found in posterior superior temporal sulcus in electrodes that exclusively indexed phrasal meaning, and not lexical meaning. These results provide direct, high-resolution signatures of minimal phrase composition in humans, a potentially species-specific computational capacity.

sentences: Photons guided by axons may enable backpropagation-based learning in the brain Despite great advances in explaining synaptic plasticity and neuron function, a complete understanding of the brains learning algorithms is still missing. Artificial neural networks provide a powerful learning paradigm through the backpropagation algorithm which modifies synaptic weights by using feedback connections. Backpropagation requires extensive communication of information back through the layers of a network. This has been argued to be biologically implausible and it is not clear whether backpropagation can be realized in the brain. Here we suggest that biophotons guided by axons provide a potential channel for backward transmission of information in the brain. Biophotons have been experimentally shown to be produced in the brain, yet their purpose is not understood. We propose that biophotons can propagate from each post-synaptic neuron to its pre-synaptic one to carry the required information backward. To reflect the stochastic character of biophoton emissions, our model includes the stochastic backward transmission of teaching signals. We demonstrate that a three-layered network of neurons can learn the MNIST handwritten digit classification task using our proposed backpropagation-like algorithm with stochastic photonic feedback. We model realistic restrictions and show that our system still learns the task for low rates of biophoton emission, information-limited (one bit per photon) backward transmission, and in the presence of noise photons. Our results suggest a new functionality for biophotons and provide an alternate mechanism for backward transmission in the brain.

sentences: Nucleoporin107 mediates female sexual differentiation via Dsx We recently identified a missense mutation in Nucleoporin107 (Nup107; D447N) underlying XX-ovarian-dysgenesis, a rare disorder characterized by underdeveloped and dysfunctional ovaries. Modelling of the human mutation in Drosophila or specific knockdown of Nup107 in the gonadal soma resulted in ovarian-dysgenesis-like phenotypes. Transcriptomic analysis identified the somatic sex-determination gene doublesex (dsx) as a target of Nup107. Establishing Dsx as a primary relevant target of Nup107, either loss or gain of Dsx in the gonadal soma is sufficient to mimic or rescue the phenotypes induced by Nup107 loss. Importantly, the aberrant phenotypes induced by compromising either Nup107 or dsx are reminiscent of BMP signaling hyperactivation. Remarkably, in this context, the metalloprotease AdamTS-A, a transcriptional target of both Dsx and Nup107, is necessary for the calibration of BMP signaling. As modulation of BMP signaling is a conserved critical determinant of soma-germline interaction, the sex and tissue specific deployment of Dsx-F by Nup107 seems crucial for the maintenance of the homeostatic balance between the germ cells and somatic gonadal cells.

sentences: The neurocomputational architecture of explore-exploit decision making Humans and other animals often make the difficult decision to try new options (exploration) and forego immediate rewards (exploitation). Novelty-seeking is an adaptive solution to this explore-exploit dilemma, but our understanding of the neural computations supporting novelty-seeking in humans is limited. Here, we presented the same explore-exploit decision making task to monkeys and humans and found evidence that the computational basis for novelty-seeking is conserved across primate species. Critically, through computational model-based decomposition of event-related functional magnetic resonance imaging (fMRI) in humans, these findings reveal a previously unidentified cortico-subcortical architecture mediating explore-exploit behavior in humans.

sentences: The evolution of ovary-specific gene expression in Hawaiian Drosophilidae 1With detailed data on gene expression accessible from an increasingly broad array of species, we can test the extent to which our developmental genetic knowledge from model organisms predicts expression patterns and variation across species. But to know when differences in gene expression across species are significant, we first need to know how much evolutionary variation in gene expression we expect to observe. Here we provide an answer by analyzing RNAseq data across twelve species of Hawaiian Drosophilidae flies, focusing on gene expression differences between the ovary and other tissues. We show that over evolutionary time, there exists a cohort of ovary specific genes that is stable and that largely corresponds to described expression patterns from laboratory model Drosophila species. However, our results provide a demonstration of the prediction that, as phylogenetic distance increases, variation between species overwhelms variation between tissue types. Using ancestral state reconstruction of expression, we describe the distribution of evolutionary changes in tissue-biased expression, and use this to identify gains and losses of ovary expression across these twelve species. We then use this distribution to calculate the evolutionary correlation in expression changes between genes, and demonstrate that genes with known interactions in D. melanogaster are significantly more correlated in their evolution than genes with no or unknown interactions. Finally, we use this correlation matrix to infer new networks of genes that share evolutionary trajectories, and we present these results as a dataset of new testable hypotheses about genetic roles and interactions in the Drosophila ovary.

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Specific Adaptive Gene Expression of Smooth Muscle Cells Challenged by Organotelluranes. Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.

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sentences: Disruption Of Proprioceptive Information During Electrical Stimulation Of The Cutaneous Afferents Restoration of proprioception with neurotechnology is critical to improve effectiveness of robotic neuro-prostheses. Unfortunately, after initial enthusiasm clinical results showed that unlike touch, proprioception could not be reliably induced. Here we show that concurrent activation of multiple sensory modalities may trigger unwanted sensory regulation mechanisms that disrupt proprioception. We recorded intra-spinal neural activity induced by stimulation of proprioceptive afferents from the radial nerve in three monkeys. Then, we superimposed stimulation of the radial nerve cutaneous branch and quantified its impact on spinal neural activity via population analysis. Proprioceptive pulses produced robust neural trajectories in the neural manifold that were disrupted by concurrent stimulation of cutaneous afferents. This disruption correlated with a reduction of afferent volleys and multi-unit activity both in the spinal cord and somatosensory cortex. Our results suggest that limited specificity not only impacts localization of artificial percepts, but also their nature to an extent that was never considered.

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Investigating the effect of obesity on adipose-derived stem cells (ASCs) using Gottingen Minipigs Obesity is associated with low grade inflammation, which may adversely impact the biological functions of adipose tissue and consequently of adipose-derived stem cells (ASCs). Studies in humans and rodents have described that obesity alters ASC properties and functionality, compromising their therapeutic prospects. The Gottingen Minipig (GM) is a commonly used obesity model. Nevertheless, there are no studies investigating the effect of obesity on ASCs from GM, which could constitute a valuable addition to both obesity modelling and adult stem cells investigations.

In this study, we isolated subcutaneous ASCs from lean and obese GM to investigate the effect of obesity on cell behavior and differentiation capacity. During culturing, we observed an inherent difference in cell morphology between lean and obese ASCs. Upon adipogenic induction, obese-ASCs readily differentiated, developing significantly larger amounts of adipocytes than corresponding lean-ASCs, hinting at a predisposition towards adipogenic differentiation. Expression profiling of obesity-related genes in cell cultures, before and after adipogenic differentiation, revealed a tendency towards up-regulation in differentiated obese-cultures. Altogether, our results indicate that stem cells from obese donors could display different therapeutic properties. In summary, our results point towards GM as a valuable model for future ASCs investigations in healthy and obese states.

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

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New insights of glycosylation role on variable domain of antibody structures N-glycosylation at antibody variable domain (FvN-glyco) has emerged as an important modification for antibody function such as stability and antigen recognition, but it is also associated with autoimmune disease and IgE-mediated hypersensitivity reaction. However, the information related to its role and regulation is still scarce. Therefore, we investigated new insights in this regarding using solved antibodies structures presenting in the Protein Data Bank (PDB). From 130 FvN-glyco structures, we observed significant findings as a higher prevalence of N-glycosylation in human and mouse antibodies containing IGHV1-8 and IGHV2-2 germline genes, respectively. We also speculate the influence of activation-induced cytidine deaminase (AID) in introducing N-glycosylation sites during somatic hypermutation, specifically on threonine to asparagine substitution. Moreover, we highlight the enrichment of anti-HIV antibodies containing N-glycosylation at antibody variable domain and where we showed a possible important role of N-glycosylation, besides to antigen-antibody interactions, in antibody chain pair and antibody-antibody interactions. These could be a positive secondary effect of glycosylation to enhance antigen binding and further neutralization, including an additional mechanism to form Fab-dimers. Overall, our findings extend the knowledge on the characteristics and diverse role of N-glycosylation at antibody variable domain.

Key PointsO_LIPrevalence of FvN-glyco in human IGHV1-8 and mouse IGHV2-2 germline genes.
C_LIO_LIEnrichment of antibody FvN-glyco against virus, especially anti-HIV-1.
C_LIO_LIFvN-glyco in the interface with antigen, antibody pair chain, and another antibody.
C_LI

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Development and validation of a mouse model of contemporary cannabis smoke exposure Cannabis is widely used for both recreational and medicinal purposes. Inhalation of combusted cannabis smoke is the most common mode of drug consumption, exposing the lungs to the pharmacologically active ingredients, including tetrahydrocannabinol (THC) and cannabidiol (CBD). While the relationship between cannabis smoke exposure and compromised respiratory health has yet to be sufficiently defined, previous investigations suggest that cannabis smoke may dysregulate pulmonary immunity. Presently, there exists few pre-clinical animal models that have been extensively validated for contemporary cannabis smoke exposure. To address this need, we developed a mouse model with readouts of total particular matter, serum cannabinoid and carboxyhemoglobin levels, lung cellular responses, and immune mediator production.

Using a commercially available smoke exposure system and a cannabis source material of documented THC/CBD composition, we exposed mice to a total particulate matter of 698.89 (SD = 66.09) {micro}g/L and demonstrate increases in serum cannabinoids and carboxyhemoglobin. We demonstrate that cannabis smoke modulates immune cell populations and mediators in both male and female BALB/c mice. This modulation is highlighted by increases in airway and lung tissue macrophage populations, including tissue-resident alveolar macrophages, monocyte-derived alveolar macrophages, and interstitial macrophage subpopulations. No changes in airway or lung tissue infiltration of neutrophils were observed. Immune mediator analysis indicated significant upregulation of MDC, TARC, and VEGF within the lung tissue of cannabis smoke exposed mice. This accessible and reproducible smoke exposure model provides a foundation to explore the impact of chronic cannabis exposures and/or co-exposures with pathogens of clinical relevance, such as influenza.

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A heterohexameric protein consisting of six linked single-domain antibodies is highly protective for BoNT/A, BoNT/B and BoNT/E exposures Botulinum neurotoxin (BoNT) serotypes A, B and E cause the vast majority of human botulism cases and pose the greatest bioterrorism threats. We previously identified multiple camelid single-domain antibodies (VHHs) that each neutralize BoNT/A, BoNT/B or BoNT/E. We also demonstrated that heterodimers of linked toxin-neutralizing VHHs are much more potent than VHH monomer pools in preventing BoNT intoxication. In this study, we expressed two different heterohexamer proteins (VNA1-ABE and VNA2-ABE) of ~100 kDa secreted from mammalian host cells, each containing the same six linked anti-BoNT VHH components ordered in two different combinations. Each heterohexamer contained two VHHs that neutralize BoNT/A, BoNT/B or BoNT/E. Both heterohexameric antitoxins displayed similar strong binding properties for the three targeted BoNT serotypes by ELISA. One ug of each heterohexameric antitoxin fully protected groups of mice co-administered with 100 LD50 of BoNT/A, BoNT/B or BoNT/E, or a pool containing 100 LD50 of each of the three toxins. The results demonstrate that long chains of at least six different linked VHHs can be expressed such that all component VHHs in the multimer retain their target binding activities. These findings make more feasible the development of a BoNT antitoxin product consisting of a small pool of proteins that, in combination, neutralize all known BoNT serotypes and subtypes.

Key ContributionHeteromultimeric proteins consisting of six linked, VHH antibodies, and including VHHs that neutralize BoNT/A, BoNT/B and BoNT/E, retain high potency to protect mice challenged with high doses of all three of these BoNT serotypes.

sentences: A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

sentences: Multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and - independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

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The renal inflammatory network of nephronophthisis STRUCTURED ABSTRACTO_ST_ABSBACKGROUNDC_ST_ABSThe majority of genetic kidney disease leading to kidney failure is caused by mutations in ciliary genes. How cilia malfunction leads to progressive kidney damage is poorly understood, but recent evidence links ciliopathy genes to CCL2 dependent macrophage recruitment in autosomal dominant polycystic kidney disease (ADPKD), the most studied renal ciliopathy. Whether or not renal inflammation is involved in other renal ciliopathies is unclear.

METHODSWe combined mice models with kidney biopsies and renal epithelial cells sampled from human urine to characterize the renal inflammatory network of nephronophthisis (NPH), the most frequent renal ciliopathy in children.

RESULTSIn human, mutations in cilia genes involved in NPH enhance urine excretion of the chemokine CCL2, causing abnormal macrophage recruitment in kidney tissues from NPH patients. Differing from ADPKD, inactivating Ccl2 specifically in mouse tubular cells does not rescue the NPH phenotype, suggesting that other inflammatory mediators are involved. Using transcriptional data from 2 NPH models, we identify a set of pro-inflammatory cytokines upregulated in this disease, independently of CCL2. The majority of detectable transcripts from this set are specifically upregulated in kidney cells from NPH patients. In line with the function of these cytokines, NPH kidneys show disproportionate neutrophils and T cells infiltrates compared to healthy subject or hypertensive and diabetic chronic kidney disease patients.

CONCLUSIONSThis study reveals that inflammation is a central aspect in human NPH and delineates a specific set of inflammatory mediators that regulates immune cell recruitment in human NPH.

SIGNIFICANCE STATEMENTMutations in genes encoding primary cilia proteins are the leading cause of genetic kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), deregulated cilia signaling leads to kidney infiltration by macrophages through the chemokine CCL2. Little is known about renal inflammation in nephronophthisis (NPH), the most frequent pediatric renal ciliopathy. Using NPH mice models, tissues and cells from NPH patients, we unveil renal inflammation as preeminent feature of NPH. Remarkably, the renal inflammatory evoked by ciliary gene mutations in NPH does not overlap with ADPKD: it is CCL2 independent, involves a prominent recruitment of neutrophils and T cells and a specific cytokine signature. This unforeseen findings strengthen the link between primary cilia and renal inflammation.

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Comparative proximity biotinylation implicates RAB18 in cholesterol mobilization and biosynthesis Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 25 RAB18- interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. Consistent with a role for RAB18 in regulating membrane contact sites (MCSs), interactors included groups of microtubule/membrane-remodelling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. We provide direct evidence validating novel interactions with SEC22A and TMCO4. We also provide functional evidence supporting identified interactions with OSBPL2/ORP2, INPP5B and the {Delta}8-{Delta}7 sterol isomerase emopamil binding protein (EBP). Notably, the cholesterol precursor and EBP-product lathosterol accumulates in RAB18-null cells. Moreover, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

SUMMARY STATEMENTWe used proximity biotinylation together with guanine nucleotide exchange factor (GEF)-null cell lines to discriminate functional RAB18-interactions. Our subsequent functional work suggests RAB18 activity influences cholesterol biosynthesis.

sentences: The autophagy initiating kinase ULK1 is required for pancreatic cancer cell growth and survival Amongst cancer subtypes, pancreatic ductal adenocarcinoma (PDA) has been demonstrated to be most sensitive to autophagy inhibition, which may be due to unique metabolic rewiring in these cells. The serine/threonine kinase ULK1 forms the catalytic center of a complex mediating the first biochemical step of autophagy. ULK1 directly recieves signals from mTORC1 and AMPK to trigger autophagy under stress and nutrient poor conditions. Studies in genetic engineered mouse models of cancer have revealed that deletion of core downstream autophagy genes (ATG5, ATG7) at the time of tumor iniation leads to a profound block in tumor progression leading to the development of autophagy inhibitors as cancer therapeutics. However, most preclinical studies and all clinical studies have relied on non-specific lysomotropic agents such as chloroquine and its derivatives, whose toxicity and off-target issues preclude further clinical development and which do not represent the impact of solely biochemically disrupting the autophagy pathway. Furthermore, druggable targets in the core autophagy pathway are quite limited, with ULK1 and ULK2 representing the only protein kinases in the pathway. Here we explore the genetic requirement for ULK1 and ULK2 in human PDA cancer cell lines and xenografts, and take advantage of new small molecule ULK1 inhibitors to demonstrate that ULK inhibition can overcome autophagy induction triggered by PDA therapeutics including chemotherapy and MEK inhibition. Finally we show that ULK inhibitors increase MHC Class I in PDA cells, suggestion a potential therapeutic avenue for such agents in combination with checkpoint immunotherapy.

sentences: Dissection of function and recognition mechanism of M. tuberculosis ESX-1 secreted virulence factor EspC Mycobacterium tuberculosis uses the ESAT-6 system-1/type VII (ESX-1) system for secretion of virulence proteins into the host cell, however the mechanism of virulence proteins secretion, molecular components and regulation of ESX-1 system are only partly understood. In the current study, we have analyzed the biological function and recognition mechanism between ESX-1 virulence EspC and EccA1 ATPase proteins. The EspC enters into A549 human lung carcinoma cells and exhibited cytotoxicity, as observed in MTT Assay. To understand the recognition mechanism between EspC and EccA1 ATPase, the EspC and EccA1 mutants were generated based on EspC~EccA1 interactions, as observed in molecular modeling. Binding analysis shows that EspC export arm interacts specifically to the {beta}-hairpin insertion motif of the TPR domain of EccA1 ATPase. Mutations in these epitopes lead to significant decrease/or abolish the binding between EspC and EccA1 ATPase. Our study provides insight into biological function and recognition mechanism between EspC and EccA1 ATPase, which can be used as target to prevent EspC secretion/ or in general virulence factor secretion by mycobacterial ESX-1 system.

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Small molecule screen employing patient-derived iPS hepatocytes identifies LRRK2 as a novel therapeutic target for Alpha1 Antitrypsin Deficiency Alpha-1 antitrypsin deficiency is a life-threatening condition caused by inheritance of the SERPINA1 gene  Z variant. This single base pair mutation leads to protein misfolding, ER entrapment and gain of toxic function. Despite the significant unmet medical need presented by this disorder, there remain no approved medicines and the only curative option is liver transplantation. We hypothesized that an unbiased screen of human hepatocytes harbouring the Z mutation (ATZ) using small molecules targeted against protein degradation pathways would uncover novel biological insights of therapeutic relevance. Here we report the results of that screen performed in a patient-derived iPSC model of ATZ. Starting from 1,041 compounds we identified 14 targets capable of reducing polymer burden, including Leucine-rich repeat kinase-2 (LRRK2), a well-studied target in Parkinsons. Genetic deletion of LRRK2 in ATZ mice reduced polymers and associated fibrotic liver disease leading us to test a library of commercially available LRRK2 kinase inhibitors in both patient iPSC and CHO cell models. One of the molecules tested, CZC-25146, reduced polymer load, increased normal AAT secretion and reduced inflammatory cytokines with pharmacokinetic properties supporting its potential use for treating liver diseases. We therefore tested CZC-25146 in the ATZ mouse model and confirmed its efficacy for polymer reduction without signs of toxicity. Mechanistically, in both human and mouse models, our data show CZC-25146 inhibits LRRK2 kinase activity and induces autophagy. Cumulatively, these findings support the use of CZC-25146 and LRRK2 inhibitors in general in hepatic proteopathy disease research and as potential new treatment approaches for patients.

One Sentence SummaryA small molecule screen in patient iPSCs with in vivo validation in mice identifies LRRK2 as a new therapeutic target for Alpha-1 Antitrypsin Deficiency.

sentences: HTSQualC is a Flexible and One-Step Quality Control Software for High-throughput Sequencing Data Analysis Use of high-throughput sequencing (HTS) has become indispensable in life science research. Raw HTS data contains several sequencing artifacts, and as a first step it is imperative to remove the artifacts for reliable downstream bioinformatics analysis. Although there are multiple stand-alone tools available that can perform the various quality control steps separately, availability of an integrated tool that can allow one-step, automated quality control analysis of HTS datasets will significantly enhance handling large number of samples parallelly. Here, we developed HTSQualC, a stand-alone, flexible, and easy-to-use software for one-step quality control analysis of raw HTS data. HTSQualC can evaluate HTS data quality and perform filtering and trimming analysis in a single run. We evaluated the performance of HTSQualC for conducting batch analysis of HTS datasets with 322 samples with an average [~]1M (paired end) sequence reads per sample. HTSQualC accomplished the QC analysis in [~]3 hours in distributed mode and [~]31 hours in shared mode, thus underscoring its utility and robust performance. In addition to command-line execution, we integrated HTSQualC into the free, open-source, CyVerse cyberinfrastructure resource as a GUI interface, for wider access to experimental biologists who have limited computational resources and/or programming abilities.

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Omicron-Based Vaccine Candidate Elicits Potent Neutralizing Antibodies in the Animal Model The B.1.1.529 (Omicron) variant of SARS-CoV-2 with multiple novel mutations has reduced the neutralization potential of vaccinated individuals sera. World Health Organization has suggested that a vaccination strategy based on repeated booster doses is unlikely to be sustainable. The objective of this commentary was to investigate the safety and neutralizing antibody induction of the Omicron-based SARS-CoV-2 vaccine candidate, BIV1-CovIran Plus, against the Omicron variant on mice and guinea pig models.

After isolation and characterization, the Omicron variant underwent chemical inactivation, purification, and was then formulated with alum adjuvant. A full human dose of BIV1-CovIran Plus was injected intraperitoneally to five female mice and two Guinea pigs for abnormal toxicity reactions evaluation and pathologic investigations. For potency evaluation, four groups of ten mice received two doses of BIV1-CovIran Plus or phosphate-buffered-saline at 7-day and 14-day intervals. The conventional virus-neutralizing test was conducted on sera acquired from vaccinated mice groups seven days after the second injection.

There was no evidence of abnormal clinical symptoms macroscopic or microscopic tissue alterations among the animal models. In all samples from the study group that received two doses of BIV1-CovIran Plus at a 7-day interval, the sera at [≥]1/32 times dilution would neutralize the Omicron variant SARS-CoV-2. Similarly, the sera of all samples from the study group, which received two doses of BIV1-CovIran Plus at a 14-day interval, at [≥]1/64 times dilution, would neutralize the Omicron variant SARS-CoV-2. Moreover, six out of ten (60%) of the samples in this group would neutralize the Omicron variant of SARS-CoV-2 at 1/128 times dilution. CPE formation was observed in all samples from the control group, and no neutralizing activity was detected at any sera dilutions.

BIV1-CovIran Plus was well-tolerated in the animal models, and no safety concerns were raised. Moreover, the vaccine candidate elicited protective neutralization against the Omicron variant. Future reports will focus on the use of the updated vaccine as a booster dose and the potency of the vaccine candidate on other SARS-CoV-2 variants.

sentences: The Streptochaeta genome and the evolution of the grasses In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size, capturing most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses ({rho}) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

sentences: Emergent periodicity in the collective synchronous flashing of fireflies Left alone, Photinus carolinus fireflies flash without an intrinsic period, making it uncertain when they may flash next. Yet when gathering at the mating lek in large swarms, these fireflies transition into predictability, synchronizing with their neighbors with a rhythmic periodicity. Here we propose a mechanism for emergence of synchrony and periodicity, and formulate the principle in a mathematical framework. Remarkably, with no fitting parameters, analytic predictions from this simple principle and framework agree strikingly well with data. Next, we add further sophistication to the framework using a computational model featuring groups of random oscillators via integrate-and-fire interactions controlled by a tunable parameter. This agent-based model of P. carolinus fireflies interacting in swarms of increasing density also shows quantitatively similar phenomenology and reduces to the analytic framework in the appropriate limit of the tunable parameter. We discuss our findings and note that the resulting dynamics follow the style of a decentralized follow-the-leader synchronization, where any of the randomly flashing individuals may take the role of the leader of any subsequent synchronized flash burst.

sentences: A deep network-based model of hippocampal memory functions under normal and Alzheimer's disease conditions We present a deep network-based model of the associative memory functions of the hippocampus. The proposed network architecture has two key modules: 1) an autoencoder module which represents the forward and backward projections of the cortico-hippocampal projections and 2) a module that computes familiarity of the stimulus and implements hill-climbing over the familiarity which represents the dynamics of the loops within the hippocampus. The proposed network is used in two simulation studies. In the first part of the study, the network is used to simulate image pattern completion by autoassociation under normal conditions. In the second part of the study, the proposed network is extended to a heteroassociative memory and is used to simulate picture naming task in normal and Alzheimers disease (AD) conditions. The network is trained on pictures and names of digits from 0 - 9. The encoder layer of the network is partly damaged to simulate AD conditions. As in case of AD patients, under moderate damage condition, the network recalls superordinate words ("odd" instead of "nine"). Under severe damage conditions, the network shows a null response ("I dont know"). Neurobiological plausibility of the model is extensively discussed.

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Diffuse optical reconstructions of NIRS data using Maximum Entropy on the Mean Functional near-infrared spectroscopy (fNIRS) measures the hemoglobin concentration changes associated with neuronal activity. Diffuse optical tomography (DOT) consists of reconstructing the optical density changes measured from scalp channels to the oxy-/deoxy-hemoglobin (i.e., HbO/HbR) concentration changes within the cortical regions. In the present study, we adapted a nonlinear source localization method developed and validated in the context of Electro- and Magneto-Encephalography (EEG/MEG): the Maximum Entropy on the Mean (MEM), to solve the inverse problem of DOT reconstruction. We first introduced depth weighting strategy within the MEM framework for DOT reconstruction to avoid biasing the reconstruction results of DOT towards superficial regions. We also proposed a new initialization of the MEM model improving the temporal accuracy of the original MEM framework. To evaluate MEM performance and compare with widely used depth weighted Minimum Norm Estimate (MNE) inverse solution, we applied a realistic simulation scheme which contained 4000 simulations generated by 250 different seeds at different locations and 4 spatial extents ranging from 3 to 40cm2 along the cortical surface. Our results showed that overall MEM provided more accurate DOT reconstructions than MNE. Moreover, we found that MEM was remained particularly robust in low signal-to-noise ratio (SNR) conditions. The proposed method was further illustrated by comparing to functional Magnetic Resonance Imaging (fMRI) activation maps, on real data involving finger tapping tasks with two different montages. The results showed that MEM provided more accurate HbO and HbR reconstructions in spatial agreement with the main fMRI cluster, when compared to MNE.

HighlightsO_LIWe introduced a new fNIRS reconstruction method - Maximum Entropy on the Mean.
C_LIO_LIWe implemented depth weighting strategy within the MEM framework.
C_LIO_LIWe improved the temporal accuracy of the original MEM reconstruction.
C_LIO_LIPerformances of MEM and MNE were evaluated with realistic simulations and real data.
C_LIO_LIMEM provided more accurate and robust reconstructions than MNE.
C_LI

sentences: Relationships between acute race-induced changes in creatine kinase activity and blood plasma myoglobin concentration and race performance in mountain bike and road cyclists The aim of this study was to determine if the changes in plasma creatine kinase (CK) activity and myoglobin (MB) concentrations as markers of muscle damage differ between competitive road (n = 14) and mountain bike (n = 11) cyclists and if these biochemical markers show a relationship with real-world race performance. CK and MB were measured from blood samples collected 2 hours before race start and 1 hour after race completion and the change in pre- and post-race difference was calculated ({Delta}CK and {Delta}MB). An incremental exercise test was used to determine maximal oxygen uptake, maximal aerobic power, and power output at the second ventilatory threshold. Post-race CK and MB increased in the whole group of cyclists. Although the magnitude of change in CK was similar in both road and mountain bike cyclists, only the increase in road cyclists was significant. MB significantly increased only in mountain bike cyclists. Multiple regression analysis revealed a significant association between both road and mountain bike race performance and {Delta}CK and {Delta}MB. The other significant predictors for mountain bike race performance were maximal aerobic power (W{middle dot}kg-1) and power output at the second ventilatory threshold (W{middle dot}kg-1) and for road race performance both maximal oxygen uptake (l{middle dot}min-1) and power output at the second ventilatory threshold (W). In conclusion, mountain bike racing was associated with an increase in MB whereas road racing with an increase in CK, with the post-race changes in CK and MB related to race performance as high {Delta}CK and low {Delta}MB were obtained by better-performing cyclists.

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A Simulator for Somatic Evolution Study Design MotivationSomatic evolution plays a key role in development, cell differentiation, and normal aging, but also diseases such as cancer, which is now mainly thought of as a disease of genetic and epigenetic modification. Understanding mechanisms of somatic mutability -- variant types and frequencies, phylogenetic structure, mutational signatures, and clonal heterogeneity -- and how they can vary between cell lineages will likely play a crucial role in biological discovery and medical applications. This need has led to a proliferation of new technologies for profiling single-cell variation, each with distinctive capabilities and limitations that can be leveraged alone or in combination with other technologies. The enormous space of options for assaying somatic variation, however, presents unsolved informatics problems with regards to selecting optimal combinations of technologies for designing appropriate studies for any particular scientific questions. Versatile simulation tools are needed to make it possible to explore and optimize potential study designs if researchers are to deploy multiomic technologies effectively.

ResultsIn this paper, we present a simulator allowing for the generation of synthetic data from a wide range of clonal lineages, variant classes, and sequencing technology choices, intended to provide a platform for effective study design in somatic lineage analysis. Our simulation framework allows for the assessment of study design setups and their statistical validity in determining different ground-truth cancer mechanisms. The user is able to input various properties of the somatic evolutionary system, mutation classes (e.g., single nucleotide polymorphisms, copy number changes, and classes of structural variation), and biotechnology options (e.g., coverage, bulk vs single cell, whole genome vs exome, error rate, number of samples) and can then generate samples of synthetic sequence reads and their corresponding ground-truth parameters for a given study design. We demonstrate the utility of the simulator for testing and optimizing study designs for various experimental queries.

Contactrussells@andrew.cmu.edu

Availabilityhttps://github.com/CMUSchwartzLab/MosaicSim

sentences: High temperature molecular motions within a model protomembrane architecture Modern phospholipid membranes are known to be in a functional, physiological state, corresponding to the liquid crystalline phase, only under very precise external conditions. The phase is characterised by specific lipid motions, which seem mandatory to permit sufficient flexibility and stability for the membrane. It can be assumed that similar principles hold for proto-membranes at the origin of life although they were likely composed of simpler, single chain fatty acids and alcohols. In the present study we investigated molecular motions of four types of model membranes to shed light on the variations of dynamics and structure as a function of temperature as protocells might have existed close to hot vents. We find a clear hierarchy among the flexibilities of the samples, where some structural parameters seem to depend on the lipids used while others do not.

sentences: A digitization theory of the Weber-Fechner law Ever since the publication of Shannons article about information theory, there have been many attempts to apply information theory to the neuroscience field. Meanwhile, the Weber-Fechner law of psychophysics states that the magnitude of a subjective sensation of a person increases in proportion to the logarithm of the intensity of the external physical-stimulus. It is hardly surprising that we assign the amount of information to the response in the Weber-Fechner law. But to date no one has succeeded in applying information theory directly to that law: the direct links between information theory and that response in the Weber-Fechner law have not yet been found. The proposed theory unveils a link between information theory and that response, and differs subtly from the field such as neural coding that involves complicated calculations and models. Because my theory targets the Weber-Fechner law which is a macroscopic phenomenon, this theory does not involve complicated calculations. My theory is expected to mark a new era in the fields of sensory perception research. My theory must be studied in parallel with the fields of microscopic scale such as neural coding. This article ultimately aims to provide the fundamental concepts and their applications so that a new field of research on stimuli and responses can be created.

sentences: Encapsulated actomyosin patterns drive cell-like membrane shape changes Cell shape changes from locomotion to cytokinesis are, to a large extent, driven by myosin-driven remodeling of cortical actin patterns. Passive crosslinkers such as -actinin and fascin as well actin nucleator Arp2/3 complex largely determine the architecture and connectivity of actin network patterns; consequently, they regulate network remodeling and membrane shape changes. Membrane constriction in animal cell cytokinesis proceeds by assembly and contraction of a contractile ring pattern rich in -actinin and myosin at the equator of the cell cortex, with which the ring is contiguous. Here we reconstitute actomyosin networks inside cell-sized lipid bilayer vesicles and show that, depending on vesicle size and concentrations of -actinin and fascin, actomyosin networks assemble into ring and aster-like patterns. Anchoring actin to the membrane enhances the interaction of the contractile networks with lipid membrane but does not change the architecture of the patterns. A membrane-bound actomyosin ring exerts force and constricts the membrane. An Arp2/3 complex-mediated actomyosin cortex is shown to assemble a ring-like pattern at the equatorial cortex and contribute to myosin-driven clustering of the cortex and consequently membrane deformation. An active gel theory unifies a model for the observed membrane constriction and protrusion induced by the membrane-bound actomyosin networks.

sentences: specificity	: an R package for generalized specificity analysis, with example analyses of Hawaiian foliar endophytic Fungi, human gut microbiome bacteria, and Antarctic glacier bacteria. Understanding the factors that influence microbes environmental distributions is important for determining drivers of microbial community composition. Species distributions are governed by parameters that influence their dispersal or survival. These include environmental variables like temperature and pH, and higher-dimensional variables like geographic distance and host species phylogenies. In microbial ecology, "specificity" is often described in the context of symbiotic or host parasitic interactions, but specificity can be more broadly used to describe the extent to which a species occupies a narrower range of an environmental variable than expected by chance. Using a standardization we describe here, Raos (1982, 2010) Quadratic Entropy can be conveniently applied to calculate specificity of a focal species to many different environmental variables, including 1-dimensional variables (vectors), dissimilarity matrices, phylogenies, and ontologies. We present our R package specificity for performing the above analyses, and apply it to four real-life microbial datasets to demonstrate its application: fungi living within the leaves of native Hawaiian plants, bacteria from the human gut microbiome, bacteria living within Antarctic glacier ice, and the Earth Microbiome Project data set. Finally, we present an interactive visualization and data exploration tool for these analyses, available as a companion R-package called specificity.shiny.

sentences: A topological approach to DNA similarity analysis from 5-dimensional representation In this paper, we propose another topological approach for DNA similarity analysis. For each DNA sequence, we transform it into a collection of vectors in 5-dimensional space in which all nucleotides of the same type, say A, C, G, T are on the same line in this 5D space. Based on this special geometric property, we combine this representation with tools in persistent homology to obtain only zeroth persistence diagrams as a topological representation of DNA sequences. Similarities between DNA sequences are signified via how close the representing zeroth persistence diagrams of the DNA sequences are, based on the Wasserstein distance of order zero, which provides a new method for analyzing similarities between DNA sequences. We test our methods on the datasets of Human rhinovirus (HRV) and Influenza A virus.

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Expansion of mutation-driven haematopoietic clones is associated with insulin resistance and low HDL-cholesterol in individuals with obesity AimsHaematopoietic clones caused by somatic mutations with [≥]2% variant allele frequency (VAF), known as clonal haematopoiesis of indeterminate potential (CHIP), increase with age and have been linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones are also associated with adverse clinical outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes, and to examine the development of clones over time in relation to age and metabolic dysregulation over up to 20 years in individuals with obesity.

Methods and ResultsWe used an ultrasensitive single-molecule molecular inversion probe sequencing assay to identify clonal haematopoiesis driver mutations (CHDMs) in blood samples from individuals with obesity from the Swedish Obese Subjects study. In a single-timepoint dataset with samples from 1050 individuals, we identified 273 candidate CHDMs in 216 individuals, with VAF ranging from 0.01% to 31.15% and CHDM prevalence and clone sizes increasing with age. Longitudinal analysis over 20 years in CHDM-positive samples from 40 individuals showed that small clones can grow over time and become CHIP. VAF increased on average by 7% (range -4% to 27%) per year. Rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low circulating levels of high-density lipoprotein-cholesterol (HDL-C) (R=-0.68, P=1.74E-05).

ConclusionOur results show that haematopoietic clones can be detected and monitored before they become CHIP and indicate that insulin resistance and low HDL-C, well-established cardiovascular risk factors, are associated with clonal expansion in individuals with obesity.

Translational perspectivesClonal haematopoiesis-driver mutations are somatic mutations in haematopoietic stem cells that lead to clones detectable in peripheral blood. Haematopoietic clones with a variant allele frequency (VAF) [≥]2%, known as clonal haematopoiesis of indeterminate potential (CHIP), are recognized as an independent cardiovascular risk factor. Here, we show that smaller clones are prevalent, and also correlate with age. Our longitudinal observations in individuals with obesity over 20 years showed that more than half of all clone-positive individuals show growing clones and clones with VAF <2% can grow and become CHIP. Importantly, clone growth was accelerated in individuals with insulin resistance and low high-density lipoprotein-cholesterol (HDL-C).

Translational outlook 1: Haematopoietic clones can be detected and monitored before they become CHIP.

Translational outlook 2: The association between insulin resistance and low HDL-C with growth of haematopoietic clones opens the possibility that treatments improving metabolism, such as weight loss, may reduce growth of clones and thereby cardiovascular risk.

One Sentence SummaryIn obesity, the growth rate of mutation-driven haematopoietic clones increased with insulin resistance and low HDL-C, both known risk factors for cardiovascular disease.

sentences: Designer Scaffolds for Bioengineered Tissue Interfaces In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Here, we study these complex living interfaces by manufacturing modular "blocks" of naturally porous decellularized plant-derived scaffolds of varying shapes and sizes with a computer numerical controlled mill. We demonstrate how each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). Cells were observed to migrate across the interface formed between an empty scaffold and a scaffold pre-seeded with cells. However, when both scaffolds contained cells, only a shallow cross-over zone of cell infiltration formed at the interface. As a proof-of-concept study, we utilized ETIs to investigate the interaction between lab grown bone and connective tissues. Consistent with the above, a cross-over zone of the two distinct cell types formed at the interface between scaffolds, otherwise the populations remained distinct. Finally, we demonstrate how ETIs are biocompatible in vivo, becoming vascularized and integrated into surrounding tissue after implantation. This work creates new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues.

sentences: Sensitive high-throughput assays for tumour burden reveal the response of a Drosophila melanogaster model of colorectal cancer to standard chemotherapies Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

sentences: Cardiac Specific Overexpression of Transcription Factor EB (TFEB) in Normal Hearts Induces Pathologic Cardiac Hypertrophy and Lethal Cardiomyopathy TFEB promotes lysosomal biogenesis, autophagy, and lysosomal exocytosis. The present study characterized the consequence of inducible TFEB overexpression in cardiomyocytes in vivo. We generated cardiomyocyte-specific doxycycline inducible (Tet off) mice to achieve spatial and temporal control of TFEB overexpression, by crossing TFEB transgenic mice with mice harboring the tTA transgene (TFEB/tTA). Two weeks after doxycycline removal, an 8-fold increase in TFEB protein expression was observed in transgenic hearts. Heart weight normalized to tibia length was increased by 2.5-fold following TFEB overexpression (TFEB/tTA), characterized by induction of markers of pathological hypertrophy, such as Nppa, Nppb and Acta1, progressive contractile dysfunction and cardiac dilatation. Overexpression of TFEB resulted in premature death, associated with high degree AV block. Reversal of TFEB overexpression normalized cardiac structure and function. Mitochondrial respiration and ATP levels were preserved after 2-weeks of TFEB induction, despite reduced mitochondrial (OXPHOS) protein expression, mtDNA content, and altered mitochondrial morphology. Signaling through mTOR was induced in TFEB/tTA mice, and when inhibited by rapamycin treatment for 4 weeks, partially offset left ventricular dysfunction. Transcriptome analysis revealed early suppression of mitochondrial metabolic pathways, induction of fibrosis and altered calcium signaling. MCOLN1, a lysosomal calcium release channel, the calcineurin target RCAN1.4, and the mitochondrial calcium uniporter (MCU) were strikingly induced in TFEB/tTA mice. In summary, persistent overexpression of TFEB at high levels (8-fold protein upregulation) in cardiomyocytes promotes pathologic cardiac hypertrophy via suppression of mitochondrial bioenergetic pathways and activation of pro-fibrotic and calcium regulatory pathways.

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Chronic toxicity of three neonicotinoid insecticides and their mixture on two daphniid species: Daphnia magna and Ceriodaphnia dubia Neonicotinoid insecticides represent nearly a quarter of the global market and are widely used in agriculture but also for lawn, garden care, and pest control. They are highly water-soluble, persistent in soil, and may enter the aquatic compartment via spray drift, runoff, or leaching, and contribute to downstream aquatic toxicity. Although insects appear to be the most sensitive group to neonicotinoids, other groups, such as crustaceans and birds, may also be affected. Furthermore, most studies focus on single-insecticide exposure and very little is known concerning the impact of neonicotinoid mixtures on aquatic invertebrates. The present study was designed to test potential toxicological effects of an environmentally relevant mixture of imidacloprid, clothianidin, and thiamethoxam on populations of Ceriodaphnia dubia and Daphnia magna under controlled conditions. Chronic toxicity tests were conducted in the laboratory, and survival and reproduction were measured for both species under exposure to nominal concentrations of imidacloprid (0.256 {micro}g/L), clothianidin (3.11 {micro}g/L), thiamethoxam (1.49 {micro}g/L), and a mixture of the three compounds at the same concentrations of the individual compounds. The neonicotinoids did not affect the survival of C. dubia and D. magna founders. Reproduction of C. dubia was affected only by the mixture. All three individual insecticides as well as the mixture caused a significant reduction in the reproduction of D. magna. Our results highlight the complexity of pesticide toxicity and show that traditional toxicological approaches such as acute mortality studies, especially tests with single compounds, can underestimate negative impacts that occur in the environment.

HighlightsO_LINeonicotinoids are currently the most frequently used insecticides worldwide.
C_LIO_LIAn environmentally relevant mixture of three neonicotinoids was evaluated on two daphniid species.
C_LIO_LIThe mixture negatively affected the reproduction of C. dubia and Daphnia magna.
C_LIO_LITraditional toxicological approaches with single compounds may underestimate the effects occurring in the environment at low concentrations.
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sentences: Mitotic checkpoint gene expression is tuned by coding sequences The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Proper functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about SAC gene expression. Here, we show in fission yeast (S. pombe) that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half-lives are supported by a high frequency of non-optimal codons. In contrast, mad1+ mRNA has a short half-life despite a low frequency of non-optimal codons and despite the lack of known destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co-translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine-tuned for proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

sentences: Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development.

sentences: Larvae and adults exhibit contrasting patterns of immune gene expression and infection resistance in wild flour beetle populations In nature, hosts face shifting patterns of parasite exposure and life history trade-offs as they develop from birth to old age. As a result, the net fitness benefit of immunological investment can change dramatically from one life stage to the next. Previous work has revealed a puzzling diversity of relative immune investment patterns among juvenile and adult stages, and it is not clear whether lessons learned from one particular population or species can be generalized to wild populations, after accounting for local adaptation and other variance-generating processes. In this study, we quantify larval and adult immune gene expression and resistance to bacterial infection in two flour beetle species (Tribolium castaneum and T. confusum) from two lab-adapted and five wild-derived populations. Our results provide a clear signal of higher infection-induced immunological investment and resistance in adults relative to larvae, despite variation among species in immune gene regulation. Better characterization of stage-specific investment in infection resistance in natural populations can inform our understanding of life history evolution and improve predictions of disease dynamics in the wild.

sentences: Direct focus sensing and shaping for high-resolution multi-photon imaging in deep tissue High-resolution optical imaging of deep tissue in-situ such as the living brain is fundamentally challenging because of the aberration and scattering of light. In this work, we develop an innovative adaptive optics three-photon microscope based on direct focus sensing and shaping that can accurately measure and effectively compensate for both low- and high-order specimen-induced aberrations and recover near-diffraction-limited performance at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 {micro}m below pia, making high-resolution microscopy in cortex near non-invasive. Functional calcium imaging with high sensitivity and accuracy, and high-precision laser-mediated microsurgery through the intact skull were demonstrated. Moreover, we also achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below pia within the intact brain.

sentences: IL-21 signaling promotes the establishment of KSHV infection in human tonsil lymphocytes by increasing early targeting of plasma cells Factors influencing Kaposis sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Here, using our unique model of KSHV infection in tonsil lymphocytes, we investigate the influence of host cytokines on the establishment of KSHV infection in human B cells. Our data demonstrate that KSHV manipulates the host cytokine microenvironment during early infection and susceptibility is generally associated with downregulation of multiple cytokines. However, we show that IL-21 signaling promotes KSHV infection by promoting both plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. Our data reveal that this phenotype is dependent upon a specific milieu of T cells, that includes IL-21 producing Th17, Tc17 and CD8+ central memory T cells. These results suggest that IL-21 plays a significant role in the early stages of KSHV infection in the human immune system and that specific immunological states favor the initial establishment of KSHV infection by increasing infection in plasma cells.

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Lossless end-to-end transport of small molecules through micron-length DNA nanochannels Designed and engineered protein and DNA nanopores can sense and characterize single molecules and control transmembrane transport of molecular species. However, designed biomolecular pores are less than 100 nm in length and are used primarily for transport across lipid membranes. Nanochannels that span longer distances could be used as conduits for molecules between non-adjacent compartments or cells. Here, we design microns-long, 7 nm diameter DNA nanochannels that small molecules can traverse according to the laws of continuum diffusion. Binding DNA origami caps to channel ends eliminates transport and demonstrates that molecules diffuse from one channel end to the other rather than permeating through channel walls. These micron-length nanochannels can also grow, form interconnects, and interface with living cells. This work thus shows how to construct multifunctional, dynamic agents that control molecular transport, opening new ways of studying intercellular signaling and modulating molecular transport between synthetic and living cells.

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sentences: Proteomic analysis of coarse and fine skin tissues of Liaoning cashmere goat Proteomics is the study of all proteins expressed by a cell or even an organism. However, knowledge of proteins that regulate the fineness of cashmere is limited. Liaoning Cashmere goat (LCG) is a valuable genetic resource of China. The skin samples of Liaoning cashmere goats during the growing period were collected performed Tandem Mass Tag (TMT) method and identified 117 differentially expressed proteins in CT_LCG (course type) and FT_LCG (fine type). To verify protein genes differentially expressed in LCG, we performed PRM validation on three candidate proteins (ALB, SDC1 and ITGB4) in CT-LCG and FT-LCG. Furthermore, primary metabolic process and lysosome are most enriched in the GO and KEGG pathways, respectively. In addition, we also derived a protein-protein interaction (PPI) regulatory network from the perspective of bioinformatics. This study sought to elucidate the molecular mechanism of differential proteins regulating cashmere fineness of Liaoning cashmere goats by using TMT quantitative proteomics analysis. Differentially expressed proteins ALB and SDC1 may regulate cashmere fineness, ITGB4 can be further studied as a promising protein. They can be used as key genes to lay a foundation for the study of cashmere fineness of Liaoning cashmere goats.

sentences: Evolutionary history of metazoan TMEM16 family Transmembrane protein 16 (TMEM16) functions as either a Ca2+-activated Cl- channel (CaCC) or phospholipid scramblase (CaPLSase) and plays diverse physiological roles. It is well conserved in eukaryotes; however, the origin and evolution of different subfamilies in Metazoa are not yet understood. To uncover the evolutionary history of the TMEM16 family, we analyzed 412 proteins from 74 invertebrate species using evolutionary genomics. We found that the TMEM16C-F and J subfamilies are vertebrate-specific, but the TMEM16A/B, G, H, and K subfamilies are ancient and present in many, but not all metazoan species. The most ancient subfamilies in Metazoa, TMEM16L and M, are only maintained in limited species. TMEM16N and O are Cnidaria- and Ecdysozoa-specific subfamilies, respectively, and Ctenophora, Xenacoelomorpha, and Rotifera contain species-specific proteins. We also identified TMEM16 genes that are closely linked together in the genome, suggesting that they have been generated via recent gene duplication. The anoctamin domain structures of invertebrate-specific TMEM16 proteins predicted by AlphaFold2 contain conserved Ca2+-binding motifs and permeation pathways with either narrow or wide inner gates. The inner gate distance of TMEM16 protein may have frequently switched during metazoan evolution, and thus determined the function of the protein as either CaCC or CaPLSase. These results demonstrate that TMEM16 family has evolved by gene gain and loss in metazoans, and the genes have been generally under purifying selection to maintain protein structures and physiological functions.

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LaminaRGeneVis: a tool to visualize gene expression across the laminar architecture of the human neocortex Application of RNA sequencing has enabled the characterization of genome-wide gene expression in the human brain, including distinct layers of the neocortex. Neuroanatomically, the molecular patterns that underlie the laminar organization of the neocortex can help link structure to circuitry and function. To advance our understanding of cortical architecture, we created LaminaRGeneVis, a web application that displays across-layer cortical gene expression from multiple datasets. These datasets were collected using bulk, single-nucleus, and spatial RNA sequencing methodologies and these data were harmonized to facilitate comparisons between datasets. The online resource facilitates single- and multi-gene analyses by providing figures and statistics for user-friendly assessment of laminar gene expression patterns in the adult human neocortex.

Availability and implementationLaminaRGeneVis is available at https://ethanhkim.shinyapps.io/laminargenevis. The source code and data is accessible at https://github.com/ethanhkim/laminargenevis.

sentences: Deforestation projections imply range-wide population decline for critically endangered Bornean orangutan Assessing where wildlife populations are at risk from future habitat loss is particularly important for land-use planning and avoiding biodiversity declines. Combining projections of future deforestation with species density information provides an improved way to anticipate such declines. Using the endemic and critically endangered Bornean orangutan (Pongo pygmaeus) as a case study we applied a spatio-temporally explicit deforestation model to forest loss data from 2001-2017 and projected future impacts on orangutans to the 2030s. Our projections point to continued deforestation across the island, amounting to a loss of forest habitat for 26,200 (CI: 19,500-34,000) orangutans. Populations currently persisting in forests gazetted for industrial timber and oil palm concessions, or unprotected forests outside of concessions, were projected to experience the worst losses within the next 15 years, amounting to 15,400 (CI: 12,000-20,500) individuals. Lowland forests with high orangutan densities in West and Central Kalimantan were also projected to be at high risk from deforestation, irrespective of land-use. In contrast, most protected areas and logging concessions currently harboring orangutans will continue to face low levels of deforestation. Our business-as-usual projections indicate the importance of protected areas, efforts to prevent the conversion of logged forests for the survival of highly vulnerable wildlife, and protecting orangutan habitat in plantation landscapes. The modeling framework could be expanded to other species with available density or occurrence data. Our findings highlight that species conservation should not only attempt to act on the current situation, but also be adapt to changes in drivers to be effective.

sentences: Biological network inference from single-cell multi-omics data using heterogeneous graph transformer We present DeepMAPS (Deep learning-based Multi-omics Analysis Platform for Single-cell data) for biological network inference from single-cell multi-omics (scMulti-omics). DeepMAPS includes both cells and genes in a heterogeneous graph to simultaneously infer cell-cell, cell-gene, and gene-gene relations. The multi-head attention mechanism in a graph transformer considers the heterogeneous relation among cells and genes within both local and global context, making DeepMAPS robust to data noise and scale. We benchmarked DeepMAPS on 18 scMulti-omics datasets for cell clustering and biological network inference, and the results showed that our method outperformed various existing tools. We further applied DeepMAPS on lung tumor leukocyte CITE-seq data and matched diffuse small lymphocytic lymphoma scRNA-seq and scATAC-seq data. In both cases, DeepMAPS showed competitive performance in cell clustering and predicted biologically meaningful cell-cell communication pathways based on the inferred gene networks. Note that we deployed a webserver using DeepMAPS implementation equipped with multiple functions and visualizations to improve the feasibility and reproducibility of scMulti-omics data analysis. Overall, DeepMAPS represents a heterogeneous graph transformer for single-cell study and may benefit the use of scMulti-omics data in various biological systems.

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A corpus for differential diagnosis: an eye diseases use case We have created a corpus for the extraction of information related to diagnosis from scientific literature focused on eye diseases. It was shown that the annotation of entities has a relatively large agreement among annotators, which translates into strong performance of the trained methods, mostly BioBERT. Furthermore it was observed that relation annotation in this domain has challenges, which might require additional exploration.

When using the trained models on MEDLINE, we could identify confirmed knowledge about the diagnosis of eye diseases and relevant new information, which supports the developments in this work. The corpus that we have developed is publicly available, thus the scientific community is able to reproduce our work and reuse the corpus in their work.

sentences: Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16 Capping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2'-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.

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Palynofacies, environments, and climate changes in the Magdalena River Basin Four environments (swamp, shallow lake, alluvial flood plain, and lagoon) from the Lower Magdalena River Basin were studied for palynofacies quantitative characterization. Each environment has been described based on four criteria: palynomorph assemblage, organic matter concentration, organic matter palynological composition, and organic particle morphology.

Shallow lakes palynological assemblages are dominated by composite and grass pollen. The POM (particulate organic matter) morphology is characterized by a maximum at {Phi} 5 class (silt), and it has a sphericity histogram with bimodal distribution (peaks at 0.1 and 0.5/0.6). From a composition point of view, POM is mainly opaque amorphous materials. POM concentration is usually lower than 0.1%.

Swamp environments palynological assemblages are dominated by grass pollen with a slightly smaller amount of composite pollen. The POM is dominated by finely dispersed amorphous and indeterminate "other" types (organo-mineral gel ?), depending on the oxidation degree. The swamp concentration of organic matter a few centimeters below the water-sediment interface varies between 0.1% and 0.3%. Below that, organic concentration is usually lower than 0.1%.

Lagoon assemblages are rich in species and specimens, but assemblages are highly variable. Main components are either finely dispersed amorphous or plant cuticular/epidermal or amorphous homogeneous and heterogeneous or fungal remains. Peat lithology is rich in mangrove pollen, while clay assemblages are dominated by composites, grass, and water plants together with Botryococcus algal remains. Lagoon sediments are the richest in POM concentration, with values between 0.13% and 1% (excluding peats). Regarding particle size and shape, in this environment, they show a trend to decrease in grain size from {Phi} 1 to {Phi} 2 class (sand) dominated assemblages to {Phi} 5 to {Phi} 6 class (silt) dominated assemblages from base to top. Elongated shapes are abundant, with 30% to 50% of particles in the tabloid to elongated tabloid classes.

Alluvial - fluvial flood basin samples are often barren in palynomorphs and organic matter. Occasionally present grass pollen and fungal remains. The POM, when present, is mainly of organo-mineral gel type and has a bimodal grain size distribution, with a minor peak at {Phi} 7 class (v.f.silt) and a major peak at {Phi} 4 to {Phi} 2 class (c. silt to f. sand).

Significant changes in quantitative palynofacies occur within the top few meters of the cores, representing the last 1000 yr of sedimentation in the area. These changes are related to shifts in climate, from colder to warmer conditions or from dry to wet periods, most probably linked with E.N.S.O. A short dry and cold period related to the "Little Ice Age" was identified in the Ayapel and Cienaga de El Medio cores.

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Specific changes in sleep oscillations after blocking human metabotropic glutamate receptor 5 in the absence of altered memory function BackgroundSleep consolidates declarative memory by repeated replay linked to the cardinal oscillations of NonREM sleep. However, there is so far little evidence of classical glutamatergic plasticity induced by this replay. Rather, we have previously reported that blocking NMDA or AMPA receptors does not affect sleep-dependent consolidation of declarative memory.

AimsInvestigate the role of metabotropic glutamate receptor 5 (mGluR5) on memory processing during sleep.

MethodsIn two placebo-controlled within-subject cross-over experiments with 20 healthy humans each, we used fenobam to block mGluR5 during sleep. In Experiment I, participants learned word-pairs (declarative task) and a finger sequence (procedural task) in the evening, then received the drug and recall was tested in the next morning. To cover possible effects on synaptic renormalization processes during sleep, in Experiment II, participants learned new word-pairs in the morning after sleep.

Results/OutcomesSurprisingly, fenobam neither reduced retention of memory across sleep nor new learning after sleep, although it severely altered sleep architecture and memoryrelevant EEG oscillations. In NonREM sleep, fenobam suppressed 12-15 Hz spindles but augmented 2-4 Hz delta waves, whereas in REM sleep it suppressed 4-8 Hz theta and 16-22 Hz beta waves. Notably, under Fenobam NonREM spindles became more consistently phase-coupled to the slow oscillation.

Conclusions/InterpretationsOur findings indicate that mGluR5-related plasticity is not essential for memory processing during sleep, even though mGlurR5 are strongly implicated in the regulation of the cardinal sleep oscillations.

Declaration of interest/FundingThe authors have nothing to disclose and funders had no influence on the research presented here.

sentences: Physiological and genetic analysis of tomato from two cultivars differing in potassium deficiency resistance Potassium (K) is one of the essential nutrients for tomato. Potassium deficiency will limit tomato growth and yield. So improving the low-K+ (LK) resistance of tomato seems important. Two tomato cultivars (JZ18 and JZ34) differing in LK resistance were obtained to analyze the plant demonstration difference under LK treatment. According to the results, JZ34 showed lower accumulation of ROS, less membrane damage and higher antioxidant enzyme activity after LK treatment. Besides, JZ34 also keeps higher K+/Na+ content, higher Ca2+ and Mg2+ content than JZ18 in both shoots and roots. Our genetic analysis revealed that the two additive-dominance-epistasis major genes plus additive-dominance polygene genetic model (E-1) was the optimum model associated with LK resistance based on root trait. The major QTL intervals were finally obtained by the bulked segregant sequencing (BSA-seq) analysis, which were 2.38 Mb at the end of chromosome 4 and 1.38 Mb at the chromosome 6. This is consistent with the analysis of the genetic model. A total of 8 genes were selected in the two candidate regions, which exhibited close related to ion and antioxidant signaling. These findings provided the inheritance pattern and foundation for further molecular mechanisms study of tomato LK resistance.

sentences: Metabolic control of type 2 innate lymphoid cells plasticity toward protective type 1-like cells during Mycobacterium tuberculosis infection Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that Mycobacterium tuberculosis infection alters the phenotype and function of immature lung ILC2 toward a protective interferon-{gamma}-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program involving the transcription factor HIF1. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen tailored immune response.

sentences: Robust IgM responses following vaccination are associated with prevention of Mycobacterium tuberculosis infection in macaques Development of an effective tuberculosis (TB) vaccine has suffered from an incomplete understanding of the correlates of protection against Mycobacterium tuberculosis (Mtb). However, recent work has shown that compared to standard intradermal Bacille Calmette-Guerin (BCG) vaccination, intravenous (IV) BCG vaccination provides nearly complete protection against TB in rhesus macaques. While studies have focused on cellular immunity in this setting, the antibody response elicited by IV BCG vaccination remains incompletely defined. Using an agnostic antibody profiling approach, here we show that IV BCG drives superior antibody responses in the plasma and the bronchoalveolar lavage fluid (BAL). While IV BCG immunization resulted in the expansion of a robust IgM, IgG, IgA, Fc-receptor binding antibodies, and antibody effector functions in the BAL, IgM titers were among the strongest markers of reduced bacterial burden in the plasma and BAL of BCG immunized animals. Moreover, IgM immunity was also enriched among animals receiving protective vaccination with an attenuated Mtb strain. Finally, a LAM-specific IgM monoclonal antibody reduced Mtb survival in vitro. Collectively, these data highlight the potential importance of IgM responses as a marker and as a functional mediator of protection against TB.

sentences: Global and gene-specific translational regulation in Escherichia coli across different conditions How well mRNA transcript levels represent protein abundances has been a controversial issue. Particularly across different environments, correlations between mRNA and protein exhibit remarkable variability from gene to gene. Translational regulation is likely to be one of the key factors contributing to mismatches between mRNA level and protein abundance in bacteria. Here, we quantified genome-wide transcriptome and relative translation efficiency (RTE) under 12 different conditions in Escherichia coli. By quantifying the mRNA-RTE correlation both across genes and across conditions, we uncovered a diversity of gene-specific translational regulations, cooperating with transcriptional regulations, in response to carbon (C), nitrogen (N), and phosphate (P) limitations. Intriguingly, we found that many genes regulating translation are themselves subject to translational regulation, suggesting possible feedbacks. Furthermore, a random forest model suggests that codon usage partially predicts a genes cross-condition variability in translation efficiency; such cross-condition variability tends to be an inherent quality of a gene, independent of the specific nutrient limitations. These findings broaden the understanding of translational regulation under different environments, and provide novel strategies for the control of translation in synthetic biology.

sentences: Where there's smoke, there's fuel: dynamic vegetation data improve predictions of wildfire hazard in the Great Basin Wildfires are a growing management concern in western US rangelands, where invasive annual grasses have altered fire regimes and contributed to an increased incidence of catastrophic large wildfires. Fire activity in arid, non-forested ecosystems is thought to be largely controlled by interannual variation in fuel amount, which in turn is controlled by antecedent weather. Thus, long-range forecasting of fire activity in rangelands should be feasible given annual estimates of fuel quantity. Using a 32 yr time series of spatial data, we employed machine learning algorithms to predict the relative probability of large (>405 ha) wildfire in the Great Basin based on fine-scale annual and 16-day estimates of cover and production of vegetation functional groups, weather, and multitemporal scale drought indices. We evaluated the predictive utility of these models with a leave-one-year-out cross-validation, building spatial hindcasts of fire probability for each year that we compared against actual footprints of large wildfires. Herbaceous aboveground biomass production, bare ground cover, and long-term drought indices were the most important predictors of burning. Across 32 fire seasons, 88% of the area burned in large wildfires coincided with the upper 3 deciles of predicted fire probabilities. At the scale of the Great Basin, several metrics of fire activity were moderately to strongly correlated with average fire probability, including total area burned in large wildfires, number of large wildfires, and maximum fire size. Our findings show that recent years of exceptional fire activity in the Great Basin were predictable based on antecedent weather-driven growth of fine fuels and reveal a significant increasing trend in fire probability over the last three decades driven by widespread changes in fine fuel characteristics.

sentences: The influence of unmeasured confounding on the MR Steiger approach The Mendelian Randomization (MR) Steiger approach is used to determine the direction of a possible causal effect between two phenotypes [1]. For two phenotypes, denoted phenotype 1 and 2, the MR Steiger approach is composed of two parts: (1) MR is performed for a set of single nucleotide polymorphisms (SNPs) that serve as instrumental variables for phenotype 1 and (2) the difference of two correlations, the correlation between the SNPs and phenotype 1 and the correlation between the SNPs and phenotype 2, is calculated. These two parts are then used to determine the direction of a possible causal effect between the two phenotypes. The original MR Steiger paper [1] shows that unmeasured confounding of the two phenotypes affects the validity of the MR Steiger approach, but does not elucidate as to how this occurs. In particular, it was argued that if the magnitude of the observational variance explained between the two phenotypes is above 0.2, the MR Steiger method may return the incorrect causal direction due to unmeasured confounding. This may initially seem surprising since unmeasured confounding does not induce spurious associations between the SNP and phenotype 2, as we demonstrate using directed acyclic graphs. In this note, we show that this is because unmeasured confounding may rescale the magnitude of a non-zero association, and thereby distort the comparison of the correlation between the SNP and phenotype 2 and the correlation between the SNP and phenotype 1. We will end with a number of cautionary remarks on the MR Steiger method, which are partly motivated by this and mentioned in the original MR Steiger paper [1].

sentences: Paternal knockdown of tRNA (cytosine-5-)-methyltransferase (Dnmt2) increases offspring susceptibility to infection in red flour beetles Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

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Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles.

ImportanceThe conoid is an enigmatic, dynamic organelle positioned at the apical tip of the coccidian subgroup of the Apicomplexa, close to the apical polar ring (APR) from which the subpellicular microtubules (SPTMs) emerge and at the site of microneme and rhoptry secretory organelles exocytosis. In Toxoplasma gondii, the conoid protrudes concomitantly to microneme secretion, during egress, motility and invasion. The conditional depletion of the apical cap structural protein AC9 or AC10 leads to a disorganization of SPTMs as well as the loss of APR and conoid that result in microneme secretion defect and block in motility, invasion and egress. We show here that depletion of the kinase ERK7 phenocopies completely AC9 and AC10 mutants. Moreover, the combination of ultrastructure expansion microscopy with an NHS ester staining revealed that ERK7 depleted parasites exhibit a dilated apical plasma membrane and a mis-positioning of the rhoptry organelles.

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Extracellular vesicles Transfer Polarized Mitochondria and Increase Cellular Energetics in Ischemic Endothelial Cells We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage- derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.

HighlightsO_LIMicrovesicles transfer mitochondria to endothelial cells and brain slice neurons
C_LIO_LIMitochondrial transfer increased ATP in ischemic brain endothelial cells (BECs)
C_LIO_LITransfer of microvesicles increased mitochondrial function in hypoxic BECs
C_LIO_LITransfer of exosomes did not affect mitochondrial function in hypoxic BECs
C_LIO_LIHomotypic BEC-derived EVs result in greater ATP levels in the recipient BECs
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Paper-and-pencil questionnaires analysis: a new automated technique to reduce analysis time and errors. Background and ObjectiveQuestionnaires are essential tools in many scientific fields, including health and medicine. However, the analysis of paper-and-pencil questionnaires is time consuming, source of errors and expensive, limiting its use in large cohort studies. Computer-based questionnaires might be a valuable alternative but they may introduce bias, especially for sensitive questions, and they require programming skills. The aim of this study is to develop a reliable and adaptable open-source technique (i.e. LightQuest) to automatically analyse various types of scanned paper-and-pencil questionnaires with closed questions, including those with inverted scale.

MethodsTo evaluate the usefulness of LightQuest, the time needed for 7 experimenters for manually code 10 sets of 4 frequently used questionnaires and the number of errors (i.e. reliability) were compared with the time and errors their made using LightQuest.

ResultsLightQuest was twice as fast as the manual analysis, even though the time to create the reference model was taken into account (933s vs. 1935s, t(2)=8.81, p<0.001). Without model creation, the reduced analysis time was more pronounced, with an average of 2.77s.question-1 for the manual technique versus 0.55s.question-1 for LightQuest (t(2)=22.5, p<0.001). Moreover, during correction of the 5180 questions performed by the 7 experimenters, LightQuest made a total of 2 errors versus 46 with the manual technique (q(2)=4.53, p<0.05).

ConclusionLightQuest demonstrated clear superiority both in terms of time and reliability. The script of this first open-source technique, which does not require programming skills, is downloadable in supplemental data and may become an asset for all studies using questionnaires.

sentences: Cortical Magnification in Human Visual Cortex Parallels Task Performance around the Visual Field Human vision has striking radial asymmetries, with performance on many tasks varying sharply with stimulus polar angle. Performance is generally better on the horizontal than vertical meridian, and on the lower than upper vertical meridian, and these asymmetries decrease gradually with deviation from the vertical meridian. Here we report cortical magnification at a fine angular resolution around the visual field. This precision enables comparisons between cortical magnification and behavior, between cortical magnification and retinal cell densities, and between cortical magnification in twin pairs. We show that cortical magnification in human primary visual cortex, measured in 163 subjects, varies substantially around the visual field, with a pattern similar to behavior. These radial asymmetries in cortex are larger than those found in the retina, and they are correlated between monozygotic twin pairs. These findings indicate a tight link between cortical topography and behavior, and suggest that visual field asymmetries are partly heritable.

sentences: Variability of social interactions in new versus established relationships in female degus When an organism explores a new environment or stimulus it varies its behavior to ensure proper sampling. As contingencies are learned, behavioral variance can give-way to routines and stereotypies. This phenomenon is common across animal species and non-biological learning systems but has not been well studied in the social domain, in which the stimulus an animal investigates, another individual, may react negatively to unexpected behaviors. Here we develop a set of generalizable measures of interaction variability to test the hypothesis that social relationship formation is associated with initially high variability, followed by stabilization of dyadic interaction patterns. We focus these tests on female degus (Octodon degus), rodents predisposed to form cooperative peer relationships. Degus were presented with a series of 20 minute, dyadic "reunion" sessions across days, interleaving exposures to familiar and unfamiliar same-sex conspecifics. We found that degu pairs could be distinguished from one-another by their session-to-session interactive behaviors, suggesting dyad-specific social relationships. Consistent with predictions, stranger interactions changed from the first to second exposure, after which they became less variable across days. Unexpectedly, however, long-term cagemates showed overall more interaction variability across sessions than strangers. Taking into account all interaction variability measures, differences between strangers and cagemates were only observed when considering many (> 10) interactions, or longer (>5 min, or session-to-session) time periods. Shorter scales, including interaction transitions, showed meaningful patterns but no reliable group differences. The results suggest that while variability of social behavior over minutes or across sessions may change as relationships develop, interactions between unfamiliar adult female degus are not systematically more variable than those of cagemates. Given known ecological pressures on female degus to form large, stable social networks, these patterns are consistent with the notion that interaction variability may be maladaptive for social coordination.

sentences: Factors that Influence Career Choice Among Different Populations of Neuroscience Trainees Enhancing the diversity of the scientific workforce is critical to achieving the mission of NIH: "To seek fundamental knowledge about the nature and behavior of living systems and the application of that knowledge to enhance health, lengthen life, and reduce the burdens of illness and disability." However, specific groups have historically been, and continue to be, underrepresented in the biomedical research workforce, especially academia. Career choice is a multi-factorial process that evolves over time; among all trainees, expressed interest in faculty research careers decreases over time in graduate school, but that trend is amplified in women and members of historically underrepresented racial and ethnic groups (Fuhrmann, Halme, OSullivan, & Lindstaedt, 2011; Gibbs, McGready, Bennett, & Griffin, 2014; C. Golde & Dore, 2004; Roach & Sauermann, 2017; Sauermann & Roach, 2012). Neuroscience as a discipline has characteristics that may exacerbate the overall trends seen in the life sciences, such as a greater growth in the number of awarded neuroscience PhDs than in other life sciences fields (US National Science Foundation, 2016b). This work was designed to investigate how career interest changes over time among recent neuroscience PhD graduates, and whether differences in career interests are associated with social identity (i.e. gender and race/ethnicity), experiences in graduate school and postdoctoral training (e.g. relationship with advisor; feelings of belonging), and personal characteristics (e.g. confidence in ones potential to be an independent researcher). We report results from a survey of 1,479 PhD neuroscientists (including 16% underrepresented (UR) and 54% female scientists). We saw repeated evidence that individual preferences about careers in general, and academic careers specifically, predict current career interest. These statistically significant preferences mostly had medium to low effect size that varied by career type. These findings were mediated by social identity and experiences in graduate school and postdoctoral training. Our findings highlight the important influence of the advisor in shaping a trainees career path, and the ways in which academic culture is perceived as unwelcoming or incongruent with the values or priorities of certain groups. For women, issues of work/life balance and structural issues of academia, and for UR women in particular, lower confidence in their ability to be an independent researcher, affected their interest in academia. Both women and UR men in our study report a lower importance of autonomy in their careers. UR respondents report feeling less like they were a part of the social and intellectual community. However, they have formed beneficial relationships with faculty outside their PhD institutions that, particularly for UR women, are associated with increased interest in academia. Our findings suggest several areas for positive growth, ways to change how we think about the impact of mentorship, and policy and programmatic interventions that extend beyond trying to change or "fix" the individual and instead recognize the systemic structures that influence career choices.

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Indigenous ancestry and admixture in the Uruguayan population The Amerindian group known as the Charruas inhabited Uruguay at the timing of European colonial contact. Even though they were extinguished as an ethnic group as a result of a genocide, Charruan heritage is part of the Uruguayan identity both culturally and genetically. While mitochondrial DNA studies have shown evidence of Amerindian ancestry in living Uruguayans, here we undertake whole-genome sequencing of 10 Uruguayan individuals with Charruan heritage. We detect chromosomal segments of Amerindian ancestry supporting the presence of indigenous genetic ancestry in living descendants. Specific haplotypes were found to be enriched in  Charruas and rare in the rest of the Amerindian groups studied. Some of these we interpret as the result of positive selection, as we identified selection signatures and they were located mostly within genes related to the infectivity of specific viruses.

Historical records describe contacts of the Charruas with other Amerindians, such as Guarani, and patterns of genomic similarity observed here concur with genomic similarity between these groups. Less expected, we found a high genomic similarity of the Charruas to Diaguita from Argentinian and Chile, which could be explained by geographically proximity.

Finally, by fitting admixture models of Amerindian and European ancestry for the Uruguayan population, we were able to estimate the timing of the first pulse of admixture between European and Uruguayan indigenous peoples in 1658 and the second migration pulse in 1683. Both dates roughly concurring with the Franciscan missions in 1662 and the foundation of the city of Colonia in 1680 by the Spanish.

sentences: The force loading rate drives cell mechanosensing through both reinforcement and fluidization Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton undergoes fluidization and softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon occurs at the organ level. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved.

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Minimal functional domains of the core polarity regulator Dlg The compartmentalized domains of polarized epithelial cells arise from mutually antagonistic actions between the apical Par complex and the basolateral Scrib module. In Drosophila, the Scrib module proteins Scribble (Scrib) and Discs-large (Dlg) are required to limit Lgl phosphorylation at the basolateral cortex, but how Scrib and Dlg could carry out such a  protection activity is not clear. We tested Protein Phosphatase 1 (PP1) as a potential mediator of this activity but demonstrate that a significant component of Scrib and Dlg regulation of Lgl is PP1-independent and found no evidence for a Scrib-Dlg-PP1 protein complex. However, the Dlg SH3 domain plays a role in Lgl protection and, in combination with the N-terminal region of the Dlg HOOK domain, in recruitment of Scrib to the membrane. We identify a  minimal Dlg comprised of the SH3 and HOOK domains that is both necessary and sufficient for Scrib localization and epithelial polarity function in vivo.

Summary StatementA minimal SH3-HOOK fragment of Dlg is sufficient to support epithelial polarity through mechanisms independent of the PP1 phosphatase.

sentences: Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9-3.1 [A], as well as the complete E. faecalis 70S ribosome at 2.2-2.5 [A]. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 [A] from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by [~]4 [A] out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

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dbGSRV: a manually curated database of genetic susceptibility to respiratory virus Human genetics has been proposed to play an essential role in inter-individual differences in respiratory virus infection occurrence and outcomes. To systematically understand human genetic contributions to respiratory virus infection, we developed the database dbGSRV, a manually curated database that integrated the host genetic susceptibility and severity studies of respiratory viruses scattered over literatures in PubMed. At present, dbGSRV contains 1932 records of genetic association studies relating 1010 unique variants and seven respiratory viruses, manually curated from 168 published articles. Users can access the records by quick searching, batch searching, advanced searching and browsing. Reference information, infection status, population information, mutation information and disease relationship are provided for each record, as well as hyper links to public databases in convenient of users accessing more information. In addition, a visual overview of the topological network relationship between respiratory viruses and associated genes is provided. Therefore, dbGSRV offers a promising avenue to facilitate researchers to dissect human factors in respiratory virus infection, define novel drug targets, conduct risk stratification of population and develop personalized medicine approaches.

Database URL: http://www.ehbio.com/dbGSRV/front/

sentences: Every cog and wheel: Unraveling biocomplexity at the genomic and phenotypic level in a population complex of Chinook salmon. Genomic diversity is the fundamental building block of biodiversity and the necessary ingredient for adaptation. Our rapidly increasing ability to quantify functional, compositional, and structural genomic diversity of populations forces the question of how to balance conservation goals - should the focus be on important functional diversity and key life history traits or on maximizing genomic diversity as a whole? Specifically, the intra-specific diversity (biocomplexity) comprised of phenotypic and genetic variation can determine the ability of a population to respond to changing environmental conditions. Here, we explore the biocomplexity of Californias Central Valley Chinook salmon (Oncorhynchus tshawytscha) population complex at the genomic level. Notably, despite apparent gene flow among individuals with the same migration (life history) phenotypes inhabiting different tributaries, each group is characterized by a surprising component of unique genomic diversity. Our results emphasize the importance of formulating conservation goals focused on maintaining biocomplexity at both the phenotypic and genotypic level. Doing so will maintain the species adaptive potential and increase the probability of persistence of the population complex despite changing environmental pressures.

sentences: Bakdrive: Identifying the Minimum Set of Bacterial Driver Species across Multiple Microbial Communities Interactions among microbes within microbial communities have been shown to play crucial roles in human health. In spite of recent progress, low-level knowledge of bacteria driving microbial interactions within microbiomes remains unknown, limiting our ability to fully understand and control microbial communities. In this study, we present a novel approach for identifying driver species within microbiomes. Bakdrive infers ecological networks of given metagenomic sequencing samples and identifies minimum sets of driver species using control theory. Bakdrive has three key innovations in this space: (i) it leverages inherent information from metagenomic sequencing samples to identify driver species, (ii) it explicitly takes host-specific variation into consideration, and (iii) it does not require a known ecological network. In extensive simulated data, we demonstrate identifying driver species identified from healthy donor samples and introducing them to the disease samples, we can restore the gut microbiome in recurrent Clostridioides difficile infection patients to a healthy state. We also applied Bakdrive to two real datasets, rCDI and Crohns disease patients, uncovering driver species consistent with previous work. In summary, Bakdrive provides a novel approach for teasing apart microbial interactions. Bakdrive is open-source and available at https://gitlab.com/treangenlab/bakdrive

sentences: In vivo physiology of foveal retinal ganglion cells in Macaca fascicularis The primate fovea is specialized for high-resolution spatial and chromatic vision, possessing a unique topography characterized by a pit in which the inner retina and associated vasculature have been excavated, the highest density of cones anywhere in the retina, and a disproportionately large representation in visual cortex. The crucial visual properties conferred by the fovea are mediated by retinal ganglion cells (RGCs) the somas of which lie at the margin of the foveal pit. Microelectrode recordings of these centermost retinal ganglion cells have been challenging due to the fragility of the fovea in the excised retina. This has been overcome in this study by combining high resolution fluorescence adaptive optics ophthalmoscopy with calcium imaging to optically record functional responses of foveal RGCs to visual stimuli in the living eye. Here we use this approach to study the chromatic responses of RGCs to uniform fields modulated in different directions in color space and achromatic responses with monochromatic drifting gratings. We repeated these recordings in many cells in each of three macaca fasicularis primates over weeks or months. We find that most of the L/M chromatically opponent cells serving the most central cones respond to high spatial frequencies, reflecting strong surround inhibition that sacrifices sensitivity at low frequencies in favor of the transmission of fine detail in the retinal image. In addition, we find the L/M cells vary in spatial frequency bandwidths, suggesting that L/M chromatic opponency, often considered largely due to input from single cones, has more diverse receptive field structure than previously thought. By characterizing the functional properties of retinal ganglion cells in vivo through adaptive optics, we provide a lower bound on the response characteristics of these cells in situ and their role in vision processing.

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Pseudomonas aeruginosa C-terminal processing protease CtpA assembles into a hexameric structure that requires activation by a spiral-shaped lipoprotein binding partner Pseudomonas aeruginosa CtpA is a carboxyl terminal-processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer-of-dimers, and an N-terminal helix of LbcA, are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of a CTP within the C-terminal processing peptidase-3 group.

IMPORTANCECarboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be theraputic targets. However, relatively little is known about their molecular mechanisms of action. In Pseudomonas aeruginosa, CtpA supports virulence by working in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we report structure-function analyses of CtpA and LbcA, which reveals that CtpA assembles into an inactive hexamer comprising a trimer of dimers. LbcA is monomeric, with an N-terminal region important for binding to and activating CtpA, followed by a spiral structure composed of 11 tetratricopetide repeats, which could wrap around a substrate for delivery to CtpA. This work provides the first structure of a CTP-3 group member, revealing a unique mutimeric arrangement and insight into how this important proteolytic system functions.

sentences: Neural Basis for Regulation of Vasopressin Secretion by Anticipated Disturbances in Osmolality Water balance, tracked by extracellular osmolality, is regulated by feedback and feedforward mechanisms. Feedback regulation is reactive, occurring as deviations in osmolality are detected. Feedforward or presystemic regulation is proactive, occurring when disturbances in osmolality are anticipated. Vasopressin (AVP) is a key hormone regulating water balance and is released during hyperosmolality to limit renal water excretion. AVP neurons are under feedback and feedforward regulation. Not only do they respond to disturbances in blood osmolality, but they are also rapidly suppressed and stimulated, respectively, by drinking and eating, which will ultimately decrease and increase osmolality. Here, we demonstrate that AVP neuron activity is regulated by multiple anatomically-and functionally-distinct neural circuits. Notably, presystemic regulation during drinking and eating are mediated by non-overlapping circuits that involve the lamina terminalis and hypothalamic arcuate nucleus, respectively. These findings reveal neural mechanisms that support differential regulation of AVP release by diverse behavioral and physiological stimuli.

sentences: The role of elections as drivers of tropical deforestation Tropical forests support immense biodiversity and provide essential ecosystem services for billions of people. Despite this value, tropical deforestation continues at a high rate. Emerging evidence suggests that elections can play an important role in shaping deforestation, for instance by incentivising politicians to allow increased utilisation of tropical forests in return for political support and votes. Nevertheless, the role of elections as a driver of deforestation has not been comprehensively tested at broad geographic scales. Here, we created an annual database from 2001 to 2018 on political elections and forest loss for 55 tropical nations and modelled the effect of elections on deforestation. In total, 1.5 million km2 of forest was lost during this time period, and the rate of deforestation increased in 37 (67%) of the analysed countries. Deforestation was significantly lower in years with presidential or lower chamber elections compared to non-election years, which is in contrast to previous local-scale studies. Moreover, deforestation was significantly higher in presidential or lower chamber elections that are competitive (i.e. when the opposition can participate in elections and has a legitimate chance to gain governmental power) compared to uncompetitive elections. Our results document a pervasive loss of tropical forests and suggest that competitive elections are potential drivers of deforestation. We recommned that organisations monitoring election transparency and fairness should also monitor environmental impacts such as forest loss, habitat destruction and resource exploitation. This would benefit the tracking of potential illegal vote buying with natural resources.

sentences: Representation of motion concepts in occipitotemporal cortex: fMRI activation, decoding and connectivity analyses Embodied theories of semantic cognition predict that brain regions involved in motion perception are engaged when people comprehend motion concepts expressed in language. Left lateral occipitotemporal cortex (LOTC) is implicated in both motion perception and motion concept processing but prior studies have produced mixed findings on which parts of this region are engaged by motion language. We scanned participants performing semantic judgements about sentences describing motion events and static events. We performed univariate analyses, multivariate pattern analyses (MVPA) and psychophysiological interaction (PPI) analyses to investigate the effect of motion on activity and connectivity in different parts of LOTC. In multivariate analyses that decoded whether a sentence described motion or not, the whole of LOTC showed above-chance level performance, with performance exceeding that of other brain regions. Univariate ROI analyses found the middle part of LOTC was more active for motion events than static ones. Finally, PPI analyses found that when processing motion events, the middle and posterior parts of LOTC (overlapping with motion perception regions), increased their connectivity with cognitive control regions. Taken together, these results indicate that the whole of the LOTC responded differently to motion vs. static events, and that these effects are most pronounced in more posterior sites. These findings are consistent with embodiment accounts of semantic processing, and suggest that understanding verbal descriptions of motion engages areas of the occipitotemporal cortex involved in perceiving motion.

sentences: Cortical waves mediate the cellular response to electric fields Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis share both similarities and differences with chemical-gradient-sensing pathways. We examined a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 minutes after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display "U-turn" behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.

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A trigger phosphodiesterase modulates the global c-di-GMP pool, motility and biofilm formation in Vibrio parahaemolyticus. Vibrio parahaemolyticus cells transit from free swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP were low or when the phosphodiesterase was catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called Trigger phosphodiesterases, hence we named the product of VP1881 TpdA, for Trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while its overexpression from an IPTG inducible promoter showed a positive effect on both swimming and swarming motility, and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes as well as the biofilm related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of this social behaviors.

ImportanceC-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm-matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, hence not many examples of these type of signaling proteins have been reported in the literature. Here we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

sentences: A fully adapted headstage for electrophysiological experiments with custom and scalable electrode arrays to record widely distributed brain regions Electrophysiological recordings lead amongst the techniques that aim to investigate the dynamics of neural activity sampled from large neural ensembles. However, the financial costs associated with the state-of-the-art technology used to manufacture probes and multi-channel recording systems make these experiments virtually inaccessible to small laboratories, especially if located in developing countries. Here, we describe a new method for implanting several tungsten electrode arrays, widely distributed over the brain. Moreover, we designed a headstage system, using the Intan(R) RHD2000 chipset, associated with a connector (replacing the expensive commercial Omnetics connector), that allows the usage of disposable and cheap cranial implants. Our results showed high-quality multichannel recording in freely moving animals (detecting local field, evoked responses and unit activities) and robust mechanical connections ensuring long-term continuous recordings. Our project represents an open source and inexpensive alternative to develop customized extracellular records from multiple brain regions.

sentences: Mapping enzyme catalysis with metabolomic biosensing Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

sentences: Fluorinated oil-surfactant mixtures with the density of water: artificial cells for synthetic biology There is a rising interest in biotechnology for the compartmentalization of biochemical reactions in water droplets. Several applications, such as the widely used digital PCR, seek to encapsulate a single molecule in a droplet to be amplified. Directed evolution, another technology with growing popularity, seeks to replicate what happens in nature by encapsulating a single gene and the protein encoded by this gene, linking genotype with phenotype. Compartmentalizing reactions in droplets also allows the experimentalist to run millions of different reactions in parallel. Compartmentalization requires a fluid that is immiscible with water and a surfactant to stabilize the droplets. While there are fluids and surfactants on the market that have been used to accomplish encapsulation, there are reported concerns with these. Span(R) 80, for example, a commonly used surfactant, has contaminants that interfere with various biochemical reactions. Similarly, synthetic fluids distributed by the cosmetic industry allow some researchers to produce experimental results that can be published, but then other researchers fail to reproduce some of these protocols due to the unreliable nature of these products, which are not manufactured with the intent of being used in biotechnology. The most reliable fluids, immiscible with water and suitable for biochemical reactions, are fluorinated fluids. Fluorinated compounds have the peculiar characteristic of being immiscible with water while at the same time not mixing with hydrophobic molecules. This peculiar characteristic has made fluorinated fluids attractive because it seems to be the basis of their being biologically inert. However, commercially available fluorinated fluids have densities between 1.4 to 1.6 g/mL. The higher-than-water density of fluorinated oils complicates handling of the droplets since these would float on the fluid since the water droplets would be less dense. This can cause aggregation and coalescence of the droplets. Here, we report the synthesis, characterization, and use of fluorinated polysiloxane oils that have densities similar to the one of water at room temperature, and when mixed with non-ionic fluorinated surfactants, can produce droplets encapsulating biochemical reactions. We show how droplets in these emulsions can host many biological processes, including PCR, DNA origami, rolling circle amplification (RCA), and Taqman(R) assays. Some of these use unnatural DNA built from an Artificially Expanded Genetic Information System (AEGIS) with six nucleotide "letters".

sentences: The balance between toxic versus nontoxic microRNAs determines platinum sensitivity in ovarian cancer Numerous micro(mi)RNAs (short noncoding RNAs that negatively regulate gene expression) have been linked to platinum (Pt) sensitivity and resistance in ovarian cancer (OC). miRNA activity occurs when the guide strand of the miRNA, with its seed sequence (pos. 2-7/8), is loaded into the RNA induced silencing complex (RISC) and targets complementary short seed matches in the 3 untranslated region of mRNAs. Toxic seeds, targeting genes critical for cancer cell survival, have been found in tumor suppressive miRNAs. Many si- and shRNAs can also kill cancer cells via toxic seeds, the most toxic carrying G-rich 6mer seed sequences. We now show that treatment of OC cells with Pt leads to an increase in RISC-bound miRNAs carrying toxic 6mer seeds and a decrease in miRNAs with nontoxic seeds. Pt-resistant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in OC patients revealed that the ratio between miRNAs with toxic versus miRNAs with nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses.

sentences: Genetic Analysis of Human RNA Binding Motif Protein 48 (RBM48) Reveals an Essential Role in U12-Type Intron Splicing U12-type or minor introns are found in most multicellular eukaryotes and constitute [~]0.5% of all introns in species with a minor spliceosome. Although the biological significance for evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome (MDS), predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA Binding Motif Protein48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5 end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron containing genes (MIGs) have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of MIGs disrupted in both species. Mutations in the majority of these orthologous MIGs have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

sentences: Attenuation of a DNA Cruciform by a Conserved Regulator Directs T3SS-1 mediated virulence in Vibrio parahaemolyticus Pathogenic Vibrio species account for 3-5 million annual life-threatening human infections. Virulence is driven by bacterial hemolysin and toxin gene expression often positively regulated by the winged helix-turn-helix (wHTH) HlyU transcriptional regulator family and silenced by Histone-like nucleoid structural protein (H-NS). In the case of Vibrio parahaemolyticus, HlyU is required for virulence gene expression associated with Type 3 Secretion System-1 (T3SS-1) although its mechanism of action is not understood. Here, we provide evidence for DNA cruciform attenuation mediated by HlyU binding to support concomitant virulence gene expression. Genetic and biochemical experiments revealed that upon HlyU mediated DNA cruciform attenuation, an intergenic cryptic promoter became accessible allowing for exsA mRNA expression and initiation of an ExsA autoactivation feedback loop at a separate ExsA-dependent promoter. Using a heterologous E. coli expression system, we reconstituted the dual promoter elements which revealed that HlyU binding and DNA cruciform attenuation were strictly required to initiate the ExsA autoactivation loop. The data indicate that HlyU acts to attenuate a transcriptional repressive DNA cruciform to support T3SS-1 virulence gene expression and reveals a non-canonical extricating gene regulation mechanism in pathogenic Vibrio species.

sentences: Characterising ontogeny of numerosity discrimination in zebrafish reveals multiple, numerical and non- numerical mechanisms. A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition.

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SARS coronavirus vaccines protect against different coronaviruses Although SARS-CoV-2 vaccines have shown efficacy against SARS-CoV-2, it is unclear if they can also protect against other coronaviruses that may infect humans in the future. Here, we show that SARS-CoV-2 vaccination in humans elicits cross-reactive antibodies against other coronaviruses. Our studies in mice demonstrate that SARS-CoV-2 vaccination protects against a common cold coronavirus, and that SARS-CoV-1 vaccination protects against SARS-CoV-2. Similarly, infection with a common cold coronavirus also conferred enhanced protection from subsequent infections with other coronaviruses. Mechanistically, both T cells and antibodies mediated cross-protection. This is the first direct demonstration that coronavirus-specific immunity can confer heterologous protection in vivo, providing a rationale for universal coronavirus vaccines.

HighlightsO_LISARS-CoV-2 vaccination elicits cross-reactive antibody against other coronaviruses in humans.
C_LIO_LICOVID-19 patients generate cross-reactive antibody against other coronaviruses.
C_LIO_LIA SARS-CoV-1 vaccine protects against SARS-CoV-2.
C_LIO_LIPrior coronavirus infections improve immune protection following heterologous coronavirus challenges.
C_LI

sentences: Interpretable deep generative models for genomics Deep neural networks implementing generative models for dimensionality reduction have been extensively used for the visualization and analysis of genomic data. One of their key limitations is lack of interpretability: it is challenging to quantitatively identify which input features are used to construct the embedding dimensions, thus preventing insight into why cells are organized in a particular data visualization, for example. Here we present a scalable, interpretable variational autoencoder (siVAE) that is interpretable by design: it learns feature embeddings that guide the interpretation of the cell embeddings in a manner analogous to factor loadings of factor analysis. siVAE is as powerful and nearly as fast to train as the standard VAE but achieves full interpretability of the embedding dimensions. Using siVAE, we exploit a number of connections between dimensionality reduction and gene network inference to identify gene neighborhoods and gene hubs, without the explicit need for gene network inference. We observe a systematic difference in the gene neighborhoods identified by dimensionality reduction methods and gene network inference algorithms in general, suggesting they provide complementary information about the underlying structure of the gene co-expression network. Finally, we apply siVAE to implicitly learn gene networks for individual iPSC lines and uncover a correlation between neuronal differentiation efficiency and loss of co-expression of several mitochondrial complexes, including NADH dehydrogenase, cytochrome C oxidase, and cytochrome b.

sentences: The gut microbiota affects the social network of honeybees The gut microbiota influences animal neurophysiology and behavior but has not previously been documented to affect emergent group-level behaviors. Here we combine gut microbiota manipulation with automated behavioral tracking of honeybee sub-colonies to show that the microbiota increases the rate and specialization of social interactions. Microbiota colonization was associated with higher abundances of one third of metabolites detected in the brain, including several amino acids, and a subset of these metabolites were significant predictors of social interactions. Colonization also affected brain transcriptional processes related to amino acid metabolism and epigenetic modification in a brain region involved in sensory perception. These results demonstrate that the gut microbiota modulates the emergent colony social network of honeybees, likely via changes in chromatin accessibility and amino acid biosynthesis.

sentences: HSC-independent definitive hematopoietic cells persist into adult life The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

sentences: The role of the angular gyrus in semantic cognition - A synthesis of five functional neuroimaging studies Semantic knowledge is central to human cognition. The angular gyrus (AG) is widely considered a key brain region for semantic cognition. However, the role of the AG in semantic processing is controversial. Key controversies concern response polarity (activation vs. deactivation) and its relation to task difficulty, lateralization (left vs. right AG), and functional-anatomical subdivision (PGa vs. PGp subregions). Here, we combined the fMRI data of five studies on semantic processing (n = 172) and analyzed the response profiles from the same anatomical regions-of-interest for left and right PGa and PGp. We found that the AG was consistently deactivated during non-semantic conditions, whereas response polarity during semantic conditions was inconsistent. However, the AG consistently showed relative response differences between semantic and non-semantic conditions, and between different semantic conditions. A combined analysis across all studies revealed that AG responses could be best explained by separable effects of task difficulty and semantic processing demand. Task difficulty effects were stronger in PGa than PGp, regardless of hemisphere. Semantic effects were stronger in left than right AG, regardless of subregion. These results suggest that the AG is engaged in both domain-general task-difficulty-related processes and domain-specific semantic processes. In semantic processing, we propose that left AG acts as a "multimodal convergence zone" that binds different semantic features associated with the same concept, enabling efficient access to task-relevant features.

sentences: Clinically-relevant T cell expansion protocols activate distinct cellular metabolic programs and phenotypes Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables including proliferation, differentiation, function, activation and mitochondrial phenotypes. T cells adapted their metabolism to match their media expansion condition as shown by glucose and glutamine uptake, and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Furthermore, T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. In one case, culturing in ascites resulted in increased glucose uptake which was insufficient to rescue T cell function. Thus, ex vivo T cell expansion conditions have profound impacts on metabolism and function.

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A species-wide genetic atlas of antimicrobial resistance in Clostridioides difficile Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI), the leading healthcare-related gastrointestinal infection in the world. An association between AMR and CDI outbreaks is well documented, however, data is limited to a few  epidemic strains in specific geographical regions. Here, through detailed analysis of 10,330 publicly-available C. difficile genomes from strains isolated worldwide (spanning 270 multilocus sequence types (STs) across all known evolutionary clades), this study provides the first species-wide snapshot of AMR genomic epidemiology in C. difficile. Of the 10,330 C. difficile genomes, 4,532 (43.9%) in 89 STs across clades 1 - 5 carried at least one genotypic AMR determinant, with 901 genomes (8.7%) carrying AMR determinants for three or more antimicrobial classes (multidrug-resistant, MDR). No AMR genotype was identified in any strains belonging to the cryptic clades. C. difficile from Australia/New Zealand had the lowest AMR prevalence compared to strains from Asia, Europe and North America (p<0.0001). Based on the phylogenetic clade, AMR prevalence was higher in clades 2 (84.3%), 4 (81.5%) and 5 (64.8%) compared to other clades (collectively 26.9%) (p<0.0001). MDR prevalence was highest in clade 4 (61.6%) which was over three times higher than in clade 2, the clade with the second-highest MDR prevalence (18.3%). There was a strong association between specific AMR determinants and three major epidemic C. difficile STs: ST1 (clade 2) with fluoroquinolone resistance (mainly T82I substitution in GyrA) (p<0.0001), ST11 (clade 5) with tetracycline resistance (various tet-family genes) (p<0.0001) and ST37 (clade 4) with macrolide-lincosamide-streptogramin B (MLSB) resistance (mainly ermB) (p<0.0001) and MDR (p<0.0001). A novel and previously overlooked tetM-positive transposon designated Tn6944 was identified, predominantly among clade 2 strains. This study provides a comprehensive review of AMR in the global C. difficile population which may aid in the early detection of drug-resistant C. difficile strains, and prevention of their dissemination world-wide.

Impact statementUtilising a publicly-available database of 10,330 sequence reads, this study provides the first species-wide evaluation of genotypic AMR in C. difficile. It reports the most common AMR determinants and their genomic neighbourhood, associations between important genotypes and specific strains or geographical regions, and rare AMR genotypes that may have been missed in earlier studies.

Data summaryThis study utilises publicly available raw sequence reads available at the NCBI Sequence Read Archive (SRA) as of January 2020. The details of all genomes are available in the Supplementary Data (10.6084/m9.figshare.14623533).

sentences: A generalized distribution interpolated between the exponential and power law distributions and applied to the walking data of the pill bug (Armadillidium vulgare) To determine whether the walking pattern of an organism is a Levy walk or a Brownian walk, it has been compared whether the frequency distribution of linear step lengths follows a power law distribution or an exponential distribution. However, there are many cases where actual data cannot be classified into either of these categories. In this paper, we propose a general distribution that includes the power law and exponential distributions as special cases. This distribution has two parameters: One represents the exponent, similar to the power law and exponential distributions, and the other is a shape parameter representing the shape of the distribution. By introducing this distribution, an intermediate distribution model can be interpolated between the power law and exponential distributions. In this study, the proposed distribution was fitted to the frequency distribution of the step length calculated from the walking data of pill bugs. The autocorrelation coefficients were also calculated from the time-series data of the step length, and the relationship between the shape parameter and time dependency was investigated. The results showed that individuals whose step-length frequency distributions were closer to the power law distribution had stronger time dependence.

sentences: A Gradient-based Spectral Explainability Method for EEG Deep Learning Classifiers The automated feature extraction capabilities of deep learning classifiers have promoted their broader application to EEG analysis. In contrast to earlier machine learning studies that used extracted features and traditional explainability approaches, explainability for classifiers trained on raw data is particularly challenging. As such, studies have begun to present methods that provide insight into the spectral features learned by deep learning classifiers trained on raw EEG. These approaches have two key shortcomings. (1) They involve perturbation, which can create out-of-distribution samples that cause inaccurate explanations. (2) They are global, not local. Local explainability approaches can be used to examine how demographic and clinical variables affected the patterns learned by the classifier. In our study, we present a novel local spectral explainability approach. We apply it to a convolutional neural network trained for automated sleep stage classification. We apply layer-wise relevance propagation to identify the relative importance of the features in the raw EEG and subsequently examine the frequency domain of the explanations to determine the importance of each canonical frequency band locally and globally. We then perform a statistical analysis to determine whether age and sex affected the patterns learned by the classifier for each frequency band and sleep stage. Results showed that {delta}, {beta}, and {gamma} were the overall most important frequency bands. In addition, age and sex significantly affected the patterns learned by the classifier for most sleep stages and frequency bands. Our study presents a novel spectral explainability approach that could substantially increase the level of insight into classifiers trained on raw EEG.

sentences: Pan-cancer mapping of single T cell profiles reveals a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity Unleashing cytotoxic CD8+ T cells for effective cancer treatment requires understanding T cell states across different tumor microenvironments. Here, we developed an algorithm to recover both shared and tumor type specific programs and used it to analyze a scRNA-seq compendium of 38,852 CD8+ T cells from 141 patients spanning nine different human cancers. We uncovered a pan-cancer T cell dysfunction program that was predictive of clinical responses to immunotherapy and highlighted CXCR6 as a pan-cancer marker of dysfunctional T cells. In mouse models, CXCR6 increased following checkpoint blockade and was repressed by TCF1. Its ligand, CXCL16, was expressed primarily by myeloid cells, and was co-regulated with antigen presentation genes. CXCR6 deletion decreased Tox, CX3CR1, and Bcl2 expression, predisposing dysfunctional PD-1+Tim3+CD8+ T cells to apoptosis, and compromising tumor growth control. Our approach discovered a TCF1:CXCR6-CXCL16 regulatory axis essential for effective anti-tumor immunity, revealing a new perspective on T cell dysfunction and new opportunities for modulating this cell state.

sentences: OViTAD: Optimized Vision Transformer to Predict Various Stages of Alzheimer's Disease Using Resting-State fMRI and Structural MRI Data Advances in applied machine learning techniques for neuroimaging have encouraged scientists to implement models to diagnose brain disorders such as Alzheimers disease at early stages. Predicting the exact stage of Alzheimers disease is challenging; however, complex deep learning techniques can manage this with precision. While successful, these complex architectures are difficult to interrogate and computationally expensive. Therefore, using novel, simpler architectures with more efficient pattern extraction capabilities, such as transformers, is of interest to neuroscientists. This study introduced an optimized vision transformer architecture to predict the group membership by separating healthy adults, mild cognitive impairment, and Alzheimers brains within the same age group (>75 years) using resting-state functional (rs-fMRI) and structural magnetic resonance imaging (sMRI) data. Our optimized architecture known as OViTAD is currently the sole vision transformer-based end-to-end pipeline and outperformed the existing transformer models and most state-of-the-art solutions. Our model achieved F1-scores of 97%{+/-}0.0 and 99.55%{+/-}0.39 from the testing sets for the rs-fMRI and sMRI modalities in the triple-class prediction experiments. Furthermore, our model reached these performances using 30% fewer parameters than a vanilla transformer. The model was robust and repeatable, producing similar estimates across three runs (we reported the averaged evaluation metrics). Finally, to challenge the model, we observed how it handled increasing noise levels by inserting varying numbers of healthy brains into the two dementia groups. Our findings suggest that optimized vision transformers are a promising and exciting new approach for neuroimaging applications, especially for Alzheimers disease prediction.

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Inhibiting microglia proliferation after spinal cord injury improves recovery in mice and nonhuman primates No curative treatment is available for any deficits induced by spinal cord injury (SCI). Following injury, microglia undergo highly diverse activation processes, including proliferation, and play a critical role on functional recovery.

In a translational objective, we investigated whether a transient pharmacological reduction of microglia proliferation after injury is beneficial for functional recovery after SCI in mice and nonhuman primates. The colony stimulating factor-1 receptor (CSF1R) regulates proliferation, differentiation, and survival of microglia, we thus used an oral administration of GW2580, a CSF1R inhibitor.

First, transient post-injury GW2580 administration in mice improves motor function recovery, promotes tissues preservation and/or reorganization (identified by coherent anti-stokes Raman scattering microscopy), and modulates glial reactivity.

Second, post-injury GW2580-treatment in nonhuman primates reduces microglia proliferation, improves functional motor function recovery, and promotes tissue protection. Notably, three months after lesion microglia reactivity returned to baseline value.

Finally, to initiate the investigation on molecular mechanisms induced by a transient post-SCI GW2580-treatment, we used microglia-specific transcriptomic analysis in mice. Notably, we detected a downregulation in the expression of inflammatory-associated genes and we identified genes that were up-regulated by SCI and further downregulated by the treatment.

Thus, a transient oral GW2580 treatment post-injury may provide a promising therapeutic strategy for SCI patients and may also be extended to other central nervous system disorders displaying microglia activation.

sentences: Genome-wide association study reveals complex genetic architecture of cadmium and mercury accumulation and tolerance traits in Medicago truncatula Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 - 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, several oxidative stress response genes and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

sentences: A computational screen for alternative genetic codes in over 250,000 genomes The genetic code has been proposed to be a "frozen accident", but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force which likely helped drive these codons to low frequency and enable their reassignment.

sentences: Untangling the cell immune response dynamic for severe and critical cases of SARS-CoV-2 infection COVID-19 is a global pandemic leading high death tolls worldwide day by day. Clinical evidence suggests that COVID-19 patients can be classified as non-severe, severe and critical cases. In particular, studies have highlighted the relationship between the lymphopenia and the severity of the illness, where CD8+ T cells have the lowest levels in critical cases. In this work, we aim to elucidate the key parameters that define the course of the disease deviating from severe to critical case. To this end, several mathematical models are proposed to represent the dynamic of the immune response in patients with SARS-CoV-2 infection. The best model had a good fit to reported experimental data, and in accordance with values found in the literature. Our results suggest that a rapid proliferation of CD8+ T cells is decisive in the severity of the disease.

sentences: Dysregulation of NRSF/REST via EHMT1 is associated with psychiatric disorders Genetic evidence indicates disrupted epigenetic regulation as a major risk factor for psychiatric disorders, but the molecular mechanisms that drive this association are undetermined. EHMT1 is an epigenetic repressor that is causal for Kleefstra Syndrome (KS), a neurodevelopmental disorder (NDD) leading to ID, and is associated with schizophrenia. Here, we show that reduced EHMT1 activity decreases NRSF/REST protein leading to abnormal neuronal gene expression and progression of neurodevelopment in human iPSC. We further show that EHMT1 regulates NRSF/REST indirectly via repression of miRNA leading to aberrant neuronal gene regulation and neurodevelopment timing. Expression of a NRSF/REST mRNA that lacks the miRNA-binding sites restores neuronal gene regulation to EHMT1 deficient cells. Importantly, the EHMT1-regulated miRNA gene set with elevated expression is enriched for NRSF/REST regulators with an association for ID and schizophrenia. This reveals a molecular interaction between H3K9 dimethylation and NSRF/REST contributing to the aetiology of psychiatric disorders.

sentences: A Novel Membrane Protein in the Rhodobacter sphaeroides LH1-RC Photocomplex We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 [A] resolution. The LH1 complex forms a C-shaped structure composed of 14 {beta}-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

sentences: Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia Stress-adaptive mechanisms enable tumor cells to overcome metabolic constraints in nutrient and oxygen poor tumors. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumor growth are poorly understood. Here, using a combination of metabolomics and CRISPR-based genetic screens, we identify GOT2-catalyzed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumor cells under hypoxic culture conditions. In contrast, GOT2-catalyzed aspartate synthesis is dispensable for pancreatic tumor formation in vivo. The dependence of pancreatic tumor cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRas-dependent, and is mediated by hypoxia inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9) through the cooption of the bicarbonate-macropinocytosis signaling axis. Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRas tumors can overcome nutrient deprivation under hypoxic conditions.

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Global and regional ecological boundaries drive abrupt changes in avian frugivory interactions Species interactions can propagate disturbances across space, though ecological and biogeographic boundaries may limit this spread. We tested whether large-scale ecological boundaries (ecoregions and biomes) and human disturbance gradients increase dissimilarity among ecological networks, while accounting for background spatial and elevational effects and differences in network sampling. We assessed network dissimilarity patterns over a broad spatial scale, using 196 quantitative avian frugivory networks (encompassing 1,496 plant and 1,003 bird species) distributed across 67 ecoregions and 11 biomes. Dissimilarity in species and interactions, but not in network structure, increased significantly across ecoregion and biome boundaries and along human disturbance gradients. Our findings suggest that ecological boundaries contribute to maintaining the worlds biodiversity of interactions and mitigating the propagation of disturbances at large spatial scales.

One-Sentence SummaryEcoregions and biomes delineate the large-scale distribution of plant-frugivore interactions.

sentences: Prenatal Stress Dysregulates Resting-State Functional Connectivity and Sensory Motifs Prenatal stress (PS) can impact fetal brain structure and function and contribute to higher vulnerability to neurodevelopmental and neuropsychiatric disorders. To understand how PS alters evoked and spontaneous neocortical activity and intrinsic brain functional connectivity, mesoscale voltage imaging was performed in adult C57BL/6NJ mice that had been exposed to auditory stress on gestational days 12-16, the age at which neocortex is developing. PS mice had a four-fold higher basal corticosterone level and reduced amplitude of cortical sensory-evoked responses to visual, auditory, whisker, forelimb, and hindlimb stimuli. Relative to control animals, PS also led to a general reduction of resting-state functional connectivity, as well as reduced inter-modular connectivity, enhanced intra-modular connectivity, and altered frequency of auditory and forelimb spontaneous sensory motifs. These resting-state changes resulted in a cortical connectivity pattern featuring disjoint but tight modules and a decline in network efficiency. The findings demonstrate that cortical connectivity is sensitive to PS and exposed offspring may be at risk for adult stress-related neuropsychiatric disorders.

sentences: Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.

sentences: Unique brewing-relevant properties of a strain of Saccharomyces jurei isolated from ash (Fraxinus excelsior) The successful application of Saccharomyces eubayanus and Saccharomyces paradoxus in brewery fermentations has highlighted the potential of wild yeast for brewing, and prompted investigation into the application potential of other members of the genus. Here, we evaluate, for the first time, the brewing potential of Saccharomyces jurei. The newly isolated strain from an ash tree (Fraxinus excelsior) in Upper Bavaria, Germany, close to the river Isar, was used to ferment a 12{degrees}P wort at 15{degrees}C. Performance was compared directly with that of a reference lager strain (TUM 34/70) and the S. eubayanus type strain. Both wild yeast rapidly depleted simple sugars and thereafter exhibited a lag phase before maltose utilization. This phase lasted for 4 and 10 days for S. eubayanus and S. jurei, respectively. S. eubayanus utilized fully the available maltose but, consistent with previous reports, did not use maltotriose. S. jurei, in contrast, utilized approx. 50% of the maltotriose available, making this the first report of maltotriose utilization in a wild Saccharomyces species. Maltotriose use was directly related to alcohol yield with 5.5, 4.9, and 4.5 % ABV produced by S. pastorianus, S. jurei and S. eubayanus. Beers also differed with respect to aroma volatiles, with a high level (0.4 mg/L) of the apple/aniseed aroma ethyl hexanoate in S. jurei beers, while S. eubayanus beers had a high level of phenylethanol (100 mg/L). A trained panel rated all beers as being of high quality, but noted clear differences. A phenolic spice/clove note was prominent in S. jurei beer. This was less pronounced in the S. eubayanus beers, despite analytical levels of 4-vinylguaiacol being similar. Tropical fruit notes were pronounced in S. jurei beers, possibly resulting from the high level of ethyl hexanoate. Herein, we present the successful results of the first intentional application of S. jurei as a yeast for beer fermentation known to us and compare its fermentation performance to other species of the genus. Results indicate considerable potential for S. jurei application in brewing, with clear advantages compared to other wild Saccharomyces species.

sentences: CytoSet: Predicting clinical outcomes via set-modeling of cytometry data AO_SCPLOWBSTRACTC_SCPLOWSingle-cell flow and mass cytometry technologies are being increasingly applied in clinical settings, as they enable the simultaneous measurement of multiple proteins across millions of cells within a multi-patient cohort. In this work, we introduce CytoSet, a deep learning model that can directly predict a patients clinical outcome from a collection of cells obtained through a blood or tissue sample. Unlike previous work, CytoSet explicitly models the cells profiled in each patient sample as a set, allowing for the use of recently developed permutation invariant architectures. We show that CytoSet achieves state-of-the-art classification performance across a variety of flow and mass cytometry benchmark datasets. The strong classification performance is further complemented by demonstrated robustness to the number of sub-sampled cells per patient and the depth of model, enabling CytoSet to scale adequately to hundreds of patient samples. The strong performance achieved by the set-based architectures used in CytoSet suggests that clinical cytometry data can be appropriately interpreted and studied as sets. The code is publicly available at https://github.com/CompCy-lab/cytoset.

sentences: TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq) for scRNA-seq based on a terminator, terminal transferase, and nanowell/bead-based scRNA-seq platform; TAS-Seq showed high tolerance to variations in the terminal transferase reaction, which complicate the handling of existing terminal transferase-based scRNA-seq methods. In murine and human lung samples, TAS-Seq yielded scRNA-seq data that were highly correlated with flow-cytometric data, showing higher gene-detection sensitivity and more robust detection of important cell-cell interactions and expression of growth factors/interleukins in cell subsets than 10X Chromium v2 and Smart-seq2. Expanding TAS-Seq application will improve understanding and atlas construction of lung biology at the single-cell level.

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Distribution and genomic variation of thermophilic cyanobacteria in diverse microbial mats at the upper temperature limits of photosynthesis Thermophilic cyanobacteria have been extensively studied in Yellowstone National Park (YNP) hot springs, particularly from decades of work on the thick laminated mats of Octopus and Mushroom Springs. However, focused studies of cyanobacteria outside of these two hot springs have been lacking, especially regarding how physical and chemical parameters along with community morphology influence the genomic makeup of these organisms. Here, we used a metagenomic approach to examine cyanobacteria existing at the upper temperature limits of photosynthesis. We examined 15 alkaline hot spring samples across six geographic areas of YNP, all with varying physical and chemical parameters, and community morphology. We recovered 22 metagenome-assembled genomes (MAGs) belonging to thermophilic cyanobacteria, notably an uncultured Synechococcus-like taxon recovered from the upper temperature limit of photosynthesis, 73{degrees}C, in addition to thermophilic Gloeomargarita. Furthermore, we found that three distinct groups of Synechococcus-like MAGs recovered from different temperature ranges vary in their genomic makeup. MAGs from the uncultured very high temperature (up to 73{degrees}C) Synechococcus-like taxon lack key nitrogen metabolism genes and have genes implicated in cellular stress responses that diverge from other Synechococcus-like MAGs. Across all parameters measured, temperature was the primary determinant of taxonomic makeup of recovered cyanobacterial MAGs. However, Fe, community morphology, and biogeography played an additional role in the distribution and abundance of upper temperature limit-adapted Synechococcus-like MAGs.These findings expand our understanding of cyanobacterial diversity in YNP and provide a basis for interrogation of understudied thermophilic cyanobacteria.

ImportanceOxygenic photosynthesis arose early in microbial evolution - approx. 2.5-3.5 billion years ago - and entirely reshaped the biological makeup of Earth. However, despite the span of time in which photosynthesis has been refined, it is strictly limited to temperatures below 73{degrees}C, a barrier that many other biological processes have been able to overcome. Furthermore, photosynthesis at temperatures above 56{degrees}C is limited to circumneutral and alkaline pH. Hot springs in Yellowstone National Park (YNP), which have a large diversity in temperatures, pH and geochemistry provide a natural laboratory to study thermophilic microbial mats, and the cyanobacteria within. While cyanobacteria in YNP microbial mats have been studied for decades, a vast majority of work has focused on two springs within the same geyser basin, both containing similar community morphologies. Thus, the drivers of cyanobacterial adaptations to the upper limits of photosynthesis across a variety of environmental parameters have been understudied. Our findings provide new insights into the influence of these parameters on both taxonomic diversity and genomic content of cyanobacteria across a range of hot spring samples.

sentences: In vitro neuronal networks show bidirectional axonal conduction with antidromic action potentials effectively depolarizing the soma Recent technological advances are revealing the complex physiology of the axon and challenging long-standing assumptions. Namely, while most action potential (AP) initiation occurs at the axon initial segment in central nervous system neurons, initiation in distal parts of the axon has been shown to occur in both physiological and pathological conditions. However, such ectopic action potential (EAP) activity has not been reported yet in studies using neuronal cultures and its functional role, if exists, is still not clear. Here, we show the spontaneous occurrence of EAPs and effective antidromic conduction in hippocampal neuronal cultures. We also observe a significant fraction of bidirectional axonal conduction in dorsal root ganglia neuronal cultures. We investigate and characterize this antidromic propagation via a combination of microfluidics, microelectrode arrays, advanced data analysis and in silico studies. We show that EAPs and antidromic conduction can occur spontaneously, and after distal axotomy or physiological changes in the axon biochemical environment. Conduction velocity is asymmetrical, with antidromic conduction being slower than orthodromic. Importantly, EAPs may carry information and can have a functional impact on the neuron, as they consistently depolarize the soma. Thus, plasticity or gene transduction mechanisms triggered by soma depolarization can also be affected by these antidromic APs. Altogether these findings have important implications for the study of neuronal function in vitro, reshaping our understanding on how information flows in neuronal cultures.

sentences: Tissue-wide coordination of calcium signaling regulates the epithelial stem cell pool during homeostasis Skin epidermal homeostasis is maintained via constant regeneration by stem cells, which must communicate to balance their self-renewal and differentiation. A key molecular pathway, Ca2+ signaling has been implicated as a signal integrator in developing and wounded epithelial tissues[1, 2, 3, 4]. Yet how stem cells carry out this signaling across a regenerative tissue remains unknown due to significant challenges in studying signaling dynamics in live mice, limiting our understanding of the mechanisms of stem cell communication during homeostasis. To interpret high dimensional signals that have complex spatial and temporal patterns, we combined optimized imaging of Ca2+ signaling in thousands of epidermal stem cells in living mice with a new machine learning tool, Geometric Scattering Trajectory Homology (GSTH). Using a combination of signal processing, data geometry, and topology, GSTH captures patterns of signaling at multiple scales, either between direct or distant stem cell neighbors. Here we show that epidermal stem cells display dynamic intercellular Ca2+ signaling among neighborhoods of up to 10 cells that is surprisingly coordinated and directed through time across a pool of thousands of stem cells. We find that this collective coordination is an emergent property of the stem cell compartment, distinct from excitatory quiescent neuronal tissues. We demonstrate that cycling stem cells, specifically G2 cells, govern homeostatic patterns of Ca2+ signaling. Stem cells in different cell cycle stages dynamically regulate localization of the gap junction component Connexin43 (Cx43). Lastly, we uncouple global from local communication and identify Cx43 as the molecular mediator necessary for connectivity between local signaling neighborhoods. This work provides resolution in how stem cells at different stages of the cell cycle communicate and how that diversity of phases is essential for tissue wide communication and signaling flow during epidermal regeneration. Our approach provides a framework to investigate stem cell populations and their signaling dynamics, previously not possible.

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A novel proneural function of Asense, integrated with of L'sc and Notch, promotes the Neuroepithelial to Neuroblast transition In the developing Drosophila optic lobe, neuroepithelial (NE) cells are transformed progressively into neurogenic progenitors called neuroblasts (NBs). The progenitors undergoing this transition are identified by the expression of the Acheate Scute Complex (AS-C) factor Lethal of Scute (Lsc).

Here we found that Asense (Ase), another AS-C factor, presents a peak of expression in the cells neighboring those transition Lsc expressing cells. This peak of Ase identifies a new transition step and it is necessary and sufficient to promote the NE to NB transition. Thus, our data provide the first direct evidence for a proneural role of Ase in CNS neurogenesis. Furthermore, we found that the peak of Ase is induced in a non-cell autonomous manner by Lsc through the activation of Notch signaling in the adjacent cells. This suggests that the two classic proneural activities, promoting neurogenesis and Notch signaling, have been split between Ase and Lsc. Thus, our data fit with a model in which the key proneural role of Ase is integrated with Notch and Lsc activities, facilitating the progressive transformation of NE cells into NBs.

SUMMARY STATEMENTThe switch of progenitor cells towards neuron production is crucial for proper brain development. The transcription factor Asense promotes this transition through a mechanism integrated with timing and neurogenic signals.

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The JAK2-STAT3 pathway controls a beneficial proteostasis response of reactive astrocytes in Huntington disease Huntingtons disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntingtons disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntingtons disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models.

Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntingtons disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntingtons disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons.

Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntingtons disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.

sentences: In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance. In vitro selection of remdesivir-resistant SARS-CoV-2 revealed the emergence of a V166L substitution, located outside of the polymerase active site of the nsp12 protein, after 9 passages. V166L remained the only nsp12 substitution after 17 passages at a final concentration of 10 {micro}M RDV, conferring a 2.3-fold increase in EC50. When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to RDV resistance.

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Synaptotagmin 7 is enriched at the plasma membrane to promote vesicle docking and control synaptic plasticity Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees.  Zap-and-freeze electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via {gamma}-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity.

O_LISYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses
C_LIO_LILocalization, trafficking, and stability of SYT7 is dependent on processing by {gamma}-secretase
C_LIO_LIShort term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation
C_LIO_LISYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms
C_LI

sentences: Disruption of the plastid-hosted iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the SUF pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. We have generated another mutant of the pathway, deficient for the SUFC ATPase, and we have investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in growth: cell division and membrane homeostasis are particularly affected. Lipidomic analysis suggests a defect in apicoplast-generated fatty acids, along with a simultaneous increase in scavenging of host-derived lipids. However, addition of exogenous lipids did not allow full restauration of growth, suggesting other more important cellular functions were impacted in addition to fatty acid synthesis. For instance, we have shown that the SUF pathway is also key for generating isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins as well as for the parasite gliding motility. Thus, plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for discovering new potential anti-Toxoplasma targets.

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Mutant-SETBP1 activates transcription of Myc programs to accelerate CSF3R-driven myeloproliferative neoplasms Colony stimulating factor 3 receptor (CSF3R) mutations lead to JAK pathway activation and are the molecular hallmark of chronic neutrophilic leukemia (CNL). Approximately half of CNL patients also have mutations in SET binding protein 1 (SETBP1). In this study, we developed models of SETBP1-mutant leukemia to understand the role that SETBP1 plays in CNL. SETBP1 mutations promote self-renewal of CSF3R-mutant hematopoietic progenitors in vitro and prevent cells from undergoing terminal differentiation. In vivo, SETBP1 mutations accelerate leukemia progression, leading to the rapid development of hepatosplenomegaly and granulocytosis. Through transcriptomic and epigenomic profiling, we found that SETBP1 enhances progenitor-associated programs--most strongly upregulating Myc and Myc target genes. This upregulation of Myc can be reversed by epigenetic modulatory drugs. In summary, we find that SETBP1 mutations promote aggressive hematopoietic cell expansion when expressed with mutant CSF3R through the upregulation of Myc-associated gene expression programs.

Statement of SignificanceSETBP1 is frequently mutated in chronic neutrophilic leukemia, but its role in the biology of this disease is unclear. We find that mutant SETBP1 enhances transcription of Myc and Myc target genes to promote aggressive disease biology, and that these oncogenic transcriptional programs can be reversed by epigenetic modulatory drugs.

sentences: Hybridization and cryptic speciation in the Tropical Eastern Pacific octocoral genus Pacifigorgia The shallow waters of the Tropical Eastern Pacific (TEP) harbor a species-rich octocoral fauna, with seven genera and 124 octocoral species described to date for the region. Of these lineages, Pacifigorgia, with 35 species, is by far the most speciose and abundant shallow-water octocoral occurring in the region. The speciation mechanisms resulting in this remarkable diversity remain speculative, despite the extensive taxonomic and molecular systematic research conducted so far in the TEP. Using genome-wide SNP markers, we provide evidence for hybridization and extensive cryptic speciation in Pacifigorgia, suggesting that the genus diversity has been underestimated by traditional and molecular systematic research. Our study highlights the difficulties faced by both traditional taxonomy and single-marker based molecular approaches to characterize octocoral diversity and evolution, and the role genome-wide molecular studies coupled to morphological research play to advance our understanding of this group.

sentences: Single-deletion-mutant, third-generation rabies viral vectors allow nontoxic retrograde targeting of projection neurons with greatly increased efficiency Rabies viral vectors have become important components of the systems neuroscience toolkit, allowing both direct retrograde targeting of projection neurons and monosynaptic tracing of inputs to defined postsynaptic populations, but the rapid cytotoxicity of first-generation ({Delta}G) vectors limits their use to short-term experiments. We recently introduced second-generation, double-deletion-mutant ({Delta}GL) rabies viral vectors, showing that they efficiently retrogradely infect projection neurons and express recombinases effectively but with little to no detectable toxicity; more recently, we have shown that {Delta}GL viruses can be used for monosynaptic tracing with far lower cytotoxicity than the first-generation system. Here we introduce third-generation ({Delta}L) rabies viral vectors, which, like first-generation vectors, have only a single gene deleted from their genomes (in this case the viral polymerase gene L) but which appear to be as nontoxic as second-generation ones: using longitudinal structural and functional two-photon imaging in mouse visual cortex in vivo, we found that they did not kill labeled neurons or noticeably perturb their response properties over the entire months-long courses of imaging. Although third-generation vectors are therefore phenotypically very similar to second-generation ones, we show that they have the major advantage of growing to much higher titers, and this key difference results in 25% - 525% increased numbers of retrogradely labeled neurons in vivo. These {Delta}L rabies viral vectors therefore constitute a new state of the art for minimally perturbative, pathway-specific expression of recombinases and transactivators in mammalian neurons selected on the basis of their axonal projections. Because replication of deletion-mutant rabies viruses within complementing cells is precisely the process that underlies monosynaptic tracing, the higher replication efficiency of this new class of rabies viral vectors furthermore suggests the potential to provide the foundation of an improved nontoxic monosynaptic tracing system.

sentences: Cell cycle exit and stem cell differentiation are coupled through regulation of mitochondrial activity in the Drosophila testis Stem cells maintain tissue homeostasis by proliferating to replace cells lost to damage or natural turnover. Whereas stem and progenitor cells proliferate, fully differentiated cells exit the cell cycle. How cell identity and cell cycle state are coordinated during this process is still poorly understood. The Drosophila testis niche supports germline stem cells and somatic cyst stem cells (CySCs), which are the only proliferating somatic cells in the testis. CySCs give rise to post-mitotic cyst cells and therefore provide a tractable model to ask how stem cell identity is linked to proliferation. We show that the G1/S cyclin, Cyclin E, is required for CySC self-renewal; however, its canonical transcriptional regulator, a complex of the E2f1 and Dp transcription factors is dispensable for self-renewal and cell cycle progression. Nevertheless, we demonstrate that E2f1/Dp activity must be silenced to allow CySCs to differentiate. We show that E2f1/Dp activity inhibits the expression of genes important for mitochondrial activity. Furthermore, promoting mitochondrial activity or biogenesis is sufficient to rescue the differentiation of CySCs with ectopic E2f1/Dp activity but not their exit from the cell cycle. Our findings together indicate that E2f1/Dp coordinates cell cycle progression with stem cell identity by regulating the metabolic state of CySCs.

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Perineuronal net receptor PTPσ regulates retention of memories Perineuronal nets (PNNs) have an important physiological role in retention of learning by restricting cognitive flexibility. Their deposition peaks after developmental periods of intensive learning, usually in late childhood, and they help in long-term preservation of new acquired skills and information. Modulation of PNN function by various techniques enhances plasticity and regulates retention of memories, which may be beneficial when memory persistence entails negative symptoms such as post-traumatic stress disorder (PTSD). In this study, we investigated the role of PTP{sigma} (receptor-type tyrosine-protein phosphatase S, a phosphatase that is activated by binding of chondroitin sulfate proteoglycans from PNNs) in retention of memories using novel object recognition and fear conditioning rodent models. We observed that mice haploinsufficient for PTPRS gene (PTP{sigma}+/-), although having improved short-term object recognition memory, display impaired long-term memory in both novel object recognition and fear conditioning paradigm, as compared to WT littermates. However, PTP{sigma}+/- mice didnt show any differences in behavioral tests that do not heavily rely on cognitive flexibility, such as elevated plus maze, open field, marble burying and forced swimming test. Since PTP{sigma} has been shown to interact with and dephosphorylate TRKB, we investigated activation of this receptor and its downstream pathways in limbic areas known to be associated with memory. We found that phosphorylation of TRKB and PLC{gamma} are increased in the hippocampus, prefrontal cortex and amygdala of PTP{sigma}+/- mice, but other TRKB-mediated signaling pathways are not affected. Our data suggest that disruption of PNN-PTP{sigma} complex facilitates short-term memory by promoting TRKB phosphorylation in different brain areas, but that PTP{sigma} activity is required for the retention of long-term memories. Inhibition of PTP{sigma} or disruption of PNN-PTP{sigma}-TRKB complex might be a potential target for disorders where negative modulation of the acquired memories can be beneficial.

Conflict of interestThe authors declare no conflict of interest.

FundingThis work was supported by Doctoral Program in Integrative Life Science, Jalmari ja Rauha Ahokkaan Saatio grant, Centre for International Mobility (CIMO) Grant TM-16-10112, and by grants from European Research Council (#322742), EU Joint Programme - Neurodegenerative Disease Research (JPND) CircProt (#301225 and #643417), Sigrid Juselius Foundation, Jane and Aatos Erkko Foundation, and the Academy of Finland (#294710 and #307416). None of the funders had a role in the data acquisition, analysis or manuscript preparation.

Author contributionsPC, CB and EC designed the study; AL, PC and CB performed the experiments and analyzed the data; AL wrote the manuscript draft; AL, PC, CB and EC revised the manuscript.

Contribution to the Field StatementPlasticity of neuronal networks increases brains ability to adapt and it is compromised in various conditions including psychiatric diseases, brain injuries, neurodevelopmental and neurodegenerative disorders. Perineuronal nets, through their receptor PTP{sigma} restrict neuronal plasticity in adult brain, which is considered important for the retention of long-term memories. We have previously shown that this restricted plasticity is mediated by inhibition by PTP{sigma} of the TRKB neurotrophin receptor. We have now investigated the biochemical and behavioral phenotype of mice with reduced PTP{sigma} expression. We observed that PTP{sigma}+/- mice have increased phosphorylation of TRKB and its downstream partner PLC{gamma}1 in the prefrontal cortex, hippocampus and amygdala, demonstrating chronic overactivation of plasticity-related pathways. Consistently, PTP{sigma}+/- mice demonstrated facilitated learning but impaired long-term memory retention. Unexpectedly, long-term memory of PTP{sigma}+/- mice was impaired, suggesting that perneuronal net-stimulated PTP{sigma} activity is important in memory retention. This effect of neuronal "hyperplasticity" induced by PTP{sigma} knockout may be both beneficial and harmful in the treatment of human patients, which should be taken into account when developing therapeutic strategies.

sentences: A homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium Interferon-lambda (IFN-{lambda}) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-{lambda} receptor (Ifnlr1), and is associated with IFN-{lambda} production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally-administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

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EEG-based neurofeedback with network components extraction: data-driven approach by multilayer ICA extension and simultaneous EEG-fMRI measurements Several studies have reported advanced treatments for depressive symptoms, such as real-time neurofeedback (NF) with functional MRI (fMRI) and/or electroencephalogram (EEG). NF focusing on a regularization of brain activity associated with the amygdala or functional connectivity (FC) between the executive control network (ECN) and default mode network (DMN) has been applied to reduce depressive symptoms. However, it is practically difficult to install the fMRI-NF system and to consistently provide treatment, because of high cost. Additionally, no practical signal processing techniques have been developed extracting FC-related features from EEG signals, particularly when no physical forward models are available. In this regard, stacked pooling and linear components estimation (SPLICE), recently proposed as a multilayer extension of independent component analysis (ICA) and related independent subspace analysis (ISA), can be a promising alternative. The resting-state EEG network features can be correlated with fMRI network activity corresponding to the DMN or ECN. This may enable the modulation of the target FC-related features in EEG-based NF.

In this study, we developed a real-time EEG NF system for improving depressive symptoms by using the SPLICE. Utilizing information from the fMRI biomarkers, we evaluated our paradigm for effectiveness with regard to upregulation of the dorsolateral prefrontal cortex /middle frontal gyrus or downregulation of the precuneus/posterior cingulate cortex. We conducted an NF experiment in participants with subclinical depression; the participants were divided into the NF group (n=8) and the sham group (n=9). We found a significant reduction and a large effect size in the rumination response scale (RRS) score (reflection) in the NF group, compared to the sham group.

However, we did not find a significant relationship between the training score and difference in symptoms. This suggests that increased controllability of the EEG signals did not directly reduce the RRS reflection score. This could be due to various reasons such as improper feature extraction, individual differences, and the targeted brain regions. In this paper, we also discuss the possible ways to modify our NF protocol including the design of the experiment, sample size, and online processing. We then discuss way to improve the NF training, based on our results.

sentences: Structures of Tweety Homolog Proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to inter-membrane dimerization Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a novel fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 [A] intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

sentences: Time to stop; agent-based modelling of chemoaffinity with competition In the classic Chemoaffinity theory, the retinotectal axon projection is thought to use pairs of orthogonal signalling gradients in the retina to specify the eventual location of synapses made on the surface of the tectum/superior colliculus. Similar orthogonal gradients in the tectum provide a coordinate system which allows the axons to match their prespecified destination with the correct location. Although the Ephrins have been shown to guide axons toward their destination, there has yet to emerge a complete account of the local interactions which halt the axonal growth cones in the correct locations to recreate the topography of the retinal cells. The model of Simpson and Goodhill (2011) provides an account of the basic topographic arrangement of cells on the tectum, as well as reproducing well known surgical and genetic manipulation experiments. However, it suffers from the absence of a local chemotactic guidance mechanism. Instead, each agent in their model is given instantaneous knowledge of the vector that would move it toward its pre specified destination. In addition to the globally supervised chemoaffinity term, Simpson and Goodhill (2011) introduced a competitive interaction for space between growth cone agents and a receptor-ligand axon-axon interaction in order to account for the full set of experimental manipulations. Here, we propose the replacement of the chemoaffinity term with a gradient following model consisting of axonal growth cone agents which carry receptor molecule expression determined by their somas location of origin on the retina. Growth cones move on the simulated tectum guided by two pairs of opposing, orthogonal signalling molecules representing the Ephrin ligands. We show that with only the chemoaffinity term and a receptor-ligand based axon-axon interaction term (meaning that all growth cone interactions are by receptor-ligand signalling), a full range of experimental manipulations to the retinotectal system can be reproduced. Furthermore, we show that the observation that competition is not and essential requirement for axons to find their way (Gosse et al., 2008) is also accounted for by the model, due to the opposing influences of signalling gradient pairs. Finally, we demonstrate that, assuming exponentially varying receptor expression in the retina, ligand expression should either be exponential if the receptor-ligand signal induces repulsion (i.e. gradient descent) or logarithmic if the signal induces attraction (gradient ascent). Thus, we find that a model analogous to the one we presented in James et al. (2020) that accounts for murine barrel patterning is also a candidate mechanism for the arrangement of the more continuous retinotectal system.

sentences: PAK1 and PAK2 in cell metabolism regulation P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. Alternatively, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms.

sentences: Effects of tendon viscoelasticity in the distribution of forces over sutures in a model tendon-to-bone repair Tears to the rotator cuff often require surgical repair. These repairs often culminate in re-tearing when sutures break through the tendon in the weeks following repair. Although numerous studies have been performed to identify suturing strategies that reduce this risk by balancing forces across sutures, none have accounted for how the viscoelastic nature of tendon influences load sharing. With the aim of providing insight into this problem, we studied how tendon viscoelasticity, tendon stiffness, and suture anchor spacing affect this balancing of forces across sutures. Results from a model of a three-row sutured re-attachment demonstrated that optimized distributions of suture stiffnesses and of the spacing of suture anchors can balance the forces across sutures to within a few percent, even when accounting for tendon viscoelasticity. Non-optimized distributions resulted in concentrated force, typically in the outermost sutures. Results underscore the importance of accounting for viscoelastic effects in the design of tendon to bone repairs.

sentences: Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth To maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis, and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

sentences: Environment and the Evolutionary Trajectory of Horizontal Gene Transfer. Horizontal Gene Transfer (HGT) is a powerful evolutionary force facilitating bacterial adaptation and emergence of novel phenotypes. Several factors, including environmental ones, are predicted to restrict HGT, but we lack data supporting these predictions. Here, we address this gap by measuring the relative fitness of 44 genes horizontally transferred from E. coli to S. Typhimurium strain 4/74 in four infection relevant environments and estimated the distribution of fitness effects (DFE) in each environment. Roughly half of the genes, 40-50%, had significant fitness effects in at least one environment, with most genes showing significant gene by environment (G x E) interactions. We further identified 12 genes with dosage-dependent effects across the four environments, indicating that intolerance of the recipient strain to high protein dosage is a significant barrier to HGT. We also found longer genes had stronger fitness detriments than shorter ones, showing that gene length can significantly hinder HGT. In summary, a substantial fraction of transferred genes had a significant fitness cost on the recipient, with both gene characteristics and the environment providing barriers to HGT.

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Initiation of organ maturation and fruit ripening in grapevine is controlled by the CARPO-NAC transcription factor Grapevine is a woody temperate perennial plant and one of the most important fruit crops with global relevance in both the fresh fruit and winemaking industries. Unfortunately, global warming is affecting viticulture by altering developmental transitions and fruit maturation processes. In this context, uncovering the molecular mechanisms controlling the onset and progression of ripening could prove essential to maintain high-quality grapes and wines. Through a deep inspection of previously published transcriptomic data we identified the NAC family member VviCARPO (Controlled Adjustment of Ripening and maturation of Plant Organs) as a key regulator of grapevine maturation whose induction precedes the expression of well-known ripening associated genes. We explored VviCARPO binding landscapes through DAP-seq and overlapped its bound genes with transcriptomics datasets from stable and transient VviCARPO overexpressing grapevine plants to define a set of high-confidence targets. Among these, we identified key molecular ripening markers. Physiological, metabolic and promoter activation analyses showed that VviCARPO induces chlorophyll degradation and anthocyanin accumulation through the up-regulation of VviSGR1 and VviMYBA1, respectively, with the latter being up-regulated through a VviCARPO-VviNAC03 regulatory complex. Despite showing a closer phylogenetic relationship to senescent-related AtNAP homologues, VviCARPO complemented the nor mutant phenotype in tomato, suggesting it may have acquired a dual role as an orchestrator of both ripening- and senescence-related processes. Our data supports CARPO as a master regulator of the grapevine vegetative-to-mature phase organ transition and therefore an essential target for insuring fruit quality and environmental resilience.

SIGNIFICANT STATEMENTCARPO is a grape NAC transcription factor central to fruit ripening and tissue senescence. This regulator influences multiple biological pathways common to both processes including cell wall metabolism, chlorophyll degradation, pigment production and hormone synthesis/signaling through regulation of their key genes. As various external stresses and changing climatic conditions influence vegetative growth and berry ripening, CARPO could prove a useful genetic and breeding target towards maintaining necessary crop performance and fruit-quality characteristics.

sentences: Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. As the largest point of contact between the X and Y, PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and associated mutational processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes and two archaic human genome sequences. We find that the PAR1 sequence is closer to nucleotide equilibrium than autosomal telomeric sequences. We detect a difference between long-term substitution patterns and extant diversity in PAR1 that is mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. Additionally, we detect excess C[->]G mutations in PAR1 of all great ape species, specific to the mutagenic effect of male recombination. Analysis of differences between frequencies of alleles segregating in females and males provided no evidence for sexually antagonistic selection in this region. Furthermore, despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence, as is the case for the X chromosome and the autosomes. Lastly, we study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. Our study provides an unprecedented quantification of population genetic forces and insight into evolutionary processes governing PAR1 biology.

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Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d Natural Killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant {beta} T cell receptors (TCR) and recognise -galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells) but our knowledge of the antigens for type II NKT cells is limited. An early study identified an NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa-drugs, but its mechanism of NKT-cell activation remained unknown. Here we demonstrate that a range of pentamethylbenzofuransulfonate (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d-tetramers identified type II NKT cell populations cells expressing {beta} and {gamma}{delta}TCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass spectrometric analysis, we detected molecules that allow binding of CD1d to TCRs, finding that both PBF and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that non-lipidic small molecules, that resemble sulfa-drugs implicated in systemic hypersensitivity and drug allergy reactions, activate a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Significance StatementWhereas T cells are known to recognize peptide, vitamin B metabolite or lipid antigens, we identify several non-lipidic small molecules, pentamethylbenzofuransulfonates (PBFs), that activate a population of CD1d-restricted NKT cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa-drug hypersensitivity.

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ORA47 is a transcriptional regulator of a general stress response hub Transcriptional regulators of general stress response (GSR) reprogram expression of selected genes to transduce informational signals into cellular events, ultimately manifested in plants ability to cope with environmental challenges. Identification of the core GSR regulatory proteins will uncover the principal modules and their mode of action in the establishment of adaptive responses. To define the GSR regulatory components, we employed a yeast-one-hybrid assay to identify the protein(s) that binds to the previously established functional GSR motif, coined Rapid Stress Response Element (RSRE). This led to the isolation of ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47), a Methyl jasmonate (MeJA) inducible protein. Subsequently, the ORA47 transcriptional activity was confirmed using RSRE-driven Luciferase (LUC) activity assay performed in the ORA47 loss- and gain-of-function lines introgressed into the 4xRSRE::Luc background. In addition, the prime contribution of CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) protein in induction of RSRE was reaffirmed by genetic studies. Moreover, exogenous application of MeJA led to enhanced levels of ORA47 and CAMTA3 transcripts, and the induction of RSRE::LUC activity. Metabolic analyses illustrated the reciprocal functional inputs of ORA47 and CAMTA3 in increasing JA levels. Lastly, transient assays identified JASMONATE ZIM-domain1 (JAZ1) as a repressor of RSRE::LUC activity.

Collectively, the report provides a fresh insight into the initial mechanistic features of transducing the informational signals into adaptive responses in part via the complex functional interplay between JA biosynthesis/signaling cascade and the transcriptional reprogramming necessary for potentiation of GSR, while offering a window into the role of intraorganellar communication in the establishment of adaptive responses.

SignificanceThe work unmasks the initial mechanistic features of adaptive responses that include tight cooperativity between JA biosynthesis and signaling cascade and the nuclear transcriptional machinery comprised of two activators (CAMTA3 and ORA47) and a suppressor JAZ1). The work further identifies CAMTA3 as a functional link between JA signaling and activation of a general stress transcriptional hub.

sentences: Bioinformatics analyses of significant genes, related pathways and candidate prognostic biomarkers in Alzheimer disease Alzheimers disease (AD) is one of the most common causes of dementia and frailty. This study aimed to use bioinformatics analysis to identify differentially expressed genes (DEGs) in AD. The Expression profiling by high throughput sequencing dataset GSE125583 was downloaded from the Gene Expression Omnibus (GEO) database and DEGs were identified. After assessment of Gene Ontology (GO) terms and pathway enrichment for DEGs, a protein-protein interaction (PPI) network, module analysis, miRNA-hub gene regulatory network construction and TF-hub gene regulatory network were conducted via comprehensive target prediction and network analyses. Finally, we validated hub genes by receiver operating characteristic curve (ROC). In total, 956 DEGs were identified in the AD samples, including 479 up regulated genes and 477 down regulated genes. Functional enrichment analysis showed that these DEGs are mainly involved in the neuronal system, GPCR ligand binding, regulation of biological quality and cell communication. The hub genes of PAK1, ELAVL2, NSF, HTR2C, TERT, UBD, MKI67, HSPB1, PYHIN1 and TES might be associated with AD. The diagnostic value and expression levels of these hub genes in AD were further confirmed by ROC analysis. In conclusion, we identified pathways and crucial candidate genes that affect the outcomes of patients with AD, and these genes might serve as potential therapeutic targets.

sentences: Structural variants are a major source of gene expression differences in humans and often affect multiple nearby genes Structural variants (SVs) are an important source of human genome diversity but their functional effects are not well understood. We mapped 61,668 SVs in 613 individuals with deep genome sequencing data from the GTEx project and measured their effects on gene expression. We estimate that common SVs are causal at 2.66% of eQTLs, which is a 10.5-fold enrichment relative to their abundance in the genome and consistent with prior work using smaller sample sizes. Duplications and deletions were the most impactful variant types, whereas the contribution of mobile element insertions was surprisingly small (0.12% of eQTLs, 1.9-fold enriched). Multi-tissue analysis of expression effects revealed that gene-altering SVs show significantly more constitutive effects than other variant types, with 62.09% of coding SV-eQTLs active in all tissues with known eQTL activity compared to 23.08% of coding SNV- and indel-eQTLs, whereas noncoding SVs, SNVs and indels show broadly similar patterns. We also identified 539 rare SVs associated with nearby gene expression outliers. Of these, 62.34% are noncoding SVs that show strong effects on gene expression yet modest enrichment at known regulatory elements, demonstrating that rare noncoding SVs are a major source of gene expression differences but remain difficult to predict from current annotations. Remarkably, both common and rare noncoding SVs often show strong regional effects on the expression of multiple genes: SV-eQTLs affect an average of 1.82 nearby genes compared to 1.09 genes affected by SNV- and indel-eQTLs, and 21.34% of rare expression-altering SVs show strong effects on 2-9 different genes. We also observe significant effects on gene expression extending 1 Mb from the SV. This provides a mechanism by which individual noncoding SVs may have strong and/or pleiotropic effects on phenotypic variation and disease.

sentences: The envelope cytoplasmic tail regulates HIV-1 assembly and spread The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT) but the function of the EnvCT and conserved domains within it remain largely uncharacterised. Here we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Strikingly we find that mutating W757 had wide-ranging consequences including altering Env mobility in the plasma membrane, preventing Env and Gag recruitment to sites of cell-cell contact for virological synapse (VS) formation and cell-cell spread, and impeding viral fusion. Notably, W757 was also required for efficient virus budding, revealing a previously unappreciated role for the EnvCT in regulating HIV-1 assembly and egress. We conclude that W757 is a key residue that stabilises the structural integrity and function of Env, consistent with the recent model that this region of the EnvCT acts as a critical supporting baseplate for Env.

sentences: Microbial Biotextiles for a Circular Materials Economy A circular materials economy can disrupt linear production models and mitigate its environmental threats. We harness extracellular biosynthesis of microbial nanocellulose (MC), coupled to a protocol that represents a serendipitous intersection between ancient textile techniques and green processing as a route to high-performance bioleather with a circular life cycle. Specifically, a plant-based lecithin phosphocholine treatment modulates cellulose cross-linking through phosphate and methylene groups, to yield a biodegradable material with superior mechanical and flame-retardant properties. A broad color palette and controlled color modulation was achieved with cultural heritage dyes and waste-to-resource strategies. Life cycle impact assessment shows up to an order of magnitude reduction in environmental impacts relative to conventional textiles, making MC relevant for widespread application in fashion, interiors, construction and insulating materials. The translational potential of this approach is tremendous, as use of microbes and green chemistry to engineer regenerative, high-performance products can accelerate a paradigm shift to a circular bioeconomy.

sentences: The Specious Art of Single-Cell Genomics Dimensionality reduction is standard practice for filtering noise and identifying relevant dimensions in large-scale data analyses. In biology, single-cell expression studies almost always begin with reduction to two or three dimensions to produce  all-in-one visuals of the data that are amenable to the human eye, and these are subsequently used for qualitative and quantitative analysis of cell relationships. However, there is little theoretical support for this practice. We examine the theoretical and practical implications of low-dimensional embedding of single-cell data, and find extensive distortions incurred on the global and local properties of biological patterns relative to the highdimensional, ambient space. In lieu of this, we propose semi-supervised dimensionality reduction to higher dimension, and show that such targeted reduction guided by the metadata associated with single-cell experiments provides useful latent space representations for hypothesis-driven biological discovery.

sentences: YAP and TAZ are transcriptional co-activators of AP-1 proteins and STAT3 during breast cellular transformation The YAP and TAZ paralogues are transcriptional co-activators recruited to target sites, primarily by TEAD proteins. Here, we show that YAP and TAZ are also recruited by JUNB and STAT3, key factors that mediate an epigenetic switch linking inflammation to cellular transformation. YAP and TAZ directly interact with JUNB and STAT3 via a WW domain important for transformation, co-occupy many target sites in vivo via AP-1 and (to a lesser extent) STAT3 sequence motifs, and stimulate transcriptional activation by AP-1 proteins. A few target sites are YAP- or TAZ-specific, and they are associated with different sequence motifs and gene classes. YAP/TAZ, JUNB, and STAT3 directly regulate a common set of target genes that overlap, but are distinct from, those regulated by YAP/TAZ and TEADs. The set of genes regulated by YAP/TAZ, STAT3, and JUNB is associated with poor survival in breast cancer patients with the triple-negative form of the disease.

sentences: SARS-CoV-2 Omicron variant is more stable than the ancestral strain on various surfaces The Omicron BA.1 SARS-CoV-2 variant of concern spreads quickly around the world and outcompetes other circulating strains. We examined the stability of this SARS-CoV-2 variant on various surfaces and revealed that the Omicron variant is more stable than its ancestral strain on smooth and porous surfaces.

sentences: Distinct phosphorylation signals drive acceptor versus self-ubiquitination selection by Parkin The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial acceptor pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Mitochondrial target proteins, Miro1 or CISD1, tethered to pUb are ubiquitinated by parkin more efficiently than if alone or Ub-tethered and ubiquitin molecules are ligated to acceptor protein lysines and not pUb. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkins role in mitochondrial homeostasis and has implications on targets for therapeutics.

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Task-specific effects of biological sex and sex hormones on object recognition memories in a 6-hydroxydopamine-lesion model of Parkinson's disease in adult male and female rats Many patients with Parkinsons disease (PD) experience impairments in cognition and memory with few therapeutic options currently available to mitigate them. This has fueled interest in determining how factors including biological sex and sex hormones might modulate higher order function in PD. Previous studies have investigated this in female rats and in gonadally intact and gonadectomized males, with and without hormone replacement, that received bilateral neostriatal 6-hydroxydopamine (6-OHDA) lesions to model PD. Barnes maze and What Where When Episodic-like memory testing showed that 6-OHDA lesions disrupted spatial working and episodic memory functions in both sexes, and that in males, androgen-sensitive behaviors could be rescued in subjects where circulating androgen levels were diminished. Here we tested similar animal groups using the Novel Object Preference (NOP) and Object-in-Place (OiP) tasks. This revealed two entirely different patterns of sex and sex hormone influence. First, for both tasks, 6-ODHA lesions impaired object discrimination in males but not females. Further, for the NOP task, 6-OHDA lesions disrupted discrimination in males rats independently of hormone status. And finally, 6-OHDA lesions impaired OiP performance in males regardless of whether androgen levels were high or low but had no effect on discrimination in gonadectomized rats given 17{beta}-estradiol. Together with previous findings, these data identify the impacts of sex and sex hormones on cognition and memory in PD as behavioral task/behavioral domain specific. This specificity could explain why a cohesive clinical picture of endocrine impacts on higher order function in PD has remained elusive.

HIGHLIGHTSO_LI6-OHDA lesions impair Novel Object performance in male but not female rats.
C_LIO_LI6-OHDA lesions impair Object-in-Place performance in male but not female rats.
C_LIO_LIGonadectomy has no effect on 6-OHDA-induced deficits in Novel Object Preference.
C_LIO_LIEstrogen replacement prevents 6-OHDA-induced Object-in-Place deficits in males.
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sentences: Deriving accurate molecular indicators of protein synthesis through Raman-based sparse classification Raman spectroscopy has the ability to retrieve molecular information from live biological samples non-invasively through optical means. Coupled with machine learning, it is possible to use the large amount of information contained in a Raman spectrum to create models that can predict the state of new samples based on statistical analysis from previous measurements. Furthermore, in case of linear models, the separation coefficients can be used to interpret which bands are contributing to the discrimination between experimental conditions, which correspond here to single-cell measurements of macrophages under in vitro immune stimulation. We here evaluate a typical linear method using discriminant analysis and PCA, and compare it to regularized logistic regression (Lasso). We find that the use of PCA is not beneficial to the classification performance. Furthermore, the Lasso approach yields sparse separation vectors, since it suppresses spectral coefficients which do not improve classification, making interpretation easier. To further evaluate the approach, we apply the Lasso technique to a well-defined case where protein synthesis is inhibited, and show that the separating features are consistent with RNA accumulation and protein levels depletion. Surprisingly, when Raman features are selected purely in terms of their classification power (Lasso), the selected coefficients are contained in side bands, while typical strong Raman peaks are not present in the discrimination vector. We propose that this occurs because large Raman bands are representative of a wide variety of cellular molecules and are therefore less suited for accurate classification.

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Osmolarity-regulated swelling initiates egg activation in Drosophila Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilisation, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well understood. For many insects, egg activation can be triggered independently of fertilisation. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte.

Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins (AQPs) and DEGenerin/Epithelial Na+ (DEG/ENaC) channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of Trpm channels to transport calcium, most likely from the perivitelline space, across the plasma membrane into the mature oocyte.

Our data establishes osmotic pressure as the mechanism that initiates egg activation in Drosophila and is consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and shows remarkable similarities to the mechanism of egg activation in some plants.

sentences: Disrupted PDZ-domain binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis and reward motivation The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors different from those seen in other genetic DAT mouse models. DA levels in striatum are reduced to [~]45% of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics.

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GinJinn2: Object detection and segmentation for ecology and evolution O_LIProper collection and preparation of empirical data still represent one of the most important, but also expensive steps in ecological and evolutionary/systematic research. Modern machine learning approaches, however, have the potential to automate a variety of tasks, which until recently could only be performed manually. Unfortunately, the application of such methods by researchers outside the field is hampered by technical difficulties, some of which, we believe, can be avoided.
C_LIO_LIHere, we present GinJinn2, a user-friendly toolbox for deep learning-based object detection and instance segmentation on image data. Besides providing a convenient command-line interface to existing software libraries, it comprises several additional tools for data handling, pre- and postprocessing, and building advanced analysis pipelines.
C_LIO_LIWe demonstrate the application of GinJinn2 for biological purposes using four exemplary analyses, namely the evaluation of seed mixtures, detection of insects on glue traps, segmentation of stomata, and extraction of leaf silhouettes from herbarium specimens.
C_LIO_LIGinJinn2 will enable users with a primary background in biology to apply deep learning-based methods for object detection and segmentation in order to automate feature extraction from image data.
C_LI

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Reconciling Conformational Heterogeneity and Substrate Recognition in Cytochrome P450 Cytochrome P450, the ubiquitous metalloenzyme involved in detoxification of foreign components, has remained one of the most popular systems for substrate-recognition process. However, despite being known for its high substrate specificity, the mechanistic basis of substrate-binding by archetypal system cytochrome P450cam has remained at odds with the contrasting reports of multiple diverse crystallographic structures of its substrate-free form. Here we address this issue by elucidating the probability of mutual dynamical transition to the other crystallographic pose of cytochrome P450cam and vice versa via unbiased all-atom computer simulation. A robust Markov state model (MSM), constructed using adaptively sampled 84 microsecond-long Molecular dynamics simulation trajectories, maps the broad and heterogenous P450cam conformational landscape into five key sub-states. In particular, the MSM identifies an intermediate-assisted dynamic equilibrium between a pair of conformations of P450cam, in which the substrate-recognition sites remain  closed and  open respectively. However, the estimate of a significantly high stationary population of closed conformation, coupled with faster rate of open [->] closed transition than its reverse process, dictates that the net conformational equilibrium would be swayed in favour of  closed conformation. Together, the investigation quantitatively infers that while a potential substrate of cytochrome P450cam would in principle explore a diverse array of conformations of substrate-free protein, it would mostly encounter a  closed or solvent-occluded conformation and hence would follow an induced-fit based recognition process. Overall, the work reconciles multiple precedent crystallographic, spectroscopic investigations and establishes how a statistical elucidation of conformational heterogeneity in protein would provide crucial insights in the mechanism of potential substrate-recognition process.

STATEMENT OF SIGNIFICANCEConformational heterogeneity plays an important role in defining the structural and functional dynamics of the enzymes. While the static three-dimensional crystallographic structures of enzymes solved in different conditions and/or environments are crucial to provide the conformational sub-states of enzymes, these are not sufficient to understand the kinetics and thermodynamics of these sub-states and their role in substrate recognition process. Cytochrome P450cam, the archtypal metalloenzyme, presents such a complex scenario due to prevalent reports of contrasting crystallographic structures of its substrate-free form. This work quantifies the conformational heterogeneity of substrate-free P450cam by exploring the possibility of mutual transition among the crystallographic poses at an atomic resolution and in the process elucidates its possible substrate-recognition mechanism.

sentences: Do host-consumed resources increase endoparasitic but decrease ectoparasitic infections? Habitat loss and disease outbreak play a major role in the decline of biodiversity. Habitat degradation is often associated with reduced food resources, which can lead to less investment in host immunity and increased infections. However, pathogens use host resources for replication and pathogen traits, such as infecting hosts internally or short generation times, might allow pathogens to rapidly capitalize on host-consumed nutrients. Thus, it is unclear whether increased food consumption by hosts should reduce or amplify pathogen levels. We conducted experiments on Cuban treefrogs (Osteopilus septentrionalis) to test how food availability affects infection levels of Ranavirus and the fungal pathogen Batrachochytrium dendrobatidis (Bd), which are both associated with mass die-offs of amphibians. Given that Ranavirus is an endoparasite with a much shorter generation time than the ectoparasitic Bd, we postulated that Ranavirus might be able to capitalize on host-consumed resources more quickly than Bd. We hypothesized that increased food availability to hosts might reduce Bd infections more than Ranavirus infections. As predicted, augmenting food access decreased Bd infection intensity, but increased Ranavirus infection intensity. Future work should assess whether pathogen traits, such as generation time and endo- versus ectoparasitism, generally affect whether food resources more positively benefit hosts or pathogens.

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Protein complex prediction using Rosetta, AlphaFold, and mass spectrometry covalent labeling Structural mass spectrometry offers several techniques for the characterization of protein structures. Covalent labeling (CL) in combination with mass spectrometry can be used as an analytical tool to study and determine structural properties of protein-protein complexes. Degrees of modification obtained from CL experiments for specific labeled residues can be compared between the unbound and bound states of complexes. This analysis can yield insights into structural features of these protein assemblies, specifically the proximity of specific residues to the protein-protein interface. However, this data is sparse and does not unambiguously elucidate protein structure. Thus, computational algorithms are needed to deduce structure from the CL data. In this work we present a novel hybrid method that combines models of protein complex subunits generated with AlphaFold with differential CL data via a CL-guided protein-protein docking in Rosetta. In a benchmark set, the RMSD (root-mean-square deviation) of the best-scoring models was below 3.6 [A] for 5/5 complexes with inclusion of CL data, whereas the same quality was only achieved for 1/5 complexes without CL data. The average improvement in RMSD observed upon inclusion of CL data was 5.2 [A]. This study suggests that our integrated approach can successfully use data obtained from CL experiments to distinguish between nativelike and non-nativelike models.

Significance StatementStructural mass spectrometry can be a powerful and versatile approach to characterize the structure of protein complexes. Data obtained from covalent labeling mass spectrometry can provide insights into higher order protein structure (particularly with respect to residue interactions and solvent accessibility) but needs to be supplemented by computational techniques to elucidate accurate, atomic-detail structural information. Here, we present a method to combine bioanalytical data obtained from covalent labeling with models generated using AlphaFold to accurately predict protein-protein complexes in Rosetta. Differential covalent labeling data can be used to determine the proximity of residues to the binding interface of complexes which we utilized to analyze computational models and improve structure prediction algorithms.

sentences: Machine-Learning-Based Olfactometry: Odor Descriptor Clustering Analysis for Olfactory Perception Prediction of Odorant Molecules Although gas chromatography/olfactometry (GC/O) has been employed as a powerful analytical tool in odor measurement, its application is limited by the variability, subjectivity, and high cost of the trained panelists who are used as detectors in the system. The advancements in data-driven science have made it possible to predict structure-odor-relationship (SOR) and thus to develop machine-learning-based olfactometry (ML-GCO) in which the human panelists may be replaced by machine learning models to obtain the sensory information of GC-separated chemical compounds. However, one challenge remained in ML-GCO is that there are too many odor descriptors (ODs) being used to describe the sensory characteristics of odorants. It is impractical to build a corresponding model for each OD. To solve this issue, we propose a SOR prediction approach based on odor descriptor clustering. 256 representative ODs are firstly classified into 20 categories using a co-occurrence Bayesian embedding model. The categorization effect is explained according to the semantic relationships using a pre-trained Word2Vec model. Various molecular structure features including molecularly parameters, molecular fingerprints, and molecular 2D graphic features extracted by convolutional neural networks, are employed to predict the aforementioned odor categories. High prediction accuracies (Area under ROC curve was 0.800{+/-}0.004) demonstrate the rationality of the proposed clustering scenario and molecular feature extraction. This study makes the ML-GCO models much closer to the practical application since they can be expected as either an auxiliary system or complete replacement of human panelists to perform the olfactory evaluation.

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

sentences: Signal complexity indicators of health status in clinical-EEG Brain signal variability changes across the lifespan in both health and disease, likely reflecting changes in information processing capacity related to development, aging and neurological disorders. While signal complexity, and multiscale entropy (MSE) in particular, has been proposed as a biomarker for neurological disorders, most observations of altered signal complexity have come from studies comparing patients with few to no comorbidities against healthy controls. In this study, we examined whether MSE of brain signals was distinguishable across individuals in a large and heterogeneous set of clinical-EEG data. Using a multivariate analysis, we found unique timescale-dependent differences in MSE across various neurological disorders. We also found MSE to differentiate individuals with non-brain comorbidities, suggesting that MSE is sensitive to brain signal changes brought about by metabolic and other non-brain disorders. Such changes were not detectable in the spectral power density of brain signals. Our findings suggest that brain signal complexity may offer complementary information to spectral power about an individuals health status and is a promising avenue for clinical biomarker development.

sentences: Multiple RNA regulatory pathways coordinate the activity and expression pattern of a conserved germline RNA-binding protein RNA regulation is essential to successful reproduction. Messenger RNAs delivered from parent to progeny govern early embryonic development. RNA-binding proteins (RBPs) are the key effectors of this process, controlling the translation and stability of parental transcripts to control cell fate specification events prior to zygotic gene activation. The KH-domain RBP MEX-3 is conserved from nematode to human. It was first discovered in Caenorhabditis elegans, where it is essential for anterior cell fate and embryo viability. Here, we show that mex-3 mRNA is itself regulated by several RBPs to define its unique germline spatiotemporal expression pattern. We also show that both poly(A) tail length control and translational regulation contribute to this expression pattern. Though the 3UTR is sufficient to establish the germline expression pattern, we show that it is not essential for reproduction. An allelic series of 3UTR deletion variants identifies repressing regions of the UTR and show that the expression pattern is not precisely coupled to reproductive health. Together, our results define the pathways that govern the spatiotemporal regulation of this highly conserved germline RBP and suggest that redundant mechanisms control MEX-3 function when RNA regulation is compromised.

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High-Intensity Interval Training Remodels the Proteome and Acetylome of Human Skeletal Muscle Exercise is an effective strategy in the prevention and treatment of metabolic diseases. Alterations in the skeletal muscle proteome, including post-translational modifications, regulate its metabolic adaptations to exercise. Here, we examined the effect of high-intensity interval training (HIIT) on the proteome and acetylome of human skeletal muscle, revealing the response of 3168 proteins and 1263 lysine acetyl-sites on 464 acetylated proteins. We identified global protein adaptations to exercise training involved in metabolism, excitation-contraction coupling, and myofibrillar calcium sensitivity. Furthermore, HIIT increased the acetylation of mitochondrial proteins, particularly those of complex V. We also highlight the regulation of exercise-responsive histone acetyl-sites. These data demonstrate the plasticity of the skeletal muscle proteome and acetylome, providing insight into the regulation of contractile, metabolic and transcriptional processes within skeletal muscle. Herein, we provide a substantial hypothesis-generating resource to stimulate further mechanistic research investigating how exercise improves metabolic health.

Impact statementHostrup and colleagues identify global proteomic and acetylomic adaptations to high-intensity interval training, demonstrating adaptations to processes regulating metabolism and contraction.

sentences: Nothing in evolution makes sense except in the light of parasites Parasitism emerges readily in models and laboratory experiments of RNA world and would lead to extinction unless prevented by compartmentalisation or spatial patterning. Modelling replication as an active computational process opens up many degrees of freedom that are exploited to meet environmental challenges, and to modify the evolutionary process itself. Here we use automata chemistry models and spatial RNA world models to study the emergence of parasitism and the complexity that evolves in response. The system is initialised with a hand-designed replicator that copies other replicators with a small chance of point mutation. Almost immediately, short parasites arise; these are copied more quickly, and so have an evolutionary advantage. The replicators also become shorter, and so are replicated faster; they evolve a mechanism to slow down replication, which reduces the difference of replication rate of replicators and parasites. They also evolve explicit mechanisms to discriminate copies of self from parasites; these mechanisms become increasingly complex. New parasite species continually arise from mutated replicators, rather than from evolving parasite lineages. Evolution itself evolves, eg by effectively increasing point mutation rates, and by generating novel emergent mutational operators. Thus, parasitism drives the evolution of complex replicators and complex ecosystems.

sentences: CONCERT: Genome-wide prediction of sequence elements that modulate DNA replication timing Proper control of replication timing (RT) is of vital importance to maintain genome and epigenome integrity. However, the genome-wide sequence determinants regulating RT remain unclear. Here, we develop a new machine learning method, named CO_SCPLOWONCERTC_SCPLOW, to simultaneously predict RT from sequence features and identify RT-modulating sequence elements in a genome-wide manner. CO_SCPLOWONCERTC_SCPLOW integrates two functionally cooperative modules, a selector, which performs importance estimationbased sampling to detect predictive sequence elements, and a predictor, which incorporates bidirectional recurrent neural networks and self-attention mechanism to achieve selective learning of longrange spatial dependencies across genomic loci. We apply CO_SCPLOWONCERTC_SCPLOW to predict RT in mouse embryonic stem cells and multiple human cell types with high accuracy. The identified RT-modulating sequence elements show novel connections with genomic and epigenomic features such as 3D chromatin interactions. In particular, CO_SCPLOWONCERTC_SCPLOW reveals a class of RT-modulating elements that are not transcriptional regulatory elements but are enriched with specific repetitive sequences. As a generic interpretable machine learning framework for predicting large-scale functional genomic profiles based on sequence features, CO_SCPLOWONCERTC_SCPLOW provides new insights into the potential sequence determinants of RT.

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High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy During the past decades, several stand-alone and combinatory methods have been developed to investigate the chemistry (i.e. mapping of elemental, isotopic and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently capable of characterizing soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows chemical and microbial identification of the rhizosphere at micro-to nano-meter spatial resolution. Our approach includes i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. Embedding quality was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil was identified by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) to avoid interferences from high levels of auto fluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy ({micro}-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to chemically map the rhizosphere and identify spatial organization of bacteria. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an ample understanding of the rhizosphere processes and their ecological significance.

Graphical Abstract

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sentences: Activation of endoplasmic reticulum stress via clustering of inner nuclear membrane proteins One of the major cellular mechanisms to ensure protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is typically triggered by accumulation of misfolded proteins in the ER lumen. Here we describe activation of ER stress via protein aggregation in the cell nucleus. We find in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) activation of ER stress due to the aggregation of the diseases-causing progerin protein at the nuclear envelope. The presence of nucleoplasmic protein aggregates is sensed and signaled to the ER lumen via immobilization and clustering of the inner nuclear membrane protein SUN2, leading to activation of the Unfolded Protein Response (UPR). These results identify a nuclear trigger of ER stress and they provide insight into the molecular disease mechanisms of HGPS.

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Phase-locking patterns underlying effective communication in exact firing rate models of neural networks Macroscopic oscillations in the brain have been observed to be involved in many cognitive tasks but their role is not completely understood. One of the suggested functions of the oscillations is to dynamically modulate communication between neural circuits. The Communication Through Coherence (CTC) theory establishes that oscillations reflect rhythmic changes in excitability of the neuronal populations. Thus, populations need to be properly phase-locked so that input volleys arrive at the peaks of excitability of the receiving population to communicate effectively. Here, we present a modeling study to explore synchronization between neuronal circuits connected with unidirectional projections. We consider an Excitatory-Inhibitory (E-I) network of quadratic integrate-and-fire neurons modeling a Pyramidal-Interneuronal Network Gamma (PING) rhythm. The network receives an external periodic input from either one or two sources, simulating the inputs from other oscillating neural groups. We use recently developed mean-field models which provide an exact description of the macroscopic activity of the spiking network. This low-dimensional mean field model allows us to use tools from bifurcation theory to identify the phase-locked states between the input and the target population as a function of the amplitude, frequency and coherence of the inputs. We identify the conditions for optimal phaselocking and selective communication. We find that inputs with high coherence can entrain the network for a wider range of frequencies. Besides, faster oscillatory inputs than the intrinsic network gamma cycle show more effective communication than inputs with similar frequency. Our analysis further shows that the entrainment of the network by inputs with higher frequency is more robust to distractors, thus giving them an advantage to entrain the network. Finally, we show that pulsatile inputs can switch between attended inputs in selective attention.

Author summaryOscillations are ubiquitous in the brain and are involved in several cognitive tasks but their role is not completely understood. The Communication Through Coherence theory proposes that background oscillations in the brain regulate the information flow between neural populations. The oscillators that are properly phase-locked so that inputs arrive at the peaks of excitability of the receiving population communicate effectively. In this paper, we study the emerging phase-locking patterns of a network generating PING oscillations under external periodic forcing, simulating the oscillatory input from other neural groups. We identify the conditions for optimal phase-locking and selective communication. Namely, we find that inputs with higher frequency and coherence have an adavantage to entrain the network and we quantify how robust are to distractors. Furthermore, we show how selective attention can be implemented by means of phase locking and we show that pulsatile inputs can switch between attended inputs.

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Single cell RNA-seq identifies developing corneal cell fates in the human cornea organoid The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here we elucidated the transcriptomic cell fate map of 4 month-old human cornea organoids and the central cornea from three donors. The organoids harbor cell clusters representing corneal epithelium, stroma and endothelium with sub populations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids develop almost equal proportion of the three major cell types. These corneal organoids offer a three-dimensional platform to model corneal diseases and integrated responses of the different cell types to treatments.

TeaserTranscriptomic Map of Cornea Organoid and Human Cornea

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Moxifloxacin-mediated killing of Mycobacterium tuberculosis involves respiratory downshift, reductive stress, and ROS accumulation Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A non-invasive redox biosensor and an ROS-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD+ ratio, and increased the labile iron pool in M. tuberculosis. Lowering the NADH/NAD+ ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and co-treatment with NAC potentiated the anti-mycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice where NAC restricted the emergence of resistance. Thus, oxidative stress, generated in a novel way, contributes to moxifloxacin-mediated killing of M. tuberculosis. The results open a way to make fluoroquinolones more effective anti-tuberculosis agents and provide a mechanistic basis for NAC-mediated enhancement of fluoroquinolone lethality in vitro and in vivo.

Author Summary

A new paradigm was revealed for stress-mediated bacterial death in which moxifloxacin treatment of M. tuberculosis decreases respiration rate (respiration increases in E. coli). Although moxifloxacin-induced, ROS-mediated bacterial death was observed, it derived from elevated levels of NADH and iron, a phenomenon not seen with antibiotic-treated E. coli. Nevertheless, stimulation of respiration and ROS by N-acetyl cysteine (NAC) enhanced moxifloxacin-mediated killing of M. tuberculosis, thereby reinforcing involvement of ROS in killing. NAC stimulation of moxifloxacin-mediated killing of M. tuberculosis and restriction of the emergence of resistance in a murine model of infection emphasize the importance of lethal action against pathogens. The work, plus published benefits of NAC to TB patients, encourage studies of NAC-based enhancement of fluoroquinolones.

sentences: Tracing the evolution of human gene regulation and its association with shifts in environment As humans spread throughout the world, they adapted to variation in many environmental factors, including climate, diet, and pathogens. Because many of these adaptations were likely mediated by multiple non-coding variants with small effects on gene regulation, it has been difficult to link genomic signals of selection to specific genes, and to describe the regulatory response to selection. To overcome this challenge, we adapted PrediXcan, a machine learning method for imputing gene regulation from genotype data, to analyze low-coverage ancient human DNA (aDNA). First, we used simulated genomes to benchmark strategies for adapting gene regulatory prediction to increase robustness to incomplete aDNA data. Applying the resulting models to 490 ancient Eurasians, we found that genes with the strongest divergent regulation among ancient populations with hunter-gatherer, pastoralist, and agricultural lifestyles are enriched for metabolic and immune functions. Next, we explored the contribution of divergent gene regulation to two traits with strong evidence of recent adaptation: dietary metabolism and skin pigmentation. We found enrichment for divergent regulation among genes previously proposed to be involved in diet-related local adaptation, and in many cases, the predicted effects on regulation provide explanations for previously observed signals of selection, e.g., at FADS1, GPX1, and LEPR. For skin pigmentation, we applied new models trained in melanocytes to a time series of 2999 ancient Europeans spanning ~38,000 years BP. In contrast to diet, skin pigmentation genes show little regulatory change over time, suggesting that adaptation mainly involved large-effect coding variants. This work demonstrates how aDNA can be combined with present-day genomes to shed light on the biological differences among ancient populations, the role of gene regulation in adaptation, and the relationship between ancient genetic diversity and the present-day distribution of complex traits.

sentences: Complex genetic architecture underlying the plasticity of maize agronomic traits Phenotypic plasticity is the property of a given genotype to produce multiple phenotypes in response to changing environmental conditions. Understanding the genetic basis of phenotypic plasticity and establishing a predictive model is highly relevant for future agriculture under changing climate. Here, we report findings on the genetic basis of phenotypic plasticity for 23 complex traits using a maize diverse population, planted at five sites with distinct environmental conditions and genotyped with ~ 6.60 million SNPs. We found that altitude-related environmental factors were main drivers for across site variation in flowering time traits but not plant architecture and yield traits. For 23 traits, we detected 109 QTLs, of which 29 was for mean, 66 was for plasticity, and 14 for both parameters, besides, 80% of the QTLs were interreacted with the environment. The effects of several QTLs changed in magnitude or sign, driving variation in phenotype plasticity, and we further experimentally validated one plastic gene ZmTPS14.1 whose effect was likely mediated by the compensation effect of ZmSPL6 which was from the downstream pathway probably. By integrating genetic diversity, environmental variation, and their interaction in a joint model, we could provide site-specific predictions with increased accuracy by as much as 15.5%, 3.8%, and 4.4% for DTT, PH, and EW, respectively. Overall, we revealed a complex genetic architecture involving multiallelic, pleiotropy, and genotype by environment interaction underlying maize complex trait mean and plasticity variation. Our study thus provided novel insights into the dynamic genetic architectures of agronomic traits in response to changing environments, paving a practical route to precision agriculture.

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Time-resolved parameterization of aperiodic and periodic brain activity Macroscopic neural dynamics comprise both aperiodic and periodic components. Recent advances in parameterizing neural power spectra offer practical tools for evaluating these features separately. Although neural signals vary dynamically and express non-stationarity in relation to ongoing behaviour and perception, current methods yield static spectral decompositions. Here, we introduce Spectral Parameterization Resolved in Time (SPRiNT) as a novel method for decomposing complex neural dynamics into periodic and aperiodic spectral elements in a time-resolved manner. First, we demonstrate, with naturalistic synthetic data, SPRiNTs capacity to reliably recover time-varying spectral features and emphasize SPRiNTs specific strengths over other time-frequency parameterization approaches based on wavelets. Second, we use SPRiNT to further illustrate how aperiodic spectral features fluctuate across time in resting-state electroencephalography (n = 178) and relate changes in aperiodic parameters to participants demographics and behaviour. Lastly, we use SPRiNT to demonstrate aperiodic dynamics related to movement behaviour in intracranial recordings in rodents. We foresee SPRiNT responding to growing neuroscientific interests in the parameterization of time-varying neural power spectra and advancing the quantitation of complex neural dynamics at the natural time scales of behaviour.

Significance StatementThe new method and reported findings address a growing interest in neuroscience for research tools that can reliably decompose brain activity at the mesoscopic scale into interpretable components. We show that the new approach proposed herein is capable of tracking transient, dynamic spectral (aperiodic and periodic) components across time, both in synthetic simulated and experimental data. We anticipate that this novel technique, SPRiNT, will enable new neuroscience inquiries that reconcile multifaceted neural dynamics with complex behaviour.

sentences: Single-Channel Permeability from Markovian Milestoning, Umbrella Sampling, and Voltage Simulations Various all-atom molecular dynamics (MD) simulation methods have been developed to compute free energies and crossing rates of ions and small molecules through ion channels. However, a systemic comparison across different methods is scarce. Using a carbon nanotube as a model of small conductance ion channel, we computed the single-channel permeability for potassium ion using umbrella sampling, Markovian milestoning, and steady-state flux under applied voltage. We show that a slightly modified inhomogeneous solubility-diffusion equation yields a single-channel permeability consistent with the mean first passage time (MFPT)-based method. For milestoning, applying cylindrical and spherical bulk boundary conditions yield consistent MFPT if factoring in the effective bulk concentration. The sensitivity of the MFPT to the output frequency of collective variables is highlighted using the convergence and symmetricity of the inward and outward MFPT profiles. The consistent transport kinetic results from all three methods demonstrated the robustness of MD-based methods in computing ion channel permeation. The advantages and disadvantages of each technique are discussed, focusing on the future applications of milestoning in more complex systems.

sentences: Identification and characterization of 19 predicted Myb-family DNA binding proteins in Magnaporthe oryzae concerning growth, conidiation, and pathogenicity Proteins with DNA binding Myb domains have been suggested to be involved in regulating development and stress responses in Eukaryotes. Magnaporthe oryzae is considered the most destructive fungal pathogen of rice, and we screened the genome for all genes encoding proteins containing Myb domains since these could be essential during pathogenesis. We found 19 genes Myb1-19. Only a few have previously been investigated, and one has been shown to be involved in pathogenesis. We tried to delete the other 18 genes and succeeded with 12. RT-qPCR showed that all 19 genes are expressed with different expression profiles during pathogenesis. Only deletions of the genes encoding proteins MoMyb2, MoMyb13, and MoMyb15 showed growth, conidiation, or infection phenotypes, indicating that they are singly needed for successful infection. Even more important, the lack of phenotypes for 9 mutants surprised us, and we extended the analysis to expression co-regulation of all 19 Myb proteins and found 5 co-regulated groups of predicted Myb-domain proteins. We point to likely compensatory regulations of other genes compensating and hiding the effect of many deletions. Further studies of the Myb-family genes are thus of interest since revealing their functions with a possible effect on pathogenicity could be targeted for future measures to control M. oryzae in rice.

sentences: Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike The SARS-CoV-2 spike protein is the target of neutralizing antibodies and the immunogen used in all currently approved vaccines. The global spread of the virus has resulted in emergence of lineages which are of concern for the effectiveness of immunotherapies and vaccines based on the early Wuhan isolate. Here we describe two SARS-CoV-2 isolates with large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis showed that the deletions result in complete reshaping of the antigenic surface of the NTD supersite. The remodeling of the NTD affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite for both spike variants. A unique escape mechanism with high antigenic impact observed in the {Delta}N135 variant was based on the loss of the Cys15-Cys136 disulfide due to the P9L-mediated shift of the signal peptide cleavage site and deletion of residues 136-144. Although the observed large loop and disulfide deletions are rare, similar modifications became independently established in several other lineages, highlighting the possibility of a general escape mechanism via the NTD supersite. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

sentences: Upregulation of CREB1 and FOXO1 transcription factor pathways in Neuregulin-1 mediated neuroprotection following ischemic stroke Neuregulin-1 (NRG-1) is growth factor that has been investigated for its neuroprotective properties following ischemic stroke. While NRG-1 has shown significant promise in preventing neuronal damage following stroke, the mechanisms behind its neuroprotective effects are unclear. The goal of this research was to investigate the effects of NRG-1 treatment on ischemia-induced gene expression profiles following a permanent middle cerebral artery occlusion (MCAO) in rats. Rats were sacrificed twelve hours following MCAO and either vehicle or NRG-1 treatment. RNA extracted from the peri-infarct cortex of the brain was hybridized to an Affymetrix Rat Genome 2.0st Microarray Gene Chip. Data were analyzed using the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software and the STRING Protein-Protein Interaction Networks database. Our results showed that NRG-1 delivery increased the regulation of pro-survival genes. Most notably, NRG-1 treatment upregulated the CREB1 and FOXO1 transcription factor pathways which are involved in increasing anti-inflammatory and cell proliferation responses and decreasing apoptosis and oxidative stress responses, respectively. Luminex multiplex transcription factor assays demonstrated that the activities of CREB1 and FOXO1 were increased by NRG-1 treatment with MCAO. These findings provide novel insight into the molecular mechanisms involved in NRG-1 mediated neuroprotection.

sentences: Extensive variation in the mouse intelectin gene family: recent duplications, deletions and inactivating variants result in diversity in laboratory strains Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57-line have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except wild-derived CAST, SPRET, SKIVE, MOLF and PANCEVO, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease.

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High-throughput Production of Diverse Xenobiotic Metabolites with P450-transduced Huh7 Hepatoma Cell Lines Precision medicine requires methods to assess drug metabolism and distribution, including the identification of known and undocumented drug and chemical exposures as well as their metabolites. Recent work demonstrated high-throughput generation of xenobiotic metabolites with human liver S-9 fractions and detection in human plasma and urine. Here, we developed a panel of lentivirally transduced human hepatoma cell lines (Huh7) that stably express individual cytochrome P450 (P450) enzymes and generate P450-specific xenobiotic metabolites. We verified protein expression by immunoblotting and demonstrated that the cell lines generate P450-specific metabolites from probe substrates. To increase analytical throughput, we used a pooling strategy where 36 chemicals were grouped into 12 unique mixtures, each mixture containing 6 randomly selected compounds, and each compound being present in two separate mixtures. Each mixture of compounds was incubated with 8 different P450 cell lines with cell extracts analyzed at 0 and 2 h. Extracts were analyzed using liquid chromatography-high resolution mass spectrometry. Cell lines selectively metabolized test substrates, with pazopanib metabolized by CYP3A4 and CYP2C8 cells, bupropion by CYP2B6, and {beta}-naphthoflavone by CYP1A2 for example, showing substrate-enzyme specificity. Predicted metabolites from the remaining 33 compounds as well as many unidentified m/z features were detected. We also show that a specific metabolite generated by CYP2B6 cells, but not detected in the S9 system, was identified in human samples. Our data show that incubating these cell lines with chemical mixtures accelerated characterization of xenobiotic chemical space, while simultaneously allowing for the contributions of specific P450 enzymes to be identified.

Significance statementHigh resolution mass spectrometry enables the identification of exposures to drugs and other xenobiotics in human samples. This paper demonstrates a workflow for high throughput production of xenobiotic metabolites using a panel of engineered cytochrome P450-expressing hepatoma cells. Active substrate-enzyme pairs can be identified using this workflow and generated metabolites can be used as surrogate standards to validate xenobiotic detection in humans.

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Pan-cancer association of DNA repair deficiencies with whole-genome mutational patterns BackgroundCancer genomes are shaped by DNA damage and repair. DNA Repair deficiencies in cancers may result in characteristic mutational patterns, as exemplified by deficiency of BRCA1/2 and efficacy prediction for PARP-inhibitors. We systematically evaluated the ability to identify and predict mono- or biallelic deficiencies of 736 DDR genes from associations with genome-wide mutational patterns, including structural variants, across 6,065 whole-genome sequenced cancers.

ResultsWe assembled 535 cancer-specific patient cohorts with shared deficiency of a DNA damage response gene. We trained and evaluated a predictive model for each cohort, and shortlisted 24 gene deficiencies that could be predicted with high accuracy from mutational patterns. These included expected gene deficiency models for BRCA1/2, MSH3/6, TP53, and CDK12. CDK12 is associated with tandem-duplications, and we demonstrate that this association can predict gene deficiency with high accuracy (AUROC=0.97) in prostate cancers.

Our novel associations include deficiencies of ATRX, IDH1, HERC2, CDKN2A, PTEN, and SMARCA4. We found that loss of ATRX or IDH1, which are often co-mutated, were associated with the same mutational phenotype in central nervous system cancers; HERC2 loss was associated with an increased number of short deletions in skin cancers and significantly co- occurring with TP53 loss; and PTEN loss was associated with decreased levels of structural rearrangements in cancers of the uterus and the central nervous system.

ConclusionOur systematic, generic approach yielded a catalogue of predictive models, which may provide targets for research and development of treatment, and potentially help guide therapy.

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Utilising TMS-EEG to assess the response to cerebellar-brain inhibition BackgroundCerebellar-brain inhibition (CBI) is a transcranial magnetic stimulation (TMS) paradigm indexing excitability of cerebellar projections to motor cortex (M1). Stimulation involved with CBI is often considered to be uncomfortable, and alternative ways to index connectivity between cerebellum and the cortex would be valuable. Utilising electroencephalography in conjunction with TMS (combined TMS-EEG) to record the response to CBI has the potential to achieve this, but has not been attempted previously.

ObjectiveTo investigate the utility of TMS-EEG for characterising cerebellar-cortical interactions recruited by CBI.

MethodsA total of 33 volunteers (25.7 {+/-} 4.9 years, 20 females) participated across three experiments. These investigated EEG responses to CBI induced with a figure-of-eight (F8; experiment 1) or double cone (DC; experiment 2) conditioning coil over cerebellum, in addition to multisensory sham stimulation (experiment 3).

ResultsBoth F8 and DC coils suppressed early TMS-evoked EEG potentials (TEPs) produced by TMS to M1 (P < 0.05). Furthermore, the TEP produced by CBI stimulation was related to the motor inhibitory response to CBI recorded in a hand muscle (P < 0.05), but only when using the DC coil. Multisensory sham stimulation failed to modify the M1 TEP.

ConclusionsCerebellar conditioning produced changes in the M1 TEP that were not apparent following sham stimulation, and that were related to the motor inhibitory effects of CBI. Our findings therefore suggest it is possible to index the response to CBI using TMS-EEG. In addition, while both F8 and DC coils appear to recruit cerebellar projections, the nature of these may be different.

sentences: A deep learning pipeline for segmentation of Proteus mirabilis colony patterns The motility mechanisms of microorganisms are critical virulence factors, enabling their spread and survival during infection. Motility is frequently characterized by qualitative analysis of macroscopic colonies, yet the standard quantification method has mainly been limited to manual measurement. Recent studies have applied deep learning for classification and segmentation of specific microbial species in microscopic images, but less work has focused on macroscopic colony analysis. Here, we advance computational tools for analyzing colonies of Proteus mirabilis, a bacterium that produces a macroscopic bullseye-like pattern via periodic swarming, a process implicated in its virulence. We present a dual-task pipeline for segmenting (1) the macroscopic colony including faint outer swarm rings, and (2) internal ring boundaries, unique features of oscillatory swarming. Our convolutional neural network for patch-based colony segmentation and U-Net with a VGG-11 encoder for ring boundary segmentation achieved test Dice scores of 93.28% and 83.24%, respectively. The predicted masks at times improved on the ground truths from our automated annotation algorithms. We demonstrate how application of our pipeline to a typical swarming assay enables ease of colony analysis and precise measurements of more complex pattern features than those which have been historically quantified.

sentences: Geminiviral genomes encode additional proteins with specific subcellular localizations and virulence function Geminiviruses are plant viruses with limited coding capacity. Geminivirus-encoded proteins were identified applying a 10-kDa arbitrary threshold; however, it is increasingly clear that small proteins play relevant roles in biological systems, which calls for the reconsideration of this criterion. Here, we show that geminiviral genomes contain additional ORFs. Using tomato yellow leaf curl virus, we demonstrate that some of these novel ORFs are expressed during the infection, and that the encoded proteins display specific subcellular localizations. We prove that the largest of these new ORFs, which we name V3, is required for full viral infection, and that the V3 protein localizes in the Golgi apparatus and functions as an RNA silencing suppressor. These results imply that the repertoire of geminiviral proteins can be expanded, and that getting a comprehensive overview of the molecular plant-geminivirus interactions will require the detailed study of small ORFs so far neglected.

sentences: Novel RNA viruses associated with avian haemosporidian parasites Avian haemosporidian parasites can cause malaria-like symptoms in their hosts and have been implicated in the demise of some bird species. The newly described Matryoshka RNA viruses (MaRNAV1 and MaRNAV2) infect parasites that in turn infect their vertebrate hosts. MaRNAV2 was the first RNA virus discovered associated with parasites of the genus Leucocytozoon. By analyzing metatranscriptomes from the NCBI SRA database with local sequence alignment tools, we detected two novel RNA viruses; we describe them as MaRNAV3 associated with Leucocytozoon and MaRNAV4 associated with Parahaemoproteus. These had ~40-60% amino acid identity to the RNA-dependent RNA-polymerase (RdRp) of MaRNAV1 and 2, respectively. These findings lead us to hypothesize that MaRNAV_like viruses are widespread and tightly associated with the order Haemosporida since they have been described in human Plasmodium vivax, and now two genera of avian haemosporidians.

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

sentences: Modular Dynamic Biomolecular Modelling with Bond Graphs: The Unification of Stoichiometry, Thermodynamics, Kinetics and Data. Renewed interest in dynamic simulation models of biomolecular systems has arisen from advances in genome-wide measurement and applications of such models in biotechnology and synthetic biology. In particular, genome-scale models of cellular metabolism beyond the steady state are required in order to represent transient and dynamic regulatory properties of the system. Development of such whole-cell models requires new modelling approaches. Here we propose the energy-based bond graph methodology, which integrates stoichiometric models with thermo-dynamic principles and kinetic modelling. We demonstrate how the bond graph approach intrinsically enforces thermodynamic constraints, provides a modular approach to modelling, and gives a basis for estimation of model parameters leading to dynamic models of biomolecular systems. The approach is illustrated using a well-established stoichiometric model of Escherichia coli (E. coli) and published experimental data.

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Global analysis of human-to-mouse intercellular RNA transfer in cell culture Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA and lncRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

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sentences: Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-induced Myeloid-Derived Suppressor Cells Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

sentences: Individual exploration and selective social learning: Balancing exploration-exploitation trade-offs in collective foraging Search requires balancing exploring for more options and exploiting the ones previously found. Individuals foraging in a group face another trade-off: whether to engage in social learning to exploit the solutions found by others or to solitarily search for unexplored solutions. Social learning can better exploit learned information and decrease the costs of finding new resources, but excessive social learning can lead to over-exploitation and too little exploration for new solutions. We study how these two trade-offs interact to influence search efficiency in a model of collective foraging under conditions of varying resource abundance, resource density, and group size. We modeled individual search strategies as Levy walks, where a power-law exponent ({micro}) controlled the trade-off between exploitative and explorative movements in individual search. We modulated the trade-off between individual search and social learning using a selectivity parameter that determined how agents responded to social cues in terms of distance and likely opportunity costs. Our results show that social learning is favored in rich and clustered environments, but also that the benefits of exploiting social information are maximized by engaging in high levels of individual exploration. We show that selective use of social information can modulate the disadvantages of excessive social learning, especially in larger groups and when individual exploration is limited. Finally, we found that the optimal combination of individual exploration and social learning gave rise to trajectories with {micro} {approx} 2 and provide support for the general optimality of such patterns in search. Our work sheds light on the interplay between individual search and social learning, and has broader implications for collective search and problem-solving.

sentences: A base-line cellular antiviral state is maintained by cGAS and its most frequent naturally occurring variant rs610913 Upon recognition of aberrantly located DNA, the innate immune sensor cGAS activates STING/IRF-3-driven antiviral responses. Here we characterized the ability of a specific variant of the cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted towards the possibility for an additional binding site for a potential cellular co-factor in cGAS dimers. However, cGAS(WT) or cGAS(P261H)-reconstituted THP-1 cGAS KO cells shared steady-state expression of interferon-stimulated genes (ISGs), as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared to cGAS KO- or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared to cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend towards a slightly reduced type I IFN response as compared to PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a base-line antiviral state rendering cells more refractory to ISG-sensitive viral infections. Even though rs610913 failed to grossly differ phenotypically from the wild-type gene, its expression potentially results in a slightly altered susceptibility to viral infections in vivo.

sentences: Soil, ocean, hot spring, and host-associated environments reveal unique selection pressures on genomic features of bacteria in microbial communities Free-living bacteria in nutrient limited environments often exhibit small genomes which curb the cost of reproduction - a phenomenon known as genomic streamlining. Streamlining has been associated with a suite of traits such as reduced GC content, fewer 16S rRNA copies, and a lower abundance of regulatory genes, such as sigma ({sigma}) factors. Here, we analyzed these traits from 116 publicly available metagenomes derived from marine, soil, host associated, and thermophilic communities. In marine and thermophilic communities, genome size and GC content declined in parallel, but GC content was higher in thermophilic communities. In soils, the relationship between genome size and GC content was negative, suggesting a different selection pressure on genome size and GC content in soil bacteria. The abundance of {sigma}-factors varied with average genome size, ecosystem type, and the specific functions regulated by the sigma factor. In marine environments, housekeeping and heat-shock {sigma}-factor genes (rpoD and rpoH respectively) increased as genome size declined, and {sigma}-factor responsible for flagella biosynthesis (fliA) decreased, suggesting a trade-off between nutrient conservation and chemotaxis. In soils, a high abundance of fliA and the stress response {sigma}-factor gene (rpoS) was associated with smaller average genome size and often located in harsh and/or carbon-limited environments such as deserts or agricultural fields - suggesting an increased capacity for stress response and mobility in nutrient-poor soils. This work showcases how ecosystem-specific environmental constraints force trade-offs which are then embedded in the genomic features of bacteria in microbial communities, specifically genome size, GC content, and regulatory genes, and further highlights the importance of considering these features in microbial community analysis.

sentences: Life histories of 9,488 bird and 4,865 mammal species I use 56,214 life history data to estimate equilibrium life history models for birds and mammals with body mass estimates. Missing parameters were estimated by allometric correlations at the lowest taxonomic level (genus, family, order, class) with data. The estimation is optimised to predict the existing data, with precision estimated separately for the different taxonomic levels of the estimator. This provides complete life history models for 9,488 species of birds, and 4,865 species of mammals. Each model includes estimates of metabolism, net assimilated energy, individual growth, mortality, fecundity, age of reproductive maturity, generation time, life span, home range, population density, biomass, population consumption, and a relative measure of intra-specific interactive competition, providing 387,531 parameter estimates in total.

sentences: Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids. Here, we studied biophysical properties of Dengue (DENV) and Zika (ZIKV) virus capsid proteins to understand the process of RNA genome encapsidation. In this route, the capsid proteins efficiently recruit the viral RNA at the ER membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids or how the nucleocapsid is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning the viral process using purified DENV and ZIKV capsids proteins, membranes mimicking the ER lipid composition and nucleic acids at in vitro conditions. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions and docked exogenous membranes and RNA molecules. When the proteins bound nucleic acids, droplet liquid-liquid phase separation was observed. We characterized these liquid condensates by measuring nucleic acid partition constant and the extent of water dipolar relaxation observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data supports a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to understand the viral nucleocapsid formation as a stable liquid-liquid phase transition, which could be relevant for Dengue and Zika gemmation, opening new avenues for antiviral intervention.

sentences: Leishmania donovani metacyclic promastigotes impair phagosome properties in inflammatory monocytes Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and to the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes were readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli was efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidified poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan was responsible for the inhibition of PV acidification, impairement of the NADPH oxidase assembly was independent of lipophosphoglycan and of the metalloprotease GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

sentences: Hybridisation and chloroplast capture between ancient Themeda triandra ecotypes in Australia Ecotypes are distinct populations within a species which are adapted to specific environmental conditions. Understanding how these ecotypes become established, and how they interact when reunited, is fundamental to elucidating how ecological adaptations are maintained. This study focuses on Themeda triandra, a dominant grassland species across Asia, Africa and Australia. It is the most widespread plant in Australia, where it has distinct ecotypes that are usually restricted to either wetter and cooler coastal regions or the drier and hotter interior. We use whole genome sequencing for over 80 Themeda accessions to reconstruct the evolutionary history of T. triandra and related taxa. A chloroplast phylogeny confirms that Australia was colonised by T. triandra twice, with the division between ecotypes predating their arrival in Australia. The nuclear genome provides evidence of gene-flow among the ecotypes, largely restricted to two geographic areas. In northern Queensland there appears to be a hybrid zone with admixed nuclear genomes and shared plastid haplotypes. Conversely, in the cracking claypans of Western Australia, there is cytonuclear discordance with individuals possessing the coastal plastid and interior clade nuclear genomes. This chloroplast capture is potentially a result of adaptive introgression, with selection detected in the rpoC2 gene which is associated with water use efficiency. A stable hybrid zone in the east, and the displacement of one ecotype in the west, highlights the unpredictable nature of hybrid zones, with repeated contacts between the same ecotypes producing different outcomes.

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The principal mitochondrial K+ uniport is associated with respiratory complex I Mitochondria generate ATP via coupling the negative electrochemical potential (proton motive force, {Delta}p), consisting of a proton gradient ({Delta}pH+) and a membrane potential ({Delta}{Psi}m) across the respiratory chain, to phosphorylation of adenosine diphosphate nucleotide. In turn,{Delta} pH+ and{Delta}{Psi} m are tightly balanced by the modulation of ionic uniporters and exchange-diffusion systems which preserve integrity of mitochondrial membranes and regulate ATP production. Here, we provide direct electrophysiological, pharmacological and genetic evidence that the main mitochondrial electrophoretic pathway for monovalent cations is associated with respiratory complex I, contrary to the long-held dogma that only H+ gradients are built across proteins of the mammalian electron transport chain.

Here we propose a theoretical framework to describe how monovalent metal cations contribute to the buildup of H+ gradients and the proton motive force, extending the classical Mitchellian view on chemiosmosis and vectorial metabolism.

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Accelerating COVID-19 research with graph mining and transformer-based learning In 2020, the White House released the, "Call to Action to the Tech Community on New Machine Readable COVID-19 Dataset," wherein artificial intelligence experts are asked to collect data and develop text mining techniques that can help the science community answer high-priority scientific questions related to COVID-19. The Allen Institute for AI and collaborators announced the availability of a rapidly growing open dataset of publications, the COVID-19 Open Research Dataset (CORD-19). As the pace of research accelerates, biomedical scientists struggle to stay current. To expedite their investigations, scientists leverage hypothesis generation systems, which can automatically inspect published papers to discover novel implicit connections. We present an automated general purpose hypothesis generation systems AGATHA-C and AGATHA-GP for COVID-19 research. The systems are based on graph-mining and the transformer model. The systems are massively validated using retrospective information rediscovery and proactive analysis involving human-in-the-loop expert analysis. Both systems achieve high-quality predictions across domains (in some domains up to 0.97% ROC AUC) in fast computational time and are released to the broad scientific community to accelerate biomedical research. In addition, by performing the domain expert curated study, we show that the systems are able to discover on-going research findings such as the relationship between COVID-19 and oxytocin hormone.

ReproducibilityAll code, details, and pre-trained models are available at https://github.com/IlyaTyagin/AGATHA-C-GP

CCS CONCEPTS* Applied computing [->] Bioinformatics; Document management and text processing; * Computing methodologies [->] Learning latent representations; Neural networks; Information extraction; Semantic networks.

sentences: Cannabidiol attenuates cognitive deficits and neuroinflammation induced by early alcohol exposure in a mice model. Foetal alcohol spectrum disorder (FASD) is the umbrella term used to describe the physical and mental disabilities induced by alcohol exposure during development. Early alcohol exposure induces cognitive impairments resulting from damage to the central nervous system (CNS). The neuroinflammatory response accompanied by neurodegenerative mechanisms contribute to those detrimental alterations. Cannabidiol (CBD) has recently emerged as an anti-inflammatory drug that might be useful to treat several neuropsychiatric disorders. In our study, we assessed the effects of CBD on long-lasting cognitive deficits induced by early alcohol exposure. Furthermore, we analysed long-term pro-inflammatory and apoptotic markers within the prefrontal cortex and hippocampus. To model alcohol binge drinking during gestational and lactation periods, we used pregnant C57BL/6 female mice with time-limited access to 20% v/v alcohol solution. Following the prenatal and lactation alcohol exposure (PLAE), we treated the male and female offspring with CBD from post-natal day (PD) 25 until PD34, and we evaluated their cognitive performance at PD60. Our results showed that CBD treatment during peri-adolescence period ameliorates cognitive deficits observed in our FASD-like mouse model, without sex differences. Moreover, CBD restores the PLAE-induced increased levels of TNF and IL-6 in the hippocampus. Thus, our study provides new insights for CBD as a therapeutic agent to counteract cognitive impairments and neuroinflammation caused by early alcohol exposure.

sentences: Multiple genes in cis mediate the effects of a single chromatin accessibility variant on aberrant synaptic development and function in human neurons Despite hundreds of risk loci from genome-wide association studies of neuropsychiatric disorders, causal variants/genes remain largely unknown. Here, in NEUROG2-induced human neurons, we identified 31 risk SNPs in 26 schizophrenia (SZ) risk loci that displayed allele-specific open chromatin (ASoC) and were likely to be functional. Editing the strongest ASoC SNP rs2027349 near vacuolar protein sorting 45 homolog (VPS45) altered the expression of VPS45, lncRNA AC244033.2, and a distal gene, C1orf54, in human neurons. Notably, the global gene expression changes in neurons were enriched for SZ risk and correlated with post-mortem brain gene expression signatures of neuropsychiatric disorders. Neurons carrying the risk allele exhibited increased dendritic complexity, synaptic puncta density, and hyperactivity, which were reversed by knocking-down distinct cis-regulated genes (VPS45, AC244033.2, or C1orf54), suggesting a phenotypic contribution from all three genes. Interestingly, transcriptomic analysis of knockdown cells suggested a non-additive effects of these genes. Our study reveals a compound effect of multiple genes at a single SZ locus on synaptic development and function, providing a mechanistic link between a non-coding SZ risk variant and disease-related cellular phenotypes.

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Intrinsic Mechanisms in the Gating of Resurgent Na+ Currents The resurgent component of the voltage-gated sodium current (INaR) is a depolarizing conductance, revealed on membrane hyperpolarizations following brief depolarizing voltage steps, which has been shown to contribute to regulating the firing properties of numerous neuronal cell types throughout the central and peripheral nervous systems. Although mediated by the same voltage-gated sodium (Nav) channels that underlie the transient and persistent Nav current components, the gating mechanisms that contribute to the generation of INaR remain unclear. Here, we characterized Nav currents in mouse cerebellar Purkinje neurons, and used tailored voltage-clamp protocols to define how the voltage and the duration of the initial membrane depolarization affect the amplitudes and kinetics of INaR. Using the acquired voltage-clamp data, we developed a novel Markov kinetic state model with parallel (fast and slow) inactivation pathways and, we show that this model reproduces the properties of the resurgent, as well as the transient and persistent, Nav currents recorded in (mouse) cerebellar Purkinje neurons. Based on the acquired experimental data and the simulations, we propose that resurgent Na+ influx occurs as a result of fast inactivating Nav channels transitioning into an open/conducting state on membrane hyperpolarization, and that the decay of INaR reflects the slow accumulation of recovered/opened Nav channels into a second, alternative and more slowly populated, inactivated state. Additional simulations reveal that extrinsic factors that affect the kinetics of fast or slow Nav channel inactivation and/or impact the relative distribution of Nav channels in the fast- and slow-inactivated states, such as the accessory Nav{beta}4 channel subunit, can modulate the amplitude of INaR.

SUMMARYThe resurgent component of the voltage-gated sodium current (INaR) is revealed on membrane hyperpolarizations following brief depolarizing voltage steps that activate the rapidly activating and inactivating, transient Nav current (INaT). To probe the mechanisms contributing to the generation and properties of INaR, we combined whole-cell voltage-clamp recordings from mouse cerebellar Purkinje neurons with computational modeling to develop a novel, blocking particle-independent, model for the gating of INaR that involves two parallel inactivation pathways, and we show that this model recapitulates the detailed biophysical properties of INaR measured in mouse cerebellar Purkinje neurons.

sentences: Graded Visual Consciousness During the Attentional Blink One of the ongoing debates about visual consciousness is whether it can be considered as an all-or-none or a graded phenomenon. This may depend on the experimental paradigm and the task used to investigate this question. The present event-related potential study (N = 32) focuses on the attentional blink paradigm for which so far only little and mixed evidence is available. Detection of T2 face targets during the attentional blink was assessed via an objective accuracy measure (reporting the faces gender), subjective visibility on a perceptual awareness scale (PAS) as well as event-related potentials time-locked to T2 onset (components P1, N1, N2, and P3). The behavioral results indicate a graded rather than an all-or-none pattern of visual awareness. Corresponding graded differences in the N1, N2, and P3 components were observed for the comparison of visibility levels. These findings suggest that conscious perception during the attentional blink can occur in a graded fashion.

sentences: A Promising Chemical Series of Positive Allosteric Modulators of the μ-Opioid Receptor that Enhance the Antinociceptive Efficacy of Opioids but not their Adverse Effects Positive allosteric modulators (PAMs) of the {micro}-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

sentences: Sleep Slow Oscillations and Spindles encode ocular dominance plasticity and promote its consolidation in adult humans Sleep and plasticity are highly interrelated, as sleep slow oscillations and sleep spindles are associated with consolidation of Hebbian-based processes. However, in adult humans, visual cortical plasticity is mainly sustained by homeostatic mechanisms, for which the role of sleep is still largely unknown. Here we demonstrate that non-REM sleep stabilizes homeostatic plasticity of ocular dominance in adult humans. We found that the effect of short-term monocular deprivation (boost of the deprived eye) was preserved at the morning awakening (>6 hours after deprivation). Subjects exhibiting stronger consolidation had increased sleep spindle density in frontopolar electrodes, suggesting distributed consolidation processes. Crucially, the individual susceptibility to visual homeostatic plasticity was encoded by changes in sleep slow oscillation rate and shape and spindle power in occipital sites, consistent with an early visual cortical site of ocular dominance homeostatic plasticity.

sentences: An ultrasensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo The purinergic transmitter ATP (adenosine 5-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. We developed an ultrasensitive GPCR Activation-Based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including primary hippocampal neurons, a zebrafish model of injury-induced ATP release, and LPS-induced ATP-release events in individual astrocytes in the mouse cortex measured using in vivo two-photon imaging. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

sentences: The role of mechanical interactions in EMT The detachment of cells from the boundary of an epithelial tissue and the subsequent invasion of these cells into surrounding tissues is important for cancer development and wound healing, and is strongly associated with the epithelial-mesenchymal transition (EMT). Chemical signals, such as TGF-{beta}, produced by surrounding tissue can be up-taken by cells and induce EMT. In this work, we present a novel cell-based discrete mathematical model of mechanical cellular relaxation, cell proliferation, and cell detachment driven by chemically-dependent EMT in an epithelial tissue. A continuum description of the model is then derived in the form of a novel nonlinear free boundary problem. Using the discrete and continuum models we explore how the coupling of chemical transport and mechanical interactions influences EMT, and postulate how this could be used to help control EMT in pathological situations.

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Simplicial and Topological Descriptions of Human Brain Dynamics Whereas brain imaging tools like functional Magnetic Resonance Imaging (fMRI) afford measurements of whole-brain activity, it remains unclear how best to interpret patterns found amid the datas apparent self-organization. To clarify how patterns of brain activity support brain function, one might identify metric spaces that optimally distinguish brain states across experimentally defined conditions. Therefore, the present study considers the relative capacities of several metric spaces to disambiguate experimentally defined brain states. One fundamental metric space interprets fMRI data topographically, i.e, as the vector of amplitudes of a multivariate signal, changing with time. Another perspective considers the condition-dependency of the brains Functional Connectivity (FC), i.e., the similarity matrix computed across the variables of a multivariate signal. More recently, metric spaces that think of the data topologically, e.g., as an abstract geometric object, have become available. In the abstract, uncertainty prevails regarding the distortions imposed by the mode of measurement upon the object under study. Features that are invariant under continuous deformations, such as rotation and inflation, constitute the features of topological data analysis. While there are strengths and weaknesses of each metric space, we find that metric spaces that track topological features are optimal descriptors of the brains experimentally defined states.

AUTHOR SUMMARYTime-Varying Functional Connectivity (TVFC) leverages brain imaging data to interpret brain function as time-varying patterns of coordinating activity among brain regions. While many questions remain regarding the organizing principles through which brain function emerges from multi-regional interactions, advances in the mathematics of Topological Data Analysis (TDA) may provide new insights into the brains functional self-organization. One tool from TDA, "persistent homology", observes the occurrence and persistence of n-dimensional holes in a sequence of simplicial complexes extracted from a weighted graph. The occurrence of such holes within the TVFC graph may indicate preferred routes of information flow among brain regions. In the present study, we compare the use of persistence homology versus more traditional metrics at the task of segmenting brain states that differ across experimental conditions. We find that the structures identified by persistence homology more accurately segment the stimuli, more accurately segment high versus low performance levels under common stimuli, and generalize better across volunteers. These findings support the topological interpretation of brain dynamics.

sentences: Computational Analysis of Protein Stability and Allosteric Interaction Networks in Distinct Conformational Forms of the SARS-CoV-2 Spike D614G Mutant: Reconciling Functional Mechanisms through Allosteric Model of Spike Regulation Structural and biochemical studies SARS-CoV-2 spike mutants with the enhanced infectivity have attracted significant attention and offered several mechanisms to explain the experimental data. The development of a unified view and a working model which is consistent with the diverse experimental data is an important focal point of the current work. In this study, we used an integrative computational approach to examine molecular mechanisms underlying functional effects of the D614G mutation by exploring atomistic modeling of the SARS-CoV-2 spike proteins as allosteric regulatory machines. We combined coarse-grained simulations, protein stability and dynamic fluctuation communication analysis along with network-based community analysis to simulate structures of the native and mutant SARS-CoV-2 spike proteins in different functional states. The results demonstrated that the D614 position anchors a key regulatory cluster that dictates functional transitions between open and closed states. Using molecular simulations and mutational sensitivity analysis of the SARS-CoV-2 spike proteins we showed that the D614G mutation can improve stability of the spike protein in both closed and open forms, but shifting thermodynamic preferences towards the open mutant form. The results offer support to the reduced shedding mechanism of S1 domain as a driver of the increased infectivity triggered by the D614G mutation. Through distance fluctuations communication analysis, we probed stability and allosteric communication propensities of protein residues in the native and mutant SARS-CoV-2 spike proteins, providing evidence that the D614G mutation can enhance long-range signaling of the allosteric spike engine. By employing network community analysis of the SARS-CoV-2 spike proteins, our results revealed that the D614G mutation can promote the increased number of stable communities and allosteric hub centers in the open form by reorganizing and enhancing the stability of the S1-S2 inter-domain interactions and restricting mobility of the S1 regions. This study provides atomistic-based view of the allosteric interactions and communications in the SARS-CoV-2 spike proteins, suggesting that the D614G mutation can exert its primary effect through allosterically induced changes on stability and communications in the residue interaction networks.

sentences: Identification of Catechins Binding Pockets in Monomeric Aβ42 Through Ensemble Docking and MD Simulations The assembly of the Amyloid-{beta} peptide (A{beta}) into toxic oligomers and fibrils is associated with Alzheimers disease and dementia. Therefore, disrupting amyloid assembly by direct targeting of the A{beta} monomeric form with small molecules or antibodies is a promising therapeutic strategy. However, given the dynamic nature of A{beta}, standard computational tools cannot be easily applied for high-throughput structure-based virtual screening in drug discovery projects. In the current study, we propose a computational pipeline - in the framework of the ensemble docking strategy - to identify catechins binding pockets in monomeric A{beta}42. It is shown that both hydrophobic aromatic interactions and hydrogen bonding are crucial for the binding of catechins to A{beta}42. Also, it has been found that all the studied ligands, especially the EGCG, can act as potent inhibitors against amyloid aggregation by blocking the central hydrophobic region of the A{beta}. Our findings are evaluated and confirmed with multi-microsecond MD simulations. Finally, it is suggested that our proposed pipeline, with low computational cost in comparison with MD simulations, is a suitable approach for the virtual screening of ligand libraries against A{beta}.

sentences: EEG-Based Focus Estimation Using Neurable's Enten Headphones and Analytics Platform We introduce Neurables research on focus using our recently developed Enten EEG headphones. First we quantify Entens performance on standard EEG protocols, including eyes-closed alpha rhythms, auditory evoked response and the P300 event-related potential paradigm. We show that Entens performance is on-par with established industry-standard hardware. We then introduce a series of experimental tasks designed to mimic how focus might be maintained or disrupted in a real-world office setting. We show that (A) these tasks induce behavioral changes that reflect underlying changes in focus levels and (B) our proprietary algorithm detects these changes across a large number of sessions without needing to adjust the model per participant or recording session. Through manipulation of our experimental protocol, we show that our algorithm is not dependent on gross EMG artifacts and it is driven by changes in EEG. Finally, we evaluated the models performance on the same subject across several days, and show that performance remained consistent over time. Our model correctly captured 80% {+/-} 4.1% of distractions present in our experiments with statistical significance. This indicates that our model generalizes across subjects, time points, and conditions. Our findings are based on EEG data collected from 132 participants across 337 sessions and 45 different experiments.

sentences: Using Progressive Context Encoders for Anomaly Detection in Digital Pathology Images Whole slide imaging (WSI) is transforming the practice of pathology, converting a qualitative discipline into a quantitative one. However, one must exercise caution in interpreting algorithm assertions, particularly in pathology where an incorrect classification could have profound impacts on a patient, and rare classes exist that may not have been seen by the algorithm during training. A more robust approach would be to identify areas of an image for which the pathologist should concentrate their effort to make a final diagnosis. This anomaly detection strategy would be ideal for WSI, but given the extremely high resolution and large file sizes, such an approach is difficult. Here, we combine progressive generative adversarial networks with a flexible adversarial autoencoder architecture capable of learning the "normal distribution" of WSIs of normal skin tissue at extremely high resolution and demonstrate its anomaly detection performance. Our approach yielded pixel-level accuracy of 89% for identifying melanoma, suggesting that our label-free anomaly detection pipeline is a viable strategy for generating high quality annotations - without tedious manual segmentation by pathologists. The code is publicly available at https://github.com/Steven-N-Hart/P-CEAD.

sentences: Generating Functional Cells Through Enhanced Interspecies Chimerism with Human Pluripotent Stem Cells Obtaining functional human cells through interspecies chimerism with human pluripotent stem cells (hPSCs) remains unsuccessful due to its extreamly low efficiency. Here, we show that hPSCs failed to differentiate and contribute teratoma in presence of mouse PSCs (mPSCs). While, MYCN, a pro-growth factor dramatically promotes hPSC contribution in teratoma co-formation by hPSCs/mPSCs. MYCN combined with BCL2 (M/B) greatly enhanced conventional hPSCs to integrate into preimplantation embryos of different species, such as mice, rabbits and pigs and substrantially contributed to mouse post-implantation chimera in embryonic and extra-embyonic tissues. Strikingly, M/B-hPSCs injected into preimplantation Flk-1+/- mouse embryos show further enhanced chimerism that allows obtaining live human CD34+ blood progenitor cells from chimeras through cell sorting. The chimera derived human CD34+ cells further gave rise to various subtype blood cells in typical colony forming unit(CFU) assay. Thus, we proof the concept to obtain functional human cells through enchanced interspecies chimerism with hPSCs.

sentences: PIVOTAL: Prioritizing variants of uncertain significance with spatial genomic patterns in the 3D proteome Variants of uncertain significance (VUS) have posed an increasingly prominent challenge to clinicians due to their growing numbers and difficulties in making clinical responses to them. Currently there are no existing methods that leverage the spatial relationship of known disease mutations and genomic properties for prioritizing variants of uncertain significance. More importantly, disease genes often associate with multiple clinically distinct diseases, but none of the existing variant prioritization methods provide clues as to the specific type of disease potentially associated with a given variant. We present PIVOTAL, a spatial neighborhood-based method using three-dimensional structural models of proteins, that significantly improves current variant prioritization tools and identifies potential disease etiology of candidate variants on a proteome scale. Using PIVOTAL, we made pathogenicity predictions for over 140,000 VUS and deployed a web application (http://pivotal.yulab.org) that enables users both to explore these data and to perform custom calculations.

sentences: COLD ATMOSPHERIC PLASMA DIFFERENTIALLY AFFECTS CELL RENEWAL AND DIFFERENTIATION OF STEM CELLS AND APC-DEFICIENT-DERIVED TUMOR CELLS IN INTESTINAL ORGANOIDS Cold atmospheric plasma (CAP) treatment has been proposed as a potentially innovative therapeutic tool in the biomedical field, notably for cancer due to its proposed toxic selectivity on cancer cells versus healthy cells. In the present study, we addressed the relevance of three-dimensional organoid technology to investigate the biological effects of CAP on normal epithelial stem cells and tumor cells isolated from mouse small intestine. CAP treatment exerted dose-dependent cytotoxicity on normal organoids and induced major transcriptomic changes associated with global response to oxidative stress, fetal-like regeneration reprogramming and apoptosis-mediated cell death. Moreover, we explored the potential selectivity of CAP on tumor-like Apc-deficient versus normal organoids in the same genetic background. Unexpectedly, tumor organoids exhibited higher resistance to CAP treatment, correlating with higher antioxidant activity at baseline as compared to normal organoids. This pilot study suggests that the ex vivo culture system could be a relevant alternative model to further investigate translational medical applications of CAP technology.

sentences: Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages 1Macrophages play fundamental roles in regulation of inflammatory responses to pathogens, resolution of inflammation and tissue repair, and maintenance of tissue homeostasis. The long (L) and short (S) isoforms of SP-R210/MYO18A, a macrophage receptor for surfactant protein A (SP-A) and C1q, regulate basal and inflammatory macrophage phenotype at multiple gene expression, translational, and subcellular levels in addition to their SP-A and C1q-mediated functions; disruption of L renders macrophages hyper-inflammatory, although the underlying mechanism had previously been unexplored. We asked whether disruption of the L isoform led to the hyper-inflammatory state via alteration of global genomic responses. RNA sequencing analysis of SP-R210L(DN) macrophages revealed basal and influenza induced upregulation of genes associated with inflammatory pathways, including TLR, RIG-I, NOD, and cytoplasmic DNA signaling, whereas knockdown of both SP-R210 isoforms (L and S) only resulted in increased RIG-I and NOD signaling. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis showed increased genome-wide deposition of the pioneer transcription factor PU.1 in SP-R210L(DN) compared to WT cells. ChIP-seq analysis of histone H3 methylation showed alterations in both repressive (H3K9me3 and H3K27me3) and transcriptionally active (H3K9me3) histone marks. Influenza A virus (IAV) infection, which stimulates an array of cytosolic and TLR-mediated antiviral mechanisms, resulted in differential redistribution between proximal promoter and distal sites and decoupling of PU.1 binding from Toll-like receptor regulated gene promoters in SP-R210L(DN) cells. Our findings suggest that SP-R210L-deficient macrophages are poised with an open PU.1-primed chromatin conformation to rapidly respond to inflammatory and metabolic stimuli.

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Tumor cell-intrinsic HFE drives glioblastoma growth BackgroundGlioblastoma (GBM) tumor cells modulate expression of iron-associated genes to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulator (HFE) gene, encoding the iron sensing HFE protein, is upregulated in GBM and correlates with poor survival outcomes. However, the molecular mechanisms underlying these observations remain unclear. Identification of pathways for targeting iron dependence in GBM tumors is therefore a critical area of investigation.

MethodsWe interrogated the impact of cell-intrinsic Hfe expression on proliferation and tumor growth through genetic loss and gain of function approaches in syngeneic mouse glioma models. We determined the expression of iron-associated genes and their relationship with survival in GBM using public datasets and identified differentially expressed pathways in Hfe knockdown cells through Nanostring transcriptional profiling.

ResultsLoss of Hfe induced apoptotic cell death in vitro and inhibited tumor growth in vivo while overexpression of Hfe accelerated both proliferation and tumor growth. Analysis of iron gene signatures in Hfe knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Analyzing differentially expressed pathways further identified oxidative stress as the top pathway upregulated with Hfe loss. Enhanced 55Fe uptake and generation of reactive oxygen species (ROS) were found with Hfe knockdown, implicating toxic iron overload resulting in apoptotic cell death.

ConclusionsCollectively, these findings identify a novel role for HFE in regulating iron homeostasis in GBM tumors and provide a potential avenue for future therapeutic development.

Key PointsO_LIHFE is an iron sensor that is upregulated in GBM and negatively impacts survival.
C_LIO_LIHFE overexpression drives proliferation and tumor growth in vivo.
C_LIO_LILoss of HFE increases production of reactive oxygen species and induces apoptosis, extending survival in vivo.
C_LI

Importance of StudyDysregulation of iron metabolism is an important feature of GBM contributing to tumor growth and negatively impacting survival. The identification of key iron regulators controlling this process is therefore important for therapeutic targeting. We identify HFE as an important regulator of iron homeostasis in GBM and suggest a role for sexual dimorphism in HFE-mediated tumor iron regulation that ultimately results in differential survival outcomes. Our findings demonstrate that HFE drives tumor cell proliferation and survival in GBM and may be a viable target for modulating tumor iron flux and inducing apoptosis in tumor cells.

sentences: SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-weeks interval between doses Continuous emergence of SARS-CoV-2 variants of concern (VOC) is fueling the COVID-19 pandemic. Omicron (B.1.1.529), is rapidly spreading worldwide. The large number of mutations in its Spike raised concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses was shown to elicit antibodies that efficiently recognize Spikes from different VOCs. Here we evaluated the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously-infected individuals that received their BNT162b2 mRNA vaccine 16-weeks apart. Omicron Spike was recognized less efficiently than D614G, Alpha, Beta, Gamma and Delta Spikes. We compared to plasma activity from participants receiving a short (4-weeks) interval regimen. Plasma from individuals of the long interval cohort recognized and neutralized better the Omicron Spike compared to those that received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.

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Assessment of CSF biomarkers and microRNA-mediated disease mechanisms in SMA ObjectiveCerebral spinal fluid (CSF) is a promising biospecimen for the detection of central nervous system (CNS) biomarkers to monitor therapeutic efficacy at the cellular level in neurological diseases. Spinal muscular atrophy (SMA) patients receiving intrathecal antisense oligonucleotide (nusinersen) therapy tend to show improved motor function, but the treatment effect on cellular function remains unknown. The objective of this study was to assess the potential of extracellular RNAs and microRNAs in SMA patient CSF as indicators of neuron and glial health following nusinersen treatment.

MethodsCSF samples from SMA Type 1-3 patients were screened using quantitative RT-PCR to assess expression of extracellular RNAs associated with inflammation and cellular stress, and microRNAs previously implicated in SMA pathogenesis. We also used mRNA sequencing and multi-electrode array approaches to assess the transcriptional and functional effects of astrocyte-associated miR-146a on healthy and SMA induced pluripotent stem cell (iPSC)-derived motor neurons.

ResultsExtracellular RNA analysis is suggestive of ongoing cellular stress, even in nusinersen treated samples. microRNAs previously associated with SMA pathology tended to show improvement in expression levels in nusinersen treated samples, with the exception of the astrocyte-secreted miR-146a. miR-146a treated iPSC-derived motor neurons showed a downregulation of extracellular matrix genes found in the synaptic perineuronal net and decreased spontaneous activity.

InterpretationExtracellular RNAs and microRNAs can be detected in SMA patient CSF samples, potentially serving as useful biomarkers to monitor cellular health during nusinersen treatment. Astrocyte health and response to nusinersen are important aspects to address in SMA pathogenesis and treatment strategies.

sentences: Clearing spheroids for 3D fluorescent microscopy:combining safe and soft chemicals with deep convolutional neural network In life sciences, there are increasing interest in 3D culture models to better reproduce the 3D environment encountered in-vivo. Imaging of such 3D culture models is instrumental for drug discovery, but face several issues before its use becomes widespread. Extensive microscopic investigation of these 3D cell models faces the challenge of light penetration in depth in opaque biological tissues. To overcome this limit, diverse clearing techniques have emerged over the past decades. However, it is not straightforward to choose the best clearing protocols, and assess quantitatively their clearing efficiency. Focusing on spheroids, we propose a combination of fast and cost-effective clearing procedure for such medium-sized samples. A generic method with local contrast metrics and deep convolutional neural network-based segmentation of nuclei is proposed to quantify the efficiency of clearing. We challenged this method by testing the possibility to transfer segmentation knowledge from a clearing protocol to another. The later results support the pertinence of training deep learning algorithms on cleared samples to further use the segmentation pipeline on non-cleared ones. This second step of the protocol gives access to digital clearing possibilities applicable to live and high-throughput optical imaging.

sentences: Tobramycin suppresses cystic fibrosis lung inflammation by increasing 5' tRNA-fMet halves secreted by P. aeruginosa Although inhaled tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of P. aeruginosa in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is unclear. Here, we demonstrate that tobramycin increases the abundance of two 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by P. aeruginosa and that the 5' tRNA-fMet halves reduce IL-8 secretion by CF bronchial epithelial cells (CF-HBECs). In mouse lung, the 5' tRNA-fMet halves attenuate KC secretion and neutrophil recruitment. We also report that the 5' tRNA-fMet halves suppress pro-inflammatory network gene expression by an Argonaut 2 (AGO2)-mediated gene silencing mechanism, thereby reducing IL-8 secretion in CF-HBECs. Moreover, tobramycin reduces the IL-8 concentration and neutrophil content in bronchoalveolar lavage fluid of pwCF. Thus, we conclude that tobramycin improves lung function in part by reducing chronic inflammation and neutrophil-mediated lung damage in pwCF.

sentences: Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo Resection of the 5-terminated strand at DNA double strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Biochemical and genetic studies have led to a multi-step model of DSB resection in which endonucleolytic cleavage mediated by Mre11 in partnership with Sae2 is coupled with exonucleolytic cleavage mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing in which molecular identifiers allow exact counting of cleaved 5 ends at base pair resolution. Mutant strains, including exo1{Delta}, mre11-H125N, exo1{Delta} and exo1{Delta} sgs1{Delta}, revealed a major Mre11-dependent cleavage position 60 to 70 bp from the DSB end whose exact position depended on local sequence and tracked a possible motif. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1{Delta} sgs1{Delta} yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo support of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

sentences: Stage-specific disruption of X chromosome expression during spermatogenesis in sterile house mouse hybrids Hybrid sterility is a complex phenotype that can result from the breakdown of spermatogenesis at multiple developmental stages. Here, we disentangle two proposed hybrid male sterility mechanisms in the house mice, Mus musculus domesticus and M. m. musculus, by comparing patterns of gene expression in sterile F1 hybrids from a reciprocal cross. We found that hybrid males from both cross directions showed disrupted X chromosome expression during prophase of meiosis I consistent with a loss of Meiotic Sex Chromosome Inactivation (MSCI) and Prdm9-associated sterility, but that the degree of disruption was greater in mice with an M. m. musculus X chromosome consistent with previous studies. During postmeiotic development, gene expression on the X chromosome was only disrupted in one cross direction, suggesting that misexpression at this later stage was genotype-specific and not a simple downstream consequence of MSCI disruption which was observed in both reciprocal crosses. Instead, disrupted postmeiotic expression may depend on the magnitude of earlier disrupted MSCI, or the disruption of particular X-linked genes or gene networks. Alternatively, only hybrids with a potential deficit of Sly copies, a Y-linked ampliconic gene family, showed overexpression in postmeiotic cells, consistent with a previously proposed model of antagonistic coevolution between the X and Y-linked ampliconic genes contributing to disrupted expression late in spermatogenesis. The relative contributions of these two regulatory mechanisms and their impact on sterility phenotypes awaits further study. Our results further support the hypothesis that X-linked hybrid sterility in house mice has a variable genetic basis, and that genotype-specific disruption of gene regulation contributes to overexpression of the X chromosome at different stages of development. Overall, these findings underscore the critical role of epigenetic regulation of the X chromosome during spermatogenesis and suggest that these processes are prone to disruption in hybrids.

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Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and removed capsular material. Sonication and glucanex digestion yield soluble CPS preparations, while French Press and modified glucanex digestion plus vortexing remove the capsule and cell wall producing polysaccharide aggregates that we call  capsule ghosts. The existence of capsule ghosts implies an inherent organization that allows it to exist independent of the cell wall surface. As sonication and glucanex digestion were noncytotoxic, it was possible to observe the cryptococcal cells rebuilding their capsule, revealing new insights into capsule architecture and synthesis consistent with a model in which the capsule is assembled from smaller polymers, which are then assemble into larger ones.

ImportanceCharacterization of the cryptococcal polysaccharide capsule relies on methods of isolation for its in vitro study. This study demonstrates that the capsule is susceptible to physical and enzymatic removal. The application of new methods yields insights into the anatomy, modular nature, and architecture of the capsule with both soluble CPS preparations and  capsule ghosts. Together these insights inform on a long-standing debate modeling capsular assembly wherein our data shows that the capsule is assembled by smaller polymers added distally rather than by proximal addition or by polymers spanning the entire capsule radius.

sentences: A Bidirectional Switch in the Shank3 Phosphorylation State Biases Synapses toward Up or Down Scaling Homeostatic synaptic plasticity requires widespread remodeling of synaptic signaling and scaffolding networks, but the role of posttranslational modifications in this process has not been systematically studied. Using deepscale, quantitative analysis of the phosphoproteome in mouse neocortical neurons, we found wide-spread and temporally complex changes during synaptic scaling up and down. We observed 424 bidirectionally modulated phosphosites that were strongly enriched for synapse-associated proteins, including S1539 in the ASD-associated synaptic scaffold protein Shank3. Using a parallel proteomic analysis performed on Shank3 isolated from rat neocortical neurons by immunoaffinity, we identified two sites that were hypo-phosphorylated during scaling up and hyper-phosphorylated during scaling down: one (rat S1615) that corresponded to S1539 in mouse, and a second highly conserved site, rat S1586. The phosphorylation status of these sites modified the synaptic localization of Shank3 during scaling protocols, and dephosphorylation of these sites via PP2A activity was essential for the maintenance of synaptic scaling up. Finally, phosphomimetic mutations at these sites prevented scaling up but not down, while phosphodeficient mutations prevented scaling down but not up. Thus, an activity-dependent switch between hypo- and hyperphosphorylation at S1586/ S1615 of Shank3 enables scaling up or down, respectively. Collectively our data show that activity-dependent phosphoproteome dynamics are important for the functional reconfiguration of synaptic scaffolds, and can bias synapses toward upward or downward homeostatic plasticity.

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MCKAT, a multi-dimensional copy numbervariant kernel association test BackgroundCopy number variants (CNVs) are the gain or loss of DNA segments in the genome. Studies have shown that CNVs are linked to various disorders, including autism, intellectual disability, and schizophrenia.

Consequently, the interest in studying a possible association of CNVs to specific disease traits is growing. However, due to the specific multi-dimensional characteristics of the CNVs, methods for testing the association between CNVs and the disease-related traits are still underdeveloped. We propose a novel multi-dimensional CNV kernel association test (MCKAT) in this paper. We aim to find significant associations between CNVs and disease-related traits using kernel-based methods.

ResultsWe address the multi-dimensionality in CNV characteristics. We first design a single pair CNV kernel, which contains three sub-kernels to summarize the similarity between two CNVs considering all CNV characteristics. Then, aggregate single pair CNV kernel to the whole chromosome CNV kernel, which summarizes the similarity between CNVs in two or more chromosomes. Finally, the association between the CNVs and disease-related traits is evaluated by comparing the similarity in the trait with kernel-based similarity using a score test in a random effect model. We apply MCKAT on genome-wide CNV datasets to examine the association between CNVs and disease-related traits, which demonstrates the potential usefulness the proposed method has for the CNV association tests. We compare the performance of MCKAT with CKAT, a uni-dimensional kernel method. Based on the results, MCKAT indicates stronger evidence, smaller p-value, in detecting significant associations between CNVs and disease-related traits in both rare and common CNV datasets.

ConclusionA multi-dimensional copy number variant kernel association test can detect significantly associated CNVs with any disease-related trait. MCKAT can help biologists detect significantly associated CNVs with any disease-related trait across a patient group instead of examining the CNVs case by case in each subject.

sentences: A Cambrian soft-bodied conical animal illustrates the origins of lophophorate phyla The origin and early evolution of lophotrochozoans remain a difficult but crucial issue in reconstructing metazoan phylogeny. Exceptionally preserved fossils have provided hopeful information for resolving this lophotrochozoan problem. Here we identify that Conicula striata, a soft-bodied conical animal from the early Cambrian Chengjiang Lagerstatte, has mosaic characteristics of different lophophorate phyla. C. striata possesses a phoronid-like vermiform trunk housing a U-shaped gut and a coiled lophophore comprising [≥]12 arms with numerous tentacles, but it also bears brachiopod-like features including an undivided mantle enclosing lophophore and a pedicle-like protrusion on annulated trunk. Phylogenetic analysis retrieves C. striata as a total-group lophophorate and as an intermediate taxon between Phoronida and Brachiopoda. This suggests that the bivalved architecture of brachiopods originated from an undivided mantle in a phoronid-like ancestor, and that the ancestral vermiform trunk became reduced during the origin of Brachiopoda, illuminating the origins of body plans in the lophophorate phyla.

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Neural tracking of the fundamental frequency of the voice: the effect of voice characteristics Traditional electrophysiological methods to study temporal auditory processing of the fundamental frequency of the voice (f0) often use unnaturally repetitive stimuli. In this study, we investigated f0 processing of meaningful continuous speech. EEG responses evoked by stories in quiet were analysed with a novel method based on linear modelling that characterizes the neural tracking of the f0. We studied both the strength and the spatio-temporal properties of the f0-tracking response. Moreover, different samples of continuous speech (six stories by four speakers: two male and two female) were used to investigate the effect of voice characteristics on the f0 response.

The results indicated that response strength is inversely related to f0 frequency and rate of f0 change throughout the story. As a result, the male-narrated stories in this study (low and steady f0) evoked stronger f0-tracking compared to female-narrated stories (high and variable f0), for which many responses were not significant. The spatio-temporal analysis revealed that f0-tracking response generators were not fixed in the brainstem but were voice-dependent as well. Voices with high and variable f0 evoked subcortically-dominated responses with a latency between 7 and 12 ms. Voices with low and steady f0 evoked responses that are both subcortically (latency of 13-15 ms) and cortically (latency of 23-26 ms) generated, with the right primary auditory cortex as a likely cortical source. Finally, additional experiments revealed that response strength greatly improves for voices with strong higher harmonics, which is particularly useful to boost the small responses evoked by voices with high f0.

sentences: Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies High-quality affinity probes are critical for sensitive and specific protein detection, in particular to detect protein biomarkers at early phases of disease development. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. In particular, clonal reagents offer opportunities for site-directed attachment of exactly one modification per affinity reagent at a site designed not to interfere with target binding to help standardize assays. The proximity extension assays (PEA) is a widely used protein assay where pairs of protein-binding reagents are modified with oligonucleotides (oligos), so that their proximal binding to a target protein generates a reporter DNA strand for DNA-assisted readout. The assays have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Here we explore nanobodies (Nb) as an alternative to polyclonal antibodies pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via Sortase A (SrtA) enzyme coupling. The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

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UniFuncNet: a flexible network annotation framework SummaryFunctional annotation is an integral part in the analysis of organisms, as well as of multi-species communities. A common way to integrate such information is using biological networks. However, current data integration network tools are heavily dependent on a single source of information, which might strongly limit the amount of relevant data contained within the network. Here we present UniFuncNet, a network annotation framework that dynamically integrates data from multiple biological databases, thereby enabling data collection from various sources based on user preference. This results in a flexible and comprehensive data retrieval framework for network based analyses of omics data. Importantly, UniFuncNets data integration methodology allows for the output of a non-redundant composite network and associated metadata. In addition, a workflow exporting UniFuncNets output to the graph database management system Neo4j was implemented, which allows for efficient querying and analysis.

AvailabilitySource code is available at https://github.com/PedroMTQ/UniFuncNet.

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Evolution of primate protomusicality via locomotion Animals communicate acoustically to report location and identity to conspecifics. More complex patterning of calls can also function as displays to potential mates and as territorial advertisement. Music and song are terms often reserved only for humans and birds, but elements of both forms of acoustic display are also found in non-human primates. While theories on proximate functions abound, ultimate drivers of specific call structures are less well understood. We hypothesized that spatio-temporal precision in landing during perilous arboreal locomotion favored the evolution of musical calling in early primates--vastly preceding the origin of more music-like behavior in hominoids and subsequent emergence of music in later hominids. We test this locomotion-based hypothesis on the origins of proto-musicality using spectrographic depictions of vocal repertoires of modern day primates and corresponding estimates of locomotor activity. Phylogenetically controlled regression analysis of 54 primate species reveals that arboreal locomotion and monogamy are robust influences on complex calling patterns while controlling for other socioecological variables. Given that these findings rest primarily upon a handful of deep branching points in the primate tree, we conclude that this coevolution likely occurred very slowly, occupying on the order of tens of millions of years.

LicenseAttribution-NoDerivatives 4.0 International (CC BY-ND 4.0)

sentences: A dynamic kissing model for enhancer-promoter communication on the surface of transcriptional condensate Enhancers are key DNA elements that regulate gene transcription by activating promoters in eukaryotes. Many potential mechanistic models have been proposed to account for enhancer-promoter communication, including the formation of the condensed enhancer-promoter clusterings that may result from liquid-liquid phase separation. However, the detailed kinetic mechanisms by which condensates may mediate enhancer-promoter contacts remain unclear. Here, we characterized enhancer-promoter communication in condensates using a self-avoiding polymer chain model under several possible spatial constraints. When the chain was confined to the spherical surface or interior, we discovered a distinct relationship between average end-to-end distance and contour length, as well as a distinct distribution of end-to-end distance. Experimental molecular tracking data showed strong preference in support of the spherical surface confinement hypothesis. Based on such observation, we proposed a novel enhancer-promoter communication model in which enhancers dynamically "kiss" promoters along the surface of transcriptional condensate. The theoretical results may guide future experiments, as researchers continue to elucidate the regulation dynamics of gene expression.

sentences: L-WNK1 is required for BK channel activation in intercalated cells BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

sentences: Dynamic proton-dependent motors power Type IX secretion and gliding adhesin movement in Flavobacterium Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMH) and show that conserved glutamate residues in GldL TMH are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

sentences: Fast processing of environmental DNA metabarcoding sequence data using convolutional neural networks 1The intensification of anthropogenic pressures have increased consequences on biodiversity and ultimately on the functioning of ecosystems. To monitor and better understand biodiversity responses to environmental changes using standardized and reproducible methods, novel high-throughput DNA sequencing is becoming a major tool. Indeed, organisms shed DNA traces in their environment and this "environmental DNA" (eDNA) can be collected and sequenced using eDNA metabarcoding. The processing of large volumes of eDNA metabarcoding data remains challenging, especially its transformation to relevant taxonomic lists that can be interpreted by experts. Speed and accuracy are two major bottlenecks in this critical step. Here, we investigate whether convolutional neural networks (CNN) can optimize the processing of short eDNA sequences. We tested whether the speed and accuracy of a CNN are comparable to that of the frequently used OBITools bioinformatic pipeline. We applied the methodology on a massive eDNA dataset collected in Tropical South America (French Guiana), where freshwater fishes were targeted using a small region (60pb) of the 12S ribosomal RNA mitochondrial gene. We found that the taxonomic assignments from the CNN were comparable to those of OBITools, with high correlation levels and a similar match to the regional fish fauna. The CNN allowed the processing of raw fastq files at a rate of approximately 1 million sequences per minute which was 150 times faster than with OBITools. Once trained, the application of CNN to new eDNA metabarcoding data can be automated, which promises fast and easy deployment on the cloud for future eDNA analyses.

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Removal of KCNQ2 from Parvalbumin-expressing Interneurons Improves Anti-Seizure Efficacy of Retigabine Anti-seizure drug (ASD) targets are widely expressed in both excitatory and inhibitory neurons. It remains unknown if the action of an ASD upon inhibitory neurons could counteract its beneficial effects on excitatory neurons (or vice versa), thereby reducing the efficacy of the ASD. Here, we examine whether the efficacy of the ASD retigabine (RTG) is altered after removal of the Kv7 potassium channel subunit KCNQ2, one of its drug targets, from parvalbumin-expressing interneurons (PV-INs). Parvalbumin-Cre (PV-Cre) mice were crossed with Kcnq2-floxed (Kcnq2fl/fl) mice to conditionally delete Kcnq2 from PV-INs. In these conditional knockout mice (cKO, PV-Kcnq2fl/fl), RTG (10 mg/kg, i.p.) significantly delayed the onset of either picrotoxin (PTX, 10 mg/kg, i.p)- or kainic acid (KA, 30mg/kg, i.p.)-induced convulsive seizures compared to vehicle, while RTG was not effective in wild-type littermates (WT). Immunostaining for KCNQ2 and KCNQ3 revealed that both subunits were enriched at axon initial segments (AISs) of hippocampal CA1 PV-INs, and their specific expression was selectively abolished in cKO mice. Accordingly, the M-currents recorded from CA1 PV-INs and their sensitivity to RTG were significantly reduced in cKO mice. While the ability of RTG to suppress CA1 excitatory neurons in hippocampal slices was unchanged in cKO mice, its suppressive effect on the spike activity of CA1 PV-INs was significantly reduced compared with WT mice. In addition, the RTG-induced suppression on intrinsic membrane excitability of PV-INs in WT mice was significantly reduced in cKO mice. These findings suggest that preventing RTG from suppressing PV-INs improves its anticonvulsant effect.

Significance StatementOne out of three patients with epilepsy remain resistant to anti-seizure drugs (ASD). Therefore, strategies that can enhance the efficacy of current ASDs or aid in the development of novel ASDs are in high demand. Here we show that by sparing a subset of inhibitory neurons, the ASD retigabine becomes more efficacious. These findings indicate that developing the ASDs that have relatively greater effects on excitatory over inhibitory neurons may be an effective strategy to improve the efficacy of ASDs.

sentences: Decadal trend of plankton community change and habitat shoaling in the Arctic gateway recorded by planktonic foraminifera The Fram Strait plays a crucial role in regulating the heat and sea-ice dynamics in the Arctic. In response to the ongoing global warming, the marine biota of this Arctic gateway is experiencing significant changes with increasing advection of Atlantic species. The footprint of this "Atlantification" has been identified in isolated observations across the plankton community, but a systematic, multi-decadal perspective on how regional climate change facilitates the invasion of Atlantic species and affects the ecology of the resident species is lacking. Here we evaluate a series of 51 depth-resolved plankton profiles collected in the Fram Strait during seven surveys between 1985 and 2015, using planktonic foraminifera as a proxy for changes in both the pelagic community composition and species vertical habitat depth. The time series reveals a progressive shift towards more Atlantic species, occurring independently of changes in local environmental conditions. We conclude that this trend is reflecting higher production of the Atlantic species in the "source" region, from where they are advected into the Fram Strait. At the same time, we observe that the ongoing extensive sea-ice export from the Arctic and associated cooling-induced decline in density and habitat shoaling of the subpolar Turborotalita quinqueloba, whereas the resident Neogloboquadrina pachyderma persists. As a result, the planktonic foraminiferal community and vertical structure in the Fram Strait shifts to a new state, driven by both remote forcing of the Atlantic invaders and local climatic changes acting on the resident species. The strong summer export of Arctic sea ice has so far buffered larger plankton transformation. We predict that if the sea-ice export will decrease, the Arctic gateway will experience rapid restructuring of the pelagic community, even in the absence of further warming. Such a large change in the gateway region will likely propagate into the Arctic proper.

sentences: A conserved role of Parkinson-associated DJ-1 metabolites in sperm motility, mitosis, and embryonic development Fertility rates in the developing world have dramatically dropped in the last decades. This drop is likely due to a decline in sperm quality and women having children at older ages. Loss of function mutations in DJ-1, a Parkinsons associated gene, are linked to alterations in multiple cellular processes such as mitochondrial activity, ROS production or sperm motility and lead to an early onset of Parkinsons disease and male infertility in humans and other species. Glycolate (GA) and D-lactate (DL), products of DJ-1 glyoxalase activity, sustain mitochondrial function and protect against environmental aggressions. We, therefore, tested whether these substances could also have a rescue effect on these phenotypes. Here, we show that DJ-1 loss of function not only affects sperm motility but also leads to defects in mitosis and an age-dependent increase in the abortion rate. Remarkably, whereas DL was only able to rescue embryonic lethality in C. elegans, GA rescued these phenotypes in all model systems tested and even increased sperm motility in wild-type sperm. These positive effects seem to be mediated through an increase in NAD(P)H production and the regulation of intracellular calcium. These findings not only strongly suggest GA as a new therapeutic candidate to improve male and female fertility but also show its potential to treat diseases associated with a decline in mitochondrial function or to improve mitochondrial function in aging.

sentences: What the odor is not: Estimation by elimination Olfactory systems use a small number of broadly sensitive receptors to combinatorially encode a vast number of odors. We propose a method of decoding such distributed representations by exploiting a statistical fact: receptors that do not respond to an odor carry more information than receptors that do because they signal the absence of all odorants that bind to them. Thus, it is easier to identify what the odor is not, rather than what the odor is. For realistic numbers of receptors, response functions, and odor complexity, this method of elimination turns an underconstrained decoding problem into a solvable one, allowing accurate determination of odorants in a mixture and their concentrations. We construct a neural network realization of our algorithm based on the structure of the olfactory pathway.

sentences: Antimicrobial resistance determinants in silage Animal products may play a role in developing and spreading antimicrobial resistance in several ways. On the one hand, residues of antibiotics not adequately used in animal farming can enter the human body via food. But resistant bacteria may also be present in animal products, which can transfer the antimicrobial resistance genes (ARG) to the bacteria in the consumers body by horizontal gene transfer. As previous studies have shown that fermented foods have a meaningful ARG content, it is indicated that such genes may also be present in silage used as mass feed in the cattle sector. In our study, we aspired to answer what ARGs occur in silage and what mobility characteristics they have? For this purpose, we have analyzed bioinformatically 52 freely available deep sequenced silage samples from shotgun metagenome next-generation sequencing. A total of 17 perfect matched ARGs occurred 55 times in the samples. More than half of these ARGs are mobile because they can be linked to integrative mobile genetic elements, prophages or plasmids. Our results point to a neglected but substantial ARG source in the food chain.

sentences: Neutralization of Omicron BA.1, BA.2, and BA.3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine The newly emerged Omicron SARS-CoV-2 has 3 distinct sublineages: BA.1, BA.2, and BA.3. BA.1 accounts for the initial surge and is being replaced by BA.2, whereas BA.3 is at a low prevalence at this time. Here we report the neutralization of BNT162b2-vaccinated sera (collected at 1 month after dose 3) against the three Omicron sublineages. To facilitate the neutralization testing, we engineered the complete BA.1, BA.2, or BA.3 spike into an mNeonGreen USA-WA1/2020 SRAS-CoV-2. All BNT162b2-vaccinated sera neutralized USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s with titers of >20; the neutralization geometric mean titers (GMTs) against the four viruses were 1211, 336, 300, and 190, respectively. Thus, the BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s were 3.6-, 4.0-, and 6.4-fold less efficiently neutralized than the USA-WA1/2020, respectively. Our data have implications in vaccine strategy and understanding the biology of Omicron sublineages.

sentences: Reduction of HDAC2 expression in human induced pluripotent stem cell derived neurons improves neuronal maturation, mitochondrial dynamics and cellular neurodegenerative disease phenotypes. Histone deacetylase 2 (HDAC2) is a major HDAC protein in the adult brain and has been shown to regulate many neuronal genes. Aberrant expression of HDAC2 and subsequent dysregulation of neuronal gene expression is implicated in neurodegeneration and brain aging. Human induced pluripotent stem cell-derived neurons (hiPSC-Ns) are widely used models for studying neurodegenerative disease mechanisms, but the role of HDAC2 in hiPSC-N differentiation and maturation has not been explored. In this study, we show that levels of HDAC2 progressively decrease as hiPSCs are differentiated towards neurons. This suppression of HDAC2 inversely corresponds to an increase in neuron-specific isoforms of Endophilin-B1, a multifunctional protein involved in mitochondrial dynamics. Expression of neuron-specific isoforms of Endophilin-B1 is accompanied by concomitant expression of a neuron-specific alternative splicing factor, SRRM4. Manipulation of HDAC2 and Endophilin-B1 using lentiviral approaches shows that knock-down of HDAC2 or overexpression of a neuron-specific Endophilin-B1 isoform promotes mitochondrial elongation and protects against cytotoxic stress in hiPSC-Ns, while HDAC2 knock-down specifically influences genes regulating mitochondrial dynamics and synaptogenesis. Furthermore, HDAC2 knock-down promotes enhanced mitochondrial respiration and reduces levels of neurotoxic amyloid beta peptides. Collectively, our study demonstrates a role for HDAC2 in hiPSC-neuronal differentiation, highlights neuron-specific isoforms of Endophilin-B1 as a marker of differentiating hiPSC-Ns, and demonstrates that HDAC2 regulates key neuronal and mitochondrial pathways in hiPSC-Ns.

sentences: Reduced variability of bursting activity during working memory Working memories have long been thought to be maintained by persistent spiking. However, mounting evidence from multiple-electrode recording (and single-trial analyses) shows that the underlying spiking is better characterized by intermittent bursts of activity. A counterargument suggested this intermittent activity is at odds with observations that spike-time variability reduces during task performance. However, this counterargument rests on assumptions, such as randomness in the timing of the bursts, that may not be correct. Thus, we analyzed spiking and LFPs from the prefrontal cortex (PFC) of monkeys to determine if task-related reductions in variability can co-exist with intermittent spiking. We found that it does because both spiking and associated gamma bursts were task-modulated, not random. In fact, the task-related reduction in spike variability could be explained by a related reduction in gamma burst variability. Our results provide further support for the intermittent activity models of working memory as well as novel mechanistic insights into how spike variability is reduced during cognitive tasks.

sentences: cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status. Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

sentences: Occurrence Of Avian Reovirus And Picobirnavirus In Wild Birds In An Environmental Protection Area In The Amazon Biome, Para, Brazil Wild birds have great prominence on transmission of diseases to humans, mainly due to their ease of access to human population, raising concerns about the potential impact of that proximity in context of the One Health. Studies referring to circulation of avian reovirus (ARV) and picobirnavirus (PBV) in wild birds are limited, in addition to reinforcing the development of researches that describe the prevalence, characterize the variants and evaluate the potential impact of these infections on the wild ecosystem and public health. The present study reports the occurrence of ARV and PBV in wild birds collected from an environmental protection area in the Amazon biome. RT-PCR analysis showed ARV infection prevalence in 0.6% (1/155) and PBV infection in 1.29% (2/155) on the samples. ARV strain isolated in this study demonstrated more phylogenetically related to other ARVs previously circulating in poultry in the same region. The two PBV strains obtained belong to genogroup I, and showed phylogenetically related to other PBV isolated from different animal species in different geographic regions. This study is a pioneer in the detection of ARV in wild birds in Brazil and presents a report of the first occurrence of PBV in wild birds of Guira guira specie. Additional studies in wild birds are required to increase the epidemiology, origin, evolution and emergence of new viruses that may provoke problems in the context of One Health.

sentences: LKB1 acts as a critical brake for the glucagon-mediated fasting response As important as the fasting response is for survival, an inability to shut it down once nutrients become available can lead to exacerbated disease and severe wasting. The liver is central to transitions between feeding and fasting states, with glucagon being a key initiator of the hepatic fasting response. However, the precise mechanisms controlling fasting are not well defined. One potential mediator of these transitions is Liver Kinase B1 (LKB1) given its role in nutrient sensing. Here, we show LKB1 knockout mice have a severe wasting and prolonged fasting phenotype despite increased food intake. By applying RNA sequencing and intravital microscopy we show that loss of LKB1 leads to a dramatic reprogramming of the hepatic lobule through robust upregulation of periportal genes and functions. This is likely mediated through the opposing effect LKB1 has on glucagon pathways and gene expression. Conclusion: our findings show that LKB1 acts as a brake to the glucagon-mediated fasting response resulting in "periportalization" of the hepatic lobule and whole-body metabolic inefficiency. These findings reveal a new mechanism by which hepatic metabolic compartmentalization is regulated by nutrient-sensing.

sentences: An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC

sentences: Neural Sources Underlying Visual Word Form Processing as Revealed by Steady State Visual Evoked Potentials (SSVEP) EEG has been central to investigations of the time course of various neural functions underpinning visual word recognition. Recently the steady-state visual evoked potential (SSVEP) paradigm has been increasingly adopted for word recognition studies due to its high signal-to-noise ratio. Such studies, however, have been typically framed around a single source in the left ventral occipitotemporal cortex (vOT). Here, we combine SSVEP recorded from 16 adult native English speakers with a data-driven spatial filtering approach--Reliable Components Analysis (RCA)--to elucidate distinct functional sources with overlapping yet separable time courses and topographies that emerge when contrasting words with pseudofont visual controls. The first component topography was maximal over left vOT regions with an early latency (approximately 180 msec). A second component was maximal over more dorsal parietal regions with a longer latency (approximately 260 msec). Both components consistently emerged across a range of parameter manipulations including changes in the spatial overlap between successive stimuli, and changes in both base and deviation frequency. We then contrasted word-in-nonword and word-in-pseudoword to test the hierarchical processing mechanisms underlying visual word recognition. Results suggest that these hierarchical contrasts fail to evoke a unitary component that might be reasonably associated with lexical access.

sentences: Hierarchical organization of rhesus macaque behavior Primatologists, psychologists and neuroscientists have long hypothesized that primate behavior is highly structured. However, fully delineating that structure has been impossible due to the difficulties of precision behavioral tracking. Here we analyzed a dataset consisting of continuous measures of the 3D position of fifteen body landmarks from two male rhesus macaques (Macaca mulatta) performing three different tasks in a large unrestrained environment over many hours. Using an unsupervised embedding approach on the tracked joints, we identified commonly repeated pose patterns, which we call postures. We found that macaques behavior is characterized by 49 distinct identifiable postures, lasting an average of 0.6 seconds each. We found evidence that behavior is hierarchically organized, in that transitions between poses tend to occur within larger modules, which correspond to intuitively identifiably actions; these actions are in turn organized hierarchically. Our behavioral decomposition allows us to identify universal (cross-individual and cross-task) and unique (specific to each individual and task) principles of behavior. These results demonstrate the hierarchical nature of primate behavior and provide a method for the automated "ethogramming" of primate behavior.

sentences: SamQL: A Structured Query Language and filtering tool for the SAM/BAM file format The Sequence Alignment/Map Format Specification (SAM) is one of the most widely used file formats in computational biology today and several tools have been developed to process it and use it. It is a flexible file format, used by many bioinformaticians on a daily basis. Despite its flexibility, SAM encoded files can often be difficult to query and understand. As genomic data are rapidly growing, structured and efficient queries on data encoded in SAM/BAM files is becoming critical. Importantly, any new tools should be able to support existing large datasets without requiring any data transformations. Here we introduce SamQL, an SQL-like query language for the SAM format with intuitive syntax that supports complex and efficient queries on top of SAM/BAM files and that can replace commonly used Bash one-liners employed by many bioinformaticians. SamQL is a complete query language that we envision as a step to a structured database engine for genomics. SamQL is written in Go, taking advantage of modern multicore compute systems and is freely available as standalone program and as an open-source library released under an MIT license, https://github.com/maragkakislab/samql/.

sentences: Spatiotemporal specificity of correlated DNA methylation and gene expression pairs across different human tissues and stages of brain development DNA methylation (DNAm) that occurs on promoter regions is primarily considered to repress gene expression. Previous studies indicated that DNAm could also show positive correlations with gene expression. Both DNAm and gene expression profiles are known to be tissue- and development-specific. This study aims to investigate how DNAm and gene expression are coordinated across different human tissues and developmental stages, as well as the biological significance of such correlations. By analyzing 2,239 samples with both DNAm and gene expression data in the same human subjects obtained from six published datasets, we evaluated the correlations between gene and CpG pairs (GCPs) at cis-regions and compared significantly correlated GCPs (cGCPs) across different tissues and brains at different age groups. A total of 37,363 cGCPs were identified in the six datasets; approximately 38% of the cGCPs were positively correlated. The majority (>90%) of cGCPs were tissue- or development-specific. We also observed that the correlation direction can be opposite in different tissues and ages. Further analysis highlighted the importance of cGCPs for their cellular functions and potential roles in complex traits and human diseases. For instance, early developmental brain possessed a highly unique set of cGCPs that were associated with neurogenesis and psychiatric disorders. By assessing the epigenetic factors involved in cGCPs, we discovered novel regulatory mechanisms of positive cGCPs distinct from negative cGCPs, which were related to multiple factors, such as H3K27me3, CTCF, and JARD2. The catalog of cGCPs compiled can be used to guide functional interpretation of genetic and epigenetic studies.

sentences: Rational domestication of a plant-based recombinant expression system expands its biosynthetic range. Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

sentences: An innovative approach for determining composite wheat quality index to identify quality enriched genotypes - insights and implications Ranking test entries or test sites on a quality basis is very difficult in wheat as value addition is perceived by several grain properties and end-products. Here, a novel approach has been developed and tested by deriving wheat quality index based on principal component analysis of 13 physico-chemical grain parameters and 3 end products of 45 wheat varieties. Depending upon the observed index range (0.15 to 0.71), the cultivars were assorted into 3 distinct classes i.e. elite, moderate and poor. The top group ascertained high quality standards of grain suited for bread and chapati whereas bottom group assured better cookies quality. This technique was also tested to differentiate quality enriched test sites within a zone or demarcate the most suited production environments to harness good quality wheat. The index will have an implication on farmers (premium price for varietal segregation), industry (product specific quality cultivars), and consumers (superior quality products).

sentences: Animal-reliance bias in publishing is a potential barrier to scientific progress Publication of scientific findings is a fundamental part of the research process that pushes innovation and generates policies and interventions that benefit society, but it is not without biases. Publication bias is generally recognized as journals preference for publishing studies based on the direction and magnitude of results. However, early evidence of a new type of bias related to publication has emerged in which editorial policy and/or peer reviewers or editors request that animal data be provided to validate studies produced using nonanimal human biology-based approaches. We describe herein "animal-reliance bias" in publishing: a reliance on or preference for animal-based methods where they may not be necessary or where nonanimal-based methods may be suitable, which affects the likelihood of a manuscript being accepted for publication. To gather evidence of the occurrence of this emerging type of publication bias, we set out to collect the experiences and perceptions of scientists and reviewers related to animal- and nonanimal-based experiments during peer review. To this end, we created a survey with 33 questions that was completed by 90 respondents working in several areas in the biological and biomedical fields. Twenty-one survey respondents indicated that they have carried out animal-based experiments for the sole purpose of anticipating reviewer requests. Thirty-one survey respondents indicated that they have been asked by peer reviewers to add animal experimental data to their nonanimal study; 14 of these felt that it was sometimes justified, and 11 did not think the request was justified. The data presented strongly support the occurrence of animal-reliance bias and indicates that status quo and conservatism biases may explain such attitudes by peer reviewers and editors. To our knowledge, this is the first study to recognize the existence of animal-reliance bias in publishing and provides the basis for developing strategies to prevent this practice from happening.

sentences: Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.

sentences: Fibrinogen and Complement Factor H are promising CSF protein biomarker(s) for Parkinson's disease with cognitive impairment- A Proteomics and ELISA based study Cognitive impairment is a debilitating non-motor symptom of Parkinsons disease (PD). The diagnosis of PD with cognitive impairment (PDCI) is essentially through clinical and neuropsychological examinations. There is an emerging need to identify biomarker(s) to foresee cognitive decline in PD patients, at an early stage. We performed label-free unbiased nontargeted proteomics (Q-TOF LC/MS-MS) in CSF of non-neurological control (NNC); PDCI; PD and normal pressure hydrocephalus (NPH), followed by targeted ELISA for validation. The diagnosis was confirmed by neuropsychological and MRI assessments prior to CSF collection. Of the 282 proteins identified by mass spectrometry, 42 were differentially altered in PD, PDCI and NPH. Further, 28 proteins were altered in PDCI and 25 in NPH. An interesting overlap of certain proteins was noted both in PDCI and NPH. Five significantly upregulated proteins in PDCI were fibrinogen, gelsolin, complement factor-H, apolipoprotein A-IV and apolipoprotein A-I. Whereas carnosine dipeptidase 1, carboxypeptidase E, dickkpof 3 and secretogranin 3 precursor proteins were down-regulated. NPH also had few uniquely altered proteins viz. insulin-like growth factor-binding protein, ceruloplasmin, -1 antitrypsin, VGF nerve growth factor, neural cell adhesion molecule L1 like protein. Interestingly, the ELISA-derived protein concentrations correlated well with the neuropsychological scores of certain cognitive domains. Executive function was affected both in PDCI and NPH. In PD, Wisconsin card sorting test (WCST) percentile correlated positively with ApoA-IV and negatively with the ratio of ApoAI: ApoA-IV. Thus assessment of a battery of proteins like fibrinogen--chain, CFAH and ApoAI: ApoA-IV ratio alongside neuropsychological could be reliable biomarkers to distinguish PDCI and NPH.

sentences: Description of Life History & Reproductive Size Threshold in Three High Elevation Puya (Bromeliaceae) AO_SCPLOWBSTRACTC_SCPLOWThe Andes are a hotspot for biodiversity and high species endemism for both plants and animals. The genus Puya (Bromeliaceae) lives throughout the Andes, including puna and the paramo ecosystems above 3500m. Here, we studied the life history and reproductive size thresholds in three species of Puya (P. raimondii, P. cryptantha and P. goudotiana): P. raimondii in the Bolivian puna, and P. cryptantha and P. goudotiana in the Colombian paramo. We collected data on threshold size at flowering and clonal reproduction. All three species were found to have a consistent minimum size at flowering, while only P. cryptantha demonstrated a minimum size for clonal reproduction. No such evidence was found for P. goudotiana. We also found a positive correlation between leaf length and fruit number for P. goudotiana and P. cryptantha. Our data supported that P. raimondii is fully semelparous and indicated that P. goudotiana and P. cryptantha may be semi-semelparous.

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Correction of the scientific production: publisher performance evaluation using a dataset of 4844 PubMed retractions. BackgroundWithdrawal of problematic scientific articles after publication is one of the mechanisms for correcting the literature available to publishers, especially in the conditions of the ever-increasing trend of publishing activity in the medical field. The market volume and the business model justify publishers involvement in the post-publication quality control(QC) of scientific production. The limited information about this subject determined us to analyze retractions and the main retraction reasons for publishers with many withdrawn articles. We also propose a score to measure the evolution of their performance. The data set used for this article consists of 4844 PubMed retracted papers published between 1.01.2009 and 31.12.2020.

MethodsWe have analyzed the retraction notes and retraction reasons, grouping them by publisher. To evaluate performance, we formulated an SDTP score whose calculation formula includes several parameters: speed (article exposure time(ET)), detection rate (percentage of articles whose retraction is initiated by the editor/publisher/institution without the authors participation), transparency (percentage of retracted articles available online and clarity of retraction notes), precision (mention of authors responsibility and percentage of retractions for reasons other than editorial errors).

ResultsThe 4844 withdrawn articles were published in 1767 journals by 366 publishers, the average number of withdrawn articles/journal being 2.74. Forty-five publishers have more than ten withdrawn articles, holding 88% of all papers and 79% of journals. Combining our data with data from another study shows that less than 7% of PubMed journals withdrew at least one article. Only 10.5% of the withdrawal notes included the individual responsibility of the authors. Nine of the top 11 publishers had the largest number of articles withdrawn in 2020, in the first 11 places finding, as expected, some big publishers. Retraction reasons analysis shows considerable differences between publishers concerning the articles ET: median values between 9 and 43 months (mistakes), 9 and 73 months (images), 10 and 42 months (plagiarism & overlap).

The SDTP score shows, between 2018 and 2020, an improvement in QC of four publishers in the top 11 and a decrease in the gap between 1st and 11th place. The group of the other 355 publishers also has a positive evolution of the SDTP score.

ConclusionsPublishers have to get involved actively and measurably in the post-publication evaluation of scientific products. The introduction of reporting standards for retraction notes and replicable indicators for quantifying publishing QC can help increase the overall quality of scientific literature.

sentences: Discovery of frog virus 3 microRNAs and their roles in evasion of host antiviral responses Frog virus 3 (FV3, genus Ranavirus) causes devastating disease in amphibian populations and is capable of subverting host immune responses. Evidence suggests that virus-encoded microRNAs (v-miRNAs) are implicated in host immunoevasion tactics. Thus, we sought to discover FV3-encoded v-miRNAs and to uncover their putative roles in immunoevasion. Small RNA libraries were generated from FV3-infected Xela DS2, a Xenopus laevis dorsal skin epithelial-like cell line, at 24- and 72-hours post-infection (hpi). We discovered 43 FV3 v-miRNAs and identified that 15 are upregulated at 24 hpi, while 18 are upregulated at 72 hpi. Target prediction analyses revealed that FV3 v-miRNAs target host genes involved in key antiviral signaling pathways, while gene ontology analyses suggest that FV3 v-miRNAs may broadly impact host cell function. This is the first study to experimentally detect mature v-miRNAs produced by FV3. Our findings highlight the possibility that ranaviral v-miRNAs facilitate immunoevasion of frog antiviral responses.

sentences: Host phenology can drive the evolution of intermediate virulence strategies in some obligate-killer parasites AO_SCPLOWBSTRACTC_SCPLOWThe traditional mechanistic trade-offs resulting in a negative correlation between transmission and virulence are the foundation of nearly all current theory on the evolution of parasite virulence. Several ecological factors have been shown to modulate the optimal virulence strategies predicted from mechanistic trade-off models, but these ecological factors have not yet been shown to be sufficient to explain the intermediate virulence strategies observed in any natural system. The timing of seasonal activity, or phenology, is a common factor that influences the types and impact of many ecological interactions but is difficult to incorporate into virulence evolution studies. We develop a mathematical model of a disease system with seasonal host activity to study the evolutionary consequences of host phenology on the virulence of obligate-killer parasite. Results from this model demonstrated that seasonal host activity is sufficient to drive the evolution of intermediate parasite virulence in some types of natural disease systems, even when a traditional mechanistic trade-off between transmission and virulence is not assumed in the modeling framework. The optimal virulence strategy in these systems can be determined by both the duration of the host activity period as well as the variation in the host emergence timing. Parasites with low virulence strategies are favored in environments with long host activity periods and in environments in which hosts emerge synchronously. The results demonstrate that host phenology can be sufficient to select for intermediate optimal virulence strategies, providing an alternative mechanism to account for virulence evolution in some natural systems.

sentences: Tuna-AI: tuna biomass estimation with Machine Learning models trained on oceanography and echosounder FAD data Echo-sounder data registered by buoys attached to drifting FADs provide a very valuable source of information on populations of tuna and their behaviour. This value increases when these data are supplemented with oceanographic data coming from CMEMS. We use these sources to develop TO_SCPLOWUNAC_SCPLOW-AI, a Machine Learning model aimed at predicting tuna biomass under a given buoy, which uses a 3-day window of echo-sounder data to capture the daily spatio-temporal patterns characteristic of tuna schools. As the supervised signal for training, we employ more than 5000 set events with their corresponding tuna catch reported by the AGAC tuna purse seine fleet.

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Atlas of tissue-specific and tissue-preferential gene expression in ecologically and economically significant conifer Pinus sylvestris Despite their ecological and economical importance, conifers genomic resources are limited, mainly due to the large size and complexity of their genomes. Additionally, the available genomic resources lack complete structural and functional annotation. Transcriptomic resources have been commonly used to compensate for these deficiencies, though for most conifer species they are limited to a small number of tissues, or capture only a fraction of the genes present in the genome.

Here we provide an atlas of gene expression patterns for conifer Pinus sylvestris across five tissues: embryo, megagametophyte, needle, phloem, and vegetative bud. We used a wide range of tissues and focused our analyses on the expression profiles of genes at tissue level. We provide comprehensive information of the per-tissue normalized expression level, indication of tissue preferential upregulation and tissue-specificity of expression. We identified a total of 48,001 tissue preferentially upregulated and tissue specifically expressed genes, of which 28% have annotation in the Swiss-Prot database. Even though most of the putative genes identified do not have functional information in current biological databases, the tissue-specific patterns discovered provide valuable information about their potential functions for further studies, as for example in the areas of plant physiology, population genetics, and genomics in general. As we provide information on tissue specificity at both diploid and haploid life stages, our data will also contribute to the understanding of evolutionary rates of different tissue types and ploidy levels.

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Coupling of NOD2 to GIV is Required for Bacterial Sensing Sensing of pathogens by Nucleotide oligomerization domain (NOD)-like 2 receptor (NOD2) induces a protective inflammatory response that coordinates bacterial clearance. Polymorphisms in NOD2 impair bacterial clearance, leading to chronic gut inflammation in Crohns disease (CD) via mechanisms that remain incompletely understood. We identify GIV/Girdin (CCDC88A) as a NOD2-interactor that shapes bacterial sensing-and-signaling in macrophages. Myeloid-specific GIV depletion exacerbated and protracted infectious colitis and abolished the protective effect of muramyl dipeptide (MDP) in both chemical colitis and severe sepsis. In the presence of GIV, macrophages enhance anti-bacterial pathways downstream of NOD2, clear microbes rapidly and concomitantly suppress inflammation. GIVs actions are mediated via its C-terminus, which directly binds the terminal leucine-rich repeat (LRR#10) of NOD2; binding is augmented by MDP and ATP, precedes receptor oligomerization, and is abolished by the 1007fs CD-risk variant which lacks LRR#10. Findings illuminate mechanisms that underlie protective NOD2 signaling and loss of function in the major 1007fs variant.

GRAPHIC ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/489574v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@d8bf25org.highwire.dtl.DTLVardef@1ed2b16org.highwire.dtl.DTLVardef@1a7c8e8org.highwire.dtl.DTLVardef@770ec2_HPS_FORMAT_FIGEXP  M_FIG C_FIG In briefThis work reveals a mechanism by which macrophages use their innate immune sensor, NOD2, to protect the host against overzealous inflammation during bacterial infections, and the consequences of its loss, as occurs in the most important Crohns disease-risk variant.

HIGHLIGHTSO_LIGIV is a functional and direct interactor of the terminal LRR repeat of NOD2
C_LIO_LIMice lacking M[FE] GIV develop dysbiosis, protracted ileocolitis and sepsis
C_LIO_LIMDP/NOD2-dependent protective host responses require GIV
C_LIO_LICD-risk NOD2 1007fs variant lacking the terminal LRR#10 cannot bind GIV
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sentences: Randomized controlled studies comparing traditional lectures versus online modules We assessed the efficacy of traditional lectures versus online modules with respect to student learning in an undergraduate introductory biochemistry course in two successive years. In the first year, students had the options of attending live lectures by the course instructor and viewing online modules pre-recorded by the instructor, with the lectures and modules covering identical content; in addition, all students had a mandatory weekly application session. Utilizing pre-course and post-course tests as an instrument with which to measure learning during the course, we observed significantly increased learning (0.7 standard deviations) with attendance of traditional lectures and decreased learning with use of online modules, even after adjustment for grade point average. In the second year, the course had the same curriculum, but students were randomized to either live lectures or online modules for the first half of the course, crossing over to the other modality during the second half. With randomization, no difference in learning was observed between the two groups. Furthermore, we found that students self-reported greater engagement when viewing online modules than when attending lectures in person. These findings suggest some aspects of the lecture experience can be shifted to online modules in STEM courses without impacts on student learning so as to use classroom time more fully for application-based active learning interventions.

sentences: Projective LDDMM: Spatially Reconstructing a Story of Rostrally-Dominant Tau in Alzheimer's Disease Since Braaks initial histological observations, it has been recognized that Alzheimers disease (AD) neurofibrillary tangles (NFTs) appear in the medial temporal lobe (MTL) of the brain very early in the disease course. MRI-based shape diffeomorphometry markers have demonstrated pre-clinical AD changes in the MTL but it has not been possible to confirm that these MRI changes correspond to the presence of NFTs. Here, we present a method termed Projective LDDMM for aligning sparse measurement profiles of AD pathology (i.e., 2D digital histology images) with 3D MRI. We reconstruct measures of 2D NFT density in the dense metric of 3D MRI, using the Mai Paxinos Atlas coordinates for two cases of advanced AD. Analyses reveal the highest levels of NFT density in the rostral third (10-15 mm) of the hippocampus and the adjoining regions of the entorhinal cortex and amygdala. These findings emphasize the selective vulnerability of MTL subregions in AD, and suggest that high resolution MRI methods might benefit from focusing on the rostral MTL to more closely link these MRI images to AD neuropathology.

sentences: Vapor mediation as a tool to control micro-nano scale dendritic crystallization and preferential bacterial distribution in drying respiratory droplets Deposits of biofluid droplets on surfaces (such as respiratory droplets formed during an expiratory event fallen on surfaces) are composed of the water-based salt-protein solution that may also contain an infection (bacterial/viral).The final patterns of the deposit formed are dictated by the composition of the fluid and flow dynamics within the droplet. This work reports the spatio-temporal, topological regulation of deposits of respiratory fluid droplets and control of motility of bacteria by tweaking flow inside droplets using non-contact vapor-mediated interactions. When evaporated on a glass surface, respiratory droplets form haphazard multiscale dendritic, cruciform-shaped precipitates--using vapor mediation as a tool to control these deposits at the level of nano-micro-millimeter scales. Wemorphologically control dendrite orientation, size and subsequently suppress cruciform-shaped crystals. The nucleation sites are controlled via preferential transfer of solutes in the droplets; thus, achieving control over crystal occurrence and growth dynamics. The active living matter like bacteria is also preferentially segregated with controlled motility without attenuation of its viability and pathogenesis. For the first time, we have experimentally presented a proof-of-concept to control the motion of live active matter like bacteria in a near non-intrusive manner. The methodology can have ramifications in biomedical applications like disease detection, controlling bacterial motility, and bacterial segregation.

sentences: Multifactorial remodeling of the cancer immunopeptidome by interferon gamma IFN{gamma} alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFN{gamma}-treated colorectal cancer patient-derived organoids (PDOs) and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFN{gamma}. IFN{gamma}-induced changes in the abundance of source proteins, switching from the constitutive- to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is up- or downregulated on HLA-I through IFN{gamma}. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFN{gamma} is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics.

sentences: Network analysis reveals a major role for 14q32 cluster miRNAs in determining transcriptional differences between IGHV-mutated and unmutated CLL Tumour cells from patients with chronic lymphocytic leukaemia (CLL) can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear to what extent these differences reflect the tumour cells innate pre/post germinal centre origin or their BCR signalling competence, we applied RNA sequencing, small RNA sequencing and DNA methylation array analysis to 38 CLL cases categorised into three groups by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. A significant proportion of the IGHV-associated miRNAs derived from chr14q32 clusters (14/38 (37%)), where all miRNAs were co-expressed with the MEG3 lncRNA, as part of the DLK1-DIO3 genomic imprinted region, a locus of known importance in the pathogenesis of other human tumours. Integrative in silico analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was predicted to be regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.

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DMRscaler: A Scale-Aware Method to Identify Regions of Differential DNA Methylation Spanning Basepair to Multi-Megabase Features BackgroundPathogenic mutations in genes that control chromatin function have been implicated in rare genetic syndromes. These chromatin modifiers exhibit extraordinary diversity in the scale of the epigenetic changes they affect, from single basepair modifications by DNMT1 to whole genome structural changes by PRM1/2. Patterns of DNA methylation are related to a diverse set of epigenetic features across this full range of epigenetic scale, making DNA methylation valuable for mapping regions of general epigenetic dysregulation. However,existing methods are unable to accurately identify regions of differential methylation across this full range of epigenetic scale directly from DNA methylation data.

ResultsTo address this, we developed DMRscaler, a novel method that uses an iterative windowing procedure to capture regions of differential DNA methylation (DMRs) ranging in size from single basepairs to whole chromosomes. We benchmarked DMRscaler against several DMR callers in simulated and natural data comparing XX and XY peripheral blood samples. DMRscaler was the only method that accurately called DMRs ranging in size from 100 bp to 1 Mb (pearsons r = 0.96) and up to 152 Mb on the X-chromosome. We then analyzed methylation data from rare-disease cohorts that harbor chromatin modifier gene mutations in NSD1, EZH2, and KAT6A where DMRscaler identified novel DMRs spanning gene clusters involved in development.

ConclusionTaken together, our results show DMRscaler is uniquely able to capture the size of DMR features across the full range of epigenetic scale and identify novel, co-regulated regions that drive epigenetic dysregulation in human disease.

sentences: Multi-curve fitting and tubulin-lattice signal removal for structure determination of large microtubule-based motors Revealing high-resolution structures of microtubule-associated proteins (MAPs) is critical for understanding their fundamental roles in various cellular activities, such as cell motility and intracellular cargo transport. Nevertheless, large molecular motors that dynamically bind and release microtubule networks are challenging for cryo-electron microscopy (cryo-EM). Traditional structure determination of MAPs bound to microtubules needs alignment information from the reconstruction of microtubules, which cannot be readily applied to large MAPs without a fixed binding pattern. Here, we developed a comprehensive approach to estimate the microtubule networks (multicurve fitting), model the tubulin-lattice signals, and remove them (tubulin-lattice subtraction) from the raw cryo-EM micrographs. The approach does not require an ordered binding pattern of MAPs on microtubules, nor does it need a reconstruction of the microtubules. We demonstrated the capability of our approach using the reconstituted outer-arm dynein bound to microtubule doublets. In addition, we applied our multi-curve fitting approach to other biological filaments and achieved accurate estimations. Our work provides a new tool to determine high-resolution structures of large MAPs bound to curved microtubule networks.

sentences: Hippocampal astroglial hypertrophy in mice subjected to early life maternal deprivation Early-life stress (ELS), including chronic deprivation of maternal care, exerts persistent life-long effects on animal physiology and behavior, and is associated with several neurodevelopmental disorders. Long-lasting changes in neuronal plasticity and electrophysiology are documented extensively in the animal models of ELS. However, the role of astroglia in the lasting effects of ELS remains elusive. Astrocytes are intricately involved in the regulation of synaptic physiology and behavior. Moreover, astrocytes play a major role in the innate and adaptive immune responses in the central nervous system (CNS). The role of immune responses and neuroinflammation in the altered brain development and persistent adverse effects of ELS are beginning to be explored. Innate immune response in the CNS is characterized by a phenomenon called astrogliosis, a process in which astrocytes undergo hypertrophy, along with changes in gene expression and function. While the immune activation and neuroinflammatory changes concomitant with ELS, or in juveniles and young adults have been reported, it is unclear whether mice subjected to ELS exhibit astrogliosis-like alterations well into late-adulthood. Here, we subjected mice to maternal separation from postnatal day 2 to day 22 and performed comprehensive morphometric analysis of hippocampal astrocytes during late-adulthood. We found that the astrocytes in the stratum radiatum region of the CA1 hippocampal subfield from maternally separated mice exhibit significant hypertrophy as late as 8 months of age, revealing the crucial changes in astrocytes that manifest long after the cessation of ELS. This study highlights the persistence of neuroinflammatory changes in mice exposed to ELS.

sentences: FRUITFULL-like genes regulate flowering time and inflorescence architecture in tomato The timing of flowering and inflorescence architecture are critical for the reproductive success of tomato, but the gene regulatory networks underlying these traits have not been fully explored. Here we show that the tomato FRUITFULL-like (FUL-like) genes FUL2 and MADS-BOX PROTEIN 20 (MBP20) induce flowering and repress inflorescence branching by promoting floral meristem determinacy. FUL1 fulfils a less prominent role and appears to depend on FUL2 and MBP20 for its upregulation in the inflorescence- and floral meristems. MBP10, the fourth tomato FUL-like gene, has probably lost its function. The tomato FUL-like proteins cannot homodimerize in in vitro assays, but heterodimerize with various other MADS-domain proteins, potentially forming distinct complexes in the transition meristem and floral meristem. Transcriptome analysis of the primary shoot meristems revealed various interesting downstream targets, including four repressors of cytokinin signalling that are upregulated during the vegetative-to-reproductive transition in ful1 ful2 mbp10 mbp20 mutants. FUL2 and MBP20 can also bind in vitro to the upstream regions of these genes, thereby probably directly stimulating cell division in the meristem upon the transition to flowering. The control of inflorescence branching does not occur via the CKXs, but appears regulated by repression of transcription factors such as TM3, APETALA 2b (AP2b) and AP2a.

sentences: ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1 There is considerable evidence that cross-talk between the Hedgehog pathway and MAPK signaling pathways occurs in several types of cancer, and contributes to the emergence of clinical resistance to Hedgehog pathway inhibitors. Here, we demonstrate that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. We show that ERK2 phosphorylates GLI1 on three evolutionarily-conserved target sites (S102, S116 and S130) located near the high-affinity binding site for the negative regulator SUFU; furthermore, these phosphorylation events cooperate to weaken the affinity of GLI1-SUFU binding by over 25 fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily-conserved SUFU-affinity-modulating region. In cultured mammalian cells, mutant GLI1 variants containing phosphomimetic substitutions of S102, S116 and S130 displayed an increased ability to drive transcription. We conclude that of multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

sentences: MT-MAG: Accurate and interpretable machine learning based taxonomic assignment of metagenome-assembled genomes, with a partial classification option We propose MT-MAG, a novel machine learning-based software tool for the complete or partial hierarchically-structured taxonomic classification of metagenome-assembled genomes (MAGs). MT-MAG is capable of classifying large and diverse metagenomic datasets: a total of 245.68 Gbp in the training sets, and 9.6 Gbp in the test sets analyzed in this study. MT-MAG is, to the best of our knowledge, the first machine learning method for taxonomic assignment of metagenomic data that offers a "partial classification" option. MT-MAG outputs complete or partial classification paths, and interpretable numerical classification confidences of its classifications, at all taxonomic ranks. To assess the performance of MT-MAG, we define a "weighted classification accuracy," with a weighting scheme reflecting the fact that partial classifications at different ranks are not equally informative. For the two benchmarking datasets analyzed (genomes from human gut microbiome species, and bacterial and archaeal genomes assembled from cow rumen metagenomic sequences), MT-MAG achieves an average of 80.13% in weighted classification accuracy. At the Species level, MT-MAG outperforms DeepMicrobes, the only other comparable software tool, by an average of 35.75% in weighted classification accuracy. In addition, MT-MAG is able to completely classify an average of 67.7% of the sequences at the Species level, compared with DeepMicrobes which only classifies 47.45%. Moreover, MT-MAG provides additional information for sequences that are not completely classified at the Species level, resulting in the partial or complete classification of 95.15%, of the genomes in the datasets analyzed. In particular, MT-MAG classifies, on average, 88.84% of the test sequences to the Phylum level, 88.39% to the Class level, 86.81% to the Order level, 81.17% to the Family level, and 71.13% to the Genus level.

sentences: RPL3L-containing ribosomes modulate mitochondrial activity in the mammalian heart The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse  specialized ribosomes is still a controversial topic. Here, we explore the biological function of RPL3L, a ribosomal protein (RP) paralog of RPL3 that is exclusively expressed in muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon Rpl3l depletion, RPL3 becomes upregulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-Seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L neither modulated translational efficiency nor ribosome affinity towards a specific subset of transcripts. By contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of mitochondrial activity fine-tuning. Our results demonstrate that the existence of tissue-specific RP paralogs does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.

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TRPM2 deficiency protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages Atherosclerosis is the major cause of ischemic heart diseases and ischemic brain stroke, which are the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable TRPM2 plays a key role in the pathogenesis of atherosclerosis. Trpm2 deletion produces a potent protective effect against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD), as evidenced by reduced atherosclerotic plaque burden, decreased macrophage load and suppressed inflammasome activation in the vessel wall. Moreover, we show that Trpm2 deletion or inhibition reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, suppresses macrophage infiltration induced by monocyte chemoattractant protein-1 (MCP1), and prevents the impairment of macrophage emigration caused by oxLDL. Intriguingly, we uncover that activation of CD36, an oxLDL receptor, can promote the activation of TRPM2, and vice versa, the CD36-mediated inflammatory cascade in atherosclerosis is dependent on TRPM2. In transfected HEK293T cells, CD36 ligands oxLDL and TSP1 induce TRPM2 activation in a CD36-dependent manner. Deleting Trpm2 or inhibiting TRPM2 activity in cultured macrophages suppresses the CD36 signaling cascade induced by oxLDL and TSP1. Our studies establish TRPM2-CD36 axis as a new mechanism underlying atherogenesis, and suggest TRPM2 as an effective therapeutic target for atherosclerosis.

HIGHLIGHTSO_LITrpm2 deletion protects against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD)
C_LIO_LITrpm2 deficiency reduces atherosclerotic lesions by minimizing foam cell formation, inhibiting macrophage infiltration and preserving macrophage emigration
C_LIO_LITRPM2 activation is required for CD36-induced oxLDL uptake and subsequent inflammatory responses
C_LIO_LIThe ligands of CD36, oxLDL and TSP1, activate TRPM2, thereby perpetuating TRPM2-CD36 inflammatory cycle in atherogenesis cascade
C_LIO_LIOur data establish TRPM2-CD36 axis as a new atherogenesis mechanism and TRPM2 as a novel therapeutic target for atherosclerosis
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O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY TRPM2-mediated Ca2+ signal is essential for CD36 induced oxLDL uptake and atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD). The activation of CD36 and TRPM2 form a positive feedback loop in atherogenesis.

C_FIG_DISPLAY

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Automated Design of Robust Genetic Circuits: Structural Variants and Parameter Uncertainty Genetic design automation methods for combinational circuits often rely on standard algorithms from electronic design automation in their circuit synthesis and technology mapping. However, those algorithms are domain-specific and are hence often not directly suitable for the biological context. In this work we identify aspects of those algorithms that require domain-adaptation. We first demonstrate that enumerating structural variants for a given Boolean specification allows us to find better performing circuits and that stochastic gate assignment methods need to be properly adjusted in order to find the best assignment. Second, we present a general circuit scoring scheme that accounts for the limited accuracy of biological device models including the variability across cells and show that circuits selected according to this score exhibit higher robustness with respect to parametric variations. If gate characteristics in a library are just given in terms of intervals, we provide means to efficiently propagate signals through such a circuit and compute corresponding scores. We demonstrate the novel design approach using the Cello gate library and 33 logic functions that were synthesized and implemented in vivo recently (1). We show that an average 1.3-fold and a peak 6.5-fold performance increase can be achieved by simply considering structural variants and that an average 1.8-fold and a peak 30-fold gain in the novel robustness score can be obtained when selecting circuits according to it.

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sentences: Age-related differences in the neural network interactions underlying the predictability gain Speech comprehension is often challenged by increased background noise, but can be facilitated via the semantic context of a sentence. This predictability gain relies on an interplay of language-specific semantic and domain-general brain regions. However, age-related differences in the interactions within and between semantic and domain-general networks remain poorly understood. Using functional neuroimaging, we investigated commonalities and differences in network interactions enabling processing of degraded speech in healthy young and old participants. Participants performed a sentence repetition task while listening to sentences with high and low predictable endings and varying intelligibility. Stimulus intelligibility was adjusted to individual hearing abilities. Older adults showed an undiminished behavioural predictability gain. Likewise, both groups recruited a similar set of semantic and cingulo-opercular brain regions. However, we observed age-related differences in effective connectivity for high predictable speech of increasing intelligibility. Young adults exhibited stronger connectivity between regions of the cingulo-opercular network and between left insula and the posterior middle temporal gyrus. Moreover, these interactions were excitatory in young adults but inhibitory in old adults. Finally, the degree of the inhibitory influence between cingulo-opercular regions was predictive of the behavioural sensitivity towards changes in intelligibility for high predictable sentences in older adults only. Our results demonstrate that the predictability gain is relatively preserved in older adults when stimulus intelligibility is individually adjusted. While young and old participants recruit similar brain regions, differences manifest in underlying network interactions. Together, these results suggest that ageing affects the network configuration rather than regional activity during successful speech comprehension under challenging listening conditions.

sentences: Vitamin D modulation of mitochondrial oxidative metabolism and mTOR enforces stress tolerance and anti-cancer responses The relationship between vitamin D and reactive oxygen species (ROS), two integral signaling and damaging molecules of the cell, is poorly understood. This is striking, given that both factors are involved in cancer cell regulation and metabolism. Mitochondria (mt) dysfunction is one of the main drivers of cancer, producing higher cellular energy and ROS that can enhance oxidative stress and stress tolerance responses. To study the effects of vitamin D on metabolic and mt dysfunction, we used the vitamin D receptor (VDR)-sensitive MG-63 osteosarcoma cell model. Using biochemical approaches, active vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D3) decreased mt ROS levels, membrane potential ({Delta}{Psi}mt), biogenesis, and translation, while enforcing endoplasmic reticulum/mitohormetic stress tolerance responses. Using a mitochondria-focused transcriptomic approach, gene set enrichment and pathway analyses show that 1,25(OH)2D3 lowered mt fusion/fission and oxidative phosphorylation (OXPHOS). By contrast, mitophagy, ROS defense, and epigenetic gene regulation were enhanced after 1,25(OH)2D3 treatment, as well as key metabolic enzymes that regulate fluxes of substrates for cellular architecture and a shift toward non-oxidative energy metabolism. ATACseq revealed putative oxi-sensitive and tumor-suppressing transcription factors that may regulate important mt functional genes such as the mTORC1 inhibitor, DDIT4/REDD1. DDIT4/REDD1 was predominantly localized to the outer mt membrane in untreated MG-63 cells yet sequestered in the cytoplasm after 1,25(OH)2D3 and rotenone treatments, suggesting a level of control by membrane depolarization to facilitate its cytoplasmic mTORC1 inhibitory function. The results show that vitamin D activates distinct adaptive metabolic responses involving mitochondria to regain redox balance and control the growth of cancer cells.

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Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B ({sigma}B), whose role in protection against acid stress is well established. However, the mechanisms leading to its activation by low pH are unknown. Here, we investigated the involvement of the stressosome, a sensory organelle, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated {sigma}B and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was present but its remaining paralogues were genetically inactivated retained the ability to induce {sigma}B activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1 T175A, T209A, T241A) and RsbS (rsbS S56A), or in the active site of the stressosome kinase RsbT (rsbT N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1 T175A variant, retained a near-wild type phenotype. The rsbR1 T209A and rsbR1 T241A mutants displayed constitutive {sigma}B activity. Mild acid shock upregulates invasion genes and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of {sigma}B in L. monocytogenes. Furthermore, RsbR1 can function independently of its paralogues and that signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.

Author summaryThe stress sensing organelle known as the stressosome, found in many bacterial and archaeal lineages, plays a crucial role in both stress tolerance and virulence in the food-borne pathogen Listeria monocytogenes. However, the mechanisms that lead to its activation and the subsequent activation of the general stress response have remained elusive. In this study, we examined the signal transduction mechanisms that operate in the stressosome in response to acid stress. We found that only one of the five putative sensory proteins present in L. monocytogenes, RsbR1, was required for effective transduction of acid tress signals. We further found that phosphorylation of RsbS and RsbR1, mediated by the RsbT kinase, is essential for signal transduction. Failure to phosphorylate RsbS on Serine 56 completely abolished acid sensing by the stressosome, which prevented the development of adaptive acid tolerance. The acid-induced activation of internalin gene expression was also abolished in mutants with defective stressosome signalling, suggesting a role for the stressosome in the invasion of host cells. Together the data provide new insights into the mechanisms that activate the stressosome in response to acid stress and highlight the role this sensory organelle plays in virulence.

sentences: Improve consensus partitioning via a hierarchical procedure Consensus partitioning is an unsupervised method widely used in high throughput data analysis for revealing subgroups and assigns stability for the classification. However, standard consensus partitioning procedures are weak to identify large numbers of stable subgroups. There are two main issues. 1. Subgroups with small differences are difficult to separate if they are simultaneously detected with subgroups with large differences. And 2. stability of classification generally decreases as the number of subgroups increases. In this work, we proposed a new strategy to solve these two issues by applying consensus partitionings in a hierarchical procedure. We demonstrated hierarchical consensus partitioning can be efficient to reveal more subgroups. We also tested the performance of hierarchical consensus partitioning on revealing a great number of subgroups with a DNA methylation dataset. The hierarchical consensus partitioning is implemented in the R package cola with comprehensive functionality for analysis and visualizations. It can also automate the analysis only with a minimum of two lines of code, which generates a detailed HTML report containing the complete analysis.

sentences: DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortiums amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

sentences: Low affinity integrin states have faster binding kinetics than the high affinity state Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins 4{beta}1 and 5{beta}1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rate measurements are similar for integrins on cell surfaces and ectodomain fragments. Although the extended-open conformations on-rate is [~]20-fold slower, its off-rate is [~]25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. These kinetic measurements, together with previous equilibrium measurements of integrin conformational state affinity and relative free energy on intact cells, are key to a definitive understanding of the mechanism of integrin activation.

sentences: Combined protein and transcript single cell RNA sequencing in human peripheral blood mononuclear cells Cryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single cell (sc)RNA sequencing in PBMCs. Among 31 participants in the WIHS Study, we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs and Louvain clustering, we identified 58 subsets among CD4 T, CD8 T, B, NK cells and monocytes. This resolution was superior to flow cytometry, mass cytometry or scRNA-sequencing without antibodies. Since the transcriptome was not needed for cell identification, combined protein and transcript scRNA-Seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportion. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease. In conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.

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Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex Antidepressants such as serotonin uptake inhibitors are the first-line pharmacological treatment for chronic stress-related psychiatric disorders. However, their late-onset therapeutic action and frequent side effects, however, are important challenges for clinicians and patients. Besides, around 30% of major depression patients are considered treatment-resistant. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid with a wide range of psychopharmacological effects, but its mechanism of action remains unclear. Here, we found that in male mice submitted to two different repeated stress protocols (chronic unpredictable and social defeat stress), low doses of CBD (7.5mg/Kg) caused an early-onset behavioral effect when combined to the antidepressant escitalopram (ESC-10mg/Kg). The behavioral effects of the ESC+CBD combination depended on the expression/activity of the N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD, responsible for synthesizing the endocannabinoid anandamide), but not the DAGL, enzyme in the ventromedial prefrontal cortex. In addition, we described a case series with three treatment-resistant depression that were successfully treated with CBD as adjuvant therapy, as evaluated by standardized clinical rating scales. After 12 weeks of treatment, two patients were considered depression remitted (MADRS score lower than 10) while one patient successfully responded to CBD as add-on treatment (more than 50% decrease from the baseline MADRS). Our results suggest that CBD might be useful as an add-on therapy for optimizing the action of antidepressants. They also suggest that CBDs beneficial actions depends on the facilitation of N-acylethanolamines actions in the medial prefrontal cortex.

HighlightsO_LIIn mice, cannabidiol (CBD), but not escitalopram, induced a fast-onset anti-stress action.
C_LIO_LICombinations of sub-effective doses of CBD and escitalopram produce anti-stress effects after only 7 days.
C_LIO_LIThe Escitalopram + CBD treatment modulated synaptic protein markers in the medial prefrontal cortex.
C_LIO_LICRISPR-Cas9-mediated knockdown of NAPE-PLD in the medial PFC prevents the anti-stress effect of the Escitalopram + CBD.
C_LIO_LIAdding CBD to an antidepressants regimen successfully treated three patients with treatment resistant depression.
C_LI

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sentences: 3D Convolutional Neural Networks for Classification of Alzheimer's and Parkinson's Disease with T1-Weighted Brain MRI Parkinsons disease (PD) and Alzheimers disease (AD) are progressive neurodegenerative disorders that affect millions of people worldwide. In this work, we propose a deep learning approach to classify these diseases based on 3D T1-weighted brain MRI. We analyzed several datasets including the Parkinsons Progression Markers Initiative (PPMI), an independent dataset from the University of Pennsylvania School of Medicine (UPenn), the Alzheimers Disease Neuroimaging Initiative (ADNI), and the Open Access Series of Imaging Studies (OASIS) dataset. The UPenn and OASIS datasets were used as independent test sets to evaluate the model performance during inference. We also implemented a random forest classifier as a baseline model by extracting key radiomics features from the same T1-weighted MRI scans. The proposed 3D convolutional neural network (CNN) model was trained from scratch for the classification tasks. For AD classification, the 3D CNN model achieved an ROC-AUC of 0.878 on the ADNI test set and an average ROC-AUC of 0.789 on the OASIS dataset. For PD classification, the proposed 3D CNN model achieved an ROC-AUC of 0.667 on the PPMI test set and an average ROC-AUC of 0.743 on the UPenn dataset. Model performance was largely maintained when using only 25% of the training dataset. The 3D CNN outperformed the random forest classifier for both the PD and AD tasks. The 3D CNN also generalized better on unseen MRI data from different imaging centers. These approaches show promise for screening of PD and AD patients using only T1-weighted brain MRI, which is relatively widely available. This model with additional validation could also be used to help differentiate between challenging cases of AD and PD when they present with similarly subtle motor and non-motor symptoms.

sentences: Enviromic assembly increases accuracy and reduces costs of the genomic prediction for yield plasticity Quantitative genetics states that phenotypic variation is a consequence of genetic and environmental factors and their subsequent interaction. Here, we present an enviromic assembly approach, which includes the use of ecophysiology knowledge in shaping environmental relatedness into whole-genome predictions (GP) for plant breeding (referred to as E-GP). We propose that the quality of an environment is defined by the core of environmental typologies (envirotype) and their frequencies, which describe different zones of plant adaptation. From that, we derive markers of environmental similarity cost-effectively. Combined with the traditional genomic sources (e.g., additive and dominance effects), this approach may better represent the putative phenotypic variation across diverse growing conditions (i.e., phenotypic plasticity). Additionally, we couple a genetic algorithm scheme to design optimized multi-environment field trials (MET), combining enviromic assembly and genomic kinships to provide in-silico realizations of the future genotype-environment combinations that must be phenotyped in the field. As a proof-of-concept, we highlight E-GP applications: (1) managing the lack of phenotypic information in training accurate GP models across diverse environments and (2) guiding an early screening for yield plasticity using optimized phenotyping efforts. Our approach was tested using two non-conventional cross-validation schemes to better visualize the benefits of enviromic assembly in sparse experimental networks. Results on tropical maize show that E-GP outperforms benchmark GP in all scenarios and cases tested. We show that for training accurate GP models, the genotype-environment combinations representativeness is more critical than the MET size. Furthermore, we discuss theoretical backgrounds underlying how the intrinsic envirotype-phenotype covariances within the phenotypic records of (MET) can impact the accuracy of GP and limits the potentialities of predictive breeding approaches. The E-GP is an efficient approach to better use environmental databases to deliver climate-smart solutions, reduce field costs, and anticipate future scenarios.

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Phylogenomic data reveal hard polytomies across the backbone of the large genus Solanum (Solanaceae) Premise of the studyEvolutionary studies require solid phylogenetic frameworks, but increased volumes of phylogenomic data have revealed incongruent topologies among gene trees in many organisms both between and within genomes. Some of these incongruences indicate polytomies that may remain impossible to resolve. Here we investigate the degree of gene-tree discordance in Solanum, one of the largest flowering plant genera that includes the cultivated potato, tomato, and eggplant, as well as 24 minor crop plants.

MethodsA densely sampled species-level phylogeny of Solanum is built using unpublished and publicly available Sanger sequences comprising 60% of all accepted species (742 spp.) and nine regions (ITS, waxy, and seven plastid markers). The robustness of this topology is tested by examining a full plastome dataset with 140 species and a nuclear target-capture dataset with 39 species of Solanum (Angiosperms353 probe set).

Key resultsWhile the taxonomic framework of Solanum remained stable, gene tree conflicts and discordance between phylogenetic trees generated from the target-capture and plastome datasets were observed. The latter correspond to regions with short internodal branches, and network analysis and polytomy tests suggest the backbone is composed of three polytomies found at different evolutionary depths. The strongest area of discordance, near the crown node of Solanum, could potentially represent a hard polytomy.

ConclusionsWe argue that incomplete lineage sorting due to rapid diversification is the most likely cause for these polytomies, and that embracing the uncertainty that underlies them is crucial to understand the evolution of large and rapidly radiating lineages.

sentences: Microbes are potential key players in the evolution of life histories and aging in Caenorhabditis elegans Microbes can have profound effects on host fitness and health and the appearance of late-onset diseases. Host-microbe interactions thus represent a major environmental context for healthy aging of the host and might also mediate trade-offs between life-history traits in the evolution of host senescence. Here we have used the nematode Caenorhadbitis elegans to examine whether host-microbe interactions might modulate the evolution of host life-history and aging. We first characterized the effects of two non-pathogenic Escherichia coli strains, a pathogenic E. coli strain and a pathogenic Serratia marcescens strain on the reproductive schedule and survival of an outbred C. elegans population, to be used in an experimental evolution study. Secondly, to investigate the dependency of these effects on host genotype, we assayed population growth rates and survival of five representative C. elegans inbred strains in response to these microbes. Our results show that host-microbe interactions have a substantial, host-genotype-dependent impact on reproductive effort and survival of the nematode host. Although pathogenic bacteria reduced host survival, as expected, they did not necessarily decrease host fertility or population growth rate. Given such microbe-specific genotypic differences in host life history, we predict that the evolution of reproductive schedules and senescence in this system might be critically contingent upon host-microbe interactions, a hypothesis which we will be testing using experimental evolution in future work.

sentences: Identification of transcription factors involved in the specification of photoreceptor subtypes During development, retinal progenitors navigate a complex landscape of fate decisions that culminates with an array of unique cell types that are required for proper vision. Here, we aim to identify factors that are required for fate decisions in photoreceptors. These factors help create a diversity of photoreceptor subtypes that sustain vision in day and night, enable the detection of colors, of prey and predators, and other aspects of vision. To identify these factors, we generate a high-quality and deep transcriptomic profile of each photoreceptor subtype in zebrafish. From these profiles, we focus on transcription factors--key players in cell-fate decisions. We apply CRISPR-FO screening as a versatile platform to explore the involvement of transcription factors in photoreceptor subtype-specification. We find that three differentially-expressed transcription factors (Foxq2, Tbx2a and Tbx2b) play unique roles in controling the identity of photoreceptor subtypes within the retina. Our results provide novel insights into the function of these factors and how photoreceptors acquire their final identities. Furthermore, we have made our transcriptomic dataset openly available and easy to explore. This dataset and the screening method will be valuable to the scientific community and will enable the exploration of genes involved in many essential aspects of photoreceptor biology.

sentences: The cnf1 gene is associated to an expanding Escherichia coli ST131 H30Rx/C2 sublineage and confers a competitive advantage for host colonization Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract acting as a source of urinary tract pathogens. We performed a high-throughput genetic screening of predominantly clinical E. coli isolates from wide geographical origins. This revealed a preferential distribution of the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, in four sequence types encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment and has known enhanced capacities to colonize the gastrointestinal tract (GIT). Statistical modeling uncovered a dominant global expansion of cnf1-positive strains within multidrug-resistant ST131 subclade H30Rx/C2. Despite the absence of phylogeographical signals, cnf1-positive isolates adopted a clonal distribution into clusters on the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. Functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2, established that a cnf1-deleted EC131GY is outcompeted by the wildtype strain in a mouse model of competitive infection of the bladder while both strains behave similarly during monoinfections. This points for positive selection of cnf1 during UTI rather than urovirulence. Wildtype EC131GY also outcompeted the mutant when concurrently inoculated into the gastrointestinal tract, arguing for selection within the gut. Whatever the site of selection, these findings support that the benefit of cnf1 enhancing host colonization by ST131-H30Rx/C2 in turn drives a worldwide dissemination of the cnf1 gene together with extended spectrum of antibiotic resistance genes.

sentences: Cell-Type Specific Profiling of Histone Post-Translational Modifications in the Adult Mouse Striatum Histone post-translational modifications (hPTMs) regulate gene expression via changes in chromatin accessibility and transcription factor recruitment. At a given locus, the coordinated enrichment of several distinct hPTMs regulate gene expression in response to external stimuli. However, neuronal hPTMs have been primarily characterized in bulk brain tissue and/or tissue pooled across subjects. This obscures both cell-type and individual variability, features essential to understand individual susceptibility to psychiatric disease. To address this limitation, we optimized a hybrid protocol, ICuRuS, to profile both activating and repressive hPTMs in neuronal subtypes from a single mouse. We report here profiling of striatal medium spiny neuron (MSN) subtypes, genetically defined by expression of Adenosine 2a Receptor (A2a) or Dopamine Receptor D1 (D1), which differentially regulate reward processing and pathophysiology. Using ICuRuS, we defined genome-wide, A2a- or D1-specific combinatorial hPTM profiles, and discovered regulatory epigenomic features at genes implicated in neurobiological function and disease.

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Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (BK2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, BK2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Funding InformationThe research was supported by US National Science Foundation Plant Genome Research Program grants (IOS-1025976 and IOS-1444448) to DMB.

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DNA Replication-transcription conflicts do not significantly contribute to spontaneous mutations due to replication errors in Escherichia coli Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch-repair (MMR) defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base-pair substitutions or small indels (insertion and deletions [≤] 4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hotspots for indels. No additional orientation bias in mutation frequencies occurred when MMR- strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress-response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus co-directional to replication. In addition, the data revealed a strong correlation of the frequency of base-pair substitutions with gene length, but no correlation with gene expression levels.

IMPORTANCEBecause DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. But, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair defective Escherichia coli strains, we find that across the genome there are no significant differences in mutation frequencies between genes oriented co-directionally versus head-on to replication. Among a subset of highly expressed genes there was a 2- to 3-fold bias for small insertions and deletions in head-on oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hotspots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and co-directionally to replication.

sentences: Evolutionary history of sexual differentiation mechanism in insects Gain of alternative splicing gives rise to functional diversity in proteins and underlies the complexity and diversity of biological aspects. However, it is still not fully understood how alternatively spliced genes develop the functional novelty. To this end, we infer the evolutionary history of the doublesex gene, the key transcriptional factor in the sexual differentiation of arthropods. doublesex is controlled by sex-specific splicing and promotes both male and female differentiation in some holometabolan insects. In contrast, doublesex promotes only male differentiation in some hemimetabolan insects. Here, we investigate ancestral states of doublesex using Thermobia domestica belonging to Zygentoma, the sister group of winged insects. We find that doublesex of T. domestica expresses sex-specific isoforms but is only necessary for male differentiation of sexual morphology. This result ensures the hypothesis that doublesex was initially only used to promote male differentiation during insect evolution. However, T. domestica doublesex has a short female-specific region and upregulates the expression of vitellogenin homologs in females, suggesting that doublesex may have already controlled some aspects of feminization in the common ancestor of winged insects. Reconstruction of the ancestral sequence and prediction of the protein structure show that the female-specific isoform of doublesex has a long C-terminal disordered region in holometabolan insects, but not in non-holometabolan species. We propose that doublesex acquired a female-specific isoform and then underwent a change in the protein motif structure, which became essential for female differentiation in sexual dimorphisms.

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A reinforcement learning algorithm shapes maternal care in mice The neural substrates for processing classical rewards such as food or drugs of abuse are well-understood. In contrast, the mechanisms by which organisms perceive social contact as rewarding and subsequently modify their interactions are unclear. Here we tracked the gradual emergence of a repetitive and highly-stereotyped parental behavior and show that trial-by-trial performance correlates with the history of midbrain dopamine (DA) neuron activity. We used a novel behavior paradigm to manipulate the subjects expectation of imminent pup contact and show that DA signals conform to reward prediction error, a fundamental component of reinforcement learning (RL). Finally, closed-loop optogenetic inactivation of DA neurons at the onset of pup contact dramatically slowed emergence of parental care. We conclude that this prosocial behavior is shaped by an RL mechanism in which social contact itself is the primary reward.

One-Sentence SummaryMaternal interactions with offspring are shaped by a dopaminergic reinforcement learning mechanism.

sentences: CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

sentences: AhNGE : A Database for Arachis hypogaea Nodule Developmental Gene Expression Symbiotic nitrogen fixation (SNF) inside root-nodules is a primary and sustainable source of soil nitrogen. Understanding nodule development and metabolism in crop legumes may lead to more effective SNF in agriculture. Peanut (Arachis hypogaea) is an economically important allotetraploid legume with non-canonical nodule developmental features. Recent genome sequencing of peanut has opened the possibility of making peanut a model for studying atypical nodule development. To help the community of nodule biologists, we have developed a database called AhNGE (Arachis hypogaea Nodule Developmental Gene Expression: http://nipgr.ac.in/AhNGE/index.php). AhNGE contains RNAseq data from six data points of nodule development in A. hypogagea cv. Tifrunner. This data represents a dynamic view of gene expression during peanut nodule development. Research in model legumes has generated a huge knowledgebase in the last twenty years. To streamline comparative genomics among legumes, we performed ortholog analysis among four legumes (Cicer, Glycine, Lotus, and Medicago) and one non-legume (Arabidopsis). This will facilitate the integration of existing knowledge in nodule development with the Arachis transcriptome. The available data can be retrieved using a single or batch query or searching using gene ID, from above mentioned five species. The output displays the gene expression pattern in graphical as well as tabular form, along with further options to download the sequence data. The database is linked with PeanutBase, the main genomic resource of peanut. Additionally, the expression level of different splicing variants can be retrieved from the database. In summary, AhNGE serves as an important resource for the scientific community working on nodule development.

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Systematic monitoring of 2-Cys peroxiredoxin-derived redox signals unveiled its role in attenuating carbon assimilation rate Transmission of reductive cues from the photosynthetic electron transport chain to redox-regulated proteins plays a crucial role in activating chloroplast metabolism. However, deciphering the role of their counterbalanced oxidative signals is challenging due to monitoring difficulties. Here, we demonstrate the light-depended redox modification of chloroplast-targeted 2-Cys peroxiredoxins and introduce peroxiredoxin-based biosensors to monitor photosynthetically-derived oxidative signals. By employing a set of genetically encoded biosensors, we show the induction of oxidative signals under habitual light intensities and their inverse relationship with NADPH levels, unraveling the combined activity of reducing and oxidizing signals in fine-tuning chloroplast metabolism. A faster increase in carbon assimilation rates during photosynthesis induction phase was measured in plants deficient in 2-Cys peroxiredoxins compared to wild-type, suggesting the involvement of oxidative signals in attenuating photosynthesis under variable light environments. We suggest that oxidative signals measured by peroxiredoxin-based biosensors reflect the limitation to photosynthesis imposed by the redox regulatory system.

One-Sentence SummaryA genetically encoded biosensor unmasked the dominant role of photosynthetically-derived oxidative signals under habitual conditions.

sentences: Neuronal hyperactivity in a LRRK2-G2019S cellular model of Parkinson's Disease Monogenic Parkinsons Disease can be caused by a mutation in the leucine-rich repeat kinase 2 (LRRK2) gene, causing a late-onset autosomal dominant inherited form of Parkinsons Disease. The function of the LRRK2 gene is incompletely understood, but several in vitro studies have reported that LRRK2-G2019S mutations affect neurite branching, calcium homeostasis and mitochondrial function, but thus far, there have been no reports of effects on electrophysiological activity. We assessed the neuronal activity of induced pluripotent stem cell derived neurons from Parkinsons Disease patients with LRRK2-G2019S mutations and isogenic controls. Neuronal activity of spontaneously firing neuronal populations was recorded with a fluorescent calcium-sensitive dye (Fluo-4) and analysed with a novel image analysis pipeline that combined semi-automated neuronal segmentation and quantification of calcium transient properties. Compared with controls, LRRK2-G2019S mutants have shortened inter-spike intervals and an increased rate of spontaneous calcium transient induction.

sentences: Global ecological and biogeochemical impacts of pelagic tunicates The pelagic tunicates, gelatinous zooplankton that include salps, doliolids, and appendicularians, are filter feeding grazers thought to produce a significant amount of particulate organic carbon (POC) detritus. However, traditional sampling methods (i.e., nets), have historically underestimated their abundance, yielding an overall underappreciation of their global biomass and contribution to ocean biogeochemical cycles relative to crustacean zooplankton. As climate change is projected to decrease the average plankton size and POC export from traditional plankton food webs, the ecological and biogeochemical role of pelagic tunicates may increase; yet, pelagic tunicates were not resolved in the previous generation of global earth system climate projections. Here we present a global ocean study using a coupled physical-biogeochemical model to assess the impact of pelagic tunicates in the pelagic food web and biogeochemical cycling. We added two tunicate groups, a large salp/doliolid and a small appendicularian to the NOAA-GFDL Carbon, Ocean Biogeochemistry, and Lower Trophics version 2 (COBALTv2) model, which was originally formulated to represent carbon flows to crustacean zooplankton. The new GZ-COBALT simulation was able to simultaneously satisfy new pelagic tunicate biomass constraints and existing ecosystem constraints, including crustacean zooplankton observations. The model simulated a global tunicate biomass of 0.10 Pg C, annual production of 0.49 Pg C y-1 in the top 100 m, and export flux of 0.7 Pg C y-1, representing 11% of the total export flux past 100 m. Overall export from the euphotic zone remained largely constant, with the GZ-COBALT pe-ratio only increasing 5.3% (from 0.112 to 0.118) compared to the COBALTv2 control. While the bulk of the tunicate-mediated export production resulted from the rerouting of phytoplankton- and mesozooplankton-mediated export, tunicates also shifted the overall balance of the upper oceans away from recycling and towards export. Our results suggest that pelagic tunicates play important trophic roles in both directly competing with microzooplankton and indirectly shunting carbon export away from the microbial loop.

sentences: PACpAInt: a deep learning approach to identify molecular subtypes of pancreatic adenocarcinoma on histology slides Pancreatic ductal adenocarcinoma (PAC) is a highly heterogeneous and plastic tumor with different transcriptomic molecular subtypes that hold great prognostic and theranostic values. We developed PACpAInt, a multistep approach using deep learning models to determine tumor cell type and their molecular phenotype on routine histological preparation at a resolution enabling to decipher complete intratumor heterogeneity on a massive scale never achieved before. PACpAInt effectively identified molecular subtypes at the slide level in three validation cohorts and had an independent prognostic value. It identified an interslide heterogeneity within a case in 39% of tumors that impacted survival. Diving at the cell level, PACpAInt identified "pure" classical and basal-like main subtypes as well as an intermediary phenotype and hybrid tumors that co-carried both classical and basal-like phenotypes. These novel artificial intelligence-based subtypes, together with the proportion of basal-like cells within a tumor had a strong prognostic impact.

sentences: Genomic sequencing confirms absence of introgression despite past hybridisation between a common and a critically endangered bird Genetic swamping resulting from interspecific hybridisation can increase extinction risk for threatened species. The development of high-throughput and reduced-representation genomic sequencing and analyses to generate large numbers of high resolution genomic markers has the potential to reveal introgression previously undetected using small numbers of genetic markers. However, few studies to date have implemented genomic tools to assess the extent of interspecific hybridisation in threatened species. Here we investigate the utility of genome-wide single nucleotide polymorphisms (SNPs) to detect introgression resulting from past interspecific hybridisation in one of the worlds rarest birds. Anthropogenic impacts have resulted in hybridisation and subsequent backcrossing of the critically endangered Aotearoa New Zealand endemic kak[i] (black stilts; Himantopus novaezelandiae) with the non-threatened self-introduced congeneric poaka (Aotearoa New Zealand population of pied stilts, Himantopus himantopus leucocephalus), yet genetic analyses with a limited set of microsatellite markers revealed no evidence of introgression of poaka genetic material in kak[i], excluding one individual. We use genomic data for [~]63% of the wild adult kak[i] population to reassess the extent of introgression resulting from hybridisation between kak[i] and poaka. Consistent with previous genetic analyses, we detected no introgression from poaka into kak[i]. These collective results indicate that, for kak[i], existing microsatellite markers provide a robust, cost-effective approach to detect cryptic hybrids. Further, for well-differentiated species, the use of genomic markers may not be required to detect admixed individuals.

sentences: The effect of a canine semen activator supplementation or addiction on the long-term refrigeration quality of dog spermatozoa Within modern biotechnology, different tools have been developed to maximize canine semen conservation protocol to optimizing reproductive results and making their handling more flexible. In the last decades, the survival of refrigerated semen has been prolonged from 2-3d with the first basic diluents, to 10-14d using the most modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Semen activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of SA was recorded by daily evaluation of chilled semen 14d. For this experiment, six adult healthy Neapolitan Mastiff dogs, were used as donors and the semen was manually collected. Spermatozoa-rich fractions of each suject was chilled using a new generation extender for long periods of time (d0) starting from the d1 to d14, different aliquot, with (experimental trial) and without SA (control trial), were evaluated daily for motility vigor, morphology and membrane integrity. The initial sperm concentration of extended semen was 417.3{+/-}170.4x106/mL (mean {+/-} SEM) with 85.89{+/-}4.76% of MNS (morphologically normal sperm), 84.47{+/-}5.22 % vital sperm and a pH of 6.2{+/-}2.8. The initial vigor was 3.83{+/-}0.48, but after one min with SA, it rose to 4.45 {+/-} 0.45 (P<0.001). The semen motility parameter increase significantly (P<0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The vigor analysis significantly increase in experimental trial (P<0.05) during the most day of the study with the exclusion of d3 and d14. For evaluate the semen characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10) and T3 (d11-d14) (P<0.001) in evaluation of morphology and membrane stability. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

sentences: Remote targeted electrical stimulation The ability to generate electric fields in deep tissues remotely, without surgically implanting electrodes, could transform diagnoses and treatments of nervous system disorders. Here, we show that focal electrostimulation effects can be elicited remotely by combining two noninvasive forms of energies--magnetic and focused ultrasonic fields. The approach, based in the Lorentz equation and referred to as Lstim, electrically stimulates specified tissue targets with the precision of deep brain or spinal cord stimulation, but does not require electrode implantation. Lstim potentiated the responses of human nerves, enhancing the neuromodulatory effects of ultrasound by 74% on average. The effects showed a double dissociation--a significant and substantial increase in nociceptive responses, yet a significant reduction in tactile responses. In line with the Lorentz equation, Lstim was only observed when nerves were oriented perpendicularly to the magnetic and ultrasonic fields. A sham condition showed no effects. Both the ultrasonic and the induced electric fields were well below the respective safety indices, and no detrimental effects were detected. Lstim uniquely integrates noninvasiveness, sharp focus, and the efficacy of electrical stimulation. The approach has the potential to provide a noninvasive tool to dissect brain function in humans and to diagnose the neural circuits involved in nervous system disorders. Moreover, this effect should be taken into account when ultrasound is applied inside MRI.

sentences: SemiBin: Incorporating information from reference genomes with semi-supervised deep learning leads to better metagenomic assembled genomes (MAGs) Metagenomic binning is the step in building metagenome-assembled genomes (MAGs) when sequences predicted to originate from the same genome are automatically grouped together. The most widely-used methods for binning are reference-independent, operating de novo and allow the recovery of genomes from previously unsampled clades. However, they do not leverage the knowledge in existing databases. Here, we propose SemiBin, an open source tool that uses neural networks to implement a semi-supervised approach, i.e. SemiBin exploits the information in reference genomes, while retaining the capability of binning genomes that are outside the reference dataset. SemiBin outperforms existing state-of-the-art binning methods in simulated and real microbiome datasets across three different environments (human gut, dog gut, and marine microbiomes). SemiBin returns more high-quality bins with larger taxonomic diversity, including more distinct genera and species. SemiBin is available as open source software at https://github.com/BigDataBiology/SemiBin/.

sentences: Engineering of Sr33 and Sr50 plant immune receptors to alter recognition specificity and autoactivity Plants possess cytoplasmic immune receptors called nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize the presence of a pathogen through a range of mechanisms: direct binding of effectors or indirect recognition of effector actions. The direct binding of effectors has been shown to be mediated through the NLRs leucine-rich repeat (LRR) domain. Accurate prediction of amino acids involved in these direct interactions can greatly enhance understanding of effector recognition and inform efforts to engineer new resistance. In this study, we utilized two homologous NLR resistance genes from wheat, Sr33 and Sr50, that recognize distinct effectors by directly binding to them through their LRR domain. While the effector recognized by Sr50 is known and described as AvrSr50, the effector recognized by Sr33 remains unknown. Through a combination of phylogenetics, allele diversity analysis in the LRR and structural modeling, we identified the amino acids in Sr50 likely to physically interact with its effector. Mutation of these sites helped identify 12 amino acids we hypothesized to be sufficient to mediate effector binding in Sr50. Changing these 12 corresponding amino acids in Sr33, we showed AvrSr50-dependent initiation of cell death in wheat protoplasts and Nicotiana benthamiana. Furthermore, we were able to pinpoint and change amino acid residues that govern autoactivity of Sr50 in the wheat protoplast cell death assay. These findings are a major advance towards the successful engineering of new effector recognition specificities in direct binder NLRs.

sentences: The Imaging and Molecular Annotation of Xenografts and Tumours (IMAXT) High Throughput Data and Analysis Infrastructure With the aim of producing a 3D representation of tumours, IMAXT uses a large variety of modalities in order to acquire tumour samples and produce a map of every cell in the tumour and its host environment. With the large volume and variety of data produced in the project we develop automatic data workflows and analysis pipelines and introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located instead of bringing data to their local computers. Here we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumour models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT.

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Systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition Epstein-Barr virus (EBV), a member of the large herpesvirus family is a very complex human{gamma} -herpesvirus. Its complexity with respect to viral gene regulation, its preferred latent life style and technical difficulties are major obstacles, which hinder efficient virus generation in vitro to mass-produce or establish recombinant wild-type EBV or mutant derivatives. To explore conditions optimizing and improving virus production, we established and tested an EBV gene library with 77 expression plasmids and a set of designed shRNAs. With this tool set we investigated the contributions of individual viral genes in the context of virus synthesis and virion functions. Engineered virus stocks were systematically analyzed with respect to physical and bioparticle concentration, virus titer and virus uptake by primary human B cells, EBVs target cells in vivo. To quantitate virus uptake by these cells, we developed a novel {beta}-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells. Based on our findings, EBV does not encode a dominant regulator that governs virus production, but our results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a transcriptional regulator of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality, and virus uptake by primary human B cells.

ImportanceFor more than 20 years HEK293 cells have been instrumental to produce virus stocks of recombinant EBV. The identification of this cell line as a source of infectious EB virions was unexpected because HEK293 cells are very distant from B cells and in particular plasma cells which produce EBV progeny in vivo. To our knowledge, no systematic analysis has addressed the fundamental question whether virus yield with respect to virus concentration, virion composition and functionality can be improved to produce recombinant EBV stocks in HEK293 cells. We tackled this question and analyzed 77 individual EBV genes for their possible contribution to virus yield. To analyze and compare different virus stocks we used established and developed novel assays to characterize important parameters of EB virions and their functions.

sentences: Interplay between bacterial clone and plasmid in the spread of antibiotic resistance genes in the gut: lessons from a temporal study in veal calves Intestinal carriage of extended spectrum {beta}-lactamase (ESBL)-producing Escherichia coli is a frequent, increasing and worrying phenomenon, but little is known about the molecular scenario and the evolutionary forces at play. We screened 45 veal calves, known to have high prevalence of carriage, for ESBL-producing E. coli on 514 rectal swabs (one randomly selected colony per sample) collected over six months. We characterized the bacterial clones and plasmids carrying blaESBL genes with a combination of genotyping methods, whole genome sequencing and conjugation assays. One hundred and seventy-three ESBL-producing E. coli isolates [blaCTX-M-1 (64.7%), blaCTX-M-14 (33.5%) or blaCTX-M-15 (1.8%)] were detected, belonging to 32 bacterial clones, mostly of phylogroup A. Calves were colonized successively by different clones with a trend in decreasing carriage. The persistence of a clone in a farm was significantly associated with the number of calves colonized. Despite a high diversity of E. coli clones and blaCTX-M-carrying plasmids, few blaCTX-M gene/plasmid/chromosomal background combinations dominated, due to (i) efficient colonization of bacterial clones and/or (ii) successful plasmid spread in various bacterial clones. The scenario  clone vs. plasmid spread depended on the farm. Thus, epistatic interactions between resistance genes, plasmids and bacterial clones contribute to optimize fitness in specific environments.

sentences: The zinc-finger transcription factor LSL-1 is a major regulator of the germline transcriptional program in C. elegans Specific gene transcriptional programs are required to ensure proper proliferation and differentiation processes underlying the production of specialized cells during development. Gene activity is mainly regulated by the concerted action of transcription factors and chromatin proteins. In the nematode C. elegans, mechanisms that silence improper transcriptional programs in germline and somatic cells have been well studied, however, how are tissue specific sets of genes turned on is less known. LSL-1 is herein defined as a novel crucial transcriptional regulator of germline genes in C. elegans. LSL-1 is first detected in the P4 blastomere and remains present at all stages of germline development, from primordial germ cell proliferation to the end of meiotic prophase. lsl-1 loss-of-function mutants exhibit many defects including meiotic prophase progression delay, a high level of germline apoptosis, and production of almost no functional gametes. Transcriptomic analysis and ChIP-seq data show that LSL-1 binds to promoters and acts as a transcriptional activator of germline genes involved in various processes, including homologous chromosome pairing, recombination, and genome stability. Furthermore, we show that LSL-1 functions by antagonizing the action of the heterochromatin proteins HPL-2/HP1 and LET-418/Mi2 known to be involved in the repression of germline genes in somatic cells. Based on our results, we propose LSL-1 to be a major regulator of the germline transcriptional program during development.

sentences: Transcriptomic profiling and microsatellite identification in cobia (Rachycentron canadum), using high throughput RNA-sequencing Cobia (Rachycentron canadum) is a marine teleost species with great productive potential worldwide. However, the genomic information currently available for this species in public databases is limited. Such lack of information hinders gene expression assessments that might bring forward novel insights into the physiology, ecology, evolution, and genetics of this potential aquaculture species. In this study, we report the first de novo transcriptome assembly of R. canadum liver, improving the availability of novel gene sequences for this species. Illumina sequencing of liver transcripts generated 1,761,965,794 raw reads, which were filtered into 1,652,319,304 high-quality reads. De novo assembly resulted in 101,789 unigenes and 163,096 isoforms, with an average length of 950.61 and 1,617.34 nt, respectively. Moreover, we found that 126,013 of these transcripts bear potentially coding sequences, and 125,993 of these elements (77.3%) correspond to functionally annotated genes found in six different databases. We also identified 701 putative ncRNA and 35,414 putative lncRNA. Interestingly, homologues for 410 of these putative lncRNAs have already been observed in previous analyzes with Danio rerio, Lates calcarifer, Seriola lalandi dorsalis, Seriola dumerili or Echeneis naucrates. Finally, we identified 7,894 microsatellites related to cobias putative lncRNAs. Thus, the information derived from the transcriptome assembly described herein will likely assist future nutrigenomics and breeding programs involving this important fish farming species.

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Glycosylated Antibiotics: New Promising Bacterial 1 Efflux Pumps Inhibitors BackgroundAntimicrobial resistance is considered a major concern problem; bacteria have evolved mechanisms to overcome antibiotics action through evolutionary process. One main resistance mechanism that bacteria developed is the pumping of the antibiotics out of bacterial cells by transmembrane transporter proteins known as efflux pumps.

Materials and methodsTo overcome bacterial resistance guided by efflux pumps, efflux pumps inhibitors (EPIs) are small molecules that obstruct efflux pumps binding sites and its structural assembly leading to disability in the efflux pumps normal function, new EPIs which under the current study are created by modifying the chemical structure of most common antibiotics including Ampicillin, Penicillin, Chloramphenicol, Ciprofloxacin and Tetracycline, such antibiotics are modified by adding N-acetyl glucose amine moiety to acceptor OH group of the respective antibiotic, the newly modified antibiotics are glycosylated EPIs. To test the effectiveness of the new EPIs in inhibiting AcrB-TolC and MexA-OprM efflux pumps functions, ADME properties for all of glycosylated antibiotics have been measured through applying Lipinskis role of 5, docking and simulation studies have been included as well.

ResultsDocked glycosylated tetracycline has given the highest binding energy in the active sites of both pumps, with -9.4 against AcrB and -8.8 against MexA. The simulation study has confirmed the binding of the glycosylated tetracycline in the active sites of both pumps, as well as its stability during the biological dynamicity of both pumps (opening and closing channels).

ConclusionThe results validation requires a long simulation time about 50 ns or more which was un applicable due to cost limitation, however, the newly glycosylated antibiotics have promising results that might make it eligible as drug candidates to overcome bacterial resistance.

sentences: Versatile patterns in the actin cortex of motile cells: Self-organized pulses can coexist with macropinocytic ring-shaped waves Self-organized patterns in the actin cytoskeleton are essential for eukaryotic cellular life. They are the building blocks of many functional structures that often operate simultaneously to facilitate, for example, nutrient uptake and movement of cells. However, to identify how qualitatively distinct actin patterns can coexist remains a challenge. Here, we use bifurcation theory to reveal a generic mechanism of pattern coexistence, showing that different types of wave patterns can simultaneously emerge in the actin system. Our theoretical analysis is complemented by live-cell imaging experiments revealing that narrow, planar, and fast-moving excitable pulses may indeed coexist with ring-shaped macropinocytic actin waves in the cortex of motile amoeboid cells.

sentences: Emergence of a recurrent insertion in the N-terminal domain of the SARS-CoV-2 spike glycoprotein Tracking the evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through genomic surveillance programs is undoubtedly one of the key priorities in the current pandemic situation. Although the genome of SARS-CoV-2 acquires mutations at a slower rate compared with other RNA viruses, evolutionary pressures derived from the widespread circulation of SARS-CoV-2 in the human population have progressively favored the global emergence, though natural selection, of several variants of concern that carry multiple non-synonymous mutations in the spike glycoprotein. These are often placed in key sites within major antibody epitopes and may therefore confer resistance to neutralizing antibodies, leading to partial immune escape, or otherwise compensate infectivity deficits associated with other non-synonymous substitutions. As previously shown by other authors, several emerging variants carry recurrent deletion regions (RDRs) that display a partial overlap with antibody epitopes located in the spike N-terminal domain (NTD). Comparatively, very little attention has been directed towards spike insertion mutations prior to the emergence of the B.1.1.529 (omicron) lineage. This manuscript describes a single recurrent insertion region (RIR1) in the N-terminal domain of SARS-CoV-2 spike protein, characterized by at least 41 independent acquisitions of 1-8 additional codons between Val213 and Leu216 in different viral lineages. Even though RIR1 is unlikely to confer antibody escape, its association with two distinct formerly widespread lineages (A.2.5 and B.1.214.2), with the quickly spreading omicron and with other VOCs and VOIs warrants further investigation concerning its effects on spike structure and viral infectivity.

sentences: The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA differentially regulates gene expression across both genomes Phage satellites exhibit various regulatory mechanisms to manipulate phage gene expression to the benefit of the satellite. While small RNAs (sRNAs) are well documented as regulators of prokaryotic gene expression, they have not been shown to play a regulatory role in satellite-phage conflicts. Vibrio cholerae encodes the phage inducible chromosomal island-like element (PLE), a phage satellite, to defend itself against the lytic phage ICP1. Here we use Hi-GRIL-seq to identify a complex RNA-RNA interactome between PLE and ICP1. Both inter- and intragenome RNA interactions were detected, headlined by the PLE-encoded trans-acting sRNA, SviR. SviR regulates both PLE and ICP1 gene expression uniquely, decreasing translation of ICP1 targets and affecting PLE mRNAs processing. The striking conservation of SviR across all known PLEs suggests the sRNA is deeply rooted in the PLE-ICP1 conflict and implicates sRNAs as unidentified regulators of phage-satellite interactions.

sentences: Generation of a novel Nkx6-1 Venus fusion reporter mouse line Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TF) that regulates motor neuron development, neuron specification and pancreatic endocrine and {beta}-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus Fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. In addition, the nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. Moreover, the levels of Nkx6-1-VF reporter is upregulated upon {beta}-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting {beta}-cells. Importantly, the Venus reporter activities allows successful tracking of {beta}-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of Nkx6-1-VF reporter line provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and isolate and track Nkx6-1-expressing cells such as pancreatic {beta}-cells, but also neurons and motor neurons in health and disease.

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Self-healing neural codes: Hebbian and homeostatic mechanisms can track evolving neural representations As an adaptive system, the brain must retain a faithful representation of the world while continuously integrating new information. Recent experiments have measured population activity in cortical and hippocampal circuits over many days, and found that patterns of neural activity associated with fixed behavioral variables and percepts change dramatically over time. Such "representational drift" raises the question of how malleable population codes can interact coherently with stable long-term representations that are found in other circuits, and with relatively rigid topographic mappings of peripheral sensory and motor signals. We explore how known plasticity mechanisms can allow single neurons to reliably read out an evolving population code without external error feedback. We find that interactions between Hebbian learning and single-cell homeostasis can exploit redundancy in a distributed population code to compensate for gradual changes in tuning. Recurrent feedback of partially stabilized readouts could allow a pool of readout cells to further correct inconsistencies introduced by representational drift. This shows how relatively simple, known mechanisms can stabilize neural tuning in the short term, and provides a plausible explanation for how plastic neural codes remain integrated with consolidated, long-term representations.

SignificanceThe brain is capable of adapting while maintaining stable long-term memories and learned skills. Recent experiments show that neural responses are highly plastic in some circuits, while other circuits maintain consistent responses over time, raising the question of how these circuits interact coherently. We show how simple, biologically motivated Hebbian and homeostatic mechanisms in single neurons can allow circuits with fixed responses to continuously track a plastic, changing representation without reference to an external learning signal.

sentences: RNA-Seq analysis identifies novel roles for the primary cilia gene SPAG17 and the SOX9 locus non-coding RNAs in systemic sclerosis Systemic sclerosis (SSc) is characterized by immune activation, vasculopathy, and unresolving fibrosis in the skin, lungs, and other organs. We performed RNA-Seq analysis on skin biopsies and peripheral blood mononuclear cells (PBMCs) from SSc patients and controls to better understand SSc pathogenesis. We analyzed these data to 1) test for case-control differences, and 2) identify genes whose expression levels correlate with SSc severity as measured by local skin score, modified Rodnan skin score (MRSS), forced vital capacity (FVC), or diffusion capacity for carbon monoxide (DLCO). We found that PBMCs from SSc patients showed a strong type 1 interferon signature. This signal replicated in the skin, with additional signals for increased extracellular matrix (ECM) genes, classical complement pathway activation, and the presence of B cells. Notably, we observed a marked decrease in the expression of SPAG17, a cilia component, in SSc skin. We identified genes that correlated with MRSS, DLCO, and FVC in SSc PBMCs and skin using weighted gene co-expression analysis (WGCNA). These genes were largely distinct from the case/control differentially expressed genes. In PBMCs, type 1 interferon signatures negatively correlated with DLCO. In SSc skin, ECM gene expression positively correlated with MRSS. Network analysis of SSc skin genes correlated with clinical features identified the non-coding RNAs SOX9-AS1 and ROCR, both near the SOX9 locus, as highly connected, "hub-like" genes in the network. These results identify non-coding RNAs and SPAG17 as novel factors potentially implicated in SSc pathogenesis.

sentences: Cap-independent translation and a precisely localized RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represseses translation and induces mRNA degradation, while the host elicits anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-meidated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5UTR of SARS-CoV-2 exhibits an IRES-like activity and promotes expression of NSP1 in an eIF4E-independent and Torin-1 resistant manner. Upon expression, NSP1 enhances cap-independent translation. However, the sub-genomic 5UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin-1. The combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

sentences: The Consequences of Differential Origin Licensing Dynamics MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

sentences: On the genetic architecture of rapidly adapting and convergent life history traits in guppies The genetic basis of traits can shape and constrain how adaptation proceeds in nature; rapid adaptation can be facilitated by polygenic traits, whereas polygenic traits may restrict re-use of the same genes in adaptation (genetic convergence). The rapidly evolving life histories of guppies in response to predation risk provide an opportunity to test this proposition. Guppies adapted to high- (HP) and low-predation (LP) environments in northern Trinidad evolve rapidly and convergently among natural populations. This system has been studied extensively at the phenotypic level, but little is known about the underlying genetic architecture. Here, we use an F2 QTL design to examine the genetic basis of seven (five female, two male) guppy life history phenotypes. We use RAD-sequencing data (16,539 SNPs) from 370 male and 267 female F2 individuals. We perform linkage mapping, estimates of genome-wide and per-chromosome heritability (multi-locus associations), and QTL mapping (single-locus associations). Our results are consistent with architectures of many-loci of small effect for male age and size at maturity and female interbrood period. Male trait associations are clustered on specific chromosomes, but female interbrood period exhibits a weak genome-wide signal suggesting a potentially highly polygenic component. Offspring weight and female size at maturity are also associated with a single significant QTL each. These results suggest rapid phenotypic evolution of guppies may be facilitated by polygenic trait architectures, but these may restrict gene-reuse across populations, in agreement with an absence of strong signatures of genetic convergence from recent population genomic analyses of wild HP-LP guppies.

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Analysis of the Neurotropic and Anti-inflammatory Effects of Neuroelectrode Functionalization with a Heparan Sulphate Mimetic Further in the search for biomimicry of the properties analogous to neural tissues, and with an ultimate goal of mitigating electrode deterioration via reactive host cell response and glial scar formation, the bio-functionalisation of PEDOT:PTS neural coating is here presented using a heparan mimetic termed (HM) F6. A sulphated mimetic polyanion, with a potential role in neuromodulation in neurodegenerative diseases, and used here for the first time as neural coating.

This work acts as a first step towards the use of HM biological dopants, to enhance neuroelectrode functionality, to promote neural outgrowth and to maintain minimal glial scar formation in vitro at the neural-interface. Further, this study opens new possibilities for the evaluation of glycan mimetics in neuroelectrode functionalisation.

sentences: PRECISE 2.0: an expanded high-quality RNA-seq compendium for Escherichia coli K-12 reveals high-resolution transcriptional regulatory structure Uncovering the structure of the transcriptional regulatory network (TRN) that modulates gene expression in prokaryotes remains an important challenge. Transcriptomics data is plentiful, necessitating the development of scalable methods for converting this data into useful knowledge about the TRN. Previously, we published the PRECISE dataset for Escherichia coli K-12 MG1655, containing 278 RNA-seq datasets created using a standardized protocol. Here, we present PRECISE 2.0, which is nearly three times the size of the original PRECISE dataset and also created using a standardized protocol. We analyze PRECISE 2.0 at multiple scales, demonstrating multiple analytical strategies for extracting knowledge from this dataset. Specifically, we: (1) highlight patterns in gene expression across the dataset; (2) utilize independent component analysis to extract 218 independently modulated groups of genes (iModulons) that describe the TRN at the systems level; (3) demonstrate the utility of iModulons over traditional differential expression analysis; and (4) uncover 6 new potential regulons. Thus, PRECISE 2.0 is a large-scale, high-quality transcriptomics dataset which may be analyzed at multiple scales to yield important biological insights.

sentences: Self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induces protective immunity in mice The spike (S), a homotrimer glycoprotein, is the most important antigen target in the research and development of SARS-CoV-2 vaccine. There is no doubt that fully simulating the advanced structure of this homotrimer in the subunit vaccine development strategy is the most likely way to improve the immune protective effect of the vaccine. In this study, the preparation strategies of S protein receptor-binding domain (RBD) trimer, S1 region trimer, and ectodomain (ECD) trimer nanoparticles were designed based on ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare these three nanoparticle vaccines with high expression levels in the silkworm. The immune results of mice show that the nanoparticle vaccine prepared by this strategy can not only induce an immune response by subcutaneous administration but also effective by oral administration. Given the stability of these ferritin-based nanoparticles vaccine, easy-to-use and low-cost oral immunization strategy can make up for the vaccination blind areas caused by the shortage of ultralow-temperature equipment and medical resources in underdeveloped areas. And the oral vaccine is also a very potential candidate to cut off the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.

sentences: BirdFlow: Learning Seasonal Bird Movements from Citizen Science Data Large-scale monitoring of seasonal animal movement is integral to science, conservation, and outreach. However, gathering representative movement data across entire species ranges is frequently intractable. Citizen science databases collect millions of animal observations throughout the year, but it is challenging to infer individual movement behavior solely from observational data. We present BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW, a probabilistic modeling framework that draws on citizen science data from the eBird database to model the population flows of migratory birds. We apply the model to 11 species of North American birds, using GPS and satellite tracking data to tune and evaluate model performance. We show that BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW models can accurately infer individual seasonal movement behavior directly from eBird relative abundance estimates. Supplementing the model with a sample of tracking data from wild birds improves performance. Researchers can extract a number of behavioral inferences from model results, including migration routes, timing, connectivty, and forecasts. The BO_SCPLOWIRDC_SCPLOWFO_SCPLOWLOWC_SCPLOW framework has the potential to advance migration ecology research, boost insights gained from direct tracking studies, and serve a number of applied functions in conservation, disease surveillance, aviation, and public outreach.

sentences: C-bouton components on rat extensor digitorum longus motoneurons are resistant to chronic functional overload Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C-boutons. Overload of Extensor Digitorum Longus (EDL) muscles was induced by removal of their synergists, Tibialis Anterior (TA) muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C-bouton complexes remained largely unaffected by overload. The C-boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post-synaptic voltage-gated potassium channel (KV2.1) was also unchanged. Small conductance calcium activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C-bouton plasticity is not necessary to increase the force output required in response to muscle overload.

sentences: SDC3 acts as a timekeeper of myogenic differentiation by regulating the insulin/AKT/mTOR axis in muscle stem cell progeny Muscle stem cells (MuSCs) are indispensable for muscle regeneration. A multitude of extracellular stimuli direct MuSC fate decisions from quiescent progenitors to differentiated myocytes. The activity of these signals is modulated by coreceptors such as syndecan-3 (SDC3). We investigated the global landscape of SDC3-mediated regulation of myogenesis using a phosphoproteomics approach which revealed, with the precision level of individual phosphosites, the large-scale extent of SDC3-mediated regulation of signal transduction in MuSCs. We then focused on INSR/AKT/mTOR as a key pathway regulated by SDC3 during myogenesis and mechanistically dissected SDC3-mediated inhibition of insulin signaling in MuSCs. SDC3 interacts with INSR limiting insulin signal transduction via AKT/mTOR. Both knockdown of INSR and inhibition of AKT rescue Sdc3-/- MuSC differentiation to wild type levels. Since SDC3 is rapidly downregulated at the onset of differentiation, our study suggests that SDC3 acts a timekeeper to restrain proliferating MuSC response to insulin and prevent premature differentiation.

sentences: Mining signaling flow to interpret mechanisms of synergy of drug combinations using deep graph neural networks Complex signaling pathways/networks are believed to be responsible for drug resistance in cancer therapy. Drug combinations inhibiting multiple signaling targets within cancer-related signaling networks have the potential to reduce drug resistance. Deep learning models have been reported to predict drug combinations. However, these models are hard to be interpreted in terms of mechanism of synergy (MoS), and thus cannot well support the human-AI based clinical decision making. Herein, we proposed a novel computational model, DeepSignalingFlow, which seeks to address the preceding two challenges. Specifically, a graph convolutional network (GCN) was developed based on a core cancer signaling network consisting of 1584 genes, with gene expression and copy number data derived from 46 core cancer signaling pathways. The novel up-stream signaling-flow (from up-stream signaling to drug targets), and the down-stream signaling-flow (from drug targets to down-stream signaling), were designed using trainable weights of network edges. The numerical features (accumulated information due to the signaling-flows of the signaling network) of drug nodes that link to drug targets were then used to predict the synergy scores of such drug combinations. The model was evaluated using the NCI ALMANAC drug combination screening data. The evaluation results showed that the proposed DeepSignalingFlow model can not only predict drug combination synergy score, but also interpret potentially interpretable MoS of drug combinations.

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Sequencing of Sars-CoV-2 genome using different Nanopore chemistries Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and Rapid Barcoding Kit (RBK) vs Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in timine and adenine homopolymeric regions than R9, at expenses of a larger rate (~10%) of mismatches in these regions.

The LSK had a larger yield than RBK, and provided longer reads than RBK. It also resulted in a larger percentage of aligned reads (99% vs 93%) and also in a complete consensus genome.

The results from this study suggest that the LSK used on a R9 flow cell could maximize the yield and accuracy of the consensus sequence when used in epidemiological surveillance of SARS-CoV-2.

KeypointsO_LISequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance
C_LIO_LINanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome
C_LIO_LILigation sequencing is preferred rather than the rapid kit using transposases
C_LI

sentences: Coming together - symbiont acquisition and early development in deep-sea Bathymodiolus mussels Symbiotic associations between animals and microorganisms are widespread and have a profound impact on the ecology, behaviour, physiology, and evolution of the host. Research on deep-sea mussels of the genus Bathymodiolus has revealed how chemosynthetic symbionts sustain their host with energy, allowing them to survive in the nutrient-poor environment of the deep ocean. However, to date, we know little about the initial symbiont colonization and how this is integrated into the early development of these mussels. Here we analysed the early developmental life stages of B. azoricus, "B". childressi and B. puteoserpentis and the changes that occur once the mussels are colonized by symbionts. We combined synchrotron-radiation based {micro}CT, correlative light and electron microscopy and fluorescence in situ hybridization to show that the symbiont colonization started when the animal settled on the sea floor and began its metamorphosis into an adult animal. Furthermore, we observed aposymbiotic life stages with a fully developed digestive system which was streamlined after symbiont acquisition. This suggests that bathymodiolin mussels change their nutritional strategy from initial filter-feeding to relying on the energy provided by their symbionts. After [~]35 years of research on bathymodiolin mussels, we are beginning to answer fundamental ecological questions concerning their life cycle and the establishment of symbiosis.

sentences: Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry DNA replication is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase onto DNA replication origins during the G1 phase is essential for initiating DNA replication. Here, we examined MCM hexamer states during the cell cycle in human hTERT-RPE1 cells using multicolor immunofluorescence-based, single-cell plot analysis, and biochemical size fractionation. Experiments involving cell-cycle arrest at the G1 phase and release from the arrest revealed that a double MCM hexamer was formed via a single hexamer during G1 progression. A single MCM hexamer was recruited to chromatin in the early G1 phase. Another single hexamer was recruited to form a double hexamer in the late G1 phase. We further examined relationship between the MCM hexamer states and the methylation levels at lysine 20 of histone H4 (H4K20) and found that the double MCM hexamer state was correlated with di/trimethyl-H4K20 (H4K20me2/3). Inhibiting the conversion from monomethyl-H4K20 (H4K20me1) to H4K20me2/3 retained the cells in the single MCM hexamer state. Non-proliferative cells, including confluent cells or Cdk4/6 inhibitor-treated cells, also remained halted in the single MCM hexamer state. We propose that the single MCM hexamer state is a halting step in the determination of cell cycle progression.

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Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus ({Delta}N30 and {Delta}N50), and 83 ({Delta}C83) or 110 residues ({Delta}C110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing {Delta}N30FliF or {Delta}N50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for {Delta}C83FliF as with wild-type FliF, although further deletion of the second transmembrane segment ({Delta}C110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.

ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

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Altered DNA methylation in ion transport and immune signalling genes is associated with severity in Pancreatic Ductal Adenocarcinoma BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the leading cancers worldwide and has a poor survival, with a relative five-year survival rate of only 8.5%. In this study we investigated epigenetic marksassociated with PDAC severity and prognosis, through studying alterations in DNA methylation patterns.

MethodsDNA methylome for tumor and adjacent normal tissue samples from PDAC patients (n=7) were generated using Illumina 450K bead chips. Differentially methylated positions (DMPs) were identified with |delta beta| > 0.2 and p-value<0.01 by comparing tumors with the adjacent normal tissues. Validation of differential methylation and associated gene expression at selected genes was carried out in an independent cohort PDAC patient.

ResultsWe identified 76 DMPs in PDAC patients that mapped to 43 genes. Among them, 44.7% (n=34) were hypo-methylated and 55.3% (n=42) were hyper-methylated DMPs in cancer samples. The trends of change in methylation at these 76 DMPs from well to moderate were like that from moderate to poorly differentiated cancer samples. The gradual trend in differential methylation was observed both in our cohort and the TCGA-PAAD cohort, suggesting methylation marks can serve as early indicators of disease pathology. Altered promoter methylation, which may affect gene expression, was observed for transcription regulators (BHLHE23, GSC2, FOXE1 and TWIST1), gated ion channels (KCNA6, and CACNB2), tumor suppressors (RASSF1, SPRED2, and NPY) and genes functioning in interferon signalling (SIGIRR, MX2, and OAS2). We also have compared the TCGA-PAAD dataset with normal pancreatic tissue data from GTEx V8 dataset leading to a confluent observation.

ConclusionsWe reported the first study on methylome in PDAC tumors from patients in India. We identified altered DNA methylation associated with increasing severity in PDAC among some genes like SIGIRR, MX2 along with other previously reported loci. We also concluded a confluence in our observation when comparing the TCGA-PAAD dataset with GTEx V8 dataset.

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Benchmarking Peptide-Protein Docking and Interaction Prediction with AlphaFold-Multimer Protein interactions are key in vital biological process. In many cases, particularly often in regulation, this interaction is between a protein and a shorter peptide fragment. Such peptides are often part of larger disordered regions of other proteins. The flexible nature of peptides enable rapid, yet specific, regulation of important functions in the cell, such as the cell-cycle. Because of this, understanding the molecular details of these interactions are crucial to understand and alter their function, and many specialized computational methods have been developed to study them.

The recent release of AlphaFold and now AlphaFold-Multimer has caused a leap in accuracy for computational modeling of proteins. Additionally, AlphaFold has proven generalizable enough that it can be adapted to a number of specialized protein modeling challenges outside of the original single-chain protein modeling it was trained for.

In this paper, the ability of AlphaFold to predict which peptides and proteins interact as well as its accuracy in modeling the resulting interaction complexes are benchmarked against established methods in the fields of peptide-protein interaction prediction and modeling. We find that AlphaFold-Multimer consistently produces predicted interaction complexes with the best DockQ-scores, with a mean DockQ of 0.49 for all 247 complexes investigated. Additionally, it can be used to separate interacting from non-interacting pairs of peptides and proteins with ROC-AUC and PR-AUC of 0.75 and 0.54, respectively, best among the method in benchmark. However, there is still room for improvement, for a decent precision of 0.8 it only recalls 0.2 of the positive examples (FPR=0.01), which means the will miss many true interactions. By combining AlphaFold-Multimer with InterPep2 the model quality for interacting proteins is increased, but it does not improve the separation of interacting from non-interacting.

sentences: Blocking unwanted sequences by target specific high-affinity binding oligonucleotides during transcriptome library preparation RNA sequencing has become the gold standard for transcriptome analysis but comes with an inherent limitation with respect to quantification of low abundant transcripts. In contrast to microarray technology, RNA-sequencing reads are proportionally divided across transcripts. Therefore, low abundant RNAs can be out-competed by highly abundant--and sometimes non-informative--RNA species. We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR-amplification of specific RNA transcripts, hereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different RNA molecules and library preparation strategies, including YRNAs in small RNA-sequencing of human plasma samples, mitochondrial rRNAs in both 3 end sequencing and long-read sequencing, and MALAT1 in single-cell 3 end sequencing. We demonstrate that the blocking strategy is extremely efficient, reproducible and specific, and generally results in better transcriptome coverage and complexity. Our method does not require modifications of the library preparation procedure apart from adding blocking oligonucleotides to the RT reaction and can thus be easily integrated in virtually any RNA sequencing library preparation protocol.

sentences: CIS calibrates GM-CSF signalling strength to regulate macrophage polarization via a STAT5-IRF8 axis The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the ability to differentiate macrophages (MOs) with opposing functions, namely proinflammatory M1-like and immunosuppressive M2-like. Despite the importance and opposing functional outcomes of these processes, the intrinsic mechanism that regulates the functional polarization of MOs under GM-CSF signaling remains elusive. Here we show that GM-CSF induced MOs polarisation resulted in the expression of the Cytokine-inducible SH2-containing protein (CIS), and that CIS deficiency diverted differentiation of monocytes into immunosuppressive M2-like MOs expression. CIS deficiency resulted in the hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting the downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss and gain of function approaches highlighted IRF8 as a critical instructor of the M1-like polarisation program. In vivo, CIS deficiency led to skewing to M2-like macrophages, which induced strong Th2 immune responses characterised by the development of severe experimental asthma. Collectively, we reveal a CIS-censored mechanism interpreting the opposing actions of GM-CSF in MO differentiation and uncovering its role in controlling allergic inflammation.

sentences: Correcting for Background Noise Improves Phenotype Prediction from Human Gut Microbiome Data The ability to predict human phenotypes accurately from metagenomic data is crucial for developing biomarkers and therapeutics for diseases. However, metagenomic data is commonly affected by technical or biological variables, unrelated to the phenotype of interest, such as sequencing protocol or host sex, which can greatly reduce or, when correlated to the phenotype of interest, inflate prediction accuracy. We perform a comparative analysis of the ability of different data transformations and existing supervised and unsupervised methods to correct microbiome data for background noise. We find that supervised methods are limited because they cannot account for unmeasured sources of variation. In addition, we observe that unsupervised approaches are often superior in addressing these issues, but existing methods developed for other  omic data types, e.g., gene expression and methylation, are restricted by parametric assumptions unsuitable for microbiome data, which is typically compositional, highly skewed, and sparse. We show that application of the centered log-ratio transformation prior to correction with unsupervised approaches improves prediction accuracy for many phenotypes while simultaneously reducing variance due to unwanted sources of variation. As new and larger metagenomic datasets become increasingly available, background noise correction will become essential for generating reproducible microbiome analyses.

sentences: Astrocyte-mediated transduction of muscle fiber contractions synchronizes hippocampal neuronal network development Exercise supports brain health in part through enhancing hippocampal function. The leading hypothesis is that muscles release factors when they contract (e.g., lactate, myokines, growth factors) that enter circulation and reach the brain where they enhance plasticity (e.g., increase neurogenesis and synaptogenesis). However, it remains unknown how the muscle signals are transduced by the hippocampal cells to modulate network activity and synaptic development. Thus, we established an in vitro model in which the media from contracting primary muscle cells (CM) is applied to developing primary hippocampal cell cultures on a microelectrode array. We found that the hippocampal neuronal network matures more rapidly (as indicated by synapse development and synchronous neuronal activity) when exposed to CM than regular media (RM). This was accompanied by a fourfold increase in the proliferation of astrocytes. Further, experiments established that the astrocytes release factors that inhibit neuronal excitability and facilitate network development. Results provide new insight into how exercise may support hippocampal function through regulating astrocyte proliferation and subsequent taming of neuronal activity into an integrated network.

sentences: Identifying tumor cells at the single cell level Tumors are highly complex tissues composed of cancerous cells, surrounded by a heterogeneous cellular microenvironment. Tumor response to treatments is governed by an interaction of cancer cell intrinsic factors with external influences of the tumor microenvironment. Disentangling the heterogeneity within a tumor is a crucial step in developing and utilization of effective cancer therapies. The single cell sequencing technology enables an effective molecular characterization of single cells within the tumor. This technology can help deconvolute heterogeneous tumor samples and thus revolutionize personalized medicine. However, a governing challenge in cancer single cell analysis is cell annotation, the assignment of a particular cell type or a cell state to each sequenced cell. One of the critical cell type annotation challenges is identification of tumor cells within single cell or spatial sequencing experiments.This is a critical limiting step for a multitude of research, clinical, and commercial applications. A reliable method addressing that challenge is a prerequisite for automatic annotation of histopathological data, profiled using multichannel immunofluorescence or spatial sequencing. Here, we propose Ikarus, a machine learning pipeline aimed at distinguishing tumor cells from normal cells at the single cell level. We have tested ikarus on multiple single cell datasets to ascertain that it achieves high sensitivity and specificity in multiple experimental contexts.

sentences: Natural variation in the maternal and zygotic mRNA complements of the early embryo in Drosophila melanogaster Maternal gene products supplied to the egg during oogenesis drive the earliest events of development in all metazoans. After the initial stages of embryogenesis, maternal transcripts are degraded as zygotic transcription is activated, this is known as the maternal to zygotic transition (MZT). Altering the abundances of maternally deposited factors in the laboratory can have a dramatic effect on development, adult phenotypes and ultimately fitness. Zygotic transcription activation is a tightly regulated process, where the zygotic genome takes over control of development from the maternal genome, and is required for the viability of the organism. Recently, it has been shown that the expression of maternal and zygotic transcripts have evolved in the Drosophila genus over the course of 50 million years of evolution. However, the extent of natural variation of maternal and zygotic transcripts within a species has yet to be determined. We asked how the maternal and zygotic pools of mRNA vary within and between populations of D. melanogaster. In order to maximize sampling of genetic diversity, African lines of D. melanogaster originating from Zambia as well as DGRP lines originating from North America were chosen for transcriptomic analysis. Single embryo RNA-seq was performed at a stage before and a stage after zygotic genome activation in order to determine which transcripts are maternally deposited and which are zygotically expressed within and between these populations. Differential gene expression analysis has been used to quantify quantitative changes in RNA levels within populations as well as fixed expression differences between populations at both stages. Generally, we find that maternal transcripts are more highly conserved, and zygotic transcripts evolve at a higher rate. We find that there is more within population variation in transcript abundance than between populations and that expression variation is highest post-MZT between African lines. Determining the natural variation of gene expression surrounding the MZT in natural populations of D. melanogaster gives insight into the extent of how a tightly regulated process may vary within a species, the extent of developmental constraint at both stages and on both the maternal and zygotic genomes, and reveals expression changes allowing this species to adapt as it spread across the world.

sentences: Frequency preference response in covalent modification cycles under substrate sequestration conditions Covalent modification cycles (CMCs) are basic units of signaling systems and their properties are well understood. However, the behavior of such systems has been mostly characterized in situations where the substrate is in excess over the modifying enzymes. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations, leading to a different scenario in which the substrate is mostly sequestered by the enzymes. In this enzyme-in-excess regime, CMCs have been shown to exhibit signal termination, the ability of the product to return to a stationary value lower than the its peak in response to constant stimulation, while this stimulation is still active, with possible implications for the ability of systems to adapt to environmental inputs. We characterize the conditions leading to signal termination in CMCs in the enzyme-in-excess regime. We also demonstrate that this behavior leads to a preferred frequency response (band-pass filters) when the cycle is subjected to periodic stimulation, while the literature reports that CMCs investigated so far behave as low pass filters. We characterize the relationship between signal termination and the preferred frequency response to periodic inputs and we explore the dynamic mechanism underlying these phenomena. Finally, we describe how the behavior of CMCs is reflected in similar types of responses in the cascades of which they are part. Evidence of protein abundance in vivo shows that enzymes and substrates are present in comparable concentrations, thus suggesting that signal termination and frequency preference response to periodic inputs are also important dynamic features of cell signaling systems, which have been overlooked.

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Immune complex solubility and size govern Fc-gamma receptor responses: A scalable cell-based reporter system Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings.

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Stereo-specific Lasofoxifene Derivatives Reveal the Interplay between Estrogen Receptor Alpha Stability and Antagonistic Activity in ESR1 Mutant Breast Cancer Cells Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend ER cellular lifetime, accumulation, and are antagonists in the breast and agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders (SERDs) reduce ER cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ER cellular lifetime and accumulation influences antagonistic activity, we synthesized a series of methylpyrollidine lasofoxifene derivatives that maintained the drugs antagonistic activities while uniquely tuning ER cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ER cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ER ligand binding domain in complex with the methylated lasofoxifene derivatives, SERMs, and SERDs show that molecules that favor a highly buried helix 12 conformation achieve the greatest transcriptional suppression activities. Together these results show that chemical reduction of ER cellular lifetime does not necessarily correlate with transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our approach shows how minor chemical additions modulate receptor cellular lifetime while maintaining other activities to achieve desired SERM or SERD profiles.

SIGNIFICANCEThis study shows that antiestrogens that enforce a wild-type-like antagonist conformation demonstrate improved therapeutic activities in hormone-resistant breast cancer cells harboring Y537S and D538G ESR1.

sentences: Triangulation of microbial fingerprinting in anaerobic digestion reveals consistent fingerprinting profiles The anaerobic digestion microbiome has been puzzling us since the dawn of molecular methods for mixed microbial community analysis. Monitoring of the anaerobic digestion microbiome can either take place via a non-targeted holistic evaluation of the microbial community through fingerprinting or by targeted monitoring of selected taxa. Here, we compared four different microbial community fingerprinting methods, i.e., amplicon sequencing, metaproteomics, metabolomics and cytomics, in their ability to characterise the full-scale anaerobic digestion microbiome. Cytometric fingerprinting through cytomics reflects a, for anaerobic digestion, novel, single cell-based approach of direct microbial community fingerprinting by flow cytometry. Three different digester types, i.e., sludge digesters, digesters treating agro-industrial waste and dry anaerobic digesters, each reflected different operational parameters. The -diversity analysis yielded inconsistent results, especially for richness, across the different methods. In contrast, {beta}-diversity analysis resulted in comparable profiles, even when translated into phyla or functions, with clear separation of the three digester types. In-depth analysis of each methods features i.e., operational taxonomic units, metaproteins, metabolites, and cytometric traits, yielded certain similar features, yet, also some clear differences between the different methods, which was related to the complexity of the anaerobic digestion process. In conclusion, cytometric fingerprinting through flow cytometry is a reliable, fast method for holistic monitoring of the anaerobic digestion microbiome, and the complementary identification of key features through other methods could give rise to a direct interpretation of anaerobic digestion process performance.

sentences: Lyophilization induces alterations in cryptococcal exopolysaccharide resulting in reduced antibody binding The structural, antigenic, and immunological characterization of microbial polysaccharides requires purification that often involves detergent precipitation and lyophilization. Here we examine physicochemical changes induced by lyophilization on exopolysaccharide (EPS) of the pathogenic fungus Cryptococcus neoformans. Solution 1H NMR reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H ssNMR shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense rosette-like aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide binds the native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

sentences: Microtubule-based perception of mechanical conflicts controls plant organ morphogenesis Precise coordination between cells and tissues is essential for differential growth in plants. During lateral root formation in Arabidopsis thaliana, the endodermis is actively remodeled to allow outgrowth of the new organ. Here, we show that microtubule arrays facing lateral root founder cells display a higher order compared to arrays on the opposite wall of the same cell, and this asymmetry is required for endodermal remodeling and lateral root initiation. We identify that MICROTUBULE ASSOCIATED PROTEIN 70-5 is necessary for the establishment of this spatially defined microtubule organization and endodermis remodeling, and thus contributes to lateral root morphogenesis. We propose that MAP70-5 and cortical microtubule arrays in the endodermis integrate the mechanical signals generated by lateral root outgrowth, facilitating the channeling of organogenesis.

sentences: Improved methods for protein and single-molecule RNA detection in C. elegans embryos Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule Fluorescence In Situ Hybridization (smFISH) and Immunofluorescence (IF) visualize the abundance and localization of mRNAs and proteins, respectively, allowing researchers to elucidate the localization, dynamics, and functions of many genes. Here, we describe several improvements and optimizations to existing IF and smFISH approaches specifically for use in C. elegans embryos. We present 1) optimized fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility, 2) a streamlined, in-tube approach that negates freeze-cracking, 3) the smiFISH (single molecule inexpensive FISH) adaptation that reduces cost, 4) an assessment of optimal anti-fade products, and 5) straightforward quantification and data analysis methods. Most importantly, published IF and smFISH protocols have predominantly been mutually exclusive, preventing exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we present methods to combine IF and smFISH protocols in C. elegans embryos including an efficient method harnessing nanobodies. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol.

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Single-cell transcriptomic and proteomic analysis of Parkinson's disease Brains Parkinsons disease (PD) is a prevalent neurodegenerative disorder where recent evidence suggests pathogenesis may be mediated by inflammatory processes. The molecular architecture of the disease remains to be fully elucidated. We performed single-nucleus transcriptomics and unbiased proteomics using postmortem tissue obtained from the prefrontal cortex of 12 individuals with late-stage PD and age-matched controls. We analyzed [~]80,000 nuclei and identified eight major cell types, including brain-resident T cells, each with distinct transcriptional changes in line with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem tissue, we found that -synuclein pathology is inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in prefrontal cortex that were preferentially downregulated in PD. Strikingly, comparing this dataset to a regionally similar published analysis for Alzheimers disease (AD), we found no common differentially expressed genes in neurons, but identified many shared differentially expressed genes in glial cells, suggesting that disease etiology in PD and AD are likely distinct. These data are presented as a resource for interrogating the molecular and cellular basis of PD and other neurodegenerative diseases.

One Sentence SummaryWe provide an extensive single cell analysis profiling nearly 80,000 brain nuclei from prefrontal cortex of late-stage Parkinsons disease brains, demonstrate that -synuclein pathology is inversely correlated with chaperone expression in excitatory neurons, found a selective abatement of neuron-astrocyte interactions with enhanced neuroinflammation, and augmented the study with proteomic analysis and cross-comparisons with Alzheimers disease datasets, providing valuable insights into the pathways of neurodegeneration and a deep definition of the underlying molecular pathology for Parkinsons disease.

sentences: Pyruvate kinase M1 suppresses development and progression of prostate adenocarcinoma Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (Pkm2). This isoform can promote anabolic metabolism to support cell proliferation; however, Pkm2 expression is dispensable for many cancers in vivo. Pyruvate kinase M1 (Pkm1) isoform expression is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied. Pkm1 is expressed in normal prostate tissue; thus, to test how differential pyruvate kinase isoform expression affects cancer initiation and progression we generated mice harboring a conditional allele of Pkm1 and crossed this allele, as well as a Pkm2 conditional allele, to a Pten loss-driven prostate cancer model. We found that Pkm1 loss leads to Pkm2 expression and accelerates prostate cancer, while deletion of Pkm2 leads to increased Pkm1 expression and suppresses cancer. Consistent with these data, a small molecule pyruvate kinase activator that mimics a PKM1-like state suppresses progression of established prostate tumors. PKM2 expression is retained in most human prostate cancers, arguing that pharmacological PKM2 activation may be beneficial for some prostate cancer patients.

sentences: Plant host shift: The viability and persistence of a polyphagous, non-native insect Assessing the effects of a plant-host shift is important for monitoring insect populations over long time periods and for interventions in a conservation or pest management framework. In a heterogeneous environment, individuals may disperse between sources and sinks in order to persist. Here we propose a single-species two-patch model that aims to capture the generational movement of an insect that exhibits density-dependent dispersal, to see how shifting between hosts could alter its viability and asymptotic dynamics. We then analyse the stability and persistence properties of the model and further validate it using parameter estimates derived from laboratory experiments. In order to evaluate the potential of this model, we applied it to Drosophila suzukii (Diptera: Drosophilidae), which has become a harmful pest in several countries around the world. Although many studies have investigated the preference and attractiveness of potential hosts on this invasive drosophilid, no studies thus far have investigated whether a shift of fruit host could affect such a species ecological viability or spatiotemporal persistence. The model results show that a shift in host choice can significantly affect the growth potential and fecundity of a species such as D. suzukii, which ultimately could aid such invasive populations in their ability to persist within a changing environment.

sentences: Isolation and Purification of Lipoarabinomannan from Urine of Adults with Active Tuberculosis Lipoarabinomannan (LAM) is a cell wall component of Mycobacterium tuberculosis that is excreted in the urine of persons with active tuberculosis (TB). Limited diagnostic sensitivity of LAM immunoassays has been due to selecting antibodies against LAM derived from in vitro cultured M. tuberculosis, rather than LAM purified from in vivo clinical urine specimens. Urinary LAM (uLAM) is critical to enable the development of and/or screening of novel uLAM-specific antibodies but is typically dilute and in heterogeneous mixtures with other urine components. We used physical, enzymatic, and chemical processes for the scaled isolation and purification of uLAM. The purified material may then be used to develop more sensitive uLAM diagnostic tests for active TB disease.

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Short latency stretch reflexes depend on the balance of activity in agonist and antagonist muscles during ballistic elbow movements The spinal stretch reflex is a fundamental building block of motor function, with a sensitivity that varies continuously during movement and when changing between movement and posture. Many have investigated task-dependent reflex sensitivity, but few have provided simple, quantitative analyses of the relationship between the volitional control and stretch reflex sensitivity throughout tasks that require coordinated activity of several muscles. Here we develop such an analysis and use it to test the hypothesis that modulation of reflex sensitivity during movement can be explained by the balance of activity within agonist and antagonist muscles better than by activity only in the muscle homonymous with the reflex. Subjects completed hundreds of flexion and extension movements as small, pseudo-random perturbations of elbow angle were applied to obtain estimates of stretch reflex amplitude throughout the movement. A subset of subjects performed a postural control task at muscle activities matched to those during movement. We found that reflex modulation during movement can be described by background activity in antagonist muscles about the elbow much better than by activity only in the muscle homonymous to the reflex (p<0.001). Agonist muscle activity enhanced reflex sensitivity whereas antagonist activity suppressed it. Surprisingly, the magnitude of these effects was similar, suggesting a balance of control between agonists and antagonists very different from the dominance of sensitivity to homonymous activity during posture. This balance is due to a large decrease in sensitivity to homonymous muscle activity during movement rather than substantial changes in the influence of antagonistic muscle activity.

New and NoteworthyThis study examined the sensitivity of the stretch reflexes elicited in elbow muscles to the background activity in these same muscles during movement and postural tasks. We found a heightened reciprocal control of reflex sensitivity during movement that was not present during maintenance of posture. These results help explain previous discrepancies in reflex sensitivity measured during movement and posture and provide a simple model for assessing their contributions to muscle activity in both tasks.

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Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection First recognized 35 years ago, glycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine cytoprotection, however, remains entirely elusive. We hypothesized that glycine targets ninjurin-1 (NINJ1), a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptotic cell death. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. Here, we first demonstrate that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in mouse and human macrophages stimulated to undergo lytic cell death. Next, we show that glycine treatment prevents NINJ1 clustering thereby preserving cellular integrity. By identifying NINJ1 as a glycine target, our data help resolve a long-standing mechanism of glycine cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death pathways.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/471765v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b9c3d9org.highwire.dtl.DTLVardef@1e903f0org.highwire.dtl.DTLVardef@1018498org.highwire.dtl.DTLVardef@a819b9_HPS_FORMAT_FIGEXP  M_FIG C_FIG SummaryGlycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury by an unknown mechanism. The authors demonstrate that NINJ1, a newly identified executioner of plasma membrane rupture across lytic cell death pathways, is a glycine target and resolve a longstanding mechanism of glycine cytoprotection.

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Distribution of multi-unit pitch responses recorded intracranially from human auditory cortex The perception of pitch requires the abstraction of stimulus properties related to the spectrotemporal structure of sound. Previous studies utilizing both animal electrophysiology and human imaging have indicated the presence of a center for pitch representation in the auditory cortex. Recent data from our own group - examining local field potentials (LFPs) in humans - indicate more widely distributed pitch-associated responses within the auditory cortex (Gander et al., 2019). To probe this with greater spatial resolution, we examined multi-unit activity related to three different auditory stimuli, in seven epilepsy patients who were implanted with high-impedance electrodes in auditory cortex for the clinical purpose of localizing seizures. The stimuli were regular-interval noise (RIN) with a pitch strength that is related to the temporal regularity, and pitch value determined by repetition rate, and harmonic complexes with missing fundamentals. We demonstrated increases in spiking activity in 69 of 104 (66%) responsive multiunit activity in auditory cortex due to pitch-associated stimuli. Importantly, these responses were distributed across the entire extent of Heschls gyrus (HG), in both primary and non-primary areas, rather than isolated to a specific region, and this finding was evident regardless of the stimulus presented. These findings are the first multi-unit pitch responses recorded from humans, and align with a recent study in macaques (Kikuchi et al., 2019) demonstrating that both local field potential and unit responses to pitch-inducing stimuli are distributed throughout auditory cortex.

Significance StatementThe perception of pitch is a fundamental acoustic attribute that is mediated by the auditory system. Despite its importance, there is still debate as to the precise areas responsible for its encoding, which may be due to differences in the recording measures and choices of stimuli used in previous studies. Here, we present the first study to measure multi-unit pitch responses in the auditory cortices of intracranially-implanted humans. Importantly, we demonstrate reliable responses to three different pitch-inducing paradigms that are distributed throughout Heschls gyrus, rather than being localized to a particular region. These data provide a bridge across animal and human studies, and aid in our understanding of the processing of a critical attribute of acoustic stimuli.

sentences: A T-cell independent universal cellular therapy strategy through antigen depletion CD19-targeting chimeric antigen receptor (CAR) T-cell therapeutics is a revolutionary, novel and successful treatment for B-cell malignancies. However, while CD19-CAR-T therapy can obtain high rates of complete responses in these patients, a significant fraction of patients may experience CD19-negative relapse. Moreover, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects such as cytokine-release syndrome (CRS). Whether CAR-T therapy can be substituted by a non-T-cell based universal cellular therapy is largely unknown. Surprisingly, we have demonstrated here that T-lymphocytic cells, as well as non-lymphocytic cells, can cause CD19 internalization and subsequent depletion when they are armed with a CD19-recognizing moiety. This CD19 antigen depletion can efficiently induce T-cell independent apoptosis in target cancer cells whose survival is dependent upon CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy. We therefore proposed a universal strategy for CRS-free cellular therapeutics, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and standardly as "off-the-shelf" mesenchymal stromal cells (MSCs) or other types of non-autologous cell expressing anergic CARs. Our results not only uncovered an unrecognized mechanism for CAR-T cytotoxicity and antigen loss, but also shed new insight into a shift in cellular therapeutics from unique patient-specific autologous therapeutics, to universal and standardized allogeneic treatment.

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

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Seasonal dynamics are the major driver of microbial diversity and composition in intensive freshwater aquaculture Aquaculture facilities such as fishponds are one of the most anthropogenically impacted freshwater ecosystems. The high fish biomass reared in aquaculture is associated with an intensive input into the water of fish-feed and fish excrements. This nutrients load may affect the microbial community in the water, which in turn can impact the fish health. To determine to what extent aquaculture practices and natural seasonal cycles affect the microbial populations, we characterized the microbiome of an inter-connected aquaculture system at monthly resolution, over three years. The system comprised two fishponds, where fish are grown, and a "control" operational water reservoir in which fish are not actively stocked. Clear natural seasonal cycles of temperature and inorganic nutrients concentration, as well as recurring cyanobacterial blooms during summer, were observed in both the fishponds and the reservoir. The structure of the aquatic bacterial communities in the system, characterized using 16S rRNA sequencing, was explained primarily by the natural seasonality, whereas aquaculture-related parameters had only a minor explanatory power. However, the cyanobacterial blooms were characterized by different cyanobacterial clades dominating at each fishpond, possibly in response to distinct nitrogen and phosphate ratios. In turn, nutrient ratios may have been by the magnitude of fish feed input. Taken together, our results show that, even in strongly anthropogenically impacted aquatic ecosystems, the structure of bacterial communities is mainly driven by the natural seasonality, with more subtle effects if aquaculture-related factors.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/433039v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@154d693org.highwire.dtl.DTLVardef@d35ae9org.highwire.dtl.DTLVardef@1f5a7a2org.highwire.dtl.DTLVardef@d07fbc_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIWe present three years of monthly microbiome data from an aquaculture facility.
C_LIO_LIThe microbiome changes seasonally, likely driven by temperature and rainwater runoff.
C_LIO_LISummer blooms of toxin-producing cyanobacteria are repeatedly observed.
C_LIO_LIFish food may impact microbiome through changes in nutrient ratios.
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sentences: Learning spatio-temporal properties of hippocampal place cells Hippocampal place cells have spatio-temporal properties: they generally respond to a single spatial location of a small environment; in addition, they also display the temporal response property of theta phase precession, namely that the phase of spiking relative to the theta wave shifts from the late phase to early phase as the animal crosses the place field. Grid cells in layer II of the medial entorhinal cortex (MEC) also have spatio-temporal properties similar to hippocampal place cells, except that grid cells respond to multiple spatial locations that form a hexagonal pattern. Other non-grid spatial cells are also abundant in the entorhinal cortex (EC). Because the EC is the upstream area that projects strongly to the hippocampus, a number of EC-hippocampus models have been proposed to explain how the spatial receptive field properties of place cells emerge. However, none of these learning models have explained how the temporal response properties of hippocampal place cells emerge as a result of the EC input. A learning model is presented here based on non-negative sparse coding in which we show that the spatial and temporal properties of hippocampal place cells can be simultaneously learnt from EC input: both MEC grid cells and other EC spatial cells contribute to the spatial properties of hippocampal place cells while MEC grid cells predominantly determine the temporal response properties of hippocampal place cells.

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Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids, including trypanosomes. Trypanosoma brucei flagella exhibit a bending wave that propagates from the flagellums tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein- associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex- type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi, rescued the knockdown with eGFP- tagged TbLC2, and quantified TbLC2s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown resulted in kinetoplast mislocalization and the formation of multiple cell clusters in cell culture. We also found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to- tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.

Author SummaryKinetoplastea is a class of ciliated protists that include parasitic trypanosomes, which cause severe disease in people and livestock in tropical regions across the globe. All trypanosomes, including Trypanosoma brucei, require a cilium to provide propulsive force for directional swimming motility, host immune evasion, and various aspects of their cell cycle. Thus, a functional cilium is essential for the virulence of the parasite.

Trypanosome cilia exhibit a unique tip-to-base beating mechanism, different from the base-to-tip beating of most other eukaryotic cilia. Multiple ciliary proteins are involved in the complex biophysical and biochemical mechanisms that underly the trypanosome ciliary beating. These include dynein motor proteins that power the beat, dynein-related light chains that regulate the beat, and many other proteins in the nexin-dynein regulatory complex, in the radial spokes, and associated with the central pair of microtubules, for example.

Here, we identify a Tctex-type dynein light chain in T. brucei that we named TbLC2 because it has sequence homology, structural similarity, and ciliary localization like LC2 homologs in other organisms. We demonstrate that TbLC2 has critical dynein regulatory functions, with implications on the unique aspects of trypanosome ciliary beating and cellular swimming motility. Our study represents an additional step toward understanding the functions of the trypanosome ciliary proteome, which could provide novel therapeutic targets against the unique aspects of trypanosome ciliary motility.

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Thiazides induce glucose intolerance through inhibition of mitochondrial carbonic anhydrase 5b in β-cells Thiazides are associated with glucose intolerance and new onset diabetes mellitus, but the molecular mechanisms remain elusive. The aim of this study was to decipher the molecular basis of thiazide-induced glucose intolerance. In mice, hydrochlorothiazide induced a pathological glucose tolerance, characterized by reduced first phase insulin secretion but normal insulin sensitivity. In vitro, thiazides inhibited glucose-and sulfonylurea-stimulated insulin secretion in islets and the murine {beta}-cell line Min6 at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO2/HCO3--dependent, not additive to unselective carbonic anhydrase (CA) inhibition with acetazolamide and independent of extracellular potassium. In contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC (SLC12A3) or NDCBE (SLC4A8). CA expression profiling with subsequent knock-down of individual CA isoforms suggested mitochondrial CA5b as molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxalacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and insulin secretion of islets isolated from CA5b KO mice was unaffected by thiazides.

In summary, our study reveals attenuated insulin secretion due to inhibition of the mitochondrial CA5b isoform in {beta}-cells as molecular mechanism of thiazide-induced glucose intolerance.

sentences: Computational analysis of the effect of SARS-CoV-2 variant Omicron Spike protein mutations on dynamics, ACE2 binding and propensity for immune escape The recently reported Omicron (B.1.1.529) SARS-CoV-2 variant has a large number of mutations in the Spike (S) protein compared to previous variants. Here we evaluate the potential effect of Omicron S mutations on S protein dynamics and ACE2 binding as contributing factors to infectivity as well as propensity for immune escape. We define a consensus set of mutations from 77 sequences assigned as Omicron in GISAID as of November 25. We create structural models of the Omicron S protein in the open and closed states, as part of a complex with ACE2 and for each of 77 complexes of S bound to different antibodies with known structures. We have previously utilized Dynamical Signatures (DS) and the Vibrational Entropy Score (VDS) to evaluate the propensity of S variants to favour the open state. Here, we introduce the Binding Influence Score (BIS) to evaluate the influence of mutations on binding affinity based on the net gain or loss of interactions within the protein-protein interface. BIS shows excellent correlation with experimental data (Pearson correlation coefficient of 0.87) on individual mutations in the ACE2 interface for the Alpha, Beta, Gamma, Delta and Omicron variants combined. On the one hand, the DS of Omicron highly favours a more rigid open state and a more flexible closed state with the largest VDS of all variants to date, suggesting a large increase in the chances to interact with ACE2. On the other hand, the BIS shows that apart from N501Y, all other mutations in the interface reduce ACE2 binding affinity. VDS and BIS show opposing effects on the overall effectiveness of Omicron mutations to promote binding to ACE2 and therefore initiate infection. To evaluate the propensity for immune escape we calculated the net change of favourable and unfavourable interactions within each S-antibody interface. The net change of interactions shows a positive score (a net increase of favourable interactions and decrease of unfavourable ones) for 41 out of 77 antibodies, a nil score for 15 and a negative score for 21 antibodies. Therefore, in only 28% of S-antibody complexes (21/77) we predict some level of immune escape due to a weakening of the interactions with Omicron S. Considering that most antibody epitopes and the mutations are within the S-ACE2 interface our results suggest that mutations within the RBD of Omicron may give rise to only partial immune escape, which comes at the expense of reduced ACE2 binding affinity. However, this reduced ACE2 affinity appears to have been offset by increasing the occupancy of the open state of the Spike protein.

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

sentences: A pigment-deficient mosquito line with fluorescent salivary glands enables in vivo imaging of individual Plasmodium sporozoites Plasmodium parasites are the causative agent of malaria, a major health burden in sub- Saharan Africa. A key step in the transmission process of Plasmodium is the colonization of the salivary glands of the female Anopheles mosquito by the parasite sporozoite stage. How sporozoites recognize and invade the salivary glands is still poorly understood. Here we generated salivary gland reporter lines in the African malaria mosquito Anopheles coluzzii using salivary gland-specific promoters of the genes encoding anopheline antiplatelet protein (AAPP), the triple functional domain protein (TRIO) and saglin (SAG). The observed expression pattern of the DsRed and roGFP2 fluorescent reporters revealed lobe-specific activity of these promoters within the salivary glands, restricted either to the distal lobes or the middle lobe. We characterized four mosquito lines (AAPP-DsRed, AAPP-roGFP2, TRIO- DsRed and SAG-EGFP) in terms of localization, expression strength and onset of expression, as well as potential influences of the expressed fluorescent reporters on the infection with Plasmodium berghei and salivary gland morphology. Furthermore, using crosses with a pigmentation deficient yellow(-) mosquito line, we demonstrate that our salivary gland reporter lines represent a valuable tool to study the process of salivary gland colonization by Plasmodium parasites in live mosquitoes.

sentences: Improved correspondence of fMRI visual field localizer data after cortex-based macroanatomical alignment Studying the visual system with fMRI often requires using localizer paradigms to define regions of interest (ROIs). However, the considerable interindividual variability of the cerebral cortex represents a crucial confound for group-level analyses. Cortex-based alignment (CBA) techniques reliably reduce interindividual macroanatomical variability. Yet, their utility has not been assessed for visual field localizer paradigms, which map specific parts of the visual field within retinotopically organized visual areas. We evaluated CBA for an attention-enhanced visual field localizer mapping homologous parts of each visual quadrant in 50 participants. We compared CBA with volume-based alignment and a surface-based analysis, which did not include macroanatomical alignment. CBA led to the strongest increase in the probability of activation overlap (up to 40 percent). On the group level, CBA led to the most consistent increase in ROI size while preserving vertical ROI symmetry. Overall, our results indicate, that in addition to the increased signal-to-noise ratio of a surface-based analysis macroanatomical alignment considerably improves statistical power. These findings confirm and extend the utility of CBA for the study of the visual system in the context of group analyses. CBA should be particularly relevant when studying neuropsychiatric disorders with abnormally increased interindividual macroanatomical variability.

sentences: The landscape of extrachromosomal circular DNA in medulloblastoma Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive tumor growth, promoting high oncogene copy number, intratumoral heterogeneity, accelerated evolution of drug resistance, enhancer rewiring, and poor outcome. ecDNA has been reported in medulloblastoma (MB), the most common malignant pediatric brain tumor, but the ecDNA landscape and its association with specific MB subgroups, its impact on enhancer rewiring, and its potential clinical implications, are not known. We assembled a retrospective cohort of 468 MB patient samples with available whole genome sequencing (WGS) data covering the four major MB subgroups WNT, SHH, Group 3 and Group 4. Using computational methods for the detection and reconstruction of ecDNA1, we find ecDNA in 82 patients (18%) and observe that ecDNA+ MB patients are more than twice as likely to relapse and three times as likely to die of disease. In addition, we find that individual medulloblastoma tumors often harbor multiple ecDNAs, each containing different amplified oncogenes along with co-amplified non-coding regulatory enhancers. ecDNA was substantially more prevalent among 31 analyzed patient-derived xenograft (PDX) models and cell lines than in our patient cohort. By mapping the accessible chromatin and 3D conformation landscapes of MB tumors that harbor ecDNA, we observe frequent candidate "enhancer rewiring" events that spatially link oncogenes with co-amplified enhancers. Our study reveals the frequency and diversity of ecDNA in a subset of highly aggressive tumors and suggests enhancer rewiring as a frequent oncogenic mechanism of ecDNAs in MB. Further, these results demonstrate that ecDNA is a frequent and potent driver of poor outcome in MB patients.

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Which lower-limb joints compensate for destabilising energy during walking in humans Maintaining stability during perturbed locomotion requires coordinated responses across multiple levels of organization (e.g., legs, joints, muscle-tendon units). However, current approaches to investigating such responses lack a "common currency" that is both shared across scales and can be directly related to perturbation demands. We used mechanical energetics to investigate the demands imposed on a leg by a transient increase in unilateral treadmill belt speed targeted to either early or late stance. We collected full body kinematics and kinetics from 7 healthy participants during 222 total perturbations. From across-subject means, we found early stance perturbations elicited no change in net work exchanged between the perturbed leg and the treadmill but net positive work at the overall leg level, and late stance perturbations elicited positive work at the leg/treadmill interface but no change in net work at the overall leg level. Across all perturbations, changes in ankle and knee work from steady state best reflected changes in overall leg work on the perturbed and contralateral sides, respectively. Broadening this paradigm to include joint level (vs. leg level) perturbations and including muscle-tendon unit mechanical energetics may reveal neuromechanical responses used in destabilizing environments which could inform design of balance-assisting devices and interventions.

Subject Areasbiomechanics, biomedical engineering, bioengineering

sentences: Facilitating population genomics of non-model organisms through optimized experimental design for reduced representation sequencing Genome-wide data are invaluable to characterize differentiation and adaptation of natural populations. Reduced representation sequencing (RRS) subsamples a genome repeatedly across many individuals. However, RRS requires careful optimization and fine-tuning to deliver high marker density while being cost-efficient. The number of genomic fragments created through restriction enzyme digestion and the sequencing library setup must match to achieve sufficient sequencing coverage per locus. Here, we present a workflow based on published information and computational and experimental procedures to investigate and streamline the applicability of RRS. In an iterative process genome size estimates, restriction enzymes and size selection windows were tested and scaled in six classes of Antarctic animals (Ostracoda, Malacostraca, Bivalvia, Asteroidea, Actinopterygii, Aves). Achieving high marker density would be expensive in amphipods, the malacostracan target taxon, due to the large genome size. We propose alternative approaches such as mitogenome or target capture sequencing for this group. Pilot libraries were sequenced for all other target taxa. Ostracods, bivalves, sea stars, and fish showed overall good coverage and marker numbers for downstream population genomic analyses. In contrast, the bird test library produced low coverage and few polymorphic loci, likely due to degraded DNA. Prior testing and optimization are important to identify which groups are amenable for RRS and where alternative methods may currently offer better cost-benefit ratios. The steps outlined here are easy to follow for other non-model taxa with little genomic resources, thus stimulating efficient resource use for the many pressing research questions in molecular ecology.

sentences: SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks progress slowly in cells depleted for SAF-A, also contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed that the boundaries between early- and late-replicating domains are blurred in cells depleted for SAF-A. Associated with these defects, SAF-A-depleted cells show elevated {gamma}H2A phosphorylation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

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Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants ability to potentially infect different animals.

ImportanceSARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

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A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. While GDP-Fucose synthesis is essential (Guo et al 2017), fucosylated glycoconjugates have not been reported in Leishmania major. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted  1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative  plasmid segregation assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null {Delta}fut1- background, established that FUT1 is essential. Similarly "plasmid shuffling" confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly a single rare {Delta}fut1-s segregant ({Delta}fut1s) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (Bandini et al 2021) joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting and their essentiality renders it an attractive target for chemotherapy of these serious human pathogens.

Significance StatementAbundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites including Leishmania. Through defining biosynthetic pathways we identified a fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical compartment for glycosyltransferases. FUT1 was essential for normal growth, requiring both mitochondrial localization and enzymatic activity. Loss of FUT1 in a unique segregant showed extensive mitochondrial defects. Enzymatic tests showed FUT1 could fucosylate glycan and peptide substrates in vitro, although as yet the native substrate is unknown. Trypanosomatid mitochondrial FUT1s may offer a facile system in the future for probing mitochondrial glycosylation in a setting uncomplicated by multiple isoforms targeted to diverse compartments, and its essentiality renders it an attractive target for chemotherapy of these deadly parasites.

sentences: Cross-modal Semantic Relationships Guide Spontaneous Orienting in Real-life Scenes In real-world scenes, the different objects and events available to our senses are interconnected within a rich web of semantic associations. These semantic links help parse information and make sense of the environment. For example, during goal-directed attention, characteristic everyday life object sounds help speed up visual search for these objects in natural and dynamic environments. However, it is not known whether semantic correspondences also play a role under spontaneous observation. Here, we investigated this question addressing whether crossmodal semantic congruence can drive spontaneous, overt visual attention in free-viewing conditions. We used eye-tracking whilst participants (N=45) viewed video clips of realistic complex scenes presented alongside sounds of varying semantic congruency with objects within the videos. We found that characteristic sounds increased the probability of looking, the number of fixations, and the total dwell time on the semantically corresponding visual objects, in comparison to when the same scenes were presented with semantically neutral sounds or just with background noise only. Our results suggest that crossmodal semantic congruence has an impact on spontaneous gaze and eye movements, and therefore on how attention samples information in a free viewing paradigm. Our findings extend beyond known effects of object-based crossmodal interactions with simple stimuli and shed new light upon how audio-visual semantically congruent relationships play out in everyday life scenarios.

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

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

sentences: Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore sequencing RNA polyadenylation plays a central role in RNA maturation, fate and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translatability and stability of mRNAs. Here we develop Nano3P-seq, a novel method that relies on nanopore sequencing to simultaneously quantify RNA abundance, tail composition and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3end, regardless of its polyadenylation status, without the need of PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide diversity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNAs, lncRNAs, sn/snoRNAs, scaRNAs and rRNAs. We find that, in addition to mRNAs and lncRNAs, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at the isoform-specific level, correlating with mRNA decay. Overall, Nano3P-seq is a simple and robust method to accurately estimate transcript levels and tail lengths in full-length individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

sentences: Dissecting Response to Cancer Immunotherapy by Applying Bayesian Network Analysis to Flow Cytometry Data Cancer immunotherapy, specifically immune checkpoint blockade therapy, has been found to be effective in the treatment of metastatic cancers. However, only a subset of patients achieve clinical responses. Consequently, elucidating immune system-related pre-treatment biomarkers that are predictive with respect to sustained clinical response is a major research priority. Another research priority is evaluating changes in the immune system before and after treatment in responders and non-responders. Our group has been studying immune signaling networks as an accurate reflection of the global immune state. Flow cytometry (FACS, Fluorescence-activated cell sorting) data characterizing immune signaling in peripheral blood mononuclear cells (PBMC) from gastroesophageal adenocarcinoma (GEA) patients were used to analyze changes in immune signaling networks in this setting. Here, we describe a novel computational pipeline developed by us to perform secondary analyses of FACS data using systems biology / machine learning techniques and concepts. It is centered around comparative Bayesian network analyses of immune signaling networks and is capable of detecting true positive signals that conventional methods (such as FlowJo manual gating) might miss. Future studies are planned to validate and follow up immune markers (and combinations / interactions thereof) associated with clinical responses that were identified by this computational pipeline.

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Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without S, M, E genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs.

IMPORTANCESARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. This achievement represents a ground-breaking discovery that will greatly accelerate the pace of developing treatments for COVID-19.

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Transfer learning improves outcome predictions for ASD from gene expression in blood BackgroundPredicting outcomes on human genetic studies is difficult because the number of variables (genes) is often much larger than the number of observations (human subject tissue samples). We investigated means for improving model performance on the types of under-constrained problems that are typical in human genetics, where the number of strongly correlated genes (features) may exceed 10,000, and the number of study participants (observations) may be limited to under 1,000.

MethodsWe created  train,  validate and  test datasets from 240 microarray observations from 127 subjects diagnosed with autism spectrum disorder (ASD) and 113  typically developing (TD) subjects. We trained a neural network model (a.k.a., the  naive model) on 10,422 genes using the  train dataset, composed of 70 ASD and 65 TD subjects, and we restricted the model to one, fully-connected hidden layer to minimize the number of trainable parameters, including a dropout layer to help prevent overfitting. We experimented with alternative network architectures and tuned the hyperparameters using the  validate dataset, and performed a single, final evaluation using the holdout  test dataset. Next, we trained a neural network model using the identical architecture and identical genes to predict tissue type in GTEx data. We transferred that learning by replacing the top layer of the GTEx model with a layer to predict ASD outcome and we retrained the new layer on the ASD dataset, again using the identical 10,422 genes.

FindingsThe  naive neural network model had AUROC=0.58 for the task of predicting ASD outcomes, which saw a statistically significant 7.8% improvement from transfer learning.

InterpretationWe demonstrated that neural network learning could be transferred from models trained on large RNA-Seq gene expression to a model trained on a small, microarray gene expression dataset with clinical utility for mitigating over-training on small sample sizes. Incidentally, we built a highly accurate classifier of tissue type with which to perform the transfer learning.

FundingThis work was supported in part by NIMH R01-MH110558 (E.C., N.E.L.)

Author SummaryImage recognition and natural language processing have enjoyed great success in reusing the computational efforts and data sources to overcome the problem of over-training a neural network on a limited dataset. Other domains using deep learning, including genomics and clinical applications, have been slower to benefit from transfer learning. Here we demonstrate data preparation and modeling techniques that allow genomics researchers to take advantage of transfer learning in order to increase the utility of limited clinical datasets. We show that a non-pre-trained,  naive model performance can be improved by 7.8% by transferring learning from a highly performant model trained on GTEx data to solve a similar problem.

sentences: Epigenetic regulation of translation repression in ferroptosis, and a role of Alternative splicing and tRNA methylation. Ferroptosis is a non-apoptotic cell death mechanism characterized by the production of lipid peroxides. Ferroptosis plays important roles in many diseases such as cancer and neurodegenerative diseases. While many effectors in the ferroptosis pathway have been mapped, its epigenetic and epitranscriptional regulatory processes are not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to Class I and Class II ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. Understanding these subtle differences is important in understanding ferroptosis as well as in developing therapeutics based on ferroptosis for various diseases. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin and the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed an important and unexplored role of Alternative splicing (AS) in regulating ferroptosis stress response and mRNA translation repression. Our results indicated that translation repression is protective against Erastin but detrimental against RSL3. We tested this theory in Alkbh1 overexpressing glioma cells. Alkbh1 demethylates tRNA and represses translation and is associated with worse outcome in glioma patients. Our results showed that Alkbh1 overexpression protected glioma cells against Erastin but was detrimental against RSL3.

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Dependable Algorithm for Visualizing Snoring Duration through Acoustic Analysis BackgroundSnoring is a nuisance for the bed partners of people who snore and is also associated with chronic diseases. Estimating the snoring duration from a whole-night-sleep period is challenging. The authors present a dependable algorithm for visualizing snoring durations through acoustic analysis.

MethodBoth instruments (Sony digital recorder and smartphones SnoreClock app) were placed within 30 cm from the examinees head during the sleep period. Subsequently, spectrograms were plotted based on audio files recorded from Sony recorders. The authors developed an algorithm to validate snoring durations through visualization of typical snoring segments.

ResultsIn total, 37 snoring recordings obtained from six individuals were analyzed. The mean age of the participants was 44.6 {+/-} 9.9 years. A 3-s segment demonstrated the typical dominant frequency bands and amplitude waves of two snores. Every recorded file was tailored to a regular 600-s segment and plotted. Visualization revealed that the typical features of the clustered snores in the amplitude domains were near-isometric spikes (most had an ascending-descending trend). The recorded snores exhibited one or more visibly fixed frequency bands. Intervals were noted between the snoring clusters and were incorporated into the whole-night snoring calculation. The correlative coefficients of snoring rates of digitally recorded files examined by Examiners A and B were higher (0.865, p < 0.001) than those with SnoreClock app (0.757, p < 0.001; 0.787, p < 0.001, respectively).

ConclusionA dependable algorithm with high reproducibility was developed for visualizing snoring durations.

sentences: Confrontation of AlphaFold2 models with cryo-EM and crystal structures enlightens alternate geometries of the CYP102A1 multidomain protein Large range structural dynamics plays a critical role for the function of electron transfer proteins. This information is generally not available from crystallographic structures, while cryo-electron microscopy (cryo-EM) can provide some elements but frequently with a degraded spatial resolution. Recently, AlphaFold-based structural modelling was extended to the prediction of protein complexes. In this work, bacterial CYP102A1 from Priestia megaterium was used as a test case to evaluate the capability of AlphaFold2 to predict alternative structures critical for catalysis. CYP102A1 monooxygenase, a NADPH-supported fatty acid hydroxylase, works as a soluble homodimer, each monomer harboring two flavins (FAD and FMN) and one heme cofactors. Large conformational changes are required during catalytic cycle to allow successive electron transfers from FAD to FMN and finally heme iron. We used the recently released AlphaFold2_advanced notebook (AF2A), to predict the possible alternate conformations supporting electron transfers in CYP102A1 homodimer. Challenging AF2A-derived models with previously reported experimental data revealed an unforeseen domain connectivity of the diflavin reductase part of the enzyme. Intermolecular crossed complex constitutes a novel type of structural organization never previously described. The predicted formation within the dimer of a stable complex between the heme containing domains was challenged and found consistent with uninterpreted features of reported crystallographic structures and cryo-EM imaging. The particularly efficient CYP102A1 catalytic mechanism was revisited to the light of the new evidenced connectivity in which the FMN-binding domain of each monomer oscillates on themselves to alternatively receive and transfer electrons without needing large structural change in the dimer. Such model was found explanatory for previously contradictory reported biochemical data. Possibility to mimic CYP102A1 structural organization into bicomponent eukaryotic P450 systems was evaluated by designing and modeling in silico synthetic reductase domains built from composite sequence segments from P. megaterium and human origins. More generally, this work illustrates how the ability of AF2A to predict alternate complex structures can enlighten and explain conformational changes critical for bio-assemblies.

sentences: Long-term ketogenic diet increases expression of SOX2-dependent oligodendrocyte- and myelination-associated genes in the aged mouse brain. Aging is associated with multiple neurodegenerative conditions that severely limit quality of life and shorten lifespan. Studies in rodents indicate that in addition to extending lifespan, the ketogenic diet improves cognitive function in aged animals, thus improving healthspan. To broadly investigate what mechanisms might be activated in the brain in response to ketogenic diet, we conducted transcriptome wide analysis on whole brain samples from 13-month-old mice, 26-month-old mice, and 26-month-old mice fed a ketogenic diet. We observed clear activation of inflammation and complement system pathways in the 26-month-old mice relative to the younger animals. Interestingly, ketogenic diet caused a modest but significant increase in the expression of SOX2-dependent oligodendrocyte/myelination markers.

sentences: Toward Quantitative Metabarcoding Amplicon-sequence data from environmental DNA (eDNA) and microbiome studies provides important information for ecology, conservation, management, and health. At present, amplicon-sequencing studies - known also as metabarcoding studies, in which the primary data consist of targeted, amplified fragments of DNA sequenced from many taxa in a mixture - struggle to link genetic observations to underlying biology in a quantitative way, but many applications require quantitative information about the taxa or systems under scrutiny. As metabarcoding studies proliferate in ecology following decades of microbial and microbiome work using similar techniques, it becomes more important to develop ways ot make them quantitative to ensure that their conclusions are adequately supported. Here we link previously disparate sets of techniques for making such data quantitative, showing that the underlying PCR mechanism explains observed patterns of amplicon data in a general way. By modeling the process through which amplicon-sequence data arises, rather than transforming the data post-hoc, we show how to estimate the starting DNA proportions from a mixture of many taxa. We illustrate how to calibrate the model using mock communities and apply the approach to simulated data and a series of empirical examples. Our approach opens the door to improve the use of metabarcoding data in a wide range of applications in ecology, public health, and related fields.

sentences: Accurate prediction of functional states of cis-regulatory modules reveals the common epigenetic rules in humans and mice We proposed a two-step approach for predicting active cis-regulatory modules (CRMs) in a cell/tissue type. We first predict a map of CRM loci in the genome using all available transcription factor binding data in the organism, and then predict functional states of all the putative CRMs in any cell/tissue type using few epigenetic marks. We have recently developed a pipeline dePCRM2 for the first step, and now presented machine-learning methods for the second step. Our approach substantially outperforms existing methods. Our results suggest common epigenetic rules for defining functional states of CRMs in various cell/tissue types in humans and mice.

sentences: The Landscape of Receptor-Mediated Precision Cancer Combination Therapy: A Single-Cell Perspective The availability of single-cell transcriptomics data opens new opportunities for rational design of combination cancer treatments. Mining such data, we employed combinatorial optimization techniques to explore the landscape of optimal combination therapies in solid tumors including brain, head and neck, melanoma, lung, breast and colon cancers. We assume that each individual therapy can target any one of 1269 genes encoding cell surface receptors, which may be targets of CAR-T, conjugated antibodies or coated nanoparticle therapies. As a baseline case, we studied the killing of at least 80% of the tumor cells while sparing more than 90% of the non-tumor cells in each patient, as a putative regimen. We find that in most cancer types, personalized combinations composed of at most four targets are then sufficient. However, the number of distinct targets that one would need to assemble to treat all patients in a cohort accordingly would be around 10 in most cases. Further requiring that the target genes be also lowly expressed in healthy tissues uncovers qualitatively similar trends. However, as one asks for more stringent and selective killing beyond the baseline regimen we focused on, we find that the number of targets needed rises rapidly. Emerging individual promising receptor targets include PTPRZ1, which is frequently found in the optimal combinations for brain and head and neck cancers, and EGFR, a recurring target in multiple tumor types. In sum, this systematic single-cell based characterization of the landscape of combinatorial receptor-mediated cancer treatments establishes first of their kind estimates on the number of targets needed, identifying promising ones for future development.

sentences: In silico assessment of missense point mutations on human cathelicidin LL-37 Cathelicidin antimicrobial peptides are a diverse family of cationic amphipathic peptides with multiple activities. In humans, cathelicidin LL-37 is one of the main host defense peptides with a remarkable medical and biotechnological potential. Deregulation of LL-37 expression has been associated with inflammatory diseases. However the effects of point mutations driven by single nucleotide polymorphisms (SNPs) on LL-37 are unknown. Here we applied an array of computational tools to investigate the effects of such mutations on LL-37 structure and activity. Due to the fact that, on cathelicidins, the prodomain is more conserved than the mature peptide, the SNP effect predictions were biased and, overall, resulted in neutral effects; and due to the slight changes in physicochemical properties, the antimicrobial predictions indicated the maintenance of such activity. Nonetheless, six out of eleven mutations altered the positive net charge, which in turn could result in less active LL-37 variants. Molecular dynamics data indicated that R07Q and N30Y mutations altered the LL-37 structure. In addition to these ones, the G03A, R07P, R07W and L31P mutations altered the helix dipole, which could also alter the antimicrobial activity. Our results indicated despite the mutations did not alter the residues from LL-37 active core, they could influence the antimicrobial activity and consequently, could be involved in inflammatory diseases.

sentences: Restricted maximum-likelihood method for learning latent variance components in gene expression data with known and unknown confounders Random effect models are popular statistical models for detecting and correcting spurious sample correlations due to hidden confounders in genome-wide gene expression data. In applications where some confounding factors are known, estimating simultaneously the contribution of known and latent variance components in random effect models is a challenge that has so far relied on numerical gradient-based optimizers to maximize the likelihood function. This is unsatisfactory because the resulting solution is poorly characterized and the efficiency of the method may be suboptimal. Here we prove analytically that maximum-likelihood latent variables can always be chosen orthogonal to the known confounding factors, in other words, that maximum-likelihood latent variables explain sample covariances not already explained by known factors. Based on this result we propose a restricted maximum-likelihood method which estimates the latent variables by maximizing the likelihood on the restricted subspace orthogonal to the known confounding factors, and show that this reduces to probabilistic PCA on that subspace. The method then estimates the variance-covariance parameters by maximizing the remaining terms in the likelihood function given the latent variables, using a newly derived analytic solution for this problem. Compared to gradient-based optimizers, our method attains greater or equal likelihood values, can be computed using standard matrix operations, results in latent factors that dont overlap with any known factors, and has a runtime reduced by several orders of magnitude. Hence the restricted maximum-likelihood method facilitates the application of random effect modelling strategies for learning latent variance components to much larger gene expression datasets than possible with current methods.

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Relict groups of spiny frogs indicate Late Paleogene-Early Neogene trans-Tibet dispersal of thermophile faunal elements BackgroundThe Himalaya-Tibet orogen (HTO) presents an outstanding geologically active formation that contributed to, and fostered, modern Asian biodiversity. However, our concepts of the historical biogeography of its biota are far from conclusive, as are uplift scenarios for the different parts of the HTO. Here, we revisited our previously published data set of the tribe Paini extending it with sequence data from the most western Himalayan spiny frogs Allopaa and Chrysopaa and using them as an indirect indicator for the paleoecological development of Tibet.

MethodsWe obtained sequence data of two mitochondrial loci (16S rRNA, COI) and one nuclear marker (Rag1) from Allopaa samples from Kashmir Himalaya as well as Chrysopaa sequence data from the Hindu Kush available from GenBank to complement our previous data set. A Maximum likelihood and dated Bayesian gene tree were generated based on the concatenated data set. To resolve the inconsistent placement of Allopaa, we performed different topology tests.

ResultsConsistent with previous results, the Southeast Asian genus Quasipaa is sister to all other spiny frogs. The results further reveal a basal placement of Chrysopaa relative to Allopaa and Nanorana with an estimated age of ca. 26 Mya. Based on the topology tests, the phylogenetic position of Allopaa as a sister clade to Chaparana seems to be most likely, resulting in a paraphyletic genus Nanorana and a separation from the latter clade around 20 Mya. Both, the placements of Chrysopaa and Allopaa support the presence of basal Paini lineages in the far north western part of the HTO, which is diametrically opposite end of the HTO with respect to the ancestral area of spiny frogs in Southeast Asia. These striking distributional patterns can be most parsimoniously explained by trans-Tibet dispersal during the late Oligocene (subtropical Chrysopaa) respectively early Miocene (warm temperate Allopaa). Within spiny frogs, only members of the monophyletic Nanorana+Paa clade are adapted to the colder temperate climates, indicating that high-altitude environments did not dominate in the HTO before ca. 15 Mya. Our results are consistent with fossil records suggesting that large parts of Tibet were characterized by subtropical to warm temperate climates at least until the early Miocene.

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Sawfish, Read in Tooth and Saw: rostral teeth as endogenous chemical records of movement and life-history in a critically endangered species O_LIThe ecology of endangered and rare species can be difficult to study due to their low abundances and legal limits on scientists ability to catch, sample, and track them. This is particularly true of sawfish (family Pristidae) whose numbers have declined precipitously, placing all five species on the IUCN Red List of Threatened Species worldwide. Best known for their distinctive, toothed rostrum, the ecology, movement, and life-history of sawfish is poorly understood.
C_LIO_LISawfish rostral teeth are modified placoid scales, which grow continuously throughout the life of the fish. This continuous growth, combined with their stable calcified makeup, makes sawfish rostral teeth a potential source of temporal records of chemical and isotopic changes through the life of the fish.
C_LIO_LIRostral teeth are often preserved in museums and as curios, potentially providing a source of life-history data to inform conservation actions without the need for field study, or as an important compliment to it. This is the first study to recover temporally explicit chemical data from sawfish rostral teeth.
C_LIO_LIUsing archived samples of largetooth sawfish (Pristis pristis) we show that multiple chemical tracers can be recovered from sawfish rostral teeth, and that these tracers can be used to understand movement across salinity gradients. We further show that sawfish rostral teeth contain repeated structures and indistinct banding which could potentially be used for aging or growth analysis of fish.
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sentences: Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

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PDXNet Portal: Patient-Derived Xenograft model, data, workflow, and tool discovery We created the PDX Network (PDXNet) Portal (https://portal.pdxnetwork.org/) to centralize access to the National Cancer Institute-funded PDXNet consortium resources (i.e., PDX models, sequencing data, treatment response data, and bioinformatics workflows), to facilitate collaboration among researchers, and to make resources easily available for research. The portal includes sections for resources, analysis results, metrics for PDXNet activities, data processing protocols, and training materials for processing PDX data.

The initial portal release highlights PDXNet model and data resources, including 334 new models across 33 cancer types. Tissue samples of these models were deposited in the NCIs Patient-Derived Model Repository (PDMR) for public access. These models have 2,822 associated sequencing files from 873 samples across 307 patients, which are hosted on the Cancer Genomics Cloud powered by Seven Bridges and the NCI Cancer Data Service for long-term storage and access with dbGaP permissions. The portal also includes results from standardized analysis workflows on PDXNet sequencing files and PDMR data (2,594 samples from 463 patients across 78 disease types). These 15 analysis workflows for whole-exome and RNA-Seq data are freely available, robust, validated, and standardized.

The model and data lists will grow substantially over the next two years and will be continuously updated as new data are available. PDXNet models support multi-agent treatment studies, determination of sensitivity and resistance mechanisms, and preclinical trials. The PDXNet portal is a centralized location for these data and resources, which we expect to be of significant utility for the cancer research community.

sentences: Electronic nicotine delivery systems exhibit reduced bronchial epithelial cells toxicity compared to cigarette: The Replica Project Cigarette smoking is the leading cause of preventable deaths worldwide. Electronic Nicotine Delivery Systems (ENDS) may reduce health risks associated with chronic exposure to smoke and their potential benefits have been the matter of intense scientific debate. Here we replicated three key published studies from the Tobacco Industry on cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol in an independent multicentric study. We aimed to establish the reliability of results and the robustness of conclusions by replicating the authors experimental protocols and further validating them with different techniques. We exposed human bronchial epithelial cell (NCI-H292) to cigarette smoke and to aerosol from ENDS. All the exposure were conducted at air-liquid interface to assess cytotoxicity effects of smoke and aerosol. Moreover, we aimed to assess different inflammatory mediators release (IL-6, IL-8 and MMP-1) from cells exposed to whole smoke and to smoke without particulate matter (vapor phase). We were able to replicate the results obtained in the original studies on cytotoxicity confirming that almost 80% of the cytotoxic effect of smoke is due to the vapor phase of smoke. Moreover, our results substantiated the reduced cytotoxic effects of ENDS aerosol in respect to cigarette smoke. However, our data are significantly different from the original ones in terms of inflammatory and remodeling activity triggered by smoke. Taken all together, the data obtained independently in different laboratories clearly demonstrate the reduced toxicity of ENDS products compared to cigarettes and thus providing a valuable tool to the harm reduction strategies in smokers.

sentences: Flexible utilization of spatial- and motor-based codes for the storage of visuo-spatial information Working memory provides flexible storage of information in service of upcoming behavioral goals. Some models propose specific fixed loci and mechanisms for the storage of visual information in working memory, such as sustained spiking in parietal and prefrontal cortex during working memory maintenance. An alternative view is that information can be remembered in a flexible format that best suits current behavioral goals. For example, remembered visual information might be stored in sensory areas for easier comparison to future sensory inputs, or might be re-coded into a more abstract action-oriented format and stored in motor areas. Here, we tested this hypothesis using a visuo-spatial working memory task where the required behavioral response was either known or unknown during the memory delay period. Using fMRI and multivariate decoding, we found that there was less information about remembered spatial position in early visual and parietal regions when the required response was known versus unknown. Further, a representation of the planned motor action emerged in primary somatosensory, primary motor, and premotor cortex during the same task condition where spatial information was reduced in early visual cortex. These results suggest that the neural networks supporting working memory can be strategically reconfigured depending on specific behavioral requirements during a canonical visual working memory paradigm.

sentences: Functional brain network community structure in childhood: Unfinished territories and fuzzy boundaries Adult cortex is organized into distributed functional communities. Yet, little is known about community architecture of childrens brains. Here, we uncovered the community structure of cortex in childhood using fMRI data from 670 children aged 9-11 years from the Adolescent Brain and Cognitive Development study. Children showed similar community structure to adults in early-developing sensory and motor communities, but differences emerged in transmodal areas. Children have more cortical territory in the limbic community, which is involved in emotion processing, than adults. Regions of association cortex interact more flexibly across communities, creating uncertainty for the model-based assignment algorithm, and perhaps reflecting cortical boundaries that are not yet solidified. Uncertainty was highest for cingulo-opercular areas involved in flexible deployment of cognitive control. Collectively, our findings suggest that community boundaries are not solidified by middle childhood, an instability that provides important context for childrens thoughts and behaviors.

sentences: Spatial working memory representations in human cortex are robust to a task-irrelevant interrupting stimulus Visual working memory (WM) enables the maintenance and manipulation of information no longer accessible in the visual world. Previous research has identified spatial WM representations in activation patterns in visual, parietal, and frontal cortex. In natural vision, the period between the encoding of information into WM and the time when it is used to guide behavior (the delay period) is rarely "empty", as is the case in most of the above laboratory experiments. In naturalistic conditions, eye movements, movement of the individual, and events in the environment result in visual signals which may overwrite or impair the fidelity of WM representations, especially in early sensory cortices. Here, we evaluated the extent to which a brief, irrelevant interrupting visual stimulus presented during a spatial WM delay period impaired behavioral performance and WM representation fidelity assayed using an image reconstruction technique (inverted encoding model). On each trial, participants (both sexes) viewed two target dots and were immediately post-cued to remember the precise spatial position of one dot. On 50% of trials, a brief interrupter stimulus appeared. While we observed strong transient univariate visual responses to the distracter stimulus, we saw no change in reconstructed neural WM representations under distraction, nor a change in behavioral performance on a continuous recall task. This suggests that spatial WM representations may be particularly robust to interference from incoming task-irrelevant visual information, perhaps related to their role in guiding movements.

sentences: Autoimmune Alleles at the Major Histocompatibility Locus Modify Melanoma Susceptibility Autoimmunity and cancer represent two different aspects of immune dysfunction. Autoimmunity is characterized by breakdowns in immune self-tolerance, while impaired immune surveillance can allow for tumorigenesis. The class I major histocompatibility complex (MHC-I), which displays derivatives of the cellular peptidome for immune surveillance by CD8+ T cells, serves as a common genetic link between these conditions. As melanoma-specific CD8+ T-cells have been shown to target melanocyte-specific peptide antigens more often than melanoma-specific antigens, we investigated whether vitiligo and psoriasis predisposing MHC-I alleles conferred a melanoma protective effect. In individuals with cutaneous melanoma from both The Cancer Genome Atlas (N = 451) and an independent validation cohort (N = 586), MHC-I autoimmune allele carriers are significantly associated with a later age of melanoma diagnosis. MHC-I autoimmune allele contributions to melanoma risk are not captured by current polygenic risk scores (PRS) and incorporation of autoimmune MHC allele presence can improve relative risk stratification. Mechanisms of autoimmune protection were neither associated with improved melanoma-driver mutation association nor improved gene-level conserved antigen presentation relative to common alleles (population frequency [≥] 1%). However, autoimmune alleles showed a marked affinity relative to common alleles for particular windows of melanocyte conserved antigens suggesting a potential relationship between antigen processing, binding, and cell-surface presentation. Overall, this study presents evidence that MHC-I autoimmune risk alleles modulate melanoma risk currently unaccounted for by current PRS.

sentences: Host- and age-dependent transcriptional changes in Mycobacterium tuberculosis cell envelope biosynthesis genes after exposure to human alveolar lining fluid Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

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GATA4 and GATA6 loss-of-expression is associated with extinction of the classical programme and poor outcome in pancreatic ductal adenocarcinoma ObjectiveGATA6 is a master regulator of pancreatic differentiation and a key regulator of the classical phenotype in pancreatic ductal adenocarcinoma (PDAC). Low GATA6 expression is associated with poor patient outcome. GATA4 is the second most expressed GATA factor in the pancreas. The aim was to assess whether, and how, GATA4 contributes to PDAC phenotype and to analyze the association of expression with clinical outcome.

DesignWe analyzed PDAC transcriptomic data, stratifying cases according to GATA4 and GATA6 expression, and identified differentially expressed genes and pathways. A multicenter TMA study to assess GATA4 and GATA6 expression in PDAC samples (n=745) from patients undergoing tumour resection was performed using immunohistochemistry with antibodies of validated specificity. GATA4 and GATA6 levels were dichotomized into high/low categorical variables; association with outcome was assessed using univariable and multivariable Cox regression models.

ResultsSubtype classification using transcriptomic data revealed that GATA4 mRNA is enriched in classical, compared to basal-like tumours. We classified samples in 4 groups as high/low for GATA4 and GATA6. Reduced expression of GATA4 did not have a major transcriptional impact. However, concomitant low expression of GATA4 enhanced the transcriptomic effects of GATA6 low expression. Reduced expression of both proteins in tumours was associated with the worst patient survival. GATA4 and GATA6 expression significantly decreased in metastases and negatively correlated with basal markers.

ConclusionsOur analyses uncover a cooperative interaction between GATA4 and GATA6 to maintain the classical PDAC phenotype and provide compelling clinical rationale for assessing their expression as biomarkers of poor prognosis.

SUMMARY BOXO_ST_ABSWhat is already known about this subject?C_ST_ABS- Patients with classical-type PDAC have a better outcome
- Retrospective analyses suggest that classical-type PDAC is more sensitive to 5-FU-based chemotherapy
- GATA6 is a surrogate biomarker of classical tumours and its expression is associated with better survival
- GATA4 and GATA6 have overlapping and unique functions during pancreatic and gastrointestinal development

What are the new findings?- Tumours displaying only low GATA4 expression have a transcriptomic profile similar to those with preserved expression of both transcription factors
- Combined low expression of GATA4 and GATA6 has the highest transcriptomic impact
- In a large multicenter tissue microarray study, patients with tumours showing low expression of both GATA4 and GATA6 have the worst overall survival
- Low expression of GATA4 and GATA6 is an independent predictor of survival in patients with resectable PDAC
- GATA4 levels are down-regulated in liver metastases and are negatively correlated with basal markers such as KRT5/6, KRT14, and TP63

How might it impact on clinical practice in the foreseeable future?- The combined assessment of GATA4 and GATA6 expression may improve prognostic stratification of patients with PDAC
- Prospective studies should confirm whether GATA4 and GATA6 expression is also predictive of response to chemotherapy in PDAC patients

sentences: A reduced-memory multi-layer perceptron withsystematic network weights generated and trainedthrough distribution hyper-parameters A multi-layer perceptron (MLP) consists of a number of forward-connected weights (Wijk) from each feeding layer node (nij) to the many initially equivalent nodes (ni+1,k) in the next layer. Exact a priori order and search space of these weights (Wijk) is random and prone to redundancy, irreproducibility and non-optimality. We demonstrate that a weight subspace (Wijk for each i and j), generated systematically using a statistical distribution with predefined breakpoints and Genetic algorithm-trained hyper-parameters substantially reduces the computational complexity of an MLP and produces comparable or better performance than similarly trained equivalent models with fully defined weights. This distribution based neural network (DBNN) provides a novel framework to create very large neural network models with currently prohibitive memory requirements.

sentences: Assessing and enhancing foldability in designed proteins Recent advances in protein-design methodology have led to a dramatic increase in reliability and scale. With these advances, dozens and even thousands of designed proteins are automatically generated and screened. Nevertheless, the success rate, particularly in design of functional proteins, is low and fundamental goals such as reliable de novo design of efficient enzymes remain beyond reach. Experimental analyses have consistently indicated that a major reason for design failure is inaccuracy and misfolding relative to the design model. To address this challenge, we describe complementary methods to diagnose and ameliorate suboptimal regions in designed proteins: first, we develop a Rosetta atomistic computational mutation scanning approach to detect energetically suboptimal positions in designs (available on a web server https://pSUFER.weizmann.ac.il); second, we demonstrate that AlphaFold2 ab initio structure prediction flags regions that may misfold in designed enzymes and binders; and third, we focus FuncLib design calculations on suboptimal positions in a previously designed low-efficiency enzyme improving its catalytic efficiency by 330 fold. Furthermore, applied to a de novo designed protein that exhibited limited stability, the same approach markedly improved stability and expressibility. Thus, foldability analysis and enhancement may dramatically increase the success rate in design of functional proteins.

sentences: Bacterial growth in multicellular aggregates leads to the emergence of complex lifecycles In response to environmental stresses such as starvation, many bacteria facultatively aggregate into multicellular structures that can attain new metabolic functions and behaviors. Despite the ubiquity and relevance of this form of collective behavior, we lack an understanding of how the spatiotemporal dynamics of aggregate development emerge from cellular physiology. Here, we show that the coupling between growth and spatial gradient formation leads to the emergence of a complex lifecycle, akin to those known for multicellular bacteria. Under otherwise carbon-limited growth conditions, the marine bacterium Vibrio splendidus 12B01 forms multicellular groups to collectively harvest carbon from the brown-algal polysaccharide alginate. This is achieved during growth on dissolved alginate polymer through formation of spherical, clonal clusters of cells that grow up to 40 {micro}m in radius. Clusters develop striking spatial patterning as they grow due to phenotypic differentiation of sub-populations into a  shell of static cells surrounding a motile  core. Combining in situ measurements of cell physiology with transcriptomics, we show that shell cells express adhesive type IV pili, while motile core cells express carbon storage granules. The emergence of shell and core phenotypes is cued by opposing gradients of carbon and nitrogen that form within cell clusters due to local metabolic activity. Eventually, the shell ruptures, releasing the carbon-storing core, and we show that carbon-storing cells more readily propagate on alginate than non-carbon storing cells. We propose that phenotypic differentiation promotes the resilience of 12B01 groups by enabling clonal groups to grow larger and propagate more effectively. Phenotypic differentiation may be a widespread, but overlooked, strategy among bacteria to enhance resilience in the context of resource limitation.

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Individualised computational modelling of immune mediated disease onset, flare and clearance in psoriasis Despite increased understanding about psoriasis pathophysiology, currently there is a lack of predictive computational models. We developed a personalisable ordinary differential equations model of human epidermis and psoriasis that incorporates immune cells and cytokine stimuli to regulate the transition between healthy and disease skin. In line with experimental data, an immune stimulus initiated transition from healthy skin to psoriasis and apoptosis induced by UVB phototherapy returned the epidermis back to the healthy state. The flexibility of our model permitted the development of a patient-specific "UVB sensitivity" parameter that enabled simulation of individual patients clinical response trajectory. In a prospective clinical study of 94 patients, serial individual UVB doses and clinical response (Psoriasis Area Severity Index) values collected over the first three weeks of UVB therapy informed estimation of the "UVB sensitivity" parameter and the prediction of individual patient outcome at the end of phototherapy. Notably, our model was able to distinguish disease flares and offers the potential for clinical application in early assessment of response to UVB therapy, and for individualised optimisation of phototherapy regimes to improve clinical outcome.

Author SummaryWe present a new computer model for psoriasis, an immune-mediated disabling skin disease which presents with red, raised scaly plaques that can appear over the whole body. Psoriasis affects millions of people in the UK alone and causes significant impairment to quality of life, and currently has no cure. Only a few treatments (including UVB phototherapy) can induce temporary remission. Despite our increased understanding about psoriasis, treatments are still given on a  trial and error basis and there are no reliable computer models that can a) elucidate the mechanisms behind psoriasis onset or flare and b) predict a patients response to a course of treatment (e.g., phototherapy) and the likelihood of inducing a period of remission. Our computer model addresses both these needs. First, it explicitly describes the interaction between the immune system and skin cells. Second, our model captures response to therapy at the individual patient level and enables personalised prediction of clinical outcomes. Notably, our model also supports prediction of amending individual UVB phototherapy regimes based on the patients initial response that include for example personalised delivery schedules (i.e., 3x weekly vs. 5x weekly phototherapy). Therefore, our work is a crucial step towards precision medicine for psoriasis treatment.

sentences: Viruses inhibit TIR gcADPR signaling to overcome bacterial defense The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

sentences: Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

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A computational method for predicting the most likely evolutionary trajectories in the stepwise accumulation of resistance mutations Pathogen evolution of drug resistance often occurs in a stepwise manner via accumulation of multiple mutations, which in combination have a non-additive impact on fitness, a phenomenon known as epistasis. Epistasis complicates the sequence-structure-function relationship and undermines our ability to predict evolution. We present a computational method to predict evolutionary trajectories that accounts for epistasis, using the Rosetta Flex ddG protocol to estimate drug binding free energy changes upon mutation and an evolutionary model based in thermodynamics and statistical mechanics. We apply this method to predict evolutionary trajectories to known multiple mutations associated with resistant phenotypes in malaria. Resistance to the combination drug sulfadoxine-pyrimethamine (SP) in malaria-causing species Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) has arisen via the accumulation of multiple point mutations in the DHFR and DHPS genes. Four known PfDHFR pyrimethamine resistance mutations are highly prevalent in field-isolates and multiple studies have shown epistatic interactions between these mutations determine the accessible evolutionary trajectories to the highly resistant quadruple mutation N51I,C59R,S108N,I164L. We simulated the possible evolutionary trajectories to this quadruple PfDHFR mutation as well as the homologous PvDHFR mutations. In both cases, our most probable pathways agreed well with those determined experimentally. We also applied this method to predict the most likely evolutionary pathways to observed multiple mutations associated with sulfadoxine resistance in PfDHPS and PvDHPS. This novel method can be applied to any drug-target system where the drug acts by binding to the target.

Significance StatementAntimicrobial resistance (AMR) is a major public health threat, resulting from the overuse and misuse of antimicrobial drugs. Our ability to monitor emerging resistance and direct the most appropriate treatment strategy (stewardship) would be strengthened by the development of methods to accurately predict the mutational pathways that lead to resistance. We present a computational method to predict the most likely resistance pathways that result from mutations that alter drug binding affinity. We utilized the Rosetta Flex ddG protocol and a thermodynamic evolutionary model. This novel approach can accurately capture known resistance trajectories and can be applied to any system where resistance arises via changes in drug binding affinity.

sentences: Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a ~50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ~15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

sentences: Water levels affect photosynthesis and nutrient use more than salinity in a scrub Red Mangrove forest of the southeastern Florida Everglades We investigated how mangrove-island micro-elevation (i.e., habitat: center vs. edge) affects tree physiology in a scrub mangrove forest of the southeastern Everglades. We measured leaf gas exchange rates of scrub Rhizophora mangle L. trees monthly during 2019, hypothesizing that CO2 assimilation (Anet) and stomatal conductance (gsw) would decline with increasing water levels and salinity, expecting more-considerable differences at mangrove-island edges than centers, where physiological stress is greatest. Water levels varied between 0 and 60 cm from the soil surface, rising during the wet season (May-October) relative to the dry season (November-April). Porewater salinity ranged from 15 to 30 ppt, being higher at mangrove-island edges than centers. Anet maximized at 15.1 {micro}mol m-2 s-1, and gsw was typically <0.2 mol m-2 s-1, both of which were greater in the dry than the wet season and greater at island centers than edges, with seasonal variability being roughly equal to variation between habitats. After accounting for season and habitat, water level positively affected Anet in both seasons but did not affect gsw. Our findings suggest that inundation stress (i.e., water level) is the primary driver of variation in leaf gas exchange rates of scrub mangroves in the Florida Everglades, while also constraining Anet more than gsw. The interaction between inundation stress due to permanent flooding and habitat varies with season as physiological stress is alleviated at higher-elevation mangrove-island center habitats during the dry season. Freshwater inflows during the wet season, increase water levels and inundation stress at higher-elevation mangrove-island centers, but also potentially alleviate salt and sulfide stress in soils. Thus, habitat heterogeneity leads to differences in nutrient and water acquisition and use between trees growing in island centers versus edges, creating distinct physiological controls on photosynthesis, which likely affect carbon flux dynamics of scrub mangroves in the Everglades.

sentences: Mechanistic Insights into the Effects of Key Mutations on SARS-CoV-2 RBD-ACE2 Binding Some recent SARS-CoV-2 variants appear to have increased transmissibility than the original strain. An underlying mechanism could be the improved ability of the variants to bind receptors on target cells and infect them. In this study, we provide atomic-level insight into the binding of the receptor binding domain (RBD) of the wild-type SARS-CoV-2 spike protein and its single (N501Y), double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants to the human ACE2 receptor. Using extensive all-atom molecular dynamics simulations and advanced free energy calculations, we estimate the associated binding affinities and binding hotspots. We observe significant secondary structural changes in the RBD of the mutants, which lead to different binding affinities. We find higher binding affinities of the double (E484Q, L452R) and triple (N501Y, E484Q, L452R) mutated variants than the wild type and the N501Y variant, which could contribute to the higher transmissibility of recent variants containing these mutations.

sentences: BCM3D 2.0: Accurate segmentation of single bacterial cells in dense biofilms using computationally generated intermediate image representations Accurate detection and segmentation of single cells in three-dimensional (3D) fluorescence time-lapse images is essential for measuring individual cell behaviors in large bacterial communities called biofilms. Recent progress in machine-learning-based image analysis is providing this capability with every increasing accuracy. Leveraging the capabilities of deep convolutional neural networks (CNNs), we recently developed bacterial cell morphometry in 3D (BCM3D), an integrated image analysis pipeline that combines deep learning with conventional image analysis to detect and segment single biofilm-dwelling cells in 3D fluorescence images. While the first release of BCM3D (BCM3D 1.0) achieved state-of-the-art 3D bacterial cell segmentation accuracies, low signal-to-background ratios (SBRs) and images of very dense biofilms remained challenging. Here, we present BCM3D 2.0 to address this challenge. BCM3D 2.0 is completely complementary to the approach utilized in BCM3D 1.0. Instead of training CNNs to perform voxel classification, we trained CNNs to translate 3D fluorescence images into intermediate 3D image representations that are, when combined appropriately later, more amenable to conventional mathematical image processing than a single experimental image. Using this approach, improved segmentation results are obtained even for very low SBRs and/or high cell density biofilm images. The improved cell segmentation accuracies in turn enable improved accuracies of tracking individual cells through 3D space and time, which opens the door to investigating time-dependent phenomena in bacterial biofilms at the cellular level.

sentences: TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress Telomeres are intrinsically difficult-to-replicate regions of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a separation-of-function TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that specifically inhibited the TRF2-ORC interaction in human cells without substantially inhibiting TRF2 interactions with its other binding partners. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions in human cells. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

sentences: A senescence-like cellular response inhibits bovine ephemeral fever virus proliferation During industrial scale production of virus for vaccine manufacturing, antiviral response of host cells can dampen maximal viral antigen yield. In addition to interferon responses, many other cellular responses such as the AMPK signaling pathway or senescence-like response may inhibit or slow down virus amplification in the cell culture system. In this study, we first performed a Gene Set Enrichment Analysis of the whole-genome mRNA transcriptome and found a senescence-like cellular response in BHK-21 cells when infected with bovine ephemeral fever virus (BEFV). To demonstrate that this senescence-like state may reduce virus growth, BHK-21 subclones showing varying degrees of senescence-like state were infected with BEFV. Results showed the BHK-21 subclones showing high senescence staining could inhibit BEFV replication while low senescence-staining subclones are permissive to virus replication. Using a different approach, a senescence-like state was induced in BHK-21 using a small molecule, camptothecin (CPT), and BEFV susceptibility was examined. Results showed that indeed CPT-treated BHK-21 is more resistant to virus infection. Overall, these results indicate that a senescence-like response may be at play in BHK-21 upon virus infection. Furthermore, cell clone selection and modulating treatments using small molecules may be tools in countering anti-viral responses.

sentences: Collagen polarization provides a structural memory for the elongation of epithelial anlage. Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here we focus on understanding cellular mechanisms for elongation, using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this loco-regional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles which were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the ECM, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry-breaking and elongation. This required {beta}1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating loco-regional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.

sentences: Microbiota-dependent and independent production of L-Dopa in the gut of Daphnia magna The host-microbiome interactions are essential for the physiological and ecological performance of the host, yet these interactions are challenging to identify. Neurotransmitters are commonly implicated in these interactions, but we know very little about the mechanisms of their involvement, especially in invertebrates. Here, we report a peripheral Catecholamine (CA) pathway involving the gut microbiome of the model species Daphnia magna. We demonstrate that: (1) tyrosine hydroxylase and dopa decarboxylase enzymes are present in the gut wall; (2) DOPA decarboxylase gene is expressed in the gut by the host, and its expression follows the molt cycle peaking after ecdysis; (3) biologically active L-Dopa, but not Dopamine, is present in the gut lumen; and (4) gut bacteria produce L-Dopa in a concentration-dependent manner when provided L-Tyrosine as a substrate. Impinging on gut bacteria involvement in host physiology and ecologically relevant traits, we suggest L-Dopa as a communication agent in the host-microbiome interactions in daphnids and, possibly, other crustaceans.

sentences: Divergence of trafficking and polarity mechanisms for PIN auxin transporters during land plant evolution The phytohormone auxin and its directional transport through tissues play a fundamental role in plant development. This polar auxin transport relies on the PIN auxin exporters, whose polarization at the plasma membrane determines the overall direction of auxin flow. Hence, PIN polarization is crucial for development, but its evolution during the rise of developmental complexity in land plants remains unclear. Here, we investigated the evolution of PIN trafficking and polarization by comparing two model bryophytes, Physcomitrella patens and Marchantia polymorpha, with the angiosperm model Arabidopsis thaliana. All examined PINs show a conserved auxin export function, which is reflected by comparable bryophytic growth defects in overexpression lines. In bryophytes, bryophytic PINs polarize to filamentous apices, while Arabidopsis PINs distribute symmetrically on the membrane with additional intracellular puncta. In the Arabidopsis root epidermis, bryophytic PINs show no defined polarity. Pharmacological interference revealed a strong cytoskeleton dependence of bryophytic but not Arabidopsis PIN polarization, which, in contrast, strongly relies on Brefeldin A-sensitive trafficking. The divergence of PIN polarization and trafficking is also observed within the bryophyte clade and between tissues in individual species. These results collectively reveal a divergence of PIN trafficking and polarity mechanisms throughout land plant evolution and suggest co-evolution of PIN sequence-based and cell-based polarity mechanisms.

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Genotype and Th2 cells control monocyte to tissue resident macrophage differentiation during nematode infection of the pleural cavity The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in the C57BL/6 strain. Here, we provide a comprehensive analysis of immune cells in the pleural cavity using both C57BL/6 and BALB/c mice. Unlike C57BL/6 mice, naive tissue-resident Large Cavity Macrophages (LCM) of BALB/c mice failed to fully implement the tissue residency program. Following infection with a pleural-dwelling nematode these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6 but not BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte to macrophage conversion required both T cells and IL-4R signalling. Host genetics are therefore a key influence on tissue resident macrophage biology, and during nematode infection Th2 cells control the differentiation pathway of tissue resident macrophages.

Graphical Abstract

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sentences: High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab strain ATCC 19977. We generated [~]51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in Mycobacterium tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, Mycobacterium smegmatis, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 Mab clinical isolates to several classes of antibiotics, including the {beta}-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

sentences: Activity budget and gut microbiome structure across reproductive states in wild, female capuchin monkeys Energy demands associated with pregnancy and lactation are significant forces in mammalian evolution. To mitigate increased energy costs associated with reproduction, female mammals have evolved behavioural and physiological responses. Some species alter activity to conserve energy during pregnancy and lactation, while others experience changes in metabolism and fat deposition. Restructuring of gut microbiota with shifting reproductive states may also help females increase energy harvest from foods, especially during pregnancy. Here, we combine life history data with >13,000 behavioural scans and >300 fecal samples collected longitudinally across multiple years from 33 white-faced capuchin monkey females to examine the relationships among behaviour, gut microbiota composition, and reproductive state. We used 16S-based amplicon sequencing and the DADA2 pipeline to analyze microbial diversity and putative functions. Reproductive state explained some variation in activity, but overall resting and foraging behaviours were relatively stable across the reproductive cycle. We found evidence for increases in biotin synthesis pathways among microbes in lactating females, and that relatoe abundance of major phyla among the states was small but significant. Otherwise, gut microbiota structure and estimated functions were not substantially different among reproductive states. These data contribute to a broader understanding of plasticity in response to physiological shifts associated with mammalian reproduction.

sentences: Salvage of Ribose from Uridine or RNA Supports Glycolysis when Glucose is Limiting Summary paragraphGlucose is vital for life, serving both as a source of energy and as a carbon building block for growth. When glucose availability is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized, genome-wide genetic screening and growth assays of 482 pooled PRISM cancer cell lines. We report that catabolism of uridine enables the growth of cells in the complete absence of glucose. While previous studies have shown that the uracil base of uridine can be salvaged to support growth in the setting of mitochondrial electron transport chain deficiency (1), our work shows that the ribose moiety of uridine can be salvaged via a pathway we call "uridinolysis" defined as: [1] the phosphorylytic cleavage of uridine by UPP1/2 into uracil and ribose-1-phosphate (R1P), [2] the conversion of R1P into fructose-6-P and glyceraldehyde-3-P by PGM2 and the non-oxidative branch of the pentose phosphate pathway (non-oxPPP), and [3] their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Intriguingly, we report that uridine nucleosides derived from RNA are also a substrate for uridinolysis and that RNA can support growth in glucose-limited conditions. Our results underscore the malleability of central carbon metabolism and raise the provocative hypothesis that RNA can also serve as a potential storage for energy.

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Integrative Genome Modeling Platform reveals essentialityof rare contact events in 3D genome organizations A multitude of sequencing-based and microscopy technologies provide the means to unravel the relationship between the three-dimensional (3D) organization of genomes and key regulatory processes of genome function. However, it remains a major challenge to systematically integrate all available data sources to characterize the nuclear organization of genomes across different spatial scales. Here, we develop a multi-modal data integration approach to produce genome structures that are highly predictive for nuclear locations of genes and nuclear bodies, local chromatin compaction, and spatial segregation of functionally related chromatin.

By performing a quantitative assessment of the predictive power of genome structures generated from different data combinations, we demonstrate that multimodal data integration can compensate for systematic errors and missing values in some of the data and thus, greatly increases accuracy and coverage of genome structure models. We also show that alternative combinations of different orthogonal data sources can converge to models with similar predictive power. Moreover, our study reveals the key contributions of low-frequency inter-chromosomal contacts (e.g., "rare" contact events) to accurately predicting the global nuclear architecture, including the positioning of genes and chromosomes. Overall, our results highlight the benefits of multi-modal data integration for genome structure analysis, available through the Integrative Genome structure Modeling (IGM) software package that we introduce here.

sentences: Mutualists construct the ecological conditions that trigger the transition from parasitism The evolution of mutualism between hosts and initially parasitic symbionts represents a major transition in evolution. Although vertical transmission of symbionts during host reproduction and partner control both favour the stability of mutualism, these mechanisms require specifically evolved features that may be absent in the first place. Therefore, the first steps of the transition from parasitism to mutualism may suffer from the cost of mutualism at the organismic level. We hypothesize that spatial structure can lead to the formation of higher selection levels favouring mutualism. This resembles the evolution of altruism, with the additional requirement that the offspring of mutualistic hosts and symbionts must co-occur often enough. Using a spatially explicit agentbased model we demonstrate that, starting from a parasitic system with global dispersal, the joint evolution of mutualistic effort and local dispersal of hosts and symbionts leads to a stable coexistence between parasites and mutualists. The evolution of local dispersal mimics vertical transmission and triggers the formation of mutualistic clusters, counteracting the organismic selection level of parasites that maintain global dispersal. The transition occurs when mutualistic symbionts increase the density of hosts, which strengthens competition between hosts and disfavours hosts inhabiting areas dominated by parasitic symbionts: mutualists construct the ecological conditions that allow their own spread. Therefore, the transition to mutualism may come from an eco-evolutionary feedback loop involving spatially structured population dynamics.

sentences: Tackling polypharmacology of kinase inhibitors and selecting chemical kinase probes by transcription factor activity profiling. Protein kinase inhibitors (PKI) evolved as promising drugs for multiple diseases. However, PKIs promiscuity causes polypharmacological effects challenging therapeutic development. We show that the polypharmacology of PKIs can be inferred from their impact on transcription factors (TF) lying at the apexes of signaling pathways. Using a high-content reporter assay, we evaluated TF activity profiles (TFAP) in cells treated with Akt, CDK, Aurora, Raf, MEK, and ERK inhibitors. For each kinase family, we found kinase-specific consensus TFAP signatures signifying the on-target PKI activity. Remarkably, proximal kinases had the highest similarity of their PKIs consensus signatures. Furthermore, we show that individual PKIs exhibited the consensus, "on-target signatures" within certain "specificity windows" and distinct "off-target signatures" at other concentrations. We also show that the TFAP signatures permit pinpointing the off-target PKI activity at irrelevant kinases and non-kinase effectors. Therefore, the TFAP approach provides clear-cut quantitative metrics for assessing PKI activity and concentration ranges where the on-target activity dominates cell response. By illuminating PKI biology invisible to target-based techniques, this effect-based approach streamlines the selection of kinase chemical probes and PKI drug prioritization.

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When Yeast Cells Change their Mind: Cell Cycle 'Start' is Reversible under Starvation Eukaryotic cells decide in late G1 whether to commit to another round of genome duplication and division. This point of irreversible cell cycle commitment is a molecular switch termed "Restriction Point" in mammals and "Start" in budding yeast. At Start, yeast cells integrate multiple signals such as pheromones, osmolarity, and nutrients. If sufficient nutrients are lacking, cells will not pass Start. However, how the cells respond to nutrient depletion after they have made the Start decision, remains poorly understood.

Here, we analyze by live cell imaging how post-Start yeast cells respond to nutrient depletion. We monitor fluorescently labelled Whi5, the cell cycle inhibitor whose export from the nucleus determines Start. Surprisingly, we find that cells that have passed Start can re-import Whi5 back into the nucleus. This occurs when cells are faced with starvation up to 20 minutes after Start. In these cells, the positive feedback loop is interrupted, Whi5 re-binds DNA, and CDK activation occurs a second time once nutrients are replenished. Cells which re-import Whi5 also become sensitive to mating pheromone again, and thus behave like pre-Start cells. In summary, we show that upon starvation the commitment decision at Start can be reversed. We therefore propose that in yeast, as has been suggested for mammalian cells, cell cycle commitment is a multi-step process, where irreversibility in face of nutrient signaling is only reached approximately 20 minutes after CDK activation at Start.

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An anchored experimental design and meta-analysis approach to address batch effects in large-scale metabolomics Large-scale untargeted metabolomics studies suffer from individual variation, batch effects and instrument variability, making comparisons of common spectral features across studies difficult. One solution is to compare studies after compound identification. However, compound identification is expensive and time consuming. We successfully identify common spectral features across multiple studies, with a generalizable experimental design approach. First, we included an anchor strain, PD1074, during sample and data collection. Second, we collected data in blocks with multiple controls. These anchors enabled us to successfully integrate three studies of Caenorhabditis elegans for nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) data from five different assays. We found 34% and 14% of features to be significant in LC-MS and NMR, respectively. Between 20-50% of spectral features differ in a mutant and among a set of genetically diverse natural strains, suggesting this reduced set of spectral features are excellent targets for compound identification.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/485859v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@84abe1org.highwire.dtl.DTLVardef@3066fforg.highwire.dtl.DTLVardef@1ba166corg.highwire.dtl.DTLVardef@164cf0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Fourteen C. elegans strains are used in three individual studies. PD1074, the anchor control strain (orange), is grown alongside test strains (green, yellow, purple). Multiple biological replicates of PD1074 captures environmental variation in growth conditions. Non-polar and polar metabolic data across the three studies (i.e., natural strains, central metabolism mutants, and UGT mutants) were collected by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Data acquisition controls in each block included biological reference material and pooled PD1074 samples. Biological replicates of PD1074 (n = 42 for LC-MS, n = 52 for NMR) were included in all batches. Meta-analysis provided comparable inferences to mixed effects models, and the estimated relative effects of each test strain to PD1074 and straightforward comparisons of test strains across experiments.

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Stromal cell sialylation suppresses T cells in inflammatory tumour microenvironments: A new tumour stromal cell immune checkpoint? Immunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells (MSC), the precursor to cancer associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Hyper-sialylation of glycans promotes immune evasion in cancer, but the role of sialyation in stromal cell-mediated immunosuppression is unknown.

Here we study changes in sialyltransferase (ST) enzymes and associated surface expressed sialic acid in stromal cells following inflammatory and tumour secretome conditioning. We show that tumour conditioned stromal cells have increased levels of sialyltransferases, 2,3/6 linked sialic acid and siglec ligands. In tumour models of solid (colorectal cancer) and haematological (multiple myeloma) stromal rich tumours, stromal cell sialylation is associated with enhanced immunosuppression. Using datasets and patient samples, we confirm that targeting sialylation in tumour stromal cells reverses immune cell exhaustion. Targeting stromal cell sialylation may represent a novel immune checkpoint to reactivate anti-tumour immunity.

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MIMOSA2: A metabolic network-based tool for inferring mechanism-supported relationships in microbiome-metabolome data MotivationRecent technological developments have facilitated an expansion of microbiome-metabolome studies, in which a set of microbiome samples are assayed using both genomic and metabolomic technologies to characterize the composition of microbial taxa and the concentrations of various metabolites. A common goal of many of these studies is to identify microbial features (species or genes) that contribute to differences in metabolite levels across samples. Previous work indicated that integrating these datasets with reference knowledge on microbial metabolic capacities may enable more precise and confident inference of such microbe-metabolite links.

ResultsWe present MIMOSA2, an R package and web application for model-based integrative analysis of microbiome-metabolome datasets. MIMOSA2 uses reference databases to construct a community metabolic model based on microbiome data and uses this model to predict differences in metabolite levels across samples. These predictions are compared with metabolomics data to identify putative microbiome-governed metabolites and specific taxonomic contributors to metabolite variation. MIMOSA2 supports various input data types and can be customized to incorporate user-defined metabolic pathways. We demonstrate MIMOSA2s ability to identify ground truth microbial mechanisms in simulation datasets, and compare its results with experimentally inferred mechanisms in a dataset describing honeybee gut microbiota. Overall, MIMOSA2 combines reference databases, a validated statistical framework, and a user-friendly interface to facilitate modeling and evaluating relationships between members of the microbiota and their metabolic products.

Availability and ImplementationMIMOSA2 is implemented in R under the GNU General Public License v3.0 and is freely available as a web server and R package from www.borensteinlab.com/software_MIMOSA2.html.

sentences: Functional Contribution of Cancer-Associated Fibroblasts in Glioblastoma The abundance and biological contribution of cancer associated fibroblasts (CAFs) in glioblastoma are poorly understood. Here, we applied single-cell RNA sequencing and spatial transcriptomics analyses to identify and characterize CAFs in human glioblastoma tumors and then performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant glioblastoma cells. We found that CAF abundance was significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling, using three large databases containing bulk RNA-sequencing data and clinical information. Proteomic analysis of the CAFs and their secretome revealed fibronectin (FN1) as a strong candidate mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF conditioned media and recombinant FN1 could facilitate the migration and invasion of glioblastoma cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of glioblastomas, while cell lines resembling the proneural-state responded to the CAF signaling better in terms of the migratory and invasive phenotypes. Investigating the in-situ expression of gene markers specifically associated with CAFs and mesenchymal malignant cells further indicated that CAFs were enriched in the perinecrotic and pseudopalisading zones of human tumors, where mesenchymal-like glioblastoma cells co-resided and thus likely interacted. Overall, this study characterized the molecular features and functional impacts of CAFs in glioblastoma, alluding to a novel cell-to-cell interaction axis mediated by CAFs in the glioblastoma microenvironment.

sentences: hibayes: An R Package to Fit Individual-Level, Summary-Level and Single-Step Bayesian Regression Models for Genomic Prediction and Genome-Wide Association Studies With the rapid development of sequencing technology, the costs of individual genotyping have been reduced dramatically, leading to genomic prediction and genome-wide association studies being widely promoted and used to predict the unknown phenotypes and to locate causal or candidate genes for animal and plant economic traits and, increasingly, for human diseases. Developing newly advanced statistical models to improve accuracy in predicting and locating for the traits with various genetic architectures has always been a hot topic in those two research domains. The Bayesian regression model (BRM) has played a crucial role in the past decade, and it has been used widely in relevant genetic analyses owing to its flexible model assumptions on the unknown genetic architecture of complex traits. To fully utilize the available data from either a self-designed experimental population or a public database, statistical geneticists have constantly extended the fitting capacity of BRM, and a series of new methodologies have been proposed for different application scenarios. Here we introduce hibayes, which is the only one tool that can implement three types of Bayesian regression models. With the richest methods achieved thus far, it covers almost all the functions involved in genetic analyses for genomic prediction and genome-wide association studies, potentially addressing a wide range of research problems, while retaining an easy-to-use and transparent interface. We believe that hibayes will facilitate the researches conducted by human geneticists, as well as plant and animal breeders. The hibayes package is freely available from CRAN at https://cran.r-project.org/package=hibayes.

sentences: Model predicts the impact of root system architecture on soil water infiltration. There is strong experimental evidence that root systems substantially change the saturated hydraulic conductivity of soil. However, the mechanisms by which roots affect soil hydraulic properties remain largely unknown. In this work, we made the hypothesis that preferential soil moisture transport occurs along the axes of roots, and that this is what changes a soils saturated hydraulic conductivity. We modified Richards equation to incorporate the preferential flow of soil moisture along the axes of roots. Using the finite element method and Bayesian optimisation, we developed a pipeline to calibrate our model with respect to a given root system. When applied to simulated root systems, the pipeline successfully predicted the pore-water pressure profiles corresponding to saturated hydraulic conductivity values, observed by Leung et al. (2018), for soils vegetated with willow and grass. Prediction accuracy improved for root systems with more realistic architectures, therefore suggesting that changes in saturated hydraulic conductivity are a result of roots enabling preferential soil moisture transport along their axes. The model proposed in this work improves our ability to predict moisture transport through vegetated soil and could help optimise irrigation, forecast flood events and plan landslide prevention strategies.

sentences: Insecticide susceptibility of Aedes (Stegomyia) aegypti (Linnaeus, 1762) and Aedes (Stegomyia) albopictus (Skuse, 1894) in Kinshasa, Democratic Republic of the Congo Aedes aegypti and Aedes albopictus are arbovirus vectors of public health concern. Although the Democratic Republic of the Congo (DRC) faces a long-standing risk of Aedes-borne viruses, data on insecticide resistance of Aedes populations are absent. To address this gap, we investigated insecticide susceptibility of Ae. aegypti and Ae. albopictus in areas with a high risk of arbovirus transmission. We also investigated the frequency of knock-down resistance (kdr) mutations in Ae. aegypti. Immature stages of Ae. aegypti and Ae. albopictus were collected from two sites in Kinshasa (Lingwala and Cite Verte) between April and July 2017 and reared to the adult stage. Wild-caught adult Ae. aegypti were collected in 2016 in another site (Ngaliema). Female Ae. aegypti (from Lingwala) and Ae. albopictus (from Cite Verte) were used in WHO tube insecticide susceptibility tests. The F1534C, V1016I and V410L kdr mutations were genotyped in Ae. aegypti from Lingwala and Ngaliema. We observed Ae. aegypti to be susceptible to bendiocarb, propoxur and malathion, suspected resistant to permethrin, and resistant to deltamethrin and DDT. Aedes albopictus was susceptible to bendiocarb, propoxur, malathion and permethrin, suspected resistant to deltamethrin and resistant to DDT. While F1534C and V1016I were not detected, a few Ae. aegypti from Lingwala were heterozygous for the mutation V410L. This study reports for the first time the insecticide resistance status of Aedes spp. and the detection of the kdr mutation V410L in Ae. aegypti in DRC. Given the resistance profile, carbamates and potentially malathion are recommended insecticide options against Ae. aegypti in Kinshasa. It will be important to develop Aedes control strategies based on the resistance patterns of Aedes in Kinshasa.

sentences: First demonstration of the FLASH effect with ultrahigh dose-rate high-energy X-rays Ultrahigh dose-rate (FLASH) radiotherapy has attracted immense attention because of its tumor control efficiency and healthy tissue protection during preclinical experiments with electrons, kilo-voltage X-rays, and protons. Using high-energy X-rays (HEXs) in FLASH is advantageous owing to its deep penetration, small divergence, and cost-effectiveness. This is the first report on the implementation of HEXs with FLASH (HEX-FLASH) and its corresponding application in vivo. With a high-current and high-energy superconducting linear accelerator, FLASH with a good dose rate and high penetration was achieved. Breast cancers artificially induced in BAL b/c mice were efficiently controlled, and normal tissues surrounding the thorax/abdomen in C57BL/6 mice were protected from radiation with HEX-FLASH. Theoretical analyses of cellular responses following HEX-FLASH irradiation were performed to interpret the experimental results and design further experiments. Thus, this study highlights the generation of HEX-FLASH for the first time and its potential in future clinical applications.

sentences: Increasing phagocytosis of microglia through targeting CD33 with liposomes displaying glycan ligands CD33 is an immunomodulatory receptor expressed on microglia and genetically linked to Alzheimers disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33. We find that CD33L liposomes increase phagocytosis of cultured monocytic cells and microglia in a CD33-dependent manner. Enhanced phagocytosis strongly correlates with loss of CD33 from the cell surface and internalization of liposomes. Increased phagocytosis by treatment with CD33L liposomes is dependent on a key intracellular signaling motif on CD33 as well as the glycan-binding ability of CD33. These effects are specific to trans engagement of CD33 by CD33L liposomes, as cis engagement through insertion of lipid-linked CD33L into cells produces the opposite effect on phagocytosis. Moreover, intracerebroventricular injection of CD33L liposomes in mice enhances phagocytosis of microglia in a CD33-dependent manner. These results demonstrate that multivalent engagement of CD33 with glycan ligands can modulate microglial cell function.

sentences: Eurasian lynx populations in Western Europe: What prospects for the next 50 years? Persistence of viable populations may be uncertain for large carnivore species, especially for those established in human-dominated landscapes. Here, we studied the Eurasian lynx in Western Europe established in the Upper Rhine metapopulation (i.e., Jura, Vosges-Palatinian and Black Forest populations) and in the Alpine population. These populations are currently considered as endangered or critically endangered due to high anthropogenic mortality and isolation. We assessed lynx persistence over a 50-year time horizon by implementing a spatially-explicit individual-based model while accounting for road mortality and habitat selection. Forecasts showed a steady growth rapidly reaching a stabilization phase with high resident female occupancy for the Alpine and Jura populations, and a positive growth for the newly re-established Vosges-Palatinian population. Moreover, this population showed a difference in female occupancy between the northern part, where a recent reintroduction program was conducted in the Palatinate Forest, and the southern part. Only the group of individuals in the Black Forest had an irregular growth probably due to the small number of only male lynx at start of the simulation and poor connectivity to surrounding populations. Exchanges of individuals between populations were limited, the Jura population playing the role of a crossroad. Persistence of lynx in Western Europe seems likely on a large scale over the next 50 years. However, lynx persistence in the southern part of the Vosges-Palatinian population and in the Black Forest appears challenging without long-term conservation management.

sentences: Two Interaction Surfaces between XPA and RPA Organize the Preincision Complex in Nucleotide Excision Repair The XPA and RPA proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair pathway. We have previously characterized the two interaction surfaces between XPA and RPA, with the RPA32 and RPA70AB subunits. Here we show that the mutations in the two individual interaction surfaces reduce NER activity in biochemical and cellular systems, and that combining mutations in two domains leads to an additive inhibition of NER, suggesting that they fulfill distinct roles. Our data suggest that the interaction between XPA and RPA32 is important for the initial association of XPA with NER complexes, while the interaction between XPA and RPA70 is needed for structural organization of the complex to license the dual incision reaction. SAXS analysis of complexes of XPA and RPA bound to ss/dsDNA junction substrates reveals the architecture of XPA and RPA in the preincision complex and shows that the two interaction domains between RPA and XPA are located at opposite sides of the two molecules. We propose a structure for the overall NER preincision complex that shows that the two strands of the NER bubble assume a U-shape with the two ss/dsDNA junctions localized in close proximity, with the interaction between XPA and RPA70 as one of the key organizing elements.

sentences: Sperm of more colorful males are better adapted to ovarian fluids in lake char (Salmonidae) Fish often spawn eggs with ovarian fluids that have been hypothesized to support sperm of some males over others (cryptic female choice). Alternatively, sperm reactions to ovarian fluids could reveal male strategies linked to their likely roles during spawning. Sperm of males who would usually be close to females during spawning are then expected to be better adapted to the presence of ovarian fluids than to water only, while the reverse would be expected for males that typically spawn at larger distance to the females. We tested these predictions with gametes and ovarian fluids from wild-caught lake char (Salvelinus umbla). We found that sperm of more colorful males showed increased sperm velocity in diluted ovarian fluids while sperm of paler males were fastest in water only. We then let equal numbers of sperm compete for fertilizations in the presence or absence of ovarian fluids and used microsatellite markers to assign in total 1,464 embryos (from 70 experimental trials) to their fathers. Overall, sperm of more colorful males reached higher fertilization success than sperm of pale males. This difference was enhanced by the presence of ovarian fluids and best explained by the increased sperm velocity. Sperm competitiveness was not enhanced with decreasing genetic distance to a given female, although parallel stress tests on embryos had revealed that females would profit more from mating with least related males rather than most colored ones. We conclude that sperm of more colorful males are best adapted to ovarian fluids, and that the observed reaction norms reveal male strategies rather than cryptic female choice.

sentences: PRDM9 directs recombination hotspots to prevent competition with meiotic transcription PRDM9 drives recombination hotspots in some mammals, including mice and apes. Non-functional orthologs of PRDM9 are present in a wide variety of vertebrates, but why it is functionally maintained in some lineages is not clear. One possible explanation is that PRDM9 plays a role in ensuring that meiosis is successful. During meiosis, available DNA may be a limiting resource given the tight packaging of chromosomes and could lead to competition between two key processes: meiotic transcription and recombination. Here we explore this potential competition and the role that PRDM9 might play in their interaction. Leveraging existing mouse genomic data, we use resampling schemes that simulate shuffled features along the genome and models that account for the rarity of features in the genome, to test if PRDM9 influences interactions between recombination hotspots and meiotic transcription in a whole genome framework. We also explored possible DNA sequence motifs associated to clusters of hotspots not tied to transcription or PRDM9. We find evidence of competition between meiotic transcription and recombination, with PRDM9 appearing to relocate recombination to avoid said conflict. We also find that retrotransposons may be playing a role in directing hotspots in the absence of other factors.

sentences: Universal features of epidemic models under social distancing guidelines Social distancing as a form of nonpharmaceutical intervention has been enacted in many countries as a form of mitigating the spread of COVID-19. There has been a large interest in mathematical modeling to aid in the prediction of both the total infected population and virus-related deaths, as well as to aid government agencies in decision making. As the virus continues to spread, there are both economic and sociological incentives to minimize time spent with strict distancing mandates enforced, and/or to adopt periodically relaxed distancing protocols, which allow for scheduled economic activity. The main objective of this study is to reduce the disease burden in a population, here measured as the peak of the infected population, while simultaneously minimizing the length of time the population is socially distanced, utilizing both a single period of social distancing as well as periodic relaxation. We derive a linear relationship among the optimal start time and duration of a single interval of social distancing from an approximation of the classic epidemic SIR model. Furthermore, we see a sharp phase transition region in start times for a single pulse of distancing, where the peak of the infected population changes rapidly; notably, this transition occurs well before one would intuitively expect. By numerical investigation of more sophisticated epidemiological models designed specifically to describe the COVID-19 pandemic, we see that all share remarkably similar dynamic characteristics when contact rates are subject to periodic or one-shot changes, and hence lead us to conclude that these features are universal in epidemic models. On the other hand, the nonlinearity of epidemic models leads to non-monotone behavior of the peak of infected population under periodic relaxation of social distancing policies. This observation led us to hypothesize that an additional single interval social distancing at a proper time can significantly decrease the infected peak of periodic policies, and we verified this improvement numerically. While synchronous quarantine and social distancing mandates across populations effectively minimize the spread of an epidemic over the world, relaxation decisions should not be enacted at the same time for different populations.

sentences: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis. Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) lack NF1 and show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. Gene expression profiling and protein expression identified P2RY14 in SCs and SC precursors (SCPs) implicating P2RY14 as a candidate upstream regulator of cAMP in EGF-dependent SCP. We found that SCP self-renewal was reduced by genetic or pharmacological inhibition of P2RY14. In NF1 deficient SCs and malignant peripheral nerve sheath tumor (MPNST) cells, P2RY14 inhibition decreased EGFR-driven phospho-Akt and increased cAMP signaling. In a neurofibroma mouse model, genetic deletion of P2RY14 increased mouse survival, delayed neurofibroma initiation and rescued cAMP signaling. Conversely, elevation of cAMP diminished SCP number in vitro and diminished SC proliferation in neurofibroma bearing mice in vivo. These studies identify the purinergic receptor P2RY14 as a critical G-protein-coupled receptor (GPCR) in NF1 mutant SCPs and SCs and suggest roles for EGFR-GPCR crosstalk in facilitating SCP self-renewal and neurofibroma initiation via cAMP and EGFR-driven phospho-Akt.

sentences: PolarMorphism enables discovery of shared genetic variants across multiple traits from GWAS summary statistics Pleiotropic SNPs are associated with multiple traits. Such SNPs can help pinpoint biological processes with an effect on multiple traits or point to a shared etiology between traits. We present PolarMorphism, a new method for the identification of pleiotropic SNPs from GWAS summary statistics. PolarMorphism can be readily applied to more than two traits or whole trait domains. PolarMorphism makes use of the fact that trait-specific SNP effect sizes can be seen as Cartesian coordinates and can thus be converted to polar coordinates r (distance from the origin) and theta (angle with the Cartesian x-axis). r describes the overall effect of a SNP, while theta describes the extent to which a SNP is shared. r and theta are used to determine the significance of SNP sharedness, resulting in a p-value per SNP that can be used for further analysis. We apply PolarMorphism to a large collection of publicly available GWAS summary statistics enabling the construction of a pleiotropy network that shows the extent to which traits share SNPs. This network shows how PolarMorphism can be used to gain insight into relationships between traits and trait domains. Furthermore, pathway analysis of the newly discovered pleiotropic SNPs demonstrates that analysis of more than two traits simultaneously yields more biologically relevant results than the combined results of pairwise analysis of the same traits. Finally, we show that PolarMorphism is more efficient and more powerful than previously published methods.

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Stability of microbiota in vineyard soils across consecutive years. Agricultural soils harbor rich and diverse microbial communities that have a deep influence on soil properties and productivity. Large scale studies have shown the impact of environmental parameters like climate or chemical composition on the distribution of bacterial and fungal species. Comparatively, little data exists documenting how soil microbial communities change between different years. Quantifying the temporal stability of soil microbial communities will allow us to better understand the relevance of the differences between environments and their impact on ecological processes on the global and local scale.

We characterized the bacterial and fungal components of the soil microbiota in ten vineyards in two consecutive years. Despite differences of species richness and diversity between the two years, we found a general stability of the taxonomic structure of the soil microbiota. Temporal differences were smaller than differences due to geographical location, vineyard land management or differences between sampling sites within the same vineyard. Using machine learning, we demonstrated that each site was characterized by a distinctive microbiota, and we identified a reduced set of indicator species that could classify samples according to their geographic origin across different years with high accuracy.

ImportanceThe temporal stability of the soil microbiota is important to understand the relevance of the differences that are found in response to a variety of environmental factors. By comparing fungal and bacterial microbiota from samples collected in the same sites in two consecutive years, we found a remarkable stability of both components, with characteristic differences between bacteria and fungi. Our work fills an important gap toward the definition of a microbial cartography of agricultural soils.

sentences: A Non-Genetic, Cell Cycle Dependent Mechanism of Platinum Resistance in Lung Adenocarcinoma We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model system and identified a non- genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

sentences: Environmental variability and longevity predict the speed of the acute glucocorticoid response across birds. The acute glucocorticoid response is a key mediator of the coordinated vertebrate response to unpredictable challenges. Rapid glucocorticoid increases initiate changes that allow animals to cope with stressors. The scope of the glucocorticoid response -- defined here as the absolute increase in glucocorticoids -- is associated with individual differences in performance and varies across species with environment and life history. In addition to varying in scope, responses can differ enormously in speed; however, relatively little is known about whether speed and absolute glucocorticoid levels covary, how selection shapes speed, or what aspects of speed are important. We used corticosterone samples collected at 5 time points from 1,750 individuals of 60 species of birds to ask i) how the speed and scope of the glucocorticoid response covary and ii) whether variation in absolute or relative speed is predicted by environmental context or life history. As predicted by a recent model, faster absolute glucocorticoid responses were strongly associated with a larger scope. Despite this covariation, the relative speed of the glucocorticoid response (standardized within species) varied independently of absolute scope, suggesting that selection could operate on both features independently. Species with faster relative glucocorticoid responses lived in locations with more variable temperature and had shorter lifespans. Our results suggest that rapid changes associated with the speed of the glucocorticoid response, such as those occurring through non-genomic receptors, might be an important determinant of coping ability and we emphasize the need for studies designed to measure speed independently of absolute glucocorticoid levels.

sentences: α-/γ-Taxilin are required for centriolar subdistal appendage assembly and microtubule organization The centrosome, composed of a pair of centrioles (mother and daughter centrioles) and pericentriolar material, is mainly responsible for microtubule nucleation and anchorage in animal cells. The subdistal appendage (SDA) is a centriolar structure located at the subdistal region on the mother centriole, and it functions in microtubule anchorage. However, the molecular composition and detailed structure of SDA remain largely unknown. Here, we identified a-taxilin and r-taxilin as new SDA components, which form a complex via their coiled-coil domains and serve as a new subgroup during SDA hierarchical assembly. Their SDA localization is dependent on ODF2, and -taxilin recruits CEP170 to the SDA. Functional analyses suggest that -taxilin and {gamma}-taxilin are responsible for centrosomal microtubule anchorage during interphase, as well as for proper spindle orientation during metaphase. Altogether, our results shed light on the molecular components and functional understanding of the SDA hierarchical assembly and microtubule organization.

sentences: T606-phosphorylation deprives the function of Kaiso as a transcription repressor It is well known that Kaiso protein encoded by ZBTB33 gene is a transcription repressor and that Kaiso-P120ctn interaction increases the shift of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalization are far from clear. Here, we reported that AKT1 could phosphorylate 606-threonine residue (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins and the depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for the pT606-Kaiso accumulation in the cytoplasm. Notably, enforced 14-3-3{sigma} (SFN) overexpression could increase the pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene CDH1. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues relative to paired normal controls. The mRNA levels of 14-3-3{sigma} and Kaiso target gene CDH1 were positively and significantly correlated with each other in bioinformatics analyses using publicly available RNA-seq datasets for human normal tissues (n=11688, r=0.60, p<0.001) in the GTEx project and for cancer cell lines (n=1156, r=0.41, p<0.001) in the CCLE project. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro as well as boosted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated by AKT1 at the T606 and the pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn that de-represses the expression of Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation may contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer.

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'Superbugs: A Pop-up Science Shop': Increasing public awareness and knowledge of antimicrobial resistance by taking science to the city Superbugs: A Pop-up Science Shop was a public engagement event in the school summer holidays of 2019, organised by members of Cardiff Universitys School of Medicine. We transformed an empty retail unit in the centre of Wales largest shopping centre into an interactive and immersive microbiology experience. We aimed to facilitate opportunities for two-way dialogue to impart positive impact on the awareness of antibiotic resistance, whilst concurrently evaluating the efficacy of an engagement strategy focused on the utilisation of public spaces to attract public demographics diverse to those who would normally engage with STEM outreach.

Over the course of 14 days, we welcomed 6,566 visitors, with 67% attending as part of the natural footfall of the shopping centre. We created 1,625 young Antibiotic Resistance Champions, located in over 200 schools. Through a multi-lateral evaluation strategy, we were able to collect quantitative and qualitative feedback on the success of our delivery model, and the impact on our stakeholders. Herein, we will discuss the evolution of  Superbugs from concept, planning and design, to the logistics of delivering an engagement event of this scale. We will focus in particular on the learning outcomes of the project, and how this will shape the future of our  Superbugs project, and engagement events beyond.

Key MessagesO_LICreating a multi-disciplinary core team is essential to the success of large-scale engagement events as well as the support and development of large numbers of colleagues/volunteers
C_LIO_LIUtilising themes of exhibition and gameplay alongside strong fear-empowerment messages is an impactful way to confer positive influence and behaviour around antimicrobial resistance (AMR) and the use of antibiotics
C_LIO_LI Pop-up shop is an effective mode of delivery to capture diverse public demographics far beyond those who would traditionally engage with scientific outreach and science engagement.
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sentences: The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase (POLRMT) is thought to be the primase for mtDNA replication. However, it is unclear how POLRMT, which normally transcribes long polycistronic RNAs, can produce short RNA oligos to initiate mtDNA replication. Here we show that the PPR domain of Drosophila POLRMT is a 3 to 5 exoribonuclease. The exoribonuclease activity is indispensable for POLRMT to synthesize short RNA oligos and to prime DNA replication in vitro. An exoribonuclease deficient POLRMT, POLRMTE423P partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in POLRMT mutant background, indicating that the exoribonuclease activity is necessary for mtDNA replication. Overexpression of POLRMTE423P in adult flies leads to severe neuromuscular defects and a marked increase of mtDNA transcripts errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription. PPR domain of human POLRMT also has exoribonuclease activity, indicating evolutionarily conserved roles of PPR domain in mitochondrial DNA and RNA metabolism.

sentences: Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes [1]. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity [2-7]. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force [8]. Instead, the kinesin-12 family motor KLP-18 performs this function [9-12]. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles [12]. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

sentences: Information maximization explains state-dependent synaptic plasticity and memory reorganization during non-rapid eye movement sleep Slow waves during the non-rapid eye movement (NREM) sleep reflect the alternating up and down states of cortical neurons; global and local slow waves promote memory consolidation and forgetting, respectively. Furthermore, distinct spike-timing-dependent plasticity (STDP) operates in these up and down states. The contribution of different plasticity rules to neural information coding and memory reorganization remains unknown. Here, we show that optimal synaptic plasticity for information maximization in a cortical neuron model provides a unified explanation for these phenomena. The model indicates that the optimal synaptic plasticity is biased towards depression as the baseline firing rate increases. This property explains the distinct STDP observed in the up and down states. Furthermore, it explains how global and local slow waves predominantly potentiate and depress synapses, respectively, if the background firing rate of excitatory neurons declines with the spatial scale of waves as the model predicts. The model provides a unifying account of the role of NREM sleep, bridging neural information coding, synaptic plasticity, and memory reorganization.

sentences: Comparison of breeding strategies for the creation of a synthetic pig line Creating a new synthetic line by crossbreeding means complementary traits from pure breeds can be combined in the new population. Although diversity is generated during the crossbreeding stage, in this study, we analyze diversity management before selection starts. Using genomic and phenotypic data from animals belonging to the first generation (G0) of a new line, different simulations were run to evaluate diversity management during the first generations of a new line and to test the effects of starting selection at two alternative times, G3 and G4. Genetic diversity was characterized by allele frequency, inbreeding coefficients based on genomic and pedigree data, and expected heterozygosity. Breeding values were extracted at each generation to evaluate differences in starting selection at G3 or G4. All simulations were run for ten generations. A scenario with genomic data to manage diversity during the first generations of a new line was compared with a random and a selection scenario. As expected, loss of diversity was higher in the selection scenario, while the scenario with diversity control preserved diversity. We also combined the diversity management strategy with different selection scenarios involving different degrees of diversity control. Our simulation results show that a diversity management strategy combining genomic data with selection starting at G4 and a moderate degree of diversity control generates genetic progress and preserves diversity.

sentences: Flow-cell based technology for massively parallelcharacterization of base-modified DNA aptamers Aptamers incorporating chemically modified bases can achieve superior affinity and specificity compared to natural aptamers, but their characterization remains a labor-intensive, low-throughput task. Here we describe the  non-natural aptamer array (N2A2) system, in which a minimally modified Illumina MiSeq instrument is used for the high-throughput generation and characterization of large libraries ([~]106) of base-modified DNA aptamer candidates on the basis of both target affinity and specificity. We first demonstrate the capability to screen multiple different base modifications to identify the optimal chemistry for high-affinity target binding. We next use N2A2 to generate aptamers that can maintain excellent specificity even in complex samples, with equally strong target affinity in both buffer and diluted human serum. Given that N2A2 requires only minor mechanical modifications to the MiSeq, we believe N2A2 offers a broadly accessible tool for generating high-quality affinity reagents for diverse applications.

sentences: Gene drives and population persistence vs elimination: the impact of spatial structure and inbreeding at low density Synthetic gene drive constructs are being developed to control disease vectors, invasive species, and other pest species. In a well-mixed random mating population a sufficiently strong gene drive is expected to eliminate a target population, but it is not clear whether the same is true when spatial processes play a role. In species with an appropriate biology it is possible that drive-induced reductions in density might lead to increased inbreeding, reducing the efficacy of drive, eventually leading to suppression rather than elimination, regardless of how strong the drive is. To investigate this question we analyse a series of explicitly solvable stochastic models considering a range of scenarios for the relative timing of mating, reproduction, and dispersal and analyse the impact of two different types of gene drive, a Driving Y chromosome and a homing construct targeting an essential gene. We find in all cases a sufficiently strong Driving Y will go to fixation and the population will be eliminated, except in the one life history scenario (reproduction and mating in patches followed by dispersal) where low density leads to increased inbreeding, in which case the population persists indefinitely, tending to either a stable equilibrium or a limit cycle. These dynamics arise because Driving Y males have reduced mating success, particularly at low densities, due to having fewer sisters to mate with. Increased inbreeding at low densities can also prevent a homing construct from eliminating a population. For both types of drive, if there is strong inbreeding depression, then the population cannot be rescued by inbreeding and it is eliminated. These results highlight the potentially critical role that low-density-induced inbreeding and inbreeding depression (and, by extension, other sources of Allee effects) can have on the eventual impact of a gene drive on a target population.

sentences: Different brain systems support the aversive and appetitive sides of human pain-avoidance learning Both unexpected pain and unexpected pain absence can drive avoidance learning, but whether they do so via shared or separate neural and neurochemical systems is largely unknown. To address this issue, we combined an instrumental pain-avoidance learning task with computational modeling, functional magnetic resonance imaging (fMRI) and pharmacological manipulations of the dopaminergic (100 mg levodopa) and opioidergic (50 mg naltrexone) systems (N=83). Computational modeling provided evidence that untreated participants learned more from received than avoided pain. Our dopamine and opioid manipulations negated this learning asymmetry by selectively increasing learning rates for avoided pain. Furthermore, our fMRI analyses revealed that pain prediction errors were encoded in subcortical and limbic brain regions, whereas no-pain prediction errors were encoded in frontal and parietal cortical regions. However, we found no effects of our pharmacological manipulations on the neural encoding of prediction errors. Together, our results suggest that human pain-avoidance learning is supported by separate threat- and safety-learning systems, and that dopamine and endogenous opioids specifically regulate learning from successfully avoided pain.

sentences: Drought is a stronger driver of plant morphology and nutritional composition than warming in two common pasture species Under warmer and drier future conditions, global livestock and dairy production are threatened by impacts on the productivity and nutritional quality of pastures. However, morphological and nutritional adjustments within plants in response to warming and drought vary among species and less is known how these relate to production and forage quality. To investigate this, we grew two common pasture species, tall fescue (Festuca arundinacea: grass) and lucerne (Medicago sativa: legume), in a climate-controlled facility, under different temperatures (ambient and elevated) and watering regimes (well-watered and droughted). We found that drought had a strong negative impact on biomass production, morphology and nutritional quality while warming only significantly affected both species when response metrics were considered in concert, although to a lesser degree than the drought. Furthermore, interactions between warming and drought were only seen for lucerne, with the greatest reduction in biomass and most dead material and dry matter content. In tall fescue, drought had bigger impacts on nutritional composition than morphological traits, while in lucerne, drought affected all morphological traits and most nutritional parameters. These findings suggest that in future climate scenarios, drought may be a stronger driver of changes in the morphology and nutritional composition of pasture grasses and legumes, compared to modest levels of warming.

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Rephine.r: a pipeline for correcting gene calls and clusters to improve phage pangenomes and phylogenies BackgroundA pangenome is the collection of all genes found in a set of related genomes. For microbes, these genomes are often different strains of the same species, and the pangenome offers a means to compare gene content variation with differences in phenotypes, ecology, and phylogenetic relatedness. Though most frequently applied to bacteria, there is growing interest in adapting pangenome analysis to bacteriophages. However, working with phage genomes presents new challenges. First, most phage families are under-sampled, and homologous genes in related viruses can be difficult to identify. Second, homing endonucleases and intron-like sequences may be present, resulting in fragmented gene calls. Each of these issues can reduce the accuracy of standard pangenome analysis tools.

MethodsWe developed an R pipeline called Rephine.r that takes as input the gene clusters produced by an initial pangenomics workflow. Rephine.r then proceeds in two primary steps. First, it identifies three common causes of fragmented gene calls: 1) indels creating early stop codons and new start codons; 2) interruption by a selfish genetic element; and 3) splitting at the ends of the reported genome. Fragmented genes are then fused to create new sequence alignments. In tandem, Rephine.r searches for distant homologs separated into different gene families using Hidden Markov Models. Significant hits are used to merge families into larger clusters. A final round of fragment identification is then run, and results may be used to infer single-copy core genomes and phylogenetic trees.

ResultsWe applied Rephine.r to three well-studied phage groups: the Tevenvirinae (e.g. T4), the Studiervirinae (e.g. T7), and the Pbunaviruses (e.g. PB1). In each case, Rephine.r recovered additional members of the single-copy core genome and increased the overall bootstrap support of the phylogeny. The Rephine.r pipeline is provided through GitHub (https://www.github.com/coevoeco/Rephine.r) as a single script for automated analysis and with utility functions and a walkthrough for researchers with specific use cases for each type of correction.

sentences: The exchange of vitamin B1 and its biosynthesis intermediates in synthetic microbial communities shapes the community composition and reveals complexities of nutrient sharing Microbial communities occupy diverse niches in nature, and exchanges of metabolites such as carbon sources, amino acids, and vitamins occur routinely among the community members. While large-scale metagenomic and metabolomic studies shed some light on these exchanges, the contribution of individual species and the molecular details of specific interactions are difficult to track. Here, we explore the molecular picture of vitamin B1 (thiamin) metabolism occurring in synthetic communities of Escherichia coli thiamin auxotrophs which engage in the exchange of thiamin and its biosynthesis intermediates. In E. coli, the two parts of thiamin - the 4-amino-5-hydroxymethyl-2-methylpyrimidine and the 4-methyl-5-(2-hydroxyethyl)thiazole - are synthesized by separate pathways using enzymes ThiC and ThiG, respectively, and are then joined by ThiE to form thiamin. We observed that even though E. coli {Delta}thiC, {Delta}thiE, and{Delta} thiG mutants are thiamin auxotrophs, co-cultures of{Delta} thiC-{Delta}thiE and{Delta} thiC-{Delta}thiG grow in a thiamin-deficient minimal medium, whereas the{Delta} thiE-{Delta}thiG co-culture does not. Analysis of the exchange of thiamin and its intermediates in Vibrio anguillarum co-cultures, and in mixed co-cultures of V. anguillarum and E. coli revealed that the general pattern of thiamin metabolism and exchange among microbes is conserved across species. Specifically, the microorganisms exchange HMP and thiamin easily among themselves but not THZ. Furthermore, we observe that the availability of exogenous thiamin in the media affects whether these strains interact with each other or grow independently. This underscores the importance of the exchange of essential metabolites as a defining factor in building and modulating synthetic or natural microbial communities.

sentences: A paternal bias in germline mutation is widespread across amniotes and can arise independently of cell divisions In humans and other mammals, germline mutations are more likely to arise in fathers than in mothers. Although this sex bias has long been attributed to DNA replication errors in spermatogenesis, recent evidence from humans points to the importance of mutagenic processes that do not depend on cell division, calling into question our understanding of this basic phenomenon. Here, we infer the ratio of paternal-to-maternal mutations, , in 42 species of amniotes, from putatively neutral substitution rates of sex chromosomes and autosomes. Despite marked differences in gametogenesis, physiologies and environments across species, fathers consistently contribute more mutations than mothers in all the species examined, including mammals, birds and reptiles. In mammals,  is as high as 4 and correlates with generation times; in birds and snakes,  appears more stable around 2. These observations can be explained by a simple model, in which mutations accrue at equal rates in both sexes during early development and at a higher rate in the male germline after sexual differentiation, with a conserved paternal-to-maternal ratio across species. Thus,  may reflect the relative contributions of two or more developmental phases to total germline mutations, and is expected to depend on generation time even if mutations do not track cell divisions.

sentences: Judgments of agency are affected by sensory noise without recruiting metacognitive processing Judgments of agency, our sense of control over our actions and the environment, often occur in noisy conditions. We examined the computations underlying judgments of agency, in particular under the influence of sensory noise. Building on previous literature, we studied whether judgments of agency incorporate uncertainty in the same way that confidence judgments do, which would imply that the former share computational mechanisms with metacognitive judgments. In two tasks, participants rated agency, or confidence in a decision about their agency, over a virtual hand that tracked their movements, either synchronously or with a delay and either under high or low noise. We compared the predictions of two computational models to participants ratings and found that agency ratings, unlike confidence, were best explained by a model involving no estimates of sensory noise. We propose that agency judgments reflect first-order measures of the internal signal, without involving metacognitive computations, challenging the assumed link between the two cognitive processes.

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Large-scale analysis of interindividual variability in single and paired-pulse TMS data: results from the 'Big TMS Data Collaboration' ObjectiveInterindividual variability of single and paired-pulse TMS data has limited the clinical and experimental applicability of these methods. This study brought together over 60 TMS researchers to create the largest known sample of individual participant single and paired-pulse TMS data to date, enabling a more comprehensive evaluation of factors driving response variability.

Methods118 corresponding authors provided deidentified individual TMS data. Mixed-effects regression investigated a range of individual and study level variables for their contribution to variability in response to single and pp TMS data.

Results687 healthy participants TMS data was pooled across 35 studies. Target muscle, pulse waveform, neuronavigation use, and TMS machine significantly predicted an individuals single pulse TMS amplitude. Baseline MEP amplitude, M1 hemisphere, and biphasic AMT significantly predicted SICI response. Baseline MEP amplitude, test stimulus intensity, interstimulus interval, monophasic RMT, monophasic AMT, and biphasic RMT significantly predicted ICF response. Age, M1 hemisphere, and TMS machine significantly predicted motor threshold.

ConclusionsThis large-scale analysis has identified a number of factors influencing participants responses to single and paired pulse TMS. We provide specific recommendations to increase the standardisation of TMS methods within and across laboratories, thereby minimising interindividual variability in single and pp TMS data.

HighlightsO_LI687 healthy participants TMS data was pooled across 35 studies
C_LIO_LISignificant relationships between age and resting motor threshold
C_LIO_LISignificant relationships between baseline MEP amplitude and SICI/ICF
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sentences: All driven by energy demand? Integrative comparison of metabolism of Enterococcus faecalis wildtype and a glutamine synthase mutant Lactic acid bacteria (LAB) play a significant role in biotechnology, e.g. food industry, but also in human health. Many LAB genera have developed a multidrug resistance in the past few years, becoming a serious problem in controlling hospital germs all around the world. Enterococcus faecalis accounts for a large part of the human infections caused by LABs. Therefore, studying its adaptive metabolism under various environmental conditions is particularly important. In this study, we investigated the effect of glutamine auxotrophy ({Delta}glnA mutant) on metabolic and proteomic adaptations of E. faecalis in response to a changing pH in its environment. Changing pH values are part of its natural environment in the human body, but also play a role in food industry. We compared the results to those of the wildtype. Our integrative method, using a genome-scale metabolic model, constrained by metabolic and proteomic data allows us to understand the bigger picture of adaptation strategies in this bacterium. The study showed that energy demand is the decisive factor in adapting to a new environmental pH. The energy demand of the mutant was higher at all conditions. It has been reported that {Delta}glnA mutants of bacteria are energetically less effective. With the aid of our data and model we are able to explain this phenomenon as a consequence of a failure to regulate glutamine uptake and the costs for the import of glutamine and the export of ammonium. Methodologically, it became apparent that taking into account the non-specificity of amino acid transporters is important for reproducing metabolic changes with genome-scale models since it affects energy balance.

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Built on sand: the shaky foundations of simulating single-cell RNA sequencing data With the emergence of hundreds of single-cell RNA-sequencing (scRNA-seq) datasets, the number of computational tools to analyse aspects of the generated data has grown rapidly. As a result, there is a recurring need to demonstrate whether newly devel-oped methods are truly performant - on their own as well as in comparison to existing tools. Benchmark studies aim to consolidate the space of available methods for a given task, and often use simulated data that provide a ground truth for evaluations. Thus, demanding a high quality standard for synthetically generated data is critical to make simulation study results credible and transferable to real data.

Here, we evaluated methods for synthetic scRNA-seq data generation in their ability to mimic experimental data. Besides comparing gene- and cell-level quality control summaries in both one- and two-dimensional settings, we further quantified these at the batch- and cluster-level. Secondly, we investigate the effect of simulators on clustering and batch correction method comparisons, and, thirdly, which and to what extent quality control summaries can capture reference-simulation similarity.

Our results suggest that most simulators are unable to accommodate complex designs without introducing artificial effects; they yield over-optimistic performance of integration, and potentially unreliable ranking of clustering methods; and, it is generally unknown which summaries are important to ensure effective simulation-based method comparisons.

sentences: Mutational hotspot in the SARS-CoV-2 Spike protein N-terminal domain conferring immune escape potential Global efforts are being taken to monitor the evolution of SARS-CoV-2, aiming at early identification of mutations with the potential of increasing viral infectivity or virulence. We report a striking increase in the frequency of recruitment of diverse substitutions at a critical residue (W152), positioned in the N-terminal domain (NTD) of the Spike protein, observed repeatedly across independent phylogenetic and geographical contexts. We investigate the impact these mutations might have on the evasion of neutralizing antibodies. Finally, we uncover that NTD is a region exhibiting particularly high frequency of mutation recruitments, suggesting an evolutionary path on which the virus maintains optimal efficiency of ACE2 binding combined with the flexibility facilitating the immune escape.

sentences: The Streptococcus agalactiae R3 surface protein is encoded by sar5 Streptococcus agalactiae (a group B streptococcus; GBS) is an important human pathogen causing pneumonia, sepsis and meningitis in neonates, as well as infections in pregnant women, immunocompromised individuals, and the elderly. For the future control of GBS-inflicted disease, GBS surface exposed proteins are particularly relevant as they may act as antigens for vaccine development and/or as serosubtype markers in epidemiological settings. Even so, the genes encoding some of the surface proteins established as serosubtype markers by antibody-based methods are still unknown. Here, we identify sar5 as the gene encoding the R3 surface protein, a serosubtype marker of hitherto unknown genetic origin.

sentences: Melanoma cells adopt features of both mesenchymal and amoeboid migration within confining channels For metastasis to occur, cancer cells must traverse a range of tissue environments. In part, this is accomplished by cells adjusting their migration mode to one that is best suited to the environment. Melanoma cells have been shown to be particularly plastic, frequently using both mesenchymal and amoeboid (bleb-based) modes of migration. It has been demonstrated that 2D confinement will promote the transition from mesenchymal to bleb-based migration. However, if melanoma cells similarly transition to bleb-based migration in response to 3D confinement, such as within narrow channels, is unknown. Here, using micro-fabricated channels, we demonstrate that melanoma cells adopt features of both mesenchymal and bleb-based migration. In narrow (8 m; height and width) channels coated with fibronectin, ~50% of melanoma cells were found to use either mesenchymal or bleb-based migration modes. In contrast, the inhibition of Src family kinases or coating channels with BSA, completely eliminated any features of mesenchymal migration. Detailed comparisons of migration parameters revealed that blebbing cells, particularly in the absence of adhesions, were faster than mesenchymal cells. In contrast to what has been previously shown under conditions of 2D confinement, pharmacologically inhibiting Arp2/3 promoted a fast filopodial-based mode of migration. Accordingly, we report that melanoma cells adopt a unique range of phenotypes under conditions of 3D confinement.

sentences: Ratiometric 4Pi single molecule localization with optimal resolution and color assignment 4Pi single molecule localization microscopy (4Pi-SMLM) with two opposing objectives achieves sub-10 nm isotropic 3D resolution with as few as 250 photons collected by each objective. Here, we developed a new ratiometric multi-color imaging strategy for 4Pi-SMLM which employed the intrinsic multi-phase interference intensity without increasing the complexity of the system and achieved both optimal 3D resolution and color separation. By partially linking the photon parameters between channels with interference difference of {pi} during global fitting of the multi-channel 4Pi single molecule data, we showed on simulated data that the loss of the localization precision is minimal compared with the theoretical minimum uncertainty, the Cramer-Rao lower bound (CRLB).

sentences: SIQ: easy quantitative measurement of mutation profiles in sequencing data Establishing mutational outcomes after genome editing is of increasing importance with the advent of highly efficient genome-targeting tools. Next-generation sequencing (NGS) has become a vital method to investigate the extent of mutagenesis at specific target sites. Thus, robust and simple-to-use software that enables researchers to retrieve mutation profiles from NGS data is needed. Here, we present Sequence Interrogation and Quantification (SIQ), a tool that can analyse sequence data of any targeted experiment (e.g. CRISPR, I-SceI, TALENs) with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using novel and insightful tornado plot visualizations as outputs we illustrate that SIQ readily identifies differences in mutational signatures obtained from various DNA-repair deficient genetic backgrounds. SIQ greatly facilitates the interpretation of complex sequence data by establishing mutational profiles at specific loci and is, to our knowledge, the first tool that can analyse Sanger sequence data as well as short and long-read NGS data (e.g. Illumina and PacBio).

sentences: Sensitive Spatiotemporal Tracking of Spontaneous Metastasis in Deep Tissues via a Genetically-Encoded Magnetic Resonance Imaging Reporter Metastasis remains a poorly understood aspect of cancer biology and the leading cause of cancer-related death, yet most preclinical cancer studies do not examine metastasis, focusing solely on the primary tumor. One major factor contributing to this paradox is a gap in available tools for accurate spatiotemporal measurements of metastatic spread in vivo. Our objective was to develop an imaging reporter system that offers sensitive three-dimensional detection of cancer cells at high resolutions in live mice. We utilized organic anion-transporting polypeptide lb3 (oatp1b3) as a magnetic resonance imaging (MRI) reporter gene to this end, and systematically optimized its framework for in vivo tracking of viable cancer cells in a spontaneous metastasis model. We were able to image metastasis on oatp1b3-MRI at the single lymph node level and continued to track its progression over time as cancer cells spread to multiple lymph nodes and different organ systems in single animals. While initial single lesions were successfully imaged in parallel via bioluminescence, later metastases were obscured by light scatter from the initial node. Importantly, we demonstrate and validate that 100-m isotropic resolution MR images could detect micrometastases in lung tissue estimated to contain fewer than 103 cancer cells. In summary, oatp1b3-MRI enables precise determination of lesion size and location over time and offers a path towards deep-tissue tracking of any oatp1b3-engineered cell type with combined high resolution, high sensitivity, 3D spatial information, and surrounding anatomical context.

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Competition is a determinant of multidrug resistant plasmid acquisition in Salmonella Fostering a balanced gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter - a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed, with Salmonella Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than fresh litter samples. We found that E. coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella.

Importance/SignificanceAntimicrobial resistance spread is a worldwide health challenge, stemming in large part, from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organization have adopted a One health approach to curtail the proliferation of antimicrobial resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella and provide the identity of the bacterial species that potentially perform this function in broiler chickens and also postulate the mechanism involved.

sentences: Investigating temperature effects on coastal microbial populations and trophic interactions with 16S and 18S rRNA metabarcoding Temperature is a universal driver of microbial life, with rising sea surface temperatures expected to differentially influence the physiology, biodiversity, and distribution of bacteria and plankton. The impact of ocean warming on microbial interactions remains unclear, despite the importance of these relationships for ecosystem functioning. We employed weekly to monthly 18S and 16S rRNA gene amplicon metabarcoding over a full year (33 d) in a subtropical estuary, investigating microbial population dynamics and network interactions with respect to a temperature gradient (9-31{degrees}C). Certain microbes (e.g., Acidimicrobiia, Nitrososphaeria, and Syndiniales) increased in relative abundance with rising temperatures (Spearman {rho} > 0.69), whereas other groups (e.g., Alpha- and Gammaproteobacteria, Bacillariophyta, and Dinophyceae) slightly decreased, became saturated, or remained stable. With network analysis, we observed an increase in 18S- 18S interactions in warm (23-31{degrees}C) vs. cold (<23{degrees}C) temperatures, largely involving Syndiniales, Bacillariophyta, and Dinophyceae ASVs. Bacteria ASVs were more connected to other microbes (higher degree and centrality) and became more prominent in the cold network, highlighted by well-established cross-domain relationships (e.g., diatom-bacteria) and positive interactions among bacteria (e.g., SAR11 and Rhodobacterales). These efforts highlight the types of interactions that may be more common under changing temperatures, with implications for modeling biogeochemistry and assessing ecosystem health.

sentences: Satellite glia are essential modulators of sympathetic neuron survival, activity, and autonomic function Satellite glia are the major glial cells in sympathetic ganglia, enveloping neuronal cell bodies. Despite this intimate association, how satellite glia contribute to sympathetic functions remain unclear. Here, we show that satellite glia are critical for metabolism, survival, and activity of sympathetic neurons and modulate autonomic behaviors in mice. Adult ablation of satellite glia results in impaired mTOR signaling, soma atrophy, reduced noradrenergic enzymes, and loss of sympathetic neurons. However, persisting neurons have elevated activity, and satellite glia-ablated mice show increased pupil dilation and heart rate, indicative of enhanced sympathetic tone. Satellite glia-specific deletion of Kir4.1, an inward-rectifying potassium channel, largely recapitulates the cellular defects observed in glia-ablated mice, suggesting that satellite glia act in part via extracellular K+ buffering. These findings highlight neuron-satellite glia as functional units in regulating sympathetic output, with implications for disorders linked to sympathetic hyper-activity such as cardiovascular disease and hypertension.

sentences: Bleeding in patients and mice with Alagille syndrome: sex differences and risk factors Spontaneous lethal bleeds are major cause of death in the pediatric liver disease Alagille syndrome (ALGS), yet risk factors and screening methods have not been established. We performed a systematic review and identified significantly more female than male patients with idiopathic intracranial hemorrhage (10:1). We investigated bleeding and vasculature in patients and a mouse model for ALGS (Jag1Ndr/Ndr mice) and asked whether phenotypes identified in mice could be detected in patients non-invasively. Jag1Ndr/Ndr mice bled spontaneously, exhibiting a thin skull and vascular defects including artery-vein crossings, tortuous vessels, capillary breakdown and CADASIL-like sparse vascular smooth muscle cell coverage which was aggravated by hypertension. Retinographs from patients confirmed tortuous blood vessels and artery-vein crossings in ALGS. In conclusion, Jag1Ndr/Ndr mice could be used to develop interventions for vascular defects in ALGS, and retinography could provide a non-invasive method for vascular analysis in these pediatric patients.

sentences: Extensive aquatic subsidies lead to territorial breakdown and high density of an apex predator Energetic subsidies between terrestrial and aquatic ecosystems can strongly influence food webs and population dynamics. Our objective was to study how aquatic subsidies affected jaguar (Panthera onca) diet, sociality, and population density in a seasonally flooded protected area in the Brazilian Pantanal. The diet (n = 138 scats) was dominated by fish (46%) and aquatic reptiles (55%), representing the first jaguar population known to feed extensively on fish and to minimally consume mammals (11%). These aquatic subsidies supported the highest jaguar population density estimate to date (12.4 per 100 km2) derived from camera traps (8,065 trap nights) and GPS collars (n = 13). Contrary to their mostly solitary behavior elsewhere, we documented social interactions previously unobserved between same-sex adults including cooperative fishing, co-traveling, and play. Our research demonstrates that aquatic subsidies seen in omnivores can be highly influential to obligate carnivores leading to high population density and altered social structure.

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Near-atomic Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex The nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-EM structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC at 4.1-4.7 [A] resolutions. The structure of an N-terminal domain of Nup358 was resolved at 3.0 [A], facilitating identification of five Nup358 molecules in each CR subunit. Aside from unveiling the assembly details of the two Y-shaped multicomponent complexes (Y complexes) in each CR subunit, the improved resolutions reveal the C-terminal fragment of Nup160 to be an organizing center at the vertex of each Y complex. Our structures show that the scaffold of a CR subunit comprises five Nup358, two Nup205 and two Nup93 molecules in addition to the previously characterized Y complexes.

One-Sentence SummaryImproved resolutions of the cytoplasmic ring (CR) of the Xenopus laevis nuclear pore complex reveal that five Nup358 molecules, together with two copies of interweaved Nup205, Nup93 and Y complexes, constitute the scaffold of each CR subunit.

sentences: Conserved cell-type specific signature of resilience to Alzheimer's disease nominates role for excitatory cortical neurons Alzheimers disease (AD), the leading cause of dementia, affects millions of people worldwide. With no disease-modifying medication currently available, the human toll and economic costs are rising rapidly. Under current standards, a patient is diagnosed with AD when both cognitive decline and pathology (amyloid plaques and neurofibrillary tangles) are present. Remarkably, some individuals who have AD pathology remain cognitively normal. Uncovering factors that lead to "cognitive resilience" to AD is a promising path to create new targets for therapies. However, technical challenges discovering novel human resilience factors limit testing, validation, and nomination of novel drugs for AD. In this study, we use single-nuclear transcriptional profiles of postmortem cortex from human individuals with high AD pathology who were either cognitively normal (resilient) or cognitively impaired (susceptible) at time of death, as well as mouse strains that parallel these differences in cognition with high amyloid. Our cross-species discovery approach highlights a novel role for excitatory layer 4/5 cortical neurons in promoting cognitive resilience to AD, and nominates several resilience genes that include ATP1A1, GABRB1, PTK2, and ROCK2. Nominated resilience genes were tested for replication in orthogonal data sets and confirmed to be correlated with cognitive resilience. Additionally, we identified several potential mechanisms of resilience, including regulation of membrane potential, axonal and dendritic growth, and general increase of protein cycle, potentially of membrane proteins. Because our discovery of resilience-associated genes in layer 4/5 cortical neurons originates from an integrated human and mouse transcriptomic space from susceptible and resilient individuals, we are positioned to test causality and perform mechanistic, validation, and pre-clinical studies in our human-relevant AD-BXD mouse panel.

sentences: Structural Basis for Accommodation of Emerging B.1.351 and B.1.1.7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented a response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for both ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies like 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.

sentences: Quantitative MHC class-I/-II expression levels in CDK12 mutated prostate cancer patients reveal intratumorally T cell adaptive immune response in tumors Treatment of advanced prostate cancer (PCa) remains a therapeutic challenge. The promising improvements provided by new immune-checkpoint blockade (ICB) therapies have shown only modest benefits in a minority of PCa patients. CDK12 defective-PCa is a recently described immune active class of advanced PCa with an elevated mutational burden, high inflammatory levels, and increased immune cell infiltrate. CDK12 has been proposed as a pre-dictive biomarker of treatment response to ICB since partial response treatment has been found among CDK12 PCa patients treated with ICB. This study was designed to investigate potential molecular causes of alterations to immune response among patient tumors with defects of CDK12 in primary (TCGA-PRAD, n = 48) and metastatic (mCRPC-SU2C, n = 10) PCa. We hypothesize that the expression of the MHC-I and -II genes would differ in the CDK12 defective tumors, which would explain the lack of response in some patients treated with ICB. Using RNA-seq data, we were able to distinguish two groups of tumors with CDK12 defective tumors based on differential MHC expression. Tumors with increased MHC levels showed the activation of several pathways associated with the immune system and elevated PD-L1, IDO1, and TIM3 expression. There was also increased composition of CD8+ T cells, B cells, {gamma}{delta} T cells, and M1 Macrophages in tumors with elevated MHC levels. These data suggest that MHC expression and associated immune response pathways may provide new biomarkers for ICB therapeutic response in CDK12-defective PCa.

sentences: Membrane-catalyzed aggregation of islet amyloid polypeptide is dominated by secondary nucleation Type II diabetes is characterized by the loss of pancreatic {beta}-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, i.e. the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent {beta}-cell death in type II diabetes.

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Controllability reveals defining features of information seeking In uncertain environments, seeking information about alternative choice options is essential for adaptive learning and decision-making. However, information seeking is usually confounded with changes-of-mind about the reliability of the preferred option. Here, we exploited the fact that information seeking requires control over which option to sample to isolate its behavioral and neurophysiological signatures. We found that changes-of-mind occurring with control require more evidence against the current option, are associated with reduced confidence, but are nevertheless more likely to be confirmed on the next decision. Multimodal neurophysiological recordings showed that these changes-of-mind are preceded by stronger activation of the dorsal attention network in magnetoencephalography (MEG), and followed by increased pupil-linked arousal during the presentation of decision outcomes. Together, these findings indicate that information seeking increases the saliency of evidence perceived as the direct consequence of ones own actions.

HighlightsO_LIExploration-exploitation dilemmas confound information seeking with changes-of-mind.
C_LIO_LIUnlike changes-of-mind, information seeking requires control over information sampling.
C_LIO_LIInformation seeking is associated with reduced confidence and active hypothesis testing.
C_LIO_LIStronger neurophysiological correlates of attention and arousal during information seeking.
C_LI

sentences: NICEgame: A workflow for annotating the knowledge gaps in metabolic reconstructions, using known and hypothetical reactions Advances in medicine and biotechnology rely on the further understanding of biological processes. Despite the increasingly available types and amounts of omics data, significant biochemical knowledge gaps remain uncharacterized. Several approaches have been developed during the past years to identify missing metabolic annotations in genome-scale. However, these approaches suggest missing metabolic reactions within a limited set of already characterized metabolic capabilities. In this study, we introduce NICEgame (Network Integrated Computational Explorer for Gap Annotation of Metabolism), a workflow to characterize missing metabolic capabilities in genome-scale metabolic models using the ATLAS of Biochemistry. NICEgame suggests alternative sets of known and hypothetical reactions to resolve gaps in metabolic networks, assesses their thermodynamic feasibility, and suggests candidate genes and proteins to catalyze the introduced reactions. We use gene essentiality data use to identify metabolic gaps in the latest genome-scale model of Escherichia coli, iML1515. We apply our gap-filling approach and further enhance its genome annotation, by suggesting reactions and putative genes to resolve 46 % of the false negative gene essentiality predictions.

sentences: Accuracies of genomic predictions for disease resistance of striped catfish to Edwardsiella ictaluri using artificial intelligence algorithms Assessments of genomic prediction accuracies using artificial intelligence (AI) algorithms (i.e., machine and deep learning methods) are currently not available or very limited in aquaculture species. The principal aim of this study was to examine the predictive performance of these new methods for disease resistance to Edwardsiella ictaluri in a population of striped catfish Pangasianodon hypophthalmus and to make comparisons with four common methods, i.e., pedigree-based best linear unbiased prediction (PBLUP), genomic-based best linear unbiased prediction (GBLUP), single-step GBLUP (ssGBLUP) and a non-linear Bayesian approach (notably BayesR). Our analyses using machine learning (i.e., ML-KAML) and deep learning (i.e., DL-MLP and DL-CNN) together with the four common methods (PBLUP, GBLUP, ssGBLUP and BayesR) were conducted for two main disease resistance traits (i.e., survival status coded as 0 and 1 and survival time, i.e., days that the animals were still alive after the challenge test) in a pedigree consisting of 560 individual animals (490 offspring and 70 parents) genotyped for 14,154 Single Nucleotide Polymorphism (SNPs). The results using 6470 SNPs after quality control showed that AI methods outperformed PBLUP, GBLUP and ssGBLUP, with the increases in the prediction accuracies for both traits by 9.1 - 15.4%. However, the prediction accuracies obtained from AI methods were comparable to those estimated using BayesR. Imputation of missing genotypes using AlphaFamImpute increased the prediction accuracies by 5.3 - 19.2% in all the methods and data used. On the other hand, there were insignificant decreases (0.3 - 5.6%) in the prediction accuracies for both survival status and survival time when multivariate models were used in comparison to univariate analyses. Interestingly, the genomic prediction accuracies based on only highly significant SNPs (P < 0.00001, 318 - 400 SNPs for survival status and 1362 - 1589 SNPs for survival time) were somewhat lower (0.3 to 15.6%) than those obtained from the whole set of 6,470 SNPs. In most of our analyses, the accuracies of genomic prediction were somewhat higher for survival time than survival status (0/1 data). It is concluded that there are prospects for the application of genomic selection to increase disease resistance to Edwardsiella ictaluri in striped catfish breeding programs.

sentences: Tonic activity in lateral habenula neurons promotes disengagement from reward-seeking behavior Survival requires both the ability to persistently pursue goals and the ability to determine when it is time to stop, an adaptive balance of perseverance and disengagement. Neural activity in the lateral habenula (LHb) has been linked to aversion and negative valence, but its role in regulating the balance between reward-seeking and disengaged behavioral states remains unclear. Here, we show that LHb neural activity is tonically elevated during minutes-long disengagements from reward-seeking behavior, whether due to repeated reward omission or following sufficient consumption of reward. Further, we show that LHb inhibition extends ongoing reward-seeking behavioral states but does not prompt re-engagement. We find no evidence for similar tonic activity fluctuations in ventral tegmental area (VTA) dopamine neurons. Our findings implicate the LHb as a key mediator of disengagement from reward-seeking behavior in multiple contexts and argue against the idea that the LHb contributes to decisions solely by signaling aversion.

sentences: Alterations in rhythmic and non-rhythmic resting-state EEG activity and their link to cognition in older age While many structural and biochemical changes in the brain have been previously associated with aging, the findings concerning electrophysiological signatures, reflecting functional properties of neuronal networks, remain rather controversial. To try resolve this issue, we took advantage of a large population study (N=1703) and comprehensively investigated the association of multiple EEG biomarkers (power of alpha and theta oscillations, individual alpha peak frequency (IAF), the slope of 1/f power spectral decay), aging, and aging and cognitive performance. Cognitive performance was captured with three factors representing processing speed, episodic memory, and interference resolution. Our results show that not only did IAF decline with age but it was also associated with interference resolution over multiple cortical areas. To a weaker extent, 1/f slope of the PSD showed age-related reductions, mostly in frontal brain regions. Finally, alpha power was negatively associated with the speed of processing in the right frontal lobe, despite the absence of age-related alterations. Our results thus demonstrate that multiple electrophysiological features, as well as their interplay, should be considered when investigating the association between age, neuronal activity, and cognitive performance.

sentences: PredicTF: a tool to predict bacterial transcription factors in complex microbial communities. Transcription Factors (TFs) are proteins that control the flow of genetic information by regulating cellular gene expression. Here we describe PredicTF, a first platform supporting the prediction and classification of novel bacterial TF in complex microbial communities. We evaluated PredicTF using a two-step approach. First, we tested PredictTFs ability to predict TFs for the genome of an environmental isolate. In the second evaluation step, PredicTF was used to predict TFs in a metagenome and 11 metatranscriptomes recovered from a community performing anaerobic ammonium oxidation (anammox) in a bioreactor. PredicTF is open source pipeline available at https://github.com/mdsufz/PredicTF.

sentences: 2-deoxyglucose inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity 2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 is central to 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced activation of protein phosphatase 1 (PP1) and a concomitant inactivation of Snf1/AMPK. We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export - thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.

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Limb, joint and pelvic kinematic control in the quail coping with step perturbations Small cursorial birds display remarkable walking skills and can negotiate complex and unstructured terrains with ease. The neuromechanical control strategies necessary to adapt to these challenging terrains are still not well understood. Here, we analyzed the 2D- and 3D pelvic and leg kinematic strategies employed by the common quail to negotiate visible step-up and step-down perturbations of 1 cm, 2.5 cm, and 5 cm. We used biplanar fluoroscopy to accurately describe joint positions in three dimensions and performed semi-automatic landmark localization using deep learning.

Quails negotiated vertical perturbations without major problems and rapidly regained steady-state locomotion. When coping with step-up perturbations, the quail mostly adapted the trailing limb to permit the leading leg to step on the elevated substrate in a similar way as it did during level locomotion. When the quail negotiated step-down perturbations, both legs showed significant adaptations. For small and moderate perturbations (not inducing aerial running) the quail kept the function of the distal joints (i.e., their kinematic pattern) largely unchanged during uneven locomotion, and most changes occurred in proximal joints. The hip regulated leg length, while the distal joints maintained the spring-damped limb patterns. However, to negotiate the largest visible step perturbations, more dramatic kinematic alterations were observed. For these large perturbations, all joints contributed to leg lengthening/ shortening in the trailing leg and both the trailing and leading legs stepped more vertically and less abducted. This indicates a shift from a dynamic walking program to strategies that are focused on maximizing safety.

sentences: Mentalizing in an economic games context is associated with enhanced activation and connectivity in left temporoparietal junction Studies in Social Neuroeconomics have consistently reported activation in social cognition regions during interactive economic games suggesting mentalizing during economic choice. It remains important to test the involvement of neural activity associated with mentalizing in an economic games context within the same sample of participants performing the same task. We designed a novel version of the classic false-belief task in which participants observed interactions between agents in the ultimatum and trust games and were subsequently asked to infer the agents beliefs. We compared activation patterns during the economic-games false-belief task to those during the classic false-belief task using conjunction analyses. We find significant overlap in left TPJ, and dmPFC, as well as temporal pole during two task phases: belief formation and belief inference. Moreover, gPPI analyses show that during belief formation right TPJ is a target of both left TPJ and right temporal pole (TP) seed regions, while during belief inferences all seed regions show interconnectivity with each other. These results indicate that across different task types and phases, mentalizing is associated with activation and connectivity across central nodes of the social cognition network. Importantly, this is the case in the context of the novel economic-games and classic false-belief tasks.

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Age-related changes in microRNAs expression in cruciate ligaments of wild-stock house mice AimCruciate ligaments (CLs) of the knee joint are commonly injured following trauma or ageing. MicroRNAs (miRs) are potential therapeutic targets in musculoskeletal disorders. This study aimed to 1) identify if wild-stock house (WSH) mice are an appropriate model to study age-related changes of the knee joint and 2) investigate expression of miRs in ageing murine CLs.

MethodsKnee joints were collected from 6 and 24 months old C57BL/6 and WSH mice (Mus musculus domesticus) for histological analysis. RNA extraction and qPCR gene expression were performed on CLs in 6, 12, 24, and 30 month WSH old mice. Expression of miR targets in CLs was determined, followed by analysis of predicted mRNA target genes and Ingenuity Pathway Analysis.

ResultsHigher CL and knee OARSI histological scores were found in 24 month old WSH mice compared to 6 and 12 month old C57BL/6 and 6 month old WSH mice (p< 0.05). miR-29a and miR-34a were upregulated in 30 month-old WSH mice in comparison to 6, 12 and 24-month-old WSH mice (p<0.05). Ingenuity Pathway Analysis on miR-29a and 34a targets was associated with inflammation through interleukins, TGF{beta} and Notch genes and p53 signalling. Collagen type I alpha 1 chain (COL1A1) correlated negatively with both miR-29a (r= -0.35) and miR-34a (r= -0.33).

ConclusionThe findings of this study support WSH house mice as an accelerated ageing model of the murine knee joint. This study also indicated that miR-29a and 34a may be important regulators of COL1A1 gene expression in murine CLs.

sentences: Effect of habitual reading direction on saccadic eye movements Cognitive processes can influence the characteristics of saccadic eye movements. Reading habits, including habitual reading direction, also affects cognitive and visuospatial processes, favouring attention to the side where reading begins. Few studies have investigated the effect of habitual reading direction on saccade directionality of low-cognitive-demand stimuli (such as dots). The current study examined horizontal prosaccade, antisaccade and self-paced saccade in subjects with two primary habitual reading directions. We hypothesised that saccades responding to the target in subjects habitual reading direction would show a longer prosaccade latency and lower antisaccade error rate (errors being a reflexive glance to a sudden-appearing target, rather than a saccade away from it). Sixteen young Chinese participants with primary habitual reading direction from left to right and sixteen young Arabic and Persian participants with primary habitual reading direction from right to left were recruited. Subjects needed to look towards a 5{degrees}/ 10{degrees}target in the prosaccade task or look towards the mirror image location of the target in the antisaccade task and look between two 10-degree targets in the self-paced saccade task. Only Arabic and Persian participants showed a shorter and directional prosaccade latency towards 5{degrees}target against their habitual reading direction. No significant effect of primary reading direction on antisaccade latency towards the correct directions was found. However, we found that Chinese readers generated significantly shorter prosaccade latencies and higher antisaccade directional errors compared with Arabic and Persian readers. The present study provides an insight into the effect of reading habits on saccadic eye movements in response to low-cognitive-demand stimuli and offers a platform for future studies to investigate the relationship between reading habits and neural mechanisms of eye movement behaviours.

sentences: The landscape of alternative polyadenylation in single cells of the developing mouse embryo 3' untranslated regions (3' UTRs) post-transcriptionally regulate mRNA stability, localization, and translation rate. While 3'-UTR isoforms have been globally quantified in limited cell types using bulk measurements, their differential usage among cell types during mammalian development remains poorly characterized. In this study, we examined a dataset comprising [~]2 million cells spanning E9.5-E13.5 of mouse embryonic development to quantify transcriptome-wide changes in alternative polyadenylation (APA). We observe a global lengthening of 3' UTRs across embryonic stages in all cell types, although we detect shorter 3' UTRs in hematopoietic lineages and longer 3' UTRs in neuronal cell types within each stage. An analysis of RBP dynamics identifies ELAV-like family members, which are concomitantly induced in neuronal lineages and developmental stages experiencing 3'-UTR lengthening, as putative regulators of APA. By measuring 3'-UTR isoforms in an expansive single cell dataset, our work provides a transcriptome-wide and organism-wide map of the dynamic landscape of alternative polyadenylation during mammalian organogenesis.

sentences: Cecropins contribute to Drosophila host defense against fungal and Gram-negative bacterial infection Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host-defence. In Drosophila, four Cecropin genes clustered in the genome (CecA1, CecA2, CecB and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion{Delta} CecA-C removing the whole Cecropin locus. Using the{Delta} CecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response.{Delta} CecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing{Delta} CecA-C in flies already lacking ten other AMP genes revealed a role for Cecropins in defence against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defence, and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line ({Delta}AMP14) that lacks fourteen immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

sentences: Ribonucleotide reductase, a novel target for gonorrhea Antibiotic resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Activity studies and negative stain electron microscopy of the Ng Ia RNR suggest that these inhibitors potentiate conversion of its active 2{beta}2 state to an inactive 4{beta}4 similar to states first identified with the Escherichia coli (Ec) Ia RNR. Resistance mutations in Ng map to the N-terminal, ATP cone domain of its  subunit and disrupt the interaction with the {beta} subunit required to form the specific quaternary inhibited state. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

sentences: The immunophilin protein FKBPL and its peptide derivatives are novel regulators of vascular integrity and inflammation via NF-kappaB signaling A breakdown in vascular integrity and excessive inflammation are hallmarks of serious pathological conditions including sepsis, acute respiratory distress syndrome (ARDs) and most recently, severe COVID-19. FK506 - binding protein like (FKBPL) is a member of the immunophilin protein superfamily with potent anti-tumor activity through inhibition of angiogenesis and cancer stemness. An FKBPL-based 23mer peptide, ALM201, displayed a good safety and pharmacokinetic profile in a Phase 1a oncology clinical trial and was subsequently designated orphan drug status by the FDA in ovarian cancer. Here we describe a novel role for FKBPL and its peptides in regulating vascular integrity and cytokine production though modulating NF-{kappa}B signaling. FKBPL knockdown promoted endothelial cell barrier permeability, which was further exacerbated upon stimulation with lipopolysaccharide (LPS) and accompanied by increased expression of TNF mRNA and phosphorylation of p65(RelA). Whilst treatment with the FKBPL based pre-clinical peptide, AD-01, increased VE-cadherin endothelial tight junctions following LPS stimulation. Bone marrow derived macrophages (BMDM) from FKBPL haploinsufficient mice (Fkbpl+/-) also demonstrated increased phosphorylation of p65(RelA) in response to LPS stimulation compared to wild-type mice. Furthermore, treatment with AD-01 inhibited p65(RelA) phosphorylation following LPS stimulation resulting in reduced NF-{kappa}B target gene expression and proinflammatory cytokine production. In an in vivo LPS survival model, Fkbpl+/- mice have reduced survival compared to wild-type mice. Moreover, treatment of wild-type mice with the clinical FKBPL-based peptide, ALM201, following LPS injection resulted in a 100% survival rate in mice at experimental endpoint, as well as an abrogation of production of pro-inflammatory cytokines, TNF and IL-6, in peritoneal lavage washings. Analysis of human genetic biobanks found an association between common genetic variants associated with FKBPL and traits associated with inflammatory disorders such as psoriasis, rheumatoid arthritis and high lymphocyte count. In summary, for the first time, we describe a novel role for FKBPL as a regulator of inflammation and vascular integrity through modulating NF-{kappa}B signaling and FKBPL based therapies demonstrate potent anti-inflammatory activity.

sentences: Conformational Flexibility in Neutralization of SARS-CoV-2 by Naturally Elicited Anti-SARS-CoV-2 Antibodies As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 [A] and 2.42 [A], respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 [A], 7.76 [A], 6.47 [A], 3.45 [A], and 7.32 [A]) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.

sentences: DeePVP: Identification and classification of phage virion protein using deep learning The poor annotation of phage virion protein (PVP) is the bottleneck of many areas of viral research, such as viral phylogenetic analysis, viral host identification and antibacterial drug design. Because of the high diversity of the PVP sequences, the PVP annotation remains a great challenging bioinformatic task. Based on deep learning, we present DeePVP that contains a main module and an extended module. The main module aims to identify the PVPs from non-PVP over a phage genome, while the extended module can further classify the predicted PVP into one of the ten major classes of PVP. Compared with the state-of-the-art tools that can distinguish PVP from non-PVP, DeePVPs main module performs much better, with an F1-score 9.05% higher in the PVP identification task. Compared with PhANNs, a tool that can further classify the predicted PVP into a specific class, the overall accuracy of DeePVPs extended module is approximately 3.72% higher in the PVP classification task. Two application cases on the genome of mycobacteriophage PDRPxv and Escherichia phage HP3 show that the predictions of DeePVP are much more reliable and can better reveal the compact PVP-enriched region, which may be conserved during the viral evolution process, over the phage genome.

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ANTIBIOFILM FORMATION ACTIVITY, RESISTANT GENES PROFILING AND DETECTION OF VIRULENCE FACTORS OF TOXIGENIC Vibrio cholerae ISOLATES FROM KISUMU COUNTY, KENYA IntroductionVibrio cholerae can switch between motile and biofilm lifestyles with some of its strains forming biofilms in addition to production of various virulence traits and possessing antimicrobial resistance traits. This study is aim to show antibiofilm formation activity, resistant genes profiling and detection of virulence factors of toxigenic vibrio cholerae isolates from Kisumu County.

MethodologyA total of 119 Vibrio cholerae O1, biotype El Tor isolates collected during 2017 cholera outbreak in Kisumu County were used for this study. The samples were cultured on TCBS and PCR assay carried out using standard procedures. Biofilm assay tests and detection of virulence factors were also done by use of standard procedures.

ResultsOf the 101 confirmed vibrio cholerae isolates, 80.2% possessed the cholera toxin gene (ctxA) whereas 19.8% did not. Analysis of the toxR gene revealed that 98.0% harbored the toxR gene and only 2.0% did not. It was also revealed that 80.2% harbored the class I integron (inDS gene) while 19.8% did not, 93.1% were confirmed to possess the SXT integrating conjugative element (ICE) while 7.0% did not. The tetracycline resistance gene was present in 96.0% of the isolates. In 7 isolants strains which were resistance to common used antibiotics were screened for biofilm formation. Three of the strains (04/17-07, 06/17-14, and 05/17-03) failed to form biofilm while four strains namely 03/17-16, 02/17-09, 04/17-13 and P. aeruginosa ATCC 10145 as a positive control formed biofilms. In addition, out of those 7 isolants 71.42% produced protease, 85.71% produced phospholipases, 71.42% of isolates has the ability to produce lipase and 100% were able to produce the haemolysin.

ConclusionAn understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

sentences: Lightning Fast and Highly Sensitive Full-Length Single-cell sequencing using FLASH-Seq In the last 10 years, single-cell RNA-sequencing (scRNA-seq) has undergone exponential growth. Emulsion droplets methods1-3, such as those commercialized by 10x Genomics, have allowed researchers to analyze tens of thousands of cells in parallel in a robust and reproducible way. However, in contrast to SMART-based full-length sequencing protocols4,5, these methods interrogate only the outer portion of the transcripts and still lack the required sensitivity for analyzing comprehensively the transcriptome of individual cells. Building upon the existing SMART-seq forerunners protocols4,5, we developed FLASH-Seq (FS), a new scRNA-seq method which displays greater sensitivity while decreasing incubation times and reducing the number of processing steps compared to its predecessors. The entire FS protocol - from lysed cells to pooled cDNA libraries - can be performed in ~4.5 hours, is automation-friendly and can be easily miniaturized to decrease costs.

sentences: Accurate estimation of cell-type resolution transcriptome in bulk tissue through matrix completion Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings.

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Relating simulation studies by provenance--Developing a family of Wnt signaling models For many biological systems, a variety of simulation models exist. A new simulation model is rarely developed from scratch, but rather revises and extends an existing one.

A key challenge, however, is to decide which model might be an appropriate starting point for a particular problem and why. To answer this question, we need to identify entities and activities that contributed to the development of a simulation model.

Therefore, we exploit the provenance data model, PROV-DM, of the World Wide Web Consortium and, building on previous work, continue developing a PROV ontology for simulation studies. Based on a case study of 19 Wnt/{beta}-catenin signaling models, we identify crucial entities and activities as well as useful metadata to both capture the provenance information from individual simulation studies and relate these forming a family of models. The approach is implemented in WebProv, a web application for inserting and querying provenance information.

Our specialization of PROV-DM contains the entities Research Question, Assumption, Requirement, Qualitative Model, Simulation Model, Simulation Experiment, Simulation Data, and Wet-lab Data as well as activities referring to building, calibrating, validating, and analyzing a simulation model. We show that most Wnt simulation models are connected to other Wnt models by using (parts of) these models. However, the overlap, especially regarding the Wet-lab Data used for calibration or validation of the models is small.

Making these aspects of developing a model explicit and queryable is an important step for assessing and reusing simulation models more effectively. Exposing this information helps to integrate a new simulation model within a family of existing ones and may lead to the development of more robust and valid simulation models.

We hope that our approach becomes part of a standardization effort and that modelers adopt the benefits of provenance when considering or creating simulation models.

Author summaryWe revise a provenance ontology for simulation studies of cellular biochemical models. Provenance information is useful for understanding the creation of a simulation model because it not only contains information about the entities and activities that have led to a simulation model but also their relations, all of which can be visualized. It provides additional structure by explicitly recording research questions, assumptions, and requirements and relating them along with data, qualitative models, simulation models, and simulation experiments through a small set of predefined but extensible activities.

We have applied our concept to a family of 19 Wnt signaling models and implemented a web-based tool (WebProv) to store the provenance information from these studies. The resulting provenance graph visualizes the story line of simulation studies and demonstrates the creation and calibration of simulation models, the successive attempts of validation and extension, and shows, beyond an individual simulation study, how the Wnt models are related. Thereby, the steps and sources that contributed to a simulation model are made explicit.

Our approach complements other approaches aimed at facilitating the reuse and assessment of simulation products in systems biology such as model repositories as well as annotation and documentation guidelines.

sentences: Two levels of selection of rhythmicity in gene expression: energy saving for rhythmic proteins and noise optimization for rhythmic transcripts Many genes have nycthemeral rhythms of expression, i.e. a 24-hours periodic variation, at either mRNA or protein level or both, and, most rhythmic genes are tissue-specific. Here, we investigate and discuss the evolutionary origins of rhythms in gene expression. Our results suggest that thythmicity of protein expression has been favored by selection for low cost. Trends are consistent in bacteria, plants and animals, and are also supported in tissue-specific patterns in mouse. Cost cannot explain rhythm at the RNA level, and we suggest instead it allows to periodically and drastically reduce expression noise. Noise control had strongest support in mouse, with limited power in other species. Genes under stronger purifying selection are rhythmically expressed at the mRNA level, probably because they are noise sensitive genes. We also suggest that mRNA rhythmicity allows to switch between optimal precision and higher stochasticity. Higher precision allows to maximize the robustness of gene expression when the function is most needed, while higher stochasticity allows to maintain oscillations and to exhibit diverse molecular phenotypes, i.e. "blind anticipation" of cells. The ability to alternate between these two states, enabled by rhythmicity at the mRNA level, might be adaptive in fluctuating environments. Finally, the adaptive role of rhythmic expression is also supported by rhythmic genes being highly expressed yet tissue-specific genes. This provides a good evolutionary explanation for the observation that nycthemeral rhythms are often tissue-specific.

sentences: High-dose drug heat map based on organoid array chip for drug selection with high safety and efficacy An organoid array chip was developed by adopting a micropillar and microwell structure to test safety and efficacy of drugs using high dose drug heat map. In the chip, we encapsulated patient-derived cells in alginate and grow them to maturity for more than 7 days to form cancer organoids. When screening drug compounds in a high-density organoid array due to lack of number of patient-derived cells, changing media without damage of organoids is a very tedious and difficult process. Organoids grown in conventional well plates needed too many cells and were also easily damaged due to multiple pipetting during maintenance culture or during experimental procedures. To solve those problem, we applied a micropillar and microwell structure to the organoid array. We used patient-derived cells from patients with Glioblastoma multiforme (GBM), the most common and lethal form of central nervous system cancer, to validate the array chip performance. After forming more than 100{micro}m-diameter organoids in 12 x 36 pillar array chip (25mm x 75mm), we tested 70 drug compounds (6 replicates) with high high-dose to find out high safety and efficacy drug candidates. Comparing the drug response of organoids derived from normal cells and cancer cells, we identified four compounds (Dacomitinib, Cediranib, Ly2835219, BGJ398) as drug candidates without toxicity to GBM cells.

sentences: The conservation of human functional variants and their effects across mammals Despite the clear potential of livestock models of human functional variants to provide important insights into the biological mechanisms driving human diseases and traits, their use to date has been limited. Generating such models via genome editing is costly and time consuming, and it is unclear which variants will have conserved effects across species. In this study we address these issues by studying naturally occurring livestock models of human functional variants. We show that orthologues of over 1.6 million human variants are already segregating in domesticated mammalian species, including several hundred previously directly linked to human traits and diseases. Models of variants linked to particular phenotypes, including metabolomic disorders and height, have been preferentially maintained across species, meaning studying the genetic basis of these phenotypes is particularly tractable in livestock. Using machine learning we demonstrate it is possible to identify human variants that are more likely to have an existing livestock orthologue, and, importantly, we show that the effects of functional variants are often conserved in livestock, acting on orthologous genes with the same direction of effect. Consequently, this work demonstrates the substantial potential of naturally occurring livestock carriers of orthologues of human functional variants to disentangle their functional impacts.

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

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The neural computation of human prosocial choices in complex motivational states Motives motivate human behavior. Most behaviors are driven by more than one motive, yet it is unclear how different motives interact and how such motive combinations affect the neural computation of the behaviors they drive. To answer this question, we induced two prosocial motives simultaneously (multi-motive condition) and separately (single motive conditions). After the different motive inductions, participants performed the same choice task in which they allocated points in favor of the other person (prosocial choice) or in favor of themselves (egoistic choice). We used fMRI to assess prosocial choice-related brain responses and drift diffusion modelling to specify how motive combinations affect individual components of the choice process. Our results showed that the combination of the two motives in the multi-motive condition increased participants choice biases prior to the behavior itself. On the neural level, these changes in initial prosocial bias were associated with neural responses in the bilateral dorsal striatum. In contrast, the efficiency of the prosocial decision process was comparable between the multi-motive and the single-motive conditions. These findings provide insights into the computation of prosocial choices in complex motivational states, the motivational setting that drives most human behaviors.

HighlightsO_LIActivating different social motives simultaneously can enhance prosocial choices
C_LIO_LIMulti-motive combinations change initial prosocial biases
C_LIO_LIDorso-striatal activation increases with larger increase of prosocial bias
C_LIO_LIMulti-motive combinations modulate relative response caution
C_LI

sentences: Geometry of neural computation unifies working memory and planning Real-world tasks require coordination of working memory, decision making, and planning, yet these cognitive functions have disproportionately been studied as independent modular processes in the brain. Here we propose that contingency representations, defined as mappings for how future behaviors depend on upcoming events, can unify working memory and planning computations. We designed a task capable of disambiguating distinct types of representations. Our experiments revealed that human behavior is consistent with contingency representations, and not with traditional sensory models of working memory. In task-optimized recurrent neural networks we investigated possible circuit mechanisms for contingency representations and found that these representations can explain neurophysiological observations from prefrontal cortex during working memory tasks. Finally, we generated falsifiable predictions for neural data to identify contingency representations in neural data and to dissociate different models of working memory. Our findings characterize a neural representational strategy that can unify working memory, planning, and context-dependent decision making.

sentences: Exploring the frame effect Probes flashed within a moving frame are dramatically displaced (Ozkan et al, 2021; Wong &Mack, 1981). The effect is much larger than that seen on static or moving probes (induced motion, Duncker, 1929; Wallach et al, 1978). These flashed probes are often perceived with the separation they have in frame coordinates -- a 100% effect. Here we explore this frame effect on flashed tests with several versions of the standard stimulus. We find that the frame effect holds for smoothly or abruptly displacing frames, even when the frame changed shape or orientation between the endpoints of its travel. The path could be non-linear, even circular. The effect was driven by perceived not physical motion. When there were competing overlapping frames, the effect was determined by which frame was attended. There were a number of constraints that limited the effect. A static anchor near the flashes suppressed the effect but an extended static texture did not. If the probes were continuous rather than flashed, the effect was abolished. The observational reports of 30 online participants suggest that the frame effect is robust to many variations in its shape and path and leads to a perception of flashed tests in their locations relative to the frame as if the frame were stationary. Our results highlight the role of frame continuity and of the grouping of the flashes with the frame in generating the frame effect.

sentences: Bridges, not walls: the hippocampus builds narrative memories across distant events Lifes events are scattered throughout time, yet we often recall different events in the context of an integrated narrative. Prior research suggests that the hippocampus, which supports memory for past events, can sometimes support integration of overlapping associations or separate events, but the conditions which lead to hippocampus-dependent memory integration are unclear. We used functional brain imaging to test whether the ability to form a larger narrative (narrative coherence) drives hippocampal memory integration. During encoding of fictional stories, the hippocampus supported patterns of activity, including activity at boundaries between events, which were more similar between distant events that formed one coherent narrative, compared with events taken from unrelated narratives. Twenty-four hours later, the hippocampus preferentially supported detailed recall of coherent narrative events, through reinstatement of hippocampal activity patterns from encoding. These findings reveal a key function of the hippocampus: the dynamic integration of events into a narrative structure for memory.

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Increased male mating success in the presence of prey and rivals in a sexually cannibalistic mantis Pre-copulatory sexual cannibalism--or cannibalism without mating--is expected to promote the evolution of male strategies that enhance mating success and reduce the risk of cannibalism, such as preferential mating with feeding females. However, sexual selection on male competitiveness may alter male courtship decisions in the face of cannibalism risk. We investigated the effect of prey availability and rival presence on male mating decisions in the highly cannibalistic Springbok mantis, Miomantis caffra. We found that males approached females more rapidly and mated more often in the presence of prey, suggesting that females distracted with foraging may be less of a threat. The presence of a rival also hastened the onset of copulation and led to higher mating success, with very large effects occurring in the presence of both prey and rivals, indicating that intrasexual competition may intensify attraction to foraging females. Taken together, our results suggest that pre-copulatory cannibalism has selected for male preference for foraging females, and that males adjust their mating strategy to both the risk of competition and the threat of cannibalism.

LAY SUMMARYDeciding when to approach a mate is critical for male mantises at risk of being cannibalised. A male might do well to pounce when a female is distracted with prey, but what if a nearby male has the same intention? In the Springbok mantis, we show that males mate faster and with greater success when both prey and a rival are present, suggesting that mating decisions depend on the dual threats of cannibalism and competition.

sentences: Directing multicellular organization by varying the aspect ratio of soft hydrogel microwells Multicellular organization with precise spatial definition is an essential step in a wide range of biological processes, including morphogenesis, development, and healing. Gradients and patterns of chemoattractants are well-described guides of multicellular organization, but the influences of three-dimensional geometry of soft hydrogels on multicellular organization are less well defined. Here, we report the discovery of a new mode of self-organization of endothelial cells in ring-like patterns on the perimeters of hydrogel microwells that is independent of protein or chemical patterning and is driven only by geometry and substrate stiffness. We observe quantitatively striking influences of both the microwell aspect ratio ({varepsilon} = perimeter/depth) and the hydrogel modulus. We systematically investigate the physical factors of cells and substrates that drive this multicellular behavior and present a mathematical model that explains the multicellular organization based upon balancing extracellular and cytoskeletal forces. These forces are determined in part by substrate stiffness, geometry, and cell density. The force balance model predicts the direction and distance of translational cell migration based on the dynamic interaction between tangential cytoskeletal tension and cell-cell and cell-substrate adhesion. We further show that the experimental observations can be leveraged to drive customized multicellular self-organization. Our observation of this multicellular behavior demonstrates the importance of the combinatorial effects of geometry and stiffness in complex biological processes. It also provides a new methodology for direction of cell organization that may facilitate the engineering of bionics and integrated model organoid systems.

sentences: Positive Feedback Regulation of fzd7 Expression Robustly Shapes Wnt Signaling Range in Early Heart Development Secreted molecules called morphogens govern tissue patterning in a concentration-dependent manner. However, it is still unclear how reproducible patterning can be achieved with diffusing molecules, especially when patterning differentiation of a thin region. Wnt is a morphogen that organizes cardiac development; especially Wnt6 patterns the cardiogenic mesoderm to induce differentiation of a thin pericardium in Xenopus. It is, however, unclear how Wnt6 can pattern such a thin tissue. In this study, we reveal that a Wnt receptor, frizzled7, is expressed in a Wnt-dependent manner in the prospective heart region, and that this receptor-feedback is essential for shaping a steep gradient of Wnt. In addition, the feedback imparts robustness against fluctuations of Wnt ligand production and allows the system to reach a steady state quickly. We also found a Wnt antagonist sFRP1, which is expressed at the opposite side of Wnt source, accumulates on a novel type of heparan sulfate (HS), N-acetyl-rich HS, which is highly presented in the outer of cardiogenic mesoderm, achieving local inhibition of Wnt signaling by restricting sFRP1 spreading. These two intricate regulatory systems restrict Wnt signaling and ensure reproducible patterning of a thin pericardium tissue.

sentences: ILnc: Prioritizing Long Non-coding RNAs for Pan-cancer Analysis of Immune Cell Infiltration The distribution and extent of immune cell infiltration into solid tumors play pivotal roles in cancer immunology and therapy. Here we introduced an immune long non-coding RNA (lncRNA) signature-based method (ILnc), for estimating the abundance of 14 immune cell types from lncRNA transcriptome data. Performance evaluation through pure immune cell data shows that our lncRNA signature sets can be more accurate than protein-coding gene signatures. We found that lncRNA signatures are significantly enriched to immune functions and pathways, such as immune response and T cell activation. In addition, the expression of these lncRNAs is significantly correlated with expression of marker genes in corresponding immune cells. Application of ILnc in 33 cancer types provides a global view of immune infiltration across cancers and we found that the abundance of most immune cells is significantly associated with patient clinical signatures. Finally, we identified six immune subtypes spanning cancer tissue types which were characterized by differences in immune cell infiltration, homologous recombination deficiency (HRD), expression of immune checkpoint genes, and prognosis. Altogether, these results demonstrate that ILnc is a powerful and exhibits broad utility for cancer researchers to estimate tumor immune infiltration, which will be a valuable tool for precise classification and clinical prediction.

sentences: An advanced lentil backcross population developed from a cross between Lens culinaris x L. ervoides for future disease resistance and genomic studies Genetically accessible variation to some of the abiotic and biotic stresses are limited in the cultivated lentil (Lens culinaris Medik.) germplasm. Introgression of novel alleles from its wild relative species might be required for enhancing the genetic improvement of the crop. L. ervoides, one of the wild relatives of lentil, is a proven source of disease resistance for the crop. Here we introduce a lentil advanced backcross population (LABC-01) developed in cultivar CDC Redberry background, based on L. ervoides alleles derived from an interspecific recombinant inbred population, LR-59-81. Two-hundred and seventeen individuals of the LABC-01 population at BC2F3:4 generation were screened for the race 0 of anthracnose (Colletotrichum lentis) and stemphylium blight (Stemphylium botryosum) under controlled conditions. The population showed significant variations for both diseases and transfer of resistance alleles into the elite cultivar was evident. It also segregated for other traits such as days to flowering, seed coat colour, seed coat pattern and flower colour. Overall, we showed that LABC-01 population can be used in breeding programs worldwide to improve disease resistance and will be available as a valuable genetic resource for future genetic analysis of desired loci introgressed from L. ervoides.

sentences: Non-destructive chemical sensing within bulk soil using 1000 biosensors per gram of matrix Gene expression can be monitored in hard-to-image environmental materials using gas-reporting biosensors, but these outputs have only been applied in autoclaved matrices that are hydrated with rich medium. To better understand the compatibility of indicator gas reporting with environmental samples, we evaluated how matrix hydration affects the gas signal of an engineered microbe added to a sieved soil. A gas-reporting microbe presented a gas signal in a forest soil (Alfisol) when hydrated to an environmentally-relevant osmotic pressure. When the gas signal was concentrated prior to analysis, a biosensor titer of 103 cells/gram soil produced a significant signal when soil was supplemented with halides. A signal was also observed without halide amendment, but a higher cell titer (106 cells/gram soil) was required. A sugar-regulated gas biosensor was able to report with a similar level of sensitivity when added to an unsterilized soil matrix, illustrating how gas concentration enables biosensing within a soil containing environmental microbes. These results establish conditions where engineered microbes can report on gene expression in living environmental matrices with minimal perturbation, using biosensor titers that are orders of magnitude lower than the number of cells typically observed in a gram of soil.

sentences: Extended genomic analyses of the broad-host-range phages vB_KmiM-2Di and vB_KmiM-4Dii reveal slopekviruses have highly conserved genomes High levels of antimicrobial resistance among members of the Klebsiella oxytoca complex (KoC) have led to renewed interest in the use of bacteriophage (phage) therapy to tackle infections caused by these bacteria. In this study we characterized two lytic phages, vB_KmiM-2Di and vB_KmiM-4Dii, that were isolated from sewage water against two GES-5-positive Klebsiella michiganensis strains (PS_Koxy2 and PS_Koxy4, respectively). ViPTree analysis showed both phages belonged to the genus Slopekvirus. rpoB gene-based sequence analysis of 108 presumptive K. oxytoca isolates (n=59 clinical, n=49 veterinary) found K. michiganensis to be more prevalent (46 % clinical and 43 % veterinary, respectively) than K. oxytoca (40 % clinical and 6 % veterinary, respectively). Host range analysis against these 108 isolates found both vB_KmiM-2Di and vB_KmiM-4Dii showed broad lytic activity against KoC species. Several putative homing endonuclease genes were encoded within the genomes of both phages, which may contribute to their broad host range. Pangenome analysis of 24 slopekviruses found that genomes within this genus are highly conserved, with more than 50 % of all predicted coding sequences representing core genes at [≥]95 % identity and [≥]70 % coverage. Given their broad host ranges, our results suggest vB_KmiM-2Di and vB_KmiM-4Dii represent attractive potential therapeutics. In addition, current recommendations for phage-based pangenome analyses may require revision.

sentences: What happened when? Brain and behavioral responses to violated expectations about the structure and content of episodic memories Episodic memories are not static but can be modified on the basis of new experiences, potentially allowing us to make valid predictions in the face of an ever-changing environment. Recent research has identified prediction errors during retrieval as a possible trigger for such changes. In the present study, we used modified episodic cues to investigate whether different types of mnemonic prediction errors modulate brain activity and subsequent memory performance. Participants encoded different episodes which consisted of short toy stories. During a subsequent functional magnetic resonance imaging (fMRI) session, participants were then presented videos showing the original episodes, or modified versions thereof. In modified videos, either the order of two subsequent action steps was changed or an object was exchanged for another. Content modifications recruited inferior frontal, parietal, and temporo-occipital areas reflecting the processing of the new object information, while brain responses to structure modifications in right dorsal premotor and inferior frontal areas remained subthreshold. In a post-fMRI memory test, the participants tendency to accept modified episodes as originally encoded increased significantly when they had been presented modified versions already during the fMRI session. Our study provides valuable initial insights into the neural processing of different types of episodic prediction errors and their influence on subsequent memory.

sentences: The first crested duck genome reveals clues to genetic compensation and crest cushion formation The Chinese crested (CC) duck is a unique indigenous waterfowl breed with a phenotypic crest trait that affects its high survival rate. Therefore, the CC duck is an ideal model to investigate the genetic compensation response to maintain genetic stability. In the present study, we first generated a chromosome-level genome of CC ducks. Comparative genomics revealed genes related to tissue repair, immune function, and tumors were under strong positive selection, which suggested that these adaptive changes might enhance cancer resistance and immune response to maintain the genetic stability of CC ducks. We sub-assembled a Chinese spot-billed duck genome and detected genome-assembled structure variants among three ducks. Functional analysis revealed that a large number of structural variants were related to the immune system, which strongly suggests the occurrence of genetic compensation in the anti-tumor and immune systems to further support the survival of CC ducks. Moreover, we confirmed that the CC duck originated from the mallard ducks. Finally, we revealed the physiological and genetic basis of crest traits and identified a causative mutation in TAS2R40 that leads to crest formation. Overall, the findings of this study provide new insights into the role of genetic compensation in adaptive evolution.

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mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the hosts intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii. Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

Significance StatementWe have previously shown that mGBP2 is an essential IFN{gamma} induced protein for defense against Toxoplasma gondii infection. mGBP2 is rapidly recruited to the parasitophorous vacuole (PV) of intracellular T. gondii and is required for the rupture and/or permeabilization of the PV membrane. Subsequently mGBP2 attacks the parasite membrane. Up to now, no data about other mGBP2 interaction partners and their molecular requirement for T. gondii control has been made available.

Here, we have identified Gal9 and Ckap4 as interacting proteins of mGBP2. Gal9 and Ckap4 are essential in the mGBP2 mediated attack of the T. gondii PV. Both proteins localize at the T. gondii PV in close association with mGBP2, as shown by state-of-the-art super resolution microscopy. In addition, T. gondii growth cannot be controlled anymore in Gal9 and Ckap4 deficient cell lines, comparable to mGBP2 deficient cells.

Thus, we have identified novel effector proteins in the intricate cell autonomous immune machinery against intracellular pathogens.

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Wildlife and Marine Mammal Spatial Observatory: Observation and automated detection of Southern Right Whales in multispectral satellite imagery The Wildlife and Marine Mammal Spatial Observatory is a joint research effort for the census of wildlife and particularly of marine mammals in satellite imagery. In that context, this paper illustrates the development of a high accuracy algorithm for the detection of right whales in sub-meter resolution multispectral satellite imagery with the constraint of a relatively small sample support of 580 southern right whale images. A significant space is devoted to exploratory data analysis to describe the statistical structure of right whale pixels and ocean surface pixels across multispectral bands.

Observations of southern right whale in satellite imagery are divided into typical and atypical right whale forms and the first observations of right whale mother and calf pairs in satellite imagery are presented. Measurements of whales are furthermore automatically extracted from whale observations (major axis length, minor axis length, etc). A significant space is also devoted to statistical data exploration, a step frequently overlooked in machine learning solutions, that yet offers interesting insight into the structure of animal detection in satellite imagery. The extracted statistics can readily be used by researchers to develop detection solutions even with low sample support. The adopted solution for detection consists of feature extraction with a convolutional neural network followed by classification with a support vector machine. 20 different convolutional neural networks were tested for feature extraction. Biostatistics parameters (accuracy, sensitivity, specificity and precision) were measured for comparison. Most architectures generally achieved high performance with low false positive and false negative rates. 100% accuracy is achieved in the case of 2 convolutional neural networks, Nasnet Large and Inception V3, and only with a specific selection of multispectral bands.

NB: This is a preprint that does not include satellite imagery due recent reviews

sentences: GNL3 is an evolutionarily-conserved stem cell gene influencing cell proliferation, animal growth, and regeneration in the hydrozoan Hydractinia Nucleostemin (NS) is a vertebrate gene preferentially expressed in stem and cancer cells, which acts to regulate cell cycle progression, genome stability, and ribosome biogenesis. NS and its paralogous gene, GNL3-like (GNL3L), arose in the vertebrate clade after a duplication event from their orthologous gene, G protein Nucleolar 3 (GNL3). Research on invertebrate GNL3, however, has been limited. To gain a greater understanding of the evolution and functions of the GNL3 gene, we have performed studies in the hydrozoan cnidarian Hydractinia symbiolongicarpus, a colonial hydroid that continuously generates pluripotent stem cells throughout its life cycle and presents impressive regenerative abilities. We show that Hydractinia GNL3 is expressed in stem and germline cells. The knockdown of GNL3 reduces the number of mitotic and S-phase cells in Hydractinia larvae of different ages. Genome editing of Hydractinia GNL3 via CRISPR/Cas9 resulted in colonies with reduced growth rates, polyps with impaired regeneration capabilities, gonadal morphological defects, and low sperm motility. Collectively, our study shows that GNL3 is an evolutionarily-conserved stem cell and germline marker involved in cell proliferation, animal growth, regeneration, and sexual reproduction in Hydractinia, and sheds new light into the evolution of GNL3 and of stem cell systems.

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Hoop-like role of the cytosolic interface helix in Vibrio PomA, an ion-conducting membrane protein, in the bacterial flagellar motor Vibrio has a polar flagellum driven by sodium ions for swimming. The force-generating stator unit consists of PomA and PomB. PomA contains four-transmembrane regions and a cytoplasmic domain of approximately 100 residues which interacts with the rotor protein, FliG, to be important for the force generation of rotation. The three-dimensional structure of the stator shows that the cytosolic interface (CI) helix of PomA is located parallel to the inner membrane. In this study, we investigated the function of CI helix and its role as stator. Systematic proline mutagenesis showed that residues K64, F66, and M67 were important for this function. The mutant stators did not assemble around the rotor. Moreover, the growth defect caused by PomB plug deletion was suppressed by these mutations. We speculate that the mutations affect the structure of the helices extending from TM3 and TM4 and reduce the structural stability of the stator complex. This study suggests that the helices parallel to the inner membrane play important roles in various processes, such as the hoop-like function in securing the stability of the stator complex and the ion conduction pathway, which may lead to the elucidation of the ion permeation and assembly mechanism of the stator.

ImportanceBacteria have a motor embedded in the membrane to rotate flagella as screw for swimming. The motor is composed rotor and stator complexes. The interaction between the rotor and stator converts the electrochemical potential gradient across the membrane into motor torque. The stator functions as an ion channel and is composed of two membrane proteins, MotA and MotB for proton or PomA and PomB for sodium ion. Based on the structural data of stator, we systematically introduce the proline replacement mutations and found that the cytosolic interface (CI) helix which is located parallel to the inner membrane between the second and third transmembrane (TM) segments, performs a hoop-like function in securing the stability of the stator complex and the ion conduction pathway. The results of this study provide novel insights into the energy conversion mechanism of the flagellar motor and the general mechanism of the ion channel function.

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Putative mobilized colistin resistance genes in the human gut microbiome BackgroundThe high incidence of bacterial genes that confer resistance to last-resort antibiotics, such as colistin caused by MCR genes, poses an unprecedented threat to our civilizations health. To understand the spread, evolution, and distribution of such genes among human populations, with the final goal of diminishing their occurrence in human environments should be a priority. To tackle this problem, we investigated the distribution and prevalence of potential mcr genes in the human gut microbiome we used a set of bioinformatics tools to screen the Unified Human Gastrointestinal Genome (UHGG) collection for the presence, synteny and phylogeny of putative mcr genes, and co-located antibiotic resistance genes.

ResultsA total of 2,079 ARGs were classified as different MCR in 2,046 Metagenome assembled genomes (MAGs), present in 1,596 individuals from 41 countries, of which 215 MCRs were identified in plasmidial contigs. The genera that presented the largest number of MCR-like genes were Suterella and Parasuterella, prevalent human gut bacteria of which Suterella wadsworthensis is associated with autism. Other potential pathogens carrying MCR genes belonged to the genus Vibrio, Escherichia and Campylobacter. Finally, we identified a total of 22,746 ARGs belonging to 21 different classes in the same 2,046 MAGs, suggesting multi-resistance potential in the corresponding bacterial strains, increasing the concern of ARGs impact in the clinical settings.

ConclusionThis study uncovers the diversity of MCR-like genes in the human gut microbiome. We showed the cosmopolitan distribution of these genes in individuals worldwide and the co-presence of other antibiotic resistance genes, including Extended-spectrum beta-lactamases (ESBL). Also, we described mcr-like genes fused to a PAP2-like domain in S. wadsworthensis. Although these novel sequences increase our knowledge about the diversity and evolution of mcr-like genes, their activity and a potential colistin resistance in the corresponding strains has to be experimentally validated.

sentences: Asymmetries around the visual field: From retina to cortex to behavior Visual performance varies around the visual field. It is best near the fovea compared to the periphery, and at iso-eccentric locations it is best on the horizontal, intermediate on the lower, and poorest on the upper meridian. The fovea-to-periphery performance decline is linked to the decreases in cone density, retinal ganglion cell (RGC) density, and V1 cortical magnification factor (CMF) as eccentricity increases. The origins of polar angle asymmetries are not well understood. Optical quality and cone density vary across the retina, but recent computational modeling has shown that these factors can only account for a small percentage of behavior. Here, we investigate how visual processing beyond the cone photon absorptions contributes to polar angle asymmetries in performance. First, we quantify the extent of asymmetries in cone density, midget RGC density, and V1 CMF. We find that both polar angle asymmetries and eccentricity gradients increase from cones to mRGCs, and from mRGCs to cortex. Second, we extend our previously published computational observer model to quantify the contribution of phototransduction by the cones and spatial filtering by mRGCs to behavioral asymmetries. Starting with photons emitted by a visual display, the model simulates the effect of human optics, cone isomerizations, phototransduction, and mRGC spatial filtering. The model performs a forced choice orientation discrimination task on mRGC responses using a linear support vector machine classifier. The model shows that asymmetries in a decision-makers performance across polar angle are greater when assessing the photocurrents than when assessing isomerizations and are greater still when assessing mRGC signals. Nonetheless, the polar angle asymmetries of the mRGC outputs are still considerably smaller than those observed from human performance. We conclude that cone isomerizations, phototransduction and the spatial filtering properties of mRGCs contribute to polar angle performance differences, but that a full account of these differences will entail additional contribution from cortical representations.

sentences: Uncertainty and precaution in hunting wolves twice in a year When humanity confronts the risk of extinction of species, many people invoke precautions, especially in the face of uncertainty. Although precautionary approaches are value judgments, the optimal design and effect of precautions or lack thereof are scientific questions. We investigated Wisconsin gray wolves Canis lupus facing a second wolf-hunt in November 2021 and use three legal thresholds as the societal value judgments about precautions: (1) the 1999 population goal, 350 wolves, (2) the threshold for statutory listing under the state threatened and endangered species act, 250 wolves; and (3) state extirpation <2 wolves. This allows us to explore the quantitative relationship between precaution and uncertainty. Working from estimates of the size wolf population in April 2021 and reproduction to November, we constructed a simple linear model with uninformative priors for the period April 2021-April 2022 including an uncertain wolf-hunt in November 2021. Our first result is that the state government under-counted wolf deaths in the year preceding both wolf-hunts. We recommend better scientific analysis be used when setting wolf-hunt quotas. We find official recommendations for a quota for the November 2021 wolf-hunt risk undesirable outcomes. Even a quota of zero has a 13% chance of crossing threshold 1. Therefore, a zero death toll would be precautionary. Proponents for high quotas bear the burden of proof that their estimates are accurate, precise, and reproducible. We discuss why our approach is transferable to non-wolves. We show how scientists have the tools and concepts for quantifying and explaining the probabilities of crossing thresholds set by laws or other social norms. We recommend that scientists grapple with data gaps by explaining what the uncertainty means for policy and the public including the consequences of being wrong.

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Profile of basal cell carcinoma mutations and copy number alterations - focus on gene-associated noncoding variants Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5UTRs, 3UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, [~]50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5UTR, and CHCHD2 frequently showed mutations in the 5UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general.

Author summaryThe study is the first systematic analysis of both coding and noncoding mutations in basal cell carcinoma (BCC), the most common and the most highly mutated human cancer. Noncoding mutations accounted for [~]50% ([~]40K mutations) of all mutations detected by the standard WES approach. Among the genes frequently mutated in noncoding regions are: BAD with a hotspot in the 3UTR, DHODH with a hotspot in the Kozak sequence, and CHCHD2 with mutations in 5UTR, all genes functionally related to cell metabolism and apoptosis. Analysis of copy number alterations showed frequent chr9q deletion, encompassing PTCH1 (the key BCC tumor suppressor), and frequent copy number gain of chr9p, encompassing the genes of the immune checkpoint proteins PD-L1 and PD-L2.

sentences: SIPA1L1/SPAR1 is a non-PSD protein involved in GPCR signaling SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the postsynaptic density (PSD)-95/N-methyl-D-aspartate receptor (NMDA-R)-complex. However, contrary to this view, our super-resolution and immunoelectron microscopic analyses demonstrate that SIPA1L1 is mainly localized to general submembranous and cytoplasmic regions in neurons, but scarcely to PSD. Our screening for native interactors of SIPA1L1 identified spinophilin and neurabin-1, regulators of G protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to 2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.

sentences: wnt16 exerts pleiotropic effects on bone and lean mass in zebrafish Osteoporosis, a disease of bone fragility, and sarcopenia, a condition of reduced muscle mass and strength, commonly occur in the same individual--a condition termed osteosarcopenia. Genetic variants at the CPED1-WNT16 locus have pleiotropic effects on human bone mineral density (BMD) and lean tissue mass. However, our current understanding of Wnt16 and its influence on bone fails to explain effects on lean mass at this locus. In this study, we sought to better understand the biological mechanism underlying pleiotropy at the CPED1-WNT16 locus on BMD and lean mass. Using single cell analysis, microCT imaging, and genetic approaches in zebrafish, we show that wnt16 exerts dual influence on muscle and bone, and provide evidence that this occurs through a function in somite development, a period in which precursors for both of these tissues are specified. We show that mutations in wnt16 alter vertebral size and lean mass during axial skeletogenesis. We show that wnt16+ cells transcriptionally resemble cells within the amniote dermomyotome, and provide evidence they function as myogenic precursors during embryonic development. We also provide evidence that WNT16 is a gene of major effect on lean mass at the CPED-WNT16 locus. Our studies support the potential for wnt16 to exert pleiotropic effects on bone and lean mass through a function in myogenic precursors.

sentences: Effect of high proportion concentrate dietary on Yak jejunal structure, physiological function and protein composition during cold season The current study aimed to investigate the damage of long-term high concentrate diet feeding pattern on Yak jejunal structure, physiological function and protein composition during cold season. Twelve Datong male Yak (Bos grunniens) with the same age from cold season were randomly selected and slaughtered to determine Yak jejunal digestive enzyme activity, morphology and protein composition on different feeding patterns in Tibetan Plateau. The results showed that Yak jejunum digestive enzyme activity and morphology of grazing reared group were better than those in the intensively reared group. A total of 96 differentially expressed proteins were identified by label-free Mass Spectrometry (MS), which could be concluded to two predominant themes: protein structure and inflammatory response. Nine differentially expressed proteins were correlated in Yak jejunum damage in different feeding patterns. According to our study, feeding patterns resulted in the differences in Yak jejunum physiological function, morphology and protein composition. Our results confirmed that feeding of long-term high concentrate diet could damage the jejunum epithelial morphology and function.

sentences: Rapid genome editing by CRISPR-Cas9-POLD3 fusion Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the particular cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

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Comparative evaluation of glomerular morphometric techniques reveals differential technical artefacts between FSGS and normal glomeruli Morphometric estimates of mean glomerular volume (MGV) have clinical implications, over and above histologic data. However, MGV estimation is time-consuming, could waste tissue sections and requires expertise limiting its utility in retrospective clinical studies.

MethodsWe evaluated MGV using both plastic and paraffin-embedded tissue from control and FSGS mice (n=10 each) using the gold-standard Disector/Cavalieri technique (Vglom-Cav) and other reported techniques [2- or 3-profile technique, Weibel-Gomez method (W-G)]. Within Vglom-Cav we examined the precision of MGV estimation while using MGVs obtained from 5- or 10-individual glomeruli measurements vs the true mean (20 glomeruli).

ResultsIn both FSGS and controls, we identified an acceptable precision of 10-glomerular sampling vs true MGV within Vglom-Cav technique [88 (79-94) % of MGV obtained were within 10% of the true MGV]. The 5-glomerular sampling was less precise [70 (56, 81) % of MGV obtained were within 10% of true MGV]. In plastic based techniques, 2- or 3-profile MGVs showed greater concordance with Vglom-Cav, than W-G MGV. The new 3-profile technique offered incremental benefit to the existing 2-profile method (improved Lins concordance in control and FSGS animals). We observed a consistent reduction of Vglom values within control animals (52+/-0.06%) in paraffin-embedded tissue (vs corresponding methods in plastic) demonstrating a clear shrinkage artefact due to tissue processing. FSGS glomeruli showed significantly less and more variable shrinkage artefact likely due to glomerular fibrosis.

ConclusionWe report the precision of 5- or 10-glomerular sampling for MGV estimation using controls and FSGS animals. We demonstrate and quantify the shrinkage bias in MGV during tissue processing for paraffin-embedding that also differentiated control animals and FSGS. Our findings have implications for experimental studies using glomerular morphometry.

sentences: Prediction of biological age by morphological staging of sarcopenia in Caenorhabditis elegans Sarcopenia encompasses a progressive decline in allover muscle quantity and quality. Given its close association with aging, it may represent a valuable healthspan marker. Given the strong commonalities with human muscle structure and the facile visualization possibilities, C. elegans represents an attractive model for studying the relationship between sarcopenia and healthspan. However, classical assessment relies on visual scoring of muscle architecture, which is subjective and inaccurate. To resolve this, we have developed an automated image analysis pipeline for the detailed quantification and classification of muscle integrity in confocal microscopy images from a cohort of aging myosin::GFP reporter strains. We then extracted a variety of morphological descriptors and found a subset to scale linearly with age. This allowed us to establish a general linear model that predicts biological age from a morphological muscle signature. To validate the model, we evaluated muscle architecture in long-lived worms that are known to experience delayed sarcopenia by targeted RNAi-mediated knockdown of the daf-2 gene. We conclude that quantitative microscopy allows for staging sarcopenia in C. elegans and will be of use for systematic screening for pharmacological or genetic modulators that mitigate age-related muscle frailty and thus improve healthspan in C. elegans.

sentences: Single nucleus pituitary transcriptomic and epigenetic landscape reveals human stem cell heterogeneity with diverse regulatory mechanisms Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility for GATA3, but with TF expression for POMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.

sentences: Single-cell RNA-sequencing reveals widespread personalized, context-specific gene expression regulation in immune cells. Gene expression and its regulation can be context-dependent. To dissect this, using samples from 120 individuals, we single-cell RNA-sequenced 1.3M peripheral blood mononuclear cells exposed to three different pathogens at two time points or left unexposed. This revealed thousands of cell type-specific expression changes (eQTLs) and pathogen-induced expression changes (response QTLs) that are influenced by genetic variation. In monocytes, the strongest responder to pathogen stimulations, genetics also affected co-expression of 71.4% of these eQTL genes. For example, the pathogen recognition receptor CLEC12A showed many such co-expression interactions, but only in monocytes after 3h pathogen stimulation. Further analysis linked this to interferon-regulating transcription factors, a finding that we recapitulated in an independent cohort of patients with systemic lupus erythematosus, a condition characterized by increased interferon activity. Altogether, this study highlights the importance of context for gaining a better understanding of the mechanisms of gene regulation in health and disease.

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Broad-spectrum antiviral activity of 3D8, a nucleic acid-hydrolyzing single chain variable fragment (scFv), targeting SARS-CoV-2 and multiple coronaviruses in vitro BackgroundThe current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of Coronavirus-induced disease 19 (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single chain variable fragment (scFv), a recombinant antibody exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property.

ObjectiveThis study is aimed to investigate an antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses.

Methods3D8, a recombinant scFv antibody was evaluated for antiviral activity against SARS-CoV-2, HCoV-OC43 and PEDV in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. Nucleic acid hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay.

Results3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles respectively in 3D8 scFv-treated cells.

ConclusionsThis data demonstrates the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

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The robustness of a simple dynamic model of island biodiversity to geological and eustatic change AimBiodiversity on islands is affected by various geo-physical processes and sea-level fluctuations. Oceanic islands (never connected to a landmass) are initially vacant with diversity accumulating via colonisation and speciation, followed by a decline as islands shrink. Continental islands have species upon formation (when disconnected from the mainland) and may have transient land-bridge connections. Theoretical predictions for the effects of these geo-processes on rates of colonisation, speciation and extinction have been proposed, but methods of phylogenetic inference assume only oceanic island scenarios without accounting for island ontogeny, sea-level changes or past landmass connections. Here, we analyse to what extent ignoring geodynamics affects the inference performance of a phylogenetic island model, DAISIE, when confronted with simulated data that violate its assumptions.

LocationSimulation of oceanic and continental islands.

MethodsWe extend the DAISIE simulation model to include: area-dependent rates of colonisation and diversification associated with island ontogeny and sea-level fluctuations, and continental islands with biota present upon separation from the mainland, and shifts in rates to mimic temporary land-bridges. We quantify the error made when geo-processes are not accounted for by applying DAISIEs inference method to geodynamic simulations.

ResultsWe find that the robustness of the model to dynamic island area is high (error is small) for oceanic islands and for continental islands that have been separated for a long time, suggesting that, for these island types, it is possible to obtain reliable results when ignoring geodynamics. However, for continental islands that have been recently or frequently connected, robustness of DAISIE is low, and inference results should not be trusted.

Main conclusions: This study highlights that under a large proportion of island biogeographic geo-scenarios (oceanic islands and ancient continental fragments) a simple phylogenetic model ignoring geodynamics is empirically applicable and informative. However, recent connection to the continent cannot be ignored, requiring development of a new inference model.

sentences: In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1{beta} have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1{beta} as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1{beta} gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1{beta} protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1{beta} were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1{beta} was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1{beta} in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1{beta}, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1{beta} has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1{beta} could be considered an effective adjuvant in future in vivo and clinical tests.

sentences: ORC binds and remodels nucleosomes to specify MCM loading onto DNA The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c.. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with new analysis of genome-wide ORC binding, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

sentences: Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 M blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

sentences: Use of an enclosed elk population to assess two non-invasive methods for estimating population size Non-invasive genetic sampling and spatially explicit capture-recapture (SCR) models are used increasingly to estimate abundance of wildlife populations, but have not been adequately tested on gregarious animals such as elk (Cervus canadensis), for which correlated space use and movements violate model assumptions of independence. To evaluate the robustness and accuracy of SCR, and to assess the utility of an alternative non-invasive method for estimating density of gregarious ungulates, we utilized a tule elk (Cervus canadensis nannodes) population of known size within a fenced enclosure on the San Luis National Wildlife Refuge in central California. We evaluated fecal genetic SCR to camera trap-based random encounter model (REM) approaches to density estimation based on comparison to the true abundance. We also subsampled the dataset to explore the effects of varying search effort and elk density on the precision and accuracy of results. We found that SCR outperformed REM methods in the full datasets, and reliably provided accurate (relative bias <10%) and reasonably precise (relative standard error [≤]20%) estimates of density at moderately low to high densities (6-17 elk/km2), when the subsampling scenarios yielded a minimum average of 20 recaptures. We also found that the number of samples used to construct detection histories was a reliable predictor of precision, and could be used to establish minimum sampling requirements in future population surveys of elk. Although field-testing in free-ranging populations is needed, our results suggest that non-invasive genetic SCR is a promising tool for future population studies and monitoring of elk and potentially other gregarious ungulates. In contrast, the REM estimate of density was highly inaccurate, imprecise, and highly sensitive to camera parameters.

sentences: Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation The mammalian NF-{kappa}B p52:p52 homodimer together with its cofactor Bcl3 activates transcription of {kappa}B sites with a central G/C base pair (bp), while it is inactive toward {kappa}B sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined its structure in complex with a P-selectin(PSel)-{kappa}B DNA (5-GGGGTGACCCC-3) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-{kappa}B-DNA complex structures, which have a central A/T bp. Molecular dynamics (MD) simulations show free DNAs exist in distinct preferred conformations, and p52:p52 homodimer induces the least amount of conformational changes on the more transcriptionally active natural PSel-{kappa}B DNA in the bound form. Our binding assays further demonstrate that the fast kinetics driven by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for {kappa}B DNA in complex with NF-{kappa}B and suggest the importance of binding kinetics, dictated by free DNA conformational and dynamic states, in controlling transcriptional activation for NF-{kappa}B.

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Large fluctuations of the fusion intermediate help SARS-CoV-2 capture host cell membranes Cell entry by SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by capture of the host cell membrane and fusion with the viral envelope. Capture and fusion require the prefusion S2 to transit to its potent, fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are unavailable, and the mechanisms and timing of membrane capture and fusion are not established. Here, we constructed a full-length model of the CoV-2 FI by extrapolating from known CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the FI was remarkably flexible and executed large bending and extensional fluctuations due to three hinges in the C-terminal base. Simulations suggested a host cell membrane capture time of [~] 2 ms. Isolated fusion peptide simulations identified an N-terminal helix that directed and maintained binding to the membrane but grossly underestimated the binding time, showing that the fusion peptide environment is radically altered when attached to its host fusion protein. The large configurational fluctuations of the FI generated a substantial exploration volume that aided capture of the target membrane, and may set the waiting time for fluctuation-triggered refolding of the FI that draws the viral envelope and host cell membrane together for fusion. These results describe the FI as a machinery designed for efficient membrane capture and suggest novel potential drug targets.

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Escaping the Fate of Sisyphus: Assessing Resistome Hybridization Baits for Antimicrobial Resistance Gene Capture Finding, characterizing, and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive, and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19,933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3,565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.

Originality-Significance StatementThis work is applicable to a wide range of research. It helps to define conditions under which hybridization capture is useful regarding not only antimicrobial resistance specifically, but also more generally how to assess the ongoing utility of existing bait sets - giving objective criteria for when and by what strategies baits should be updated. We also provide a method for quantifying and comparing antimicrobial resistance genes (ARGs) similar to what is used for RNAseq experiments. This approach improves comparison of ARGs across environments. Thus, the work provides an improved foundation for ARG future studies, while cutting across traditional areas of microbiology and extending beyond.

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Overexpression of Zinc finger (GpZF) promotes drought tolerance in grass pea (Lathyrus sativus) The genes encoding Cys2/His2-type zinc finger proteins constitute a large family in higher plants consisting of a family of plant transcription factors. GpZF encodes a Cys2/His2-type zinc finger protein. The purposes of this study are to elucidate further the functions of a novel zinc finger transcription factor in the grass pea (GpZF) gene involved in the drought stress response in the grass pea (Lathyrus sativus) and to investigate its biochemical and physiological parameters under stress conditions. GpZF was expressed in grass pea. Relative gene expression analysis showed the GpZF gene in independent transgenic lines was more under drought mild and severe treatments (50% and 25% field capacity-FC). Furthermore, overexpression of this gene in grass pea results in more relative water content, free proline, and soluble sugars than the wild-type (WT) plants under 50% and 25% FC stresses. Moreover, in 25% FC, the independent transgenic lines revealed an increase in survival rates and dry weight than the WT plants. GpZF thus implies the positive role in drought stress tolerance in Lathyrus sativus. In conclusion, the transgenic grass pea plants generated in this study could be used to farm arid areas.

HighlightGpZF is a positive regulator in drought stress tolerance in grass pea.

sentences: Mapping the vocal circuitry of Alston's singing mouse with pseudorabies virus Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alstons singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

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Topic: Hyper immune Bovine Colostrum as a Low-Cost, Large-Scale Source of Antibodies against COVID-19 Many different strategies have been used to fight against COVID-19 pandemic as a therapeutics or prophylaxis approaches. However, none of them so far have used, passive immune transfer using products from immunized farm animals. Hyper immune bovine colostrums (HBC) have been used against many different respiratory and gastrointestinal tracts infections during past decades.

Six mixed Holstein X Semental dairy cattles in their 6-7 months of gestation period years were chosen for hyper immunization with COVID-19 vaccine. An isolated and very well protected site was selected and equipped according to animal husbandry code of practice, used for animal experimentation. Specific IgG level against SARS-CoV-2 virus was measured before and after vaccination in the sera, and in the colostrum following parturition. Very high specific IgG level was detected one week following second vaccination in the sera and in first colostrums after parturition. Safety of the product was approved following phase 1 of clinical trials in 40 healthy volunteers. Phase 2 of the clinical trials is underway. Early results show effectiveness of the product in reducing sore throat and cough in early stages of SARS-CoV-2 infection.

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Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications Current methods to identify RNA modifications with short-read sequencing are laborious and direct RNA sequencing gets proclaimed as the viable alternative. Herein, we harness the selective reactivity of the acrylonitrile towards the Inosine (I) and pseudouridine ({Psi}) modifications and developed a chemical probe-based direct RNA sequencing method. We first demonstrated that the chemical probe-induced differential signature profile using nanopore sequencing could facilitate the selective assessment of I and{Psi} in the in vitro synthesized RNA. Furthermore, we verified the I and{Psi} modification with single-nucleotide resolution using RNA derived from mouse brain without the need for a null dataset using knockouts. Our chemical probe-based nanopore sequencing strategy can be extended to profile multiple RNA modifications on a single RNA and may facilitate the diagnosis of disease-associated epitranscriptome markers by generating a comparative dataset in clinical scenarios.

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sentences: Single-cell protein analysis reveals metastable states during the transition to a sensory organ fate Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make to undergo sensory organ differentiation has been sucessfully described as such. We have extended these studies by focusing on the Senseless protein, which orchestrates the sensory fate transition. Wing cells contain intermediate Senseless numbers prior to their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are not consistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the four states are stable over time, the number of molecules in each state vary with time. Remarkably, the fold-change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.

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Genetically-Modified Macrophages Accelerate Myelin Repair Preventing neurodegeneration-associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents degeneration of demyelinated axons, promoting this process in patients might halt the development of permanent disability. In demyelinating mouse lesions, local overexpression of Semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases OPC recruitment and remyelination. However, molecular targeting to MS lesions is a challenge because these are disseminated in the central nervous system.

We hypothesized that a clinically-relevant paradigm to deliver Sema3F to demyelinating lesions and increase OPC recruitment may be to use blood-derived macrophages as vehicles. Thus, we chose transplantation of genetically-modified hematopoietic stem cells (HSCs) as means of obtaining circulating monocytes that overexpress Sema3F. We first demonstrated that the supernatant from Sema3F-lentiviral vector transduced HSCs stimulates OPC migration in Neuropilin 2 (Nrp2, Sema3F receptor)-dependent fashion. We then investigated whether OPCs remain responsive to Sema3F with age. While Sema3F expression in the lesions of middle-aged and old mice (characterized by decreased efficiency of OPC recruitment and remyelination) was decreased, middle-aged OPCs retained Nrp2 expression and migrated in response to both recombinant Sema3F and Sema3F-transduced cell supernatant in vitro. We then investigated whether blood cells engineered to overexpress Sema3F can target demyelinating CNS lesions and improve remyelination. Thus, we transplanted Sema3F-transduced HSCs and obtained chimeric mice (with Sema3F overexpression in blood cells), in which we induced demyelinating spinal cord lesions. Transgene-carrying cells, predominantly macrophages, quickly infiltrated lesions in both control and Sema3F chimeras. While infiltration of Sema3F-expressing cells did not alter the inflammatory status of the lesions nor OPC survival, it increased OPC recruitment, which accelerated the onset of remyelination. Our results provide a proof-of-concept that blood cells, particularly monocyte-derived macrophages, can be used to deliver pro-remyelinating agents "at the right time and place", suggesting novel means for remyelination-promoting strategies.

sentences: A re-emerging arbovirus in livestock in China: Early genomic surveillance and phylogeographic analysis Viruses in livestock represent a great risk to public health due to the close contact of their hosts with humans and the potential long-range transportation though animal trade. Here, we show how to predict outbreaks of potential zoonotic pathogens and use spatially-explicit phylogeographic and phylodynamic approaches to provide estimates of key epidemiological parameters. First, we use metagenomic next generation sequencing (mNGS) to identify a Getah virus (GETV) as the pathogen responsible for a re-emerging swine disease in China. The GETV isolate is able to replicate in a variety of cell lines including human cells and shows high pathogenicity in a mouse model suggesting a potential public health risk. We obtained 16 complete genomes and 78 E2 gene sequences from viral strains collected within China from 2017 to 2021 through large-scale surveillance among livestock, pets and mosquitoes. Moreover, phylogenetic analysis reveals that three major GETV lineages are responsible for the current epidemic in livestock in China. We identify three potential positively selected sites and mutations of interest in E2, which may impact the transmissibility and pathogenicity of the virus. We then reconstruct the evolutionary and dispersal history of the virus and test the impact of several environmental factors on the viral genetic diversity through time and on the dispersal dynamic of viral lineages. Of note, we identify temporal variation in livestock meat consumption as a main predictor of viral genetic diversity through time. Finally, phylogeographic analyses indicate that GETV preferentially circulates within areas associated with relatively higher mean annual temperature and pig population density. Our results highlight the importance of continuous surveillance of GETV among livestock in southern Chinese regions associated with relatively high temperatures, and the need to control mosquitoes in the livestock farms. Our analyses of GETV also provide a baseline for future studies of the molecular epidemiology and early warning of emerging arboviruses in China.

sentences: Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions--lacking somatic gonadal cell coverage of germ cells--are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

sentences: Sequence determinants and evolution of constitutive and alternative splicing in yeast species RNA splicing is a key process in eukaryotic gene expression. Most Intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Using a combinatorial design of synthetic introns we demonstrate how non-consensus splice site sequences affect splicing efficiency in each of the three splice sites. We then show that S. cerevisiae splicing machinery tends to select alternative 3 splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns 3 ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organisms splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, exposing intronic features that control and facilitate alternative splicing. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome.

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Structural and functional determinants inferred from deep mutational scans Mutations that affect protein binding to a cognate partner primarily occur either at buried residues or at exposed residues directly involved in partner binding. Distinguishing between these two categories based solely on mutational phenotypes is challenging. The bacterial toxin CcdB kills cells by binding to DNA Gyrase. Cell death is prevented by binding to its cognate antitoxin CcdA, at an extended interface that partially overlaps with the GyrA binding site. Using the CcdAB toxin-antitoxin (TA) system as a model, a comprehensive site-saturation mutagenesis library of CcdB was generated in its native operonic context. The mutational sensitivity of each mutant was estimated by evaluating the relative abundance of each mutant in two strains, one resistant and the other sensitive to the toxic activity of the CcdB toxin, through deep sequencing. The ability to bind CcdA was inferred through a RelE reporter gene assay, since the CcdAB complex binds to its own promoter, repressing transcription. By analysing mutant phenotypes in the CcdB sensitive, CcdB resistant and RelE reporter strains, it was possible to assign residues to buried, CcdA interacting or GyrA interacting sites. A few mutants were individually constructed, expressed, and biophysically characterised to validate molecular mechanisms responsible for the observed phenotypes. Residues inferred to be important for antitoxin binding, are also likely to be important for rejuvenating CcdB from the CcdB-Gyrase complex. Therefore, even in the absence of structural information, when coupled to appropriate genetic screens, such high-throughput strategies can be deployed for predicting structural and functional determinants of proteins.

Broader Impact StatementPartial loss-of-function mutations predominantly occur either at buried-site or exposed, active-site residues. We report a facile method to identify multiple binding sites for different interacting partners for a protein, and distinguish them from buried site and exposed non active-site residues, solely from mutational data.

sentences: Episodic memory in aspects of brain information transfer by resting-state network topology Studies suggest that resting-state functional connectivity conveys cognitive information; also, activity flow mediates cognitive information transfer. However, the exact mechanism of interregional interactions underlying episodic memory remains unclear. We performed a combined analysis of task-evoked activity and resting-state functional connectivity by activity flow mapping to estimate the information transfer mechanism of episodic memory. We found that the cognitive control and attentional networks were the most recruited structures in information transfers during both encoding and retrieval processes; these networks were correlated with task-evoked activation. Differences in information transfer intensity between encoding and retrieval mainly existed in the visual, somatomotor and hippocampal systems. Furthermore, information transfer showed high predictive power for episodic memory ability and mediated relationships between task-evoked activation and memory performance. Additional analysis indicated that structural connectivity had a transportive role in information transfer. Finally, our study presented the information transfer mechanism of episodic memory from multiple neural perspectives.

sentences: Automated identification and registration of anatomical landmarks in C. elegans The physiology of the nematode C. elegans can be visualized with many microscopy techniques. However, quantitative microscopy of C. elegans is complicated by the flexible and deformable nature of the nematode. These differences in posture and shape must be addressed in some fashion in any automated or manual analysis. Manual approaches are time intensive and require hand-labeling anatomical regions of interest. Automated tools exist, but generally rely on high-magnification imaging using labeled nuclei as fiducial markers. Here we describe a suite of new tools that allows for high-throughput analysis of whole-body images, aligned using anatomical landmarks identified from brightfield images. We show how these tools can be used in basic morphometric tasks and examine anatomical variation and morphological changes in a population over time.

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Expression of GDNF Receptors Within Nucleus Ambiguus During Rat Development ObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervation after nerve injury. In contrast, little is known regarding the expression of GDNF receptors in the formation of the nucleus ambiguus (NA) and its innervation of the larynx during embryogenesis. Differences may suggest therapeutic targets after nerve injury.

Study DesignLaboratory experiment.

MethodsRat brainstems at E14, E16, E18, E20, adult (4 animals/timepoint) were sectioned and stained for GDNF receptors: GFR-1, GFR-2, GFR-3, and Ret. Islet1 and ChAT were used as markers for motoneuron cell bodies. Sections were observed using Zeiss Axio Imager M2 Microscope and quantified using Image J.

ResultsExpression of all four GDNF receptors was identified within the nucleus ambiguus, as well as hypoglossal and facial nuclei of the adult rat brainstem. During rat development, GFR-3 and Ret exhibited upregulation within the nucleus ambiguus at E14 whereas GFR-1 began showing upregulation at E20. GFR-2 exhibited no upregulation at embryonic timepoints. Conclusion: Upregulation of the GDNF receptors within the nucleus ambiguus occur after laryngeal muscles innervation during development and may associated with maturation and maintenance of the neuromuscular synapses of the larynx before and after birth. No differences among ambiguus, hypoglossal, and facial nuclei was observed.

sentences: Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutations in genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

sentences: Visualizing cell-cell interactions between immune effector and tumor cells induced by antibodies using a proximity-dependent biosensor system Therapeutic antibodies can engage innate (NK) and adaptive (T) immune cells to eliminate pathogenic or malignant cells. Activated NK or T cells exert cytotoxic effects dependent upon direct cell-cell contact and subsequent formation of immunological synapses (IS) with target cells. Therefore, understanding antibody-mediated cell-cell interactions is crucial to optimizing antibody pharmacology and efficacy across all therapeutic areas. To date, most investigations around antibody-mediated cell-cell interactions have primarily focused on molecular-scale interactions. In this study, we developed a biosensor system to evaluate direct cell-cell contact and interactions and the formation of IS at the cell population level. In this biosensor system, two structural complementary luciferase units (SmBit and LgBit) were respectively expressed on immune effector and target cell membranes with optimized spacer lengths. Upon cell-cell contact, two subunits come together to form active NanoLuc, generating a luminescent signal for longitudinal monitoring. The selectivity of the system were optimized by adjusting the spacer lengths to assure the signal is from stable cell-cell interactions with minimal interference from nonspecific and transient intercellular contact. This system was then applied to longitudinally quantify cell-cell interactions between NK and target cells induced by an anti-CD20 antibody (rituximab) and between T and target cells induced by a bispecific (anti-CD3/CD19) T cell engaging antibody (blinatumomab) in a three-dimensional cell culture system. This biosensor system shows promise for monitoring cell-cell interactions in physiologically relevant environments and provides insights into the environmental factors that impede cell-cell interaction and antibody efficacy.

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Multiparametric assessment of cellular lipid metabolism in hypercholesterolemia Systematic insight into cellular dysfunctions can improve understanding of disease etiology, risk assessment and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validated this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrated lipid storage data into a novel readout, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlated positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explained hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking from a few mls of blood and is applicable to dissect metabolic disorders, such as hypercholesterolemia.

MotivationWe have limited information on how cellular lipid uptake and processing differ between individuals and influence the development of metabolic diseases, such as hypercholesterolemia. Available assays are labor intensive, require skilled personnel and are difficult to scale to higher throughput, making it challenging to obtain systematic functional cell-based data from individuals. To overcome this problem, we established a scalable automated analysis pipeline enabling reliable quantification of multiple cellular readouts, including lipid uptake, storage and mobilization, from different white blood cell populations. This approach provides new personalized insights into the cellular basis of hypercholesterolemia and obesity.

Graphical Abstract

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sentences: Site-Specific Synthesis of N4-Acetylcytidine in RNA Reveals Physiological Duplex Stabilization N4-acetylcytidine (ac4C) is a post-transcriptional modification of RNA that is conserved across all domains of life. All characterized sites of ac4C in eukaryotic RNA occur in the central nucleotide of a 5-CCG-3 consensus sequence. However, the thermodynamic consequences of cytidine acetylation in this context have never been assessed due to its challenging synthesis. Here we report the synthesis and biophysical characterization of ac4C in its endogenous eukaryotic sequence context. First, we develop a synthetic route to homogenous RNAs containing electrophilic acetyl groups. Next, we use thermal denaturation to interrogate the effects of ac4C on duplex stability and mismatch discrimination in a native sequence found in human ribosomal RNA. Finally, we demonstrate the ability of this chemistry to incorporate ac4C into the complex modification landscape of human tRNA, and use duplex melting combined with sequence analysis to highlight a potentially unique enforcing role for ac4C in this setting. By enabling the analysis of nucleic acid acetylation in its physiological sequence context, these studies establish a chemical foundation for understanding the function of a universally-conserved nucleobase in biology and disease.

sentences: Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a critical regulator of cytokinesis predominantly in transformed cells and its deregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals have not been clearly defined. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated perinatal lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3{beta}/{beta}-Catenin/Myc axis in an Akt-dependent manner. The phenotype was recapitulated using human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3{beta}. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting its novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.

sentences: Automated Landmarking via Multiple Templates Geometric morphometrics based on landmark data has been increasingly used in biomedical and biological research for quantifying complex phenotypes. However, manual landmarking can be laborious and subject to intra and interobserver errors. This has motivated the development of automated landmarking methods. We have recently introduced ALPACA (Automated Landmarking through Point Cloud Alignment and Correspondence), a fast method to automatically annotate landmarks via the use of a landmark template as a part of the SlicerMorph toolkit. Yet, using a single template may not consistently perform well for large study samples, especially when the sample consists of specimens with highly variable morphology, as it is common in evolutionary studies. In this study, we introduce a variation on our ALPACA pipeline that supports multiple specimen templates, which we call MALPACA. We show that MALPACA outperforms ALPACA consistently by testing on two different datasets. We also introduce a method of choosing the templates that can be used in conjunction with MALPACA, when no prior information is available. This K-means method uses an approximation of the total morphological variation in the dataset to suggest samples within the population to be used as landmark templates. While we advise investigators to pay careful attention to the template selection process in any of the template-based automated landmarking approaches, our analyses show that the introduced K-means based method of templates selection is better than randomly choosing the templates. In summary, MALPACA can accommodate larger morphological disparity commonly found in evolutionary studies with performance comparable to human observer.

sentences: Estimating the conjugative transfer rate of antibiotic resistance genes: Effect of model structural errors The spread of antibiotic resistance genes (ARG) occurs widely through plasmid transfer majorly facilitated via bacterial conjugation. To assess the spread of these mobile ARG, it is necessary to develop appropriate tools to estimate plasmid transfer rates under different environmental conditions. Process-based models are widely used for the estimation of plasmid transfer rate constants. Empirical studies have repeatedly highlighted the importance of subtle processes like delayed growth, the maturation of transconjugants, the physiological cost of plasmid carriage, and the dependence of conjugation on the cultures growth stage. However, models used for estimating the transfer rates typically neglect them. We conducted virtual mating experiments to quantify the impact of these four typical structural model deficits on the estimated plasmid transfer rate constants. We found that under all conditions, the plasmid cost and the lag phase in growth must be taken into account to obtain unbiased estimates of plasmid transfer rate constants. We observed a tendency towards the underestimation of plasmid transfer rate constants when structurally deficient models were fitted to virtual mating data. This holds for all the structural deficits and mating conditions tested in our study. Our findings might explain an important component of the negative bias in model predictions known as the plasmid paradox. We also discuss other structural deficits that could lead to an overestimation of plasmid transfer rate constants and we demonstrate the impact of ill-fitted parameters on model predictions.

sentences: The role of mental simulation in primate physical inference abilities Primates can richly parse sensory inputs to infer latent information, and adjust their behavior accordingly. It has been hypothesized that such flexible inferences are aided by simulations of internal models of the external world. However, evidence supporting this hypothesis has been based on behavioral models that do not emulate neural computations. Here, we test this hypothesis by directly comparing the behavior of humans and monkeys in a ball interception task to that of recurrent neural network (RNN) models with or without the capacity to "simulate" the underlying latent variables. Humans and monkeys had strikingly similar behavioral patterns suggesting common underlying neural computations. Comparison between primates and a large class of RNNs revealed that only RNNs that were optimized to simulate the position of the ball were able to accurately capture key features of the behavior such as systematic biases in the inference process. These results are consistent with the hypothesis that primates use mental simulation to make flexible inferences. Moreover, our work highlights a general strategy for using model neural systems to test computational hypotheses of higher brain function.

sentences: Protein sequence profile prediction using ProtAlbert transformer1 Protein profiles have many applications in bioinformatics. To construct the profile from a protein sequence, the sequence is aligned with database. However, sometimes there are no similar sequences with the query. This paper proposes a method based on pre-trained ProtAlbert transformer to predict the profile for a single protein sequence without alignment. The performance of transformers on natural languages is impressive. Protein sequences can be viewed as a language; therefore, we can benefit from using these models. We analyze the attention heads in different layers of ProtAlbert to show that the transformer can capture five essential protein characteristics of the family from a single protein sequence. These assessments are performed on the CASP13 dataset to find representative heads for each of five protein characteristics. Then, these heads are investigated on one thermophilic and two mesophilic proteins as case studies. The results show the significant attention heads for protein family properties extracted from a single protein sequence. This analysis led us to propose an algorithm called PA_SPP for profile prediction using only a single protein sequence as input. In our algorithm, we apply the masked language modeling method of ProtAlbert. The results display high similarity between the predicted profiles and HSSP profiles.

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Proximity to human settlement is directly related to carriage of critically important antimicrobial-resistant Escherichia coli and Klebsiella pneumoniae in Silver Gulls Human population and activities play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of critically important antimicrobial-resistant (CIA-R) Escherichia coli and Klebsiella pneumoniae by Silver Gulls and their proximity to human populations. Faecal swabs (n=229) were collected from Silver Gulls across 10 southern coastline locations in Western Australia (WA). The sampling locations included main town centres and remote areas. Fluoroquinolone and extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae were isolated and tested for antimicrobial sensitivity. Genome sequencing was performed to validate phenotypic resistance profiles and determine the molecular characteristics of strains. CIA-R E. coli and K. pneumoniae were detected in 69 (30.1%) and 20 (8.73%) of the faecal swabs respectively. Two large urban locations tested positive for CIA-R E. coli (frequency ranging from 34.3%-84.3%), and/or for CIA-R K. pneumoniae (frequency ranging from 12.5%-50.0%). A small number of CIA-R E. coli (3/31, 9.7%) were identified at a small tourist town, but no CIA-R bacteria were recovered from gulls at remote sites. Commonly detected E. coli sequence types (STs) included ST131 (12.5%) and ST1193 (10.0%), and five K. pneumoniae STs were found. Resistance genes including blaCTX-M-3, blaCTX-M-15 and blaCTX-M-27 were identified in both bacterial species. High-level colonisation of CIA-R E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that anthropogenic activities are strongly associated with acquisition of resistant bacteria by gulls.

ImportanceHumans play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of resistant bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) among Silver Gulls and their proximity to human populations. The frequency of resistant E. coli carriage was high (ranging from 34.3 - 84.3%) in the samples collected from areas with high human population density while resistant K. pneumoniae frequencies at these sites varied from 0 to 50%. However, resistant E. coli and K. pneumoniae were not recovered from any of the remote sites that did not have a permanent human population. This study, conducted across a large stretch of the southwestern Australian coastline, indicated that seagulls act as vectors in carrying and disseminating antimicrobial resistant bacteria, including clinically significant strains. High-level colonisation of resistant E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that human activities are strongly associated with acquisition of resistant bacteria by Silver gulls.

sentences: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin Psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition remain unknown. Here we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveal that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

sentences: Genetic diversity of local and introduced cassava germplasm in Burundi using DArTseq molecular analyses In Burundi, most of small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Steak Disease (CBSD) and Cassava Mosaic Disease (CMD) with production annual losses of US$1 billion annually. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships of local landraces and introduced elite germplasm using morphological and simple nucleotide polymorphism (SNP) markers as well as identifying a core set of germplasm from the local varieties to be used in the cassava breeding program. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Wards Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.

sentences: Variation in female songbird state determines signal strength needed to evoke copulation It is the female response to male signals that determines courtship success. In most songbirds, females control reproduction via the copulation solicitation display (CSD), an innate, stereotyped posture produced in direct response to male displays. Because CSD can be elicited in the absence of males by the presentation of recorded song, CSD production enables investigations into the effects of underlying signal features and behavioral state on female mating preferences. Using computer vision to quantify CSD trajectory in female brown-headed cowbirds (Molothrus ater), we show that both song quality and a females internal state predict CSD production, as well as the onset latency and duration of the display. We also show that CSD can be produced in a graded fashion based on both signal strength and internal state. These results emphasize the importance of underlying receiver state in determining behavioral responses and suggest that female responsiveness acts in conjunction with male signal strength to determine the efficacy of male courtship.

sentences: Controlling one's world: identification of sub-regions of primate PFC underlying goal-directed behavior Impaired detection of causal relationships between actions and their outcomes can lead to maladaptive behavior. However, causal roles of specific prefrontal cortex (PFC) sub-regions and the caudate nucleus in mediating such relationships in primates are unclear. We inactivated and over-activated five PFC sub-regions, reversibly and pharmacologically: areas 24 (perigenual anterior cingulate cortex), 32 (medial PFC), 11 (anterior orbitofrontal cortex, OFC), 14 (rostral ventromedial PFC/medial OFC) and 14-25 (caudal ventromedial PFC), and the anteromedial caudate, to examine their role in expressing learned action-outcome contingencies using a contingency degradation paradigm in marmosets. Area 24 or caudate inactivation impaired the response to contingency change, while area 11 inactivation enhanced it, and inactivation of areas 14, 32 or 14-25 had no effect. Over-activation of areas 11 and 24 impaired this response. These findings demonstrate distinct roles of PFC sub-regions in goal-directed behavior and illuminate the candidate neurobehavioral substrates of psychiatric disorders including obsessive-compulsive disorder.

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Signatures of Discriminative Copy Number Aberrations in 31 Cancer Subtypes Copy number aberrations (CNA) are one of the most important classes of genomic mutations related to oncogenetic effects. In the past three decades, a vast amount of CNA data has been generated by molecular-cytogenetic and genome sequencing based methods. While this data has been instrumental in the identification of cancer-related genes and promoted research into the relation between CNA and histo-pathologically defined cancer types, the heterogeneity of source data and derived CNV profiles pose great challenges for data integration and comparative analysis. Furthermore, a majority of existing studies have been focused on the association of CNA to pre-selected "driver" genes with limited application to rare drivers and other genomic elements.

In this study, we developed a bioinformatics pipeline to integrate a collection of 44,988 high-quality CNA profiles of high diversity. Using a hybrid model of neural networks and attention algorithm, we generated the CNA signatures of 31 cancer subtypes, depicting the uniqueness of their respective CNA landscapes. Finally, we constructed a multi-label classifier to identify the cancer type and the organ of origin from copy number profiling data. The investigation of the signatures suggested common patterns, not only of physiologically related cancer types but also of clinico-pathologically distant cancer types such as different cancers originating from the neural crest. Further experiments of classification models confirmed the effectiveness of the signatures in distinguishing different cancer types and demonstrated their potential in tumor classification.

sentences: Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating a de novo peroxidases activity. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.

sentences: Artificial selection methods from evolutionary computing show promise for directed evolution of microbes Directed microbial evolution harnesses evolutionary processes in the laboratory to construct microorganisms with enhanced or novel functional traits. Attempting to direct evolutionary processes for applied goals is fundamental to evolutionary computation, which harnesses the principles of Darwinian evolution as a general purpose search engine for solutions to challenging computational problems. Despite their overlapping approaches, artificial selection methods from evolutionary computing are not commonly applied to living systems in the laboratory. In this work, we ask if parent selection algorithms--procedures for choosing promising progenitors--from evolutionary computation might be useful for directing the evolution of microbial populations when selecting for multiple functional traits. To do so, we introduce an agent-based model of directed microbial evolution, which we used to evaluate how well three selection algorithms from evolutionary computing (tournament selection, lexicase selection, and non-dominated elite selection) performed relative to methods commonly used in the laboratory (elite and top-10% selection). We found that multi-objective selection techniques from evolutionary computing (lexicase and non-dominated elite) generally outperformed the commonly used directed evolution approaches when selecting for multiple traits of interest. Our results motivate ongoing work transferring these multi-objective selection procedures into the laboratory. Additionally, our findings suggest that more sophisticated artificial selection methods from evolutionary computation should also be evaluated for use in directed microbial evolution.

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

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Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation like intestinal stem cells, RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. Our results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete development by RSC activation.

One-Sentence SummaryActivated Drosophila renal stem cells rebuild the adult Malphigian tubules using a less efficient but more stone-resistant design.

sentences: Generative power of a protein language model trained on multiple sequence alignments Computational models starting from large ensembles of evolutionarily related protein sequences capture a representation of protein families and learn constraints associated to protein structure and function. They thus open the possibility for generating novel sequences belonging to protein families. Protein language models trained on multiple sequence alignments, such as MSA Transformer, are highly attractive candidates to this end. We propose and test an iterative method that directly uses the masked language modeling objective to generate sequences using MSA Transformer. We demonstrate that the resulting sequences generally score better than those generated by Potts models, and even than natural sequences, for homology, coevolution and structure-based measures. Moreover, MSA Transformer better reproduces the higher-order statistics and the distribution of sequences in sequence space of natural data than Potts models, although Potts models better reproduce first- and second-order statistics. MSA Transformer is thus a strong candidate for protein sequence generation and protein design.

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Environmental enrichment mitigatesthe long-lasting sequelae of perinatal fentanyl exposure The opioid epidemic is a rapidly evolving societal issue driven, in part, by a surge in synthetic opioid use. A rise in fentanyl use among pregnant women has led to a 40-fold increase in the number of perinatally-exposed infants in the past decade. These children are more likely to develop mood- and somatosensory-related conditions later in life, suggesting that fentanyl may permanently alter neural development. Here, we examined the behavioral and synaptic consequences of perinatal fentanyl exposure in adolescent male and female C57BL/6J mice and assessed the therapeutic potential of environmental enrichment to mitigate these effects. Dams were given ad libitum access to fentanyl (10 {micro}g/mL, per os) across pregnancy and until weaning (PD 21). Perinatally-exposed adolescent mice displayed hyperactivity (PD 45), enhanced sensitivity to anxiogenic environments (PD 46), and sensory maladaptation (PD 47) - sustained behavioral effects that were completely normalized by environmental enrichment (PD 21-45). Additionally, environmental enrichment normalized the fentanyl-induced changes in the frequency of miniature excitatory postsynaptic currents of layer 2/3 neurons in the primary somatosensory cortex (S1). We also demonstrate that fentanyl impairs short- and long-term potentiation in S1 layer 2/3 neurons which, instead, exhibit a sustained depression of synaptic transmission that is restored by environmental enrichment. On its own, environmental enrichment suppressed long-term depression of control S1 neurons from vehicle-treated mice subjected to standard housing conditions. These results demonstrate that the lasting effects of fentanyl can be ameliorated with a non-invasive intervention introduced during early development.

Significance StatementIllicit use of fentanyl accounts for a large proportion of opioid-related overdose deaths. Children exposed to opioids during development have a higher risk of developing neuropsychiatric disorders later in life. Here, we employ a preclinical model of perinatal fentanyl exposure that recapitulates these long-term impairments and show, for the first time, that environmental enrichment can reverse deficits in somatosensory circuit function and behavior. These findings have the potential to directly inform and guide ongoing efforts to mitigate the consequences of perinatal opioid exposure.

sentences: Evolution of class-structured populations in fluctuating environments What is the influence of periodic environmental fluctuations on life-history evolution? We present a general theoretical framework to understand and predict the long-term evolution of lifehistory traits under a broad range of ecological scenarios. Specifically, we investigate how periodic fluctuations affect selection when the population is also structured in distinct classes. This analysis yields time-varying selection gradients that clarify the influence of the fluctuations of the environment on the competitive ability of a specific life-history mutation. We use this framework to analyse the evolution of key life-history traits of pathogens. We examine three different epidemiological scenarios and we show how periodic fluctuations of the environment can affect the evolution of virulence and transmission as well as the preference for different hosts. These examples yield new and testable predictions on pathogen evolution, and illustrate how our approach can provide a better understanding of the evolutionary consequences of time-varying environmental fluctuations in a broad range of scenarios.

sentences: Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA. We mapped the mutational sensitivity of the T4 bacteriophage sliding clamp and clamp loader by deep mutagenesis, and found that residues not involved in catalysis or binding display remarkable tolerance to mutation. An exception is a glutamine residue in the AAA+ module (Gln 118) that is not located at a catalytic or interfacial site. Gln 118 forms a hydrogen-bonded junction in a helical unit that we term the central coupler, because it connects the catalytic centers to DNA and the sliding clamp. A suppressor mutation indicates that hydrogen bonding in the junction is important, and molecular dynamics simulations reveal that it maintains rigidity in the central coupler. The glutamine-mediated junction is preserved in diverse AAA+ ATPases, suggesting that a connected network of hydrogen bonds that links ATP molecules is an essential aspect of allosteric communication in these proteins.

sentences: Automated, high-accuracy, remote measurement of heart rate Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces.

sentences: The human GID complex engages two independent modules for substrate recruitment The human GID (hGID) complex is an evolutionary conserved E3 ubiquitin ligase regulating diverse biological processes including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remains poorly understood. While the yeast GID complex recognizes Pro/N-end rule substrates via yeast Gid4, the human GID complex requires a WDR26/Gid7-dependent module to trigger proteasomal degradation of mammalian HBP1. Here, using biochemical assays, crosslinking-mass spectrometry and cryo-electron microscopy, we show that hGID unexpectedly engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 together with RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end rule degron. GID4 substrate binding and ligase activity is regulated by ARMC8, while the shorter ARMC8{beta} isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provide insights into the regulation of its ligase activity.

sentences: Emergent evolutionary forces in spatial models of luminal growth in the human gut microbiota The genetic composition of the gut microbiota is constantly reshaped by ecological and evolutionary forces. These strain-level dynamics can be challenging to understand because they emerge from complex spatial growth processes that take place within a host. Here we introduce a general population genetic framework to predict how stochastic evolutionary forces emerge from simple models of microbial growth in spatially extended environments like the intestinal lumen. Our framework shows how fluid flow and longitudinal variation in growth rate combine to shape the frequencies of genetic variants in sequenced fecal samples, yielding analytical expressions for the effective generation times, selection coefficients, and rates of genetic drift. We find that the emergent evolutionary dynamics can often be captured by well-mixed models that lack explicit spatial structure, even when there is substantial spatial variation in species-level composition. By applying these results to the human colon, we find that continuous fluid flow and simple forms of wall growth are unlikely to create sufficient bottlenecks to allow large fluctuations in mutant frequencies within a host. We also find that the effective gener-ation times may be significantly shorter than expected from traditional average growth rate estimates. Our results provide a starting point for qua ntifying genetic turnover in spatially extended settings like the gut microbiota, and may be relevant for other microbial ecosystems where unidirectional fluid flow plays an important role.

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A severe form of maternal separation in rat consisting of nineteen-day 6-hour daily separation at unpredictable times minimally affects behavior across lifespan and possibly confers protection against some maladaptive outcomes Maternal separation (MS), a type of early life stress, has been associated with adverse socioemotional and behavioral outcomes throughout the lifespan across multiple species. Comprehensive longitudinal biobehavioral characterization of MS in rats is sparse and conflicting, warranting more studies. We conducted an MS paradigm involving 6-hour daily separation at unpredictable start times from P2 to P21. We hypothesized this severe form of MS would lead to developmentally emerging maladaptive biobehavioral consequences from juvenile through adult periods compared to Controls (C), especially in social behaviors. We tested: (1) own dam odor preference shortly after weaning; (2) juvenile and adult anxiety-like, sociability, and play behaviors using the light-dark test, three-chambered social interaction test, and video-coded juvenile play behavior; and (3) adult coping behaviors and neuroendocrine response using the forced swim test and blood corticosterone responses. Our results show minimal effects on biobehavioral outcomes across the lifespan. Recently weaned MS male rats had a stronger preference for their dams odor. Juvenile MS females spent more time in rough-and-tumble play than C female rats. No differences in sociability were found in the juvenile or adult periods. MS promoted a decrease in anxiety-like behavior that persisted from juvenile to adult periods. Finally, MS led to deficits in coping behavior in male adults, but basal and reactive corticosterone levels were unaltered by MS. More studies are needed to validate our surprising findings and probe the neural mechanisms underlying some of the observed protective effects.

Significance StatementMaternal separation (MS), a type of early life stress, has been shown to lead to short and long-term adverse socioemotional consequences in humans and biobehavioral outcomes in rodents. Available data on MS exposed rats are sparse, conflicting, and often lack longitudinal outcomes. We designed a 6-hour unpredictable daily MS paradigm from postnatal day (P2) to P21 to severely disrupt normal dam-pup interactions. Our results show MS leads to increased preference for own dam odor, persisting reductions in anxiety-like behaviors from juvenile to adulthood, and no effects on sociability. Adverse effects on coping behaviors were uniquely identified in males. The present study warrants reevaluation of the central dogma that MS leads to maladaptive biobehavioral outcomes.

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

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Direct Modulation Index: a measure of phase amplitude coupling for neurophysiology data Neural communication across different spatial and temporal scales is a topic of great interest in clinical and basic science. Phase-amplitude coupling (PAC) has attracted particular interest due to its functional role in a wide range of cognitive and motor functions. Here, we introduce a novel measure termed the direct modulation index (dMI). Based on the classical modulation index, dMI provides an estimate of PAC that is bound to an absolute interval between 0 and +1, resistant against noise, and reliable even for small amounts of data. To highlight the properties of this newly-proposed measure, we evaluated dMI by comparing it to the classical modulation index, mean vector length, and phase-locking value using simulated data. We ascertained that dMI provides a more accurate estimate of PAC than the existing methods and that is resilient to varying noise levels and signal lengths. As such, dMI permits a reliable investigation of PAC, which may reveal insights crucial to our understanding of functional brain architecture in key contexts such as behaviour and cognition. A Python toolbox that implements dMI and other measures of PAC is freely available at https://github.com/neurophysiological-analysis/FiNN.

Highlights- Neural coupling measures are sensitive to higher harmonics of the target oscillation.
- dMI achieves frequency-specificity by sine-fitting the phase-amplitude histogram.
- Increased robustness to noise and signal duration in comparison to other measures.
- dMI allows for reliable estimation of phase-amplitude coupling.

sentences: Heterocyclic aromatic amines (HAAs) target mitochondrial physiology Heterocyclic aromatic amines (HAA) may be found naturally in plants or are formed through the Maillard reaction when meat is cooked at high temperatures. Previous studies have indicated HAAs are especially toxic to dopaminergic neurons, whereas specific exposures may also affect other neuronal populations such as cholinergic neurons. Biochemical mechanisms of neurotoxic action implicate elevated oxidative stress and afflicted mitochondria. Notably, these mechanisms are of importance in Alzheimers disease (AD) and Parkinsons disease (PD). Most neurodegenerative disease cases are sporadic, where environmental and dietary factors may modulate risk. To further investigate HAA neurotoxicity, we tested the effect of common HAA (harmane, harmine, norharmane, PhIP, and HONH-PhIP) exposure on mitochondrial physiology, a key pathogenic target in AD and PD. Upon assessing mitochondrial bioenergetics, we observed a significant reduction in basal respiration, ATP production, maximal respiratory capacity, and non-mitochondrial respiration, indicating that HAA negatively impacts mitochondrial respiration. Followed by more specific studies on individual mitochondrial complexes, it was found that harmane, harmine, norharmane specifically inhibit complex I enzyme activity, whereas HONH-PhiP, a reactive metabolite of PhIP, inhibits Complex III enzyme activity. Our findings provide significant advancement with respect to underlying mechanisms of toxicity, though also validating HAA exposure as a potential risk factor for AD and PD.

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Ebola virus disease outbreak in the Republic of Guinea 2021: hypotheses of origin. In 2013-2016, a large-scale Ebola virus diseas (EVD) outbreak hit the countries of West Africa for the first time. Thus provoking a health crisis. The most affected countries were Sierra Leone, Liberia and the Republic of Guinea. Since the end of the outbreak in 2016, there have been no new cases until recently. In February 2021, the Republic of Guinea announced about laboratory-confirmed EVD cases in its territory.

By April 12, 2021, an Ebola virus disease outbreak in the province of NZerekore (Gueke sub-prefecture) of the Republic of Guinea had affected 16 people, 9 of whom had died. The origin of this outbreak is still unknown. This paper considers various hypotheses of its emergence. Within the frames of the study sequencing and analysis of the whole genome sequence of the strain that initiated the epidemic process have been carried out. The most likely cause of this outbreak, according to the results obtained, should be deemed a long-term persistence of the virus in the body of survivors, followed by a relapse of the disease and transmission among contact persons.

sentences: Regulation of sedimentation rate shapes the evolution of multicellularity in a unicellular relative of animals. Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves, are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma - close unicellular relatives of animals with brief transient multicellular life stages - we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least two distinct physical mechanisms. First, we find extensive (>300x) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular S. arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in four additional species from the Sphaeroforma genus, suggesting variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodelling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.

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SALARECON connects the Atlantic salmon genome to growth and feed efficiency Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

Author summaryAtlantic salmon aquaculture generates billions of euros annually, but faces challenges of sustainability. Salmon are carnivores by nature, and fish oil and fish meal have become scarce resources in fish feed production. Novel, sustainable feedstuffs are being trialed hand in hand with studies of the genetics of growth and feed efficiency. This calls for a mathematical-biological framework to integrate data with understanding of the effects of novel feeds on salmon physiology and its interplay with genetics. We have developed the SALARECON model of the core salmon metabolic reaction network, linking its genome to metabolic fluxes and growth. Computational analyses show good agreement with observed growth, amino acid limitations, and feed efficiencies, illustrating the potential for in silico studies of potential feed mixtures. In particular, in silico screening of possible diets will enable more efficient animal experiments with improved knowledge gain. We have adopted best practices for test-driven development, virtual experiments to assay metabolic capabilities, revision control, and FAIR data and model management. This facilitates fast, collaborative, reliable development of the model for future applications in sustainable production biology.

sentences: Experimental demonstration of prenatal programming of mitochondrial aerobic metabolism lasting until adulthood It is increasingly being postulated that among-individual variation in mitochondrial function underlies variation in individual performance (e.g. growth rate) and state of health. It has been suggested (but not adequately tested) that environmental conditions experienced before birth have been suggested to programme postnatal mitochondrial function, with persistent effects potentially lasting into adulthood. We tested this hypothesis in an avian model by experimentally manipulating prenatal conditions (incubation temperature and stability), then measuring mitochondrial aerobic metabolism in blood cells from the same individuals during the middle of the growth period and at adulthood. Mitochondrial aerobic metabolism changed markedly across life stages, and part of these age-related changes were influenced by the prenatal temperature conditions. A high incubation temperature induced a consistent and long-lasting increase in mitochondrial aerobic metabolism. Postnatal mitochondrial aerobic metabolism was positively associated with oxidative damage on DNA but not telomere length. While we detected significant within-individual consistency in mitochondrial aerobic metabolism across life-stages, the prenatal temperature regime only accounted for a relatively small proportion (<20%) of the consistent among-individual differences we observed. Our results demonstrate that prenatal conditions can program consistent and long-lasting differences in mitochondrial function, which could potentially underlie among-individual variation in performance and health state.

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SlKIX8 and SlKIX9 are negative regulators of leaf and fruit growth in tomato Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is still unknown. We used CRISPR-Cas9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. We found that loss of function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and that these leaves exhibited increased expression of putative SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can provide strategies to improve important traits in produce such as thickness of the fruit flesh.

One sentence summaryTwo transcriptional repressors negatively regulate organ growth in tomato with loss-of-function lines producing enlarged fruits due to an appearance of more expanded cells in the fruit flesh.

sentences: An LR framework incorporating sensitivity analysis to model multiple direct and secondary transfer events on skin surface Bayesian logistic regression is used to model the probability of DNA recovery following direct and secondary transfer and persistence over a 24 hour period between deposition and sample collection. Sub-source level likelihood ratios provided the raw data for activity-level analysis. Probabilities of secondary transfer are typically low, and there are challenges with small data-sets with low numbers of positive observations. However, the persistence of DNA over time can be modelled by a single logistic regression for both direct and secondary transfer, except that the time since deposition must be compensated by an offset value for the latter. This simplifies the analysis. Probabilities are used to inform an activity-level Bayesian Network that takes account of alternative propositions e.g. time of assault and time of social activities. The model is extended in order to take account of multiple contacts between person of interest and   victim. Variables taken into account include probabilities of direct and secondary transfer, along with background DNA from unknown individuals. The logistic regression analysis is Bayesian -for each analysis, 4000 separate simulations were carried out. Quantile assignments enable calculation of a plausible range of probabilities and sensitivity analysis is used to describe the corresponding variation of LRs that occur when modelled by the Bayesian network. It is noted that there is need for consistent experimental design, and analysis, to facilitate inter-laboratory comparisons. Appropriate recommendations are made. The open-source program written in R-code ALTRaP (Activity Level, Transfer, Recovery and Persistence) enables analysis of complex multiple transfer propositions that are commonplace in cases-work e.g. between those who cohabit. A number of case examples are provided. ALTRaP can be used to replicate the results and can easily be modified to incorporate different sets of data and variables.

sentences: Enhanced Mirror Neuron Network Activity and Effective Connectivity during Live Interaction Facial expressions are indispensable for daily communication. Previous neuroimaging studies investigating facial expression processing presented pre-recorded stimuli and lacked live face-to-face interactions. Our paradigm alternated between presentations of models real-time performance and pre-recorded videos of dynamic facial expressions to participants. Simultaneous fMRI and facial electromyography activity recordings, as well as post-scan valence and arousal ratings were acquired. Live facial expressions enhanced the subjective valence and arousal ratings as well as facial muscular responses. Live performances showed greater engagement of the right posterior superior temporal sulcus (pSTS) and right inferior frontal gyrus (IFG) more, and modulated the effective connectivity between them. A support vector machine reliably classified multivoxel activation patterns in brain regions involved in dynamic facial expression processing in mentalizing and salience networks. The results suggest that live social interaction modulates the activity and connectivity of the right mirror neuron system and enhances spontaneous mimicry, further facilitating emotional contagion.

sentences: Identifying key federal, state, and private lands strategies for achieving 30x30 in the US Achieving ambitious conservation goals to conserve at least 30% of US lands and waters by 2030 ("30x30") will require a multi-scale baseline understanding of current protections, key decision makers, and policy tools for moving forward. To help conservationists and decision makers support the science-based call to address the biodiversity and climate crises, we analyze the current spatial patterns of imperiled species biodiversity and carbon stores in the U.S. relative to protected areas. Analyses demonstrate that 30x30 is numerically achievable nationally, but high spatial heterogeneity highlights the need for tailored approaches from a mix of authorities at federal, regional, and state scales. Critically, current land protections rarely overlap with areas essential for conserving imperiled species biodiversity and mitigating climate change. Nearly one-fifth of unprotected biodiversity hotspots and carbon-rich areas are also at risk of either land conversion or climate exposure by 2050. We discuss this baseline relative to key policy considerations for making practical, substantive progress toward the goal.

sentences: Chromosome X-wide analysis of positive selection in human populations: from common and private signals to selection impact on inactivated genes and enhancers-like signatures The ability of detecting adaptive (positive) selection in the genome has opened the possibility of understanding the genetic bases of population-specific adaptations genome-wide. Here we present the analysis of recent selective sweeps specifically in the X chromosome in different human populations from the third phase of the 1000 Genomes Project using three different haplotype-based statistics. We describe numerous instances of genes under recent positive selection that fit the regimes of hard and soft sweeps, showing a higher amount of detectable sweeps in sub-Saharan Africans than in non-Africans (Europe and East Asia). A global enrichment is seen in neural-related processes while numerous genes related to fertility appear among the top candidates, reflecting the importance of reproduction in human evolution. Commonalities with previously reported genes under positive selection are found, while particularly strong new signals are reported in specific populations or shared across different continental groups. We report an enrichment of signals in genes that escape X chromosome inactivation, which may contribute to the differentiation between sexes. We also provide evidence of a widespread presence of soft-sweep-like signatures across the chromosome and a global enrichment of highly scoring regions that overlap potential regulatory elements. Among these, enhancers-like signatures seem to present putative signals of positive selection that might be in concordance with selection in their target genes. Also, particularly strong signals appear in regulatory regions that show differential activities, which might point to population-specific regulatory adaptations.

sentences: GPT-2's activations predict the degree of semantic comprehension in the human brain Language transformers, like GPT-2, have demonstrated remarkable abilities to process text, and now constitute the backbone of deep translation, summarization and dialogue algorithms. However, whether these models encode information that relates to human comprehension remains controversial. Here, we show that the representations of GPT-2 not only map onto the brain responses to spoken stories, but also predict the extent to which subjects understand narratives. To this end, we analyze 101 subjects recorded with functional Magnetic Resonance Imaging while listening to 70 min of short stories. We then fit a linear model to predict brain activity from GPT-2s activations, and correlate this mapping with subjects comprehension scores as assessed for each story. The results show that GPT-2s brain predictions significantly correlate with semantic comprehension. These effects are bilaterally distributed in the language network and peak with a correlation of R=0.50 in the angular gyrus. Overall, this study paves the way to model narrative comprehension in the brain through the lens of modern language algorithms.

sentences: Self-association configures the NAD+-binding site of plant NLR TIR domains TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the "AE interface" (mediated by helices A and E), the "BB-loop" (connecting {beta}-strand B and helix B in the structure), and the "BE interface" (mediated by the BB-loop from one TIR and the "DE surface" of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact "DE surface" is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

sentences: Discovery of a Novel Non-Narcotic Analgesic Derived from the CL-20 Explosive: Synthesis, Pharmacology and Target Identification of Thio-wurtzine, a Potent Inhibitor of the Opioid Receptors and the Voltage-Dependent Calcium Channels The number of candidate molecules for new non-narcotic analgesics is extremely limited. Here we report the identification of thiowurtzine, a new potent analgesic molecule with promising application in chronic pain treatment. We describe the chemical synthesis of this unique compound derived from the hexaazaisowurtzitane (CL-20) explosive molecule. Then we use animal experiments to assess its analgesic activity in vivo upon chemical, thermal and mechanical exposures, compared to the effect of several reference drugs. Finally, we investigate the potential receptors of thiowurtzine in order to better understand its complex mechanism of action. We use docking, molecular modeling and molecular dynamics simulations to identify and characterize the potential targets of the drug and confirm the results of the animal experiments. Our findings finally indicate that thiowurtzine may have a complex mechanism of action, by targeting the mu, kappa, delta and ORL1 opioid receptors, and the voltage-gated calcium channels as well.

sentences: Genomic dissection of the microevolution of Australian epidemic Bordetella pertussis Whooping cough (pertussis) is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccine coverage, pertussis has re-emerged in many countries and caused two large epidemics in Australia since 2007. Here, we undertook a genomic and phylogeographic study of 385 Australian B. pertussis isolates collected from 2008 to 2017. The Australian B. pertussis population was found to be composed of mostly ptxP3 strains carrying different fim3 alleles, with ptxP3-fim3A genotype expanded far more than ptxP3-fim3B. Within the former, there were six co-circulating epidemic lineages (EL1 to EL6). The multiple ELs emerged, expanded, and then declined at different time points over the two epidemics, likely driven by immune selection from pertussis vaccination and natural infection in addition to local and global transmission events. Both hard and soft selective sweeps through vaccine selection pressures determined the current B. pertussis population dynamics. Relative risk analysis found that once a new B. pertussis lineage emerged, it was more likely to spread locally within the first 1.5 years. However, after 1.5 years, any new lineage was likely to expand to a wider region and became no longer spatially structured across the country. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunised population.

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SimBu: Bias-aware simulation of bulk RNA-seq data with variable cell type composition MotivationAs complex tissues are typically composed of various cell types, deconvolution tools have been developed to computationally infer their cellular composition from bulk RNA sequencing (RNA-seq) data. To comprehensively assess deconvolution performance, gold-standard datasets are indispensable. Gold-standard, experimental techniques like flow cytometry or immunohistochemistry are resource-intensive and cannot be systematically applied to the numerous cell types and tissues profiled with high-throughput transcriptomics. The simulation of  pseudo-bulk data, generated by aggregating single-cell RNA-seq (scRNA-seq) expression profiles in pre-defined proportions, offers a scalable and cost-effective alternative. This makes it feasible to create in silico gold standards that allow fine-grained control of cell-type fractions not conceivable in an experimental setup. However, at present, no simulation software for generating pseudo-bulk RNA-seq data exists.

ResultsWe developed SimBu, an R package capable of simulating pseudo-bulk samples based on various simulation scenarios, designed to test specific features of deconvolution methods. A unique feature of SimBu is the modelling of cell-type-specific mRNA bias using experimentally-derived or data-driven scaling factors. Here, we show that SimBu can generate realistic pseudo-bulk data, recapitulating the biological and statistical features of real RNA-seq data. Finally, we illustrate the impact of mRNA bias on the evaluation of deconvolution tools and provide recommendations for the selection of suitable methods for estimating mRNA content.

ConclusionSimBu is a user-friendly and flexible tool for simulating realistic pseudo-bulk RNA-seq datasets serving as in silico gold-standard for assessing cell-type deconvolution methods.

AvailabilitySimBu is freely available at https://github.com/omnideconv/SimBu as an R package under the GPL-3 license.

Contactalex.dietrich@tum.de and markus.list@tum.de

Supplementary informationSupplementary data are available at Bioinformatics online.

sentences: Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the  replication fork trap region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the non-permissive interface of Tus-ter and termination occurs when a converging replisome meets the non-permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15-24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the inclusion of lagging strand processing enzymes as well as several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

sentences: Feasibility evaluation of transtympanic laser stimulation of the cochlea from the outer ear Infrared laser stimulation has been studied as an alternative approach to auditory prostheses. This study evaluated the feasibility of infrared laser stimulation of the cochlea from the outer ear bypassing the middle ear function. An optic fiber was inserted into the ear canal and a laser was used to irradiate the cochlea through the tympanic membrane in Mongolian gerbil. A pulsed infrared laser (10.1 mJ/cm2) and clicking sound (70 peak-to-peak equivalent sound pressure level) were presented to the animals. The amplitude of the laser-evoked cochlear response was systematically decreased following insertion of a filter between the tympanic membrane and cochlea; however, the auditory-evoked cochlear response did not decrease. The filter was removed and the laser-evoked response returned to around the original level. The amplitude ratio and the relative change in response amplitude before and during filter insertion significantly decreased as the absorbance of the infrared filter increased. These results indicate that laser irradiation could bypass the function of the middle ear and directly activate the cochlea. Therefore, an auditory prosthesis based on laser stimulation represents a possible noninvasive alternative to conventional auditory prostheses requiring surgical implants.

sentences: Forest wildflowers bloom earlier as Europe warms - but not everywhere equally. Some of the most striking biological responses to climate change are the observed shifts in the timing of life-history events of many organisms. Plants, in particular, often flower earlier in response to climate warming, and herbarium specimens are excellent witnesses of such long-term changes. However, in large-scale analyses the magnitude of phenological shifts may vary geographically, and the data are often clustered, and it is thus necessary to account for spatial correlation to avoid geographical biases and pseudoreplication. Here, we analysed herbarium specimens of 20 spring-flowering forest understory herbs to estimate how their flowering phenology shifted across Europe during the last century. Our analyses show that on average these forest wildflowers now bloom over six days earlier than at the beginning of the last century. These changes were strongly associated with warmer spring temperatures. Plants flowered on average of 3.6 days earlier per 1{degrees}C warming. However, in some parts of Europe plants flowered earlier or later than expected. This means, there was significant residual spatial variation in flowering time across Europe, even after accounting for the effects of temperature, precipitation, elevation and year. Including this spatial autocorrelation into our statistical models significantly improved model fit and reduced bias in coefficient estimates. Our study indicates that forest wildflowers in Europe strongly advanced their phenology in response to climate change during the last century, with potential severe consequences for their associated ecological communities. It also demonstrates the power of combining herbarium data with spatial modelling when testing for long-term phenology trends across large spatial scales.

sentences: Conformational Changes in the Negative Arm of the Circadian Clock Correlate with Dynamic Interactomes Involved in Post-transcriptionally Regulated Processes The circadian clock employs a transcriptional/translational negative feedback loop (TTFL) to anticipate environmental changes due to the Earths diurnal cycle, with regulation of organismal physiology believed to stem from temporal transcriptional activation by the positive arm. However, up to 80% of oscillating proteins do not have rhythmic mRNA, establishing circadian post-transcriptional regulation through unknown mechanisms. Given the pervasive conservation of the intrinsically disordered nature of negative-arm clock proteins, we hypothesized that post-transcriptional regulation may stem from conformational shifts in negative-arm proteins that time vacillations in the constituents of negative-arm macromolecular complexes to time cellular physiology. Our investigation of the negative arm clock protein in Neurospora crassa, FREQUENCY (FRQ), demonstrated temporal conformational fluidity correlated with daily changes in physiologically diverse macromolecular complex components. A parallel investigation of the macromolecular complexes centered around Drosophila melanogaster PERIOD (dPER) and human PERIOD (hPER2) found a similar number and physiological diversity of interacting partners in higher eukaryotes. Short linear motifs (SLiMs) associated with the interactors localized to disordered and phosphorylated regions on the PERs and FRQ, with disordered interactors oscillating in the macromolecular complexes over circadian time. This oscillation correlated with oscillations in post-transcriptionally regulated proteins, suggesting the negative arm may tune cellular physiology and proteostasis post-transcriptionally via vacillations in the circadian negative-arm macromolecular protein complexes.

sentences: Simulation of the omicron variant of SARS-CoV-2 shows broad antibody escape, weakened ACE2 binding, and modest increase in furin binding The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is significant reduction of antibody binding strength corresponding to escape, the omicron spike pays a cost in terms of weaker receptor binding. The furin cleavage domain is the same or weaker binding than the alpha variant, suggesting less viral load and disease intensity than the extant delta variant.

sentences: Detection of Small Fiber Neuronal Activity with Optically Pumped Magnetometers Electrodiagnosis is routinely integrated into clinical neurophysiology practice for peripheral nerve disease diagnoses such as neuropathy, demyelinating disorders, nerve entrapment/impingement, plexopathy or radiculopathy. Measured with conventional surface electrodes, the propagation of peripheral nerve action potentials along a nerve is the result of ionic current flow which, according to Amperes Law, generates a small magnetic field that is also detected as an "action current" by magnetometers such as superconducting quantum interference device (SQUID) Magnetoencephalography (MEG) systems. Optically pumped magnetometers (OPMs) are an emerging class of quantum magnetic sensors with a demonstrated sensitivity at the 1 fT/{surd}Hz level, capable of cortical action current detection. But OPMs were ostensibly constrained to low bandwidth therefore precluding their use in peripheral nerve electrodiagnosis. With careful OPM bandwidth characterization, we hypothesized OPMs may also detect compound action current signatures consistent with both Sensory Nerve Action Potential (SNAP) and the Hoffmann Reflex (H-Reflex). In as much, our work confirms OPMs enabled with expanded bandwidth can detect the magnetic signature of both the SNAP and H-Reflex. Taken together, OPMs now show potential as an emerging electrodiagnostic tool.

sentences: Hybridization In The Late Pleistocene: Merging Morphological And Genetic Evidence This is an exciting time for our understanding of the origin of our species. Previous scientific consensus saw human evolution as defined by adaptive differences (behavioural and/or biological) and the emergence of Homo sapiens as the ultimate replacement of non-modern groups by a modern, adaptively more competitive one. However, recent research has shown that the process underlying our origins was considerably more complex. While archaeological and fossil evidence suggests that behavioural complexity may not be confined to the modern human lineage, recent paleogenomic work shows that gene flow between distinct lineages (e.g. Neanderthals, Denisovans, early H. sapiens) occurred repeatedly in the Late Pleistocene, likely contributing elements to our genetic make-up that might have been crucial to our success as a diverse, adaptable species. Following these advances, the prevailing human origins model has shifted from one of near-complete replacement to a more nuanced view of partial replacement with considerable reticulation. Here we provide a brief introduction to the current genetic evidence for hybridization among hominins, its prevalence in, and effects on, comparative mammal groups, and especially how it manifests in the skull. We then explore the degree to which cranial variation seen in the fossil record of Late Pleistocene hominins from Western Eurasia corresponds with our current genetic and comparative data. We are especially interested in understanding the degree to which skeletal data can reflect admixture. Our findings indicate some correspondence between these different lines of evidence, flag individual fossils as possibly admixed, and suggest that different cranial regions may preserve hybridisation signals differentially. We urge further studies of the phenotype in order to expand our ability to detect the ways in which migration, interaction and genetic exchange have shaped the human past, beyond what is currently visible with the lens of ancient DNA.

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Forecasting the future risk of dengue epidemics facing climate change in New Caledonia, South Pacific. BackgroundDengue dynamics result from the complex interaction between the virus, the host and the vector, all being under the influence of the environment. Several studies have explored the link between climate and dengue outbreaks in New Caledonia. None of them have explored the evolution of the dengue outbreak risk facing climate change.

Methodology/Principal FindingsIn this study we chose the threshold time dependent reproduction number (Rt) as the modeling target to focus on time frames suitable for outbreak growths. A weekly statistical model of dengue outbreak risk (i.e. dengue outbreak probability) based on climate variables was developed using support vector machines (SVM) and then used in combination with CMIP5 projections of rainfall and temperature to estimate the future evolution of seasonal weekly risk and the inter-annual yearly risk of dengue outbreak up to the year 2100. The weekly risk of dengue outbreak is estimated using the number of days with maximal temperature exceeding 30.8{degrees}C during 80 days preceding the predicted week and the mean of precipitation during 60 days preceding the predicted week. According to the SVM model and to the worst greenhouse gas emission scenario projection (RCP8.5), the time frame suitable for epidemic growth will gain one month starting in November instead of December and the yearly risk of dengue outbreak occurrence increases regularly up to 2100 and reach a probability of 1 around 2080, making the dynamic of dengue fever endemic in New Caledonia.

Conclusions/SignificanceA complete method to assess seasonal and inter annual variability of the risk of dengue outbreaks with respect to climate change is proposed. We conclude that climate change is likely to increase the risk of dengue in New-Caledonia (the other non climatic parameters remaining constant) in terms of both frequency of outbreak and temporal spread of the outbreak.

Author summaryDengue virus is transmitted to human through the bite of an Aedes mosquito vector. Dengue fever is a worldwide public health concern, especially on tropical and subtropical countries. Over the last decade, the toll of dengue fever has increased in New Caledonia, raising questions about the future of the disease in this French island territory located in the South Pacific. Climate has a strong influence on dengue through its influence on the ecology of the vector and the viral cycle. Several studies have explored the link between climate and dengue in New Caledonia, with the aim of explaining and predicting dengue outbreaks. None of these studies have explored the possible outcome climate change will have on the risk of dengue fever in New Caledonia. This is the goal of this study, through projections of rainfall and temperature and the selection of an appropriate prediction target for our statistical model, we assess the climate-induced risk of dengue outbreaks up to the 2100 horizon. We prove that the inter-annual risk of dengue outbreaks in New Caledonia will raise, according to all the greenhouse gas emission scenarios and according to the high emission scenario, dengue fever will become an endemic disease in New Caledonia.

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CIAlign - A highly customisable command line tool to clean, interpret and visualise multiple sequence alignments. BackgroundThroughout biology, multiple sequence alignments (MSAs) form the basis of much investigation into biological features and relationships. These alignments are at the heart of many bioinformatics analyses. However, sequences in MSAs are often incomplete or very divergent, which leads to poorly aligned regions or large gaps in alignments. This slows down computation and can impact conclusions without being biologically relevant. Therefore, cleaning the alignment by removing these regions can substantially improve analyses. Manual editing of MSAs is very widespread but is time-consuming and difficult to reproduce.

ResultsWe present a comprehensive, user-friendly MSA trimming tool with multiple visualisation options. Our highly customisable command line tool aims to give intervention power to the user by offering various options, and outputs graphical representations of the alignment before and after processing to give the user a clear overview of what has been removed.

The main functionalities of the tool include removing regions of low coverage due to insertions, removing gaps, cropping poorly aligned sequence ends and removing sequences that are too divergent or too short. The thresholds for each function can be specified by the user and parameters can be adjusted to each individual MSA. CIAlign is designed with an emphasis on solving specific and common alignment problems and on providing transparency to the user.

ConclusionCIAlign effectively removes problematic regions and sequences from MSAs and provides novel visualisation options. This tool can be used to refine alignments for further analysis and processing. The tool is aimed at anyone who wishes to automatically clean up parts of an MSA and those requiring a new, accessible way of visualising large MSAs.

sentences: Immediate predation risk alters the relationship between potential and realised selection on male traits in the Trinidad guppy Poecilia reticulata Predation risk perception can alter mating behaviours in males and females, but the consequences for sexual selection remain underexplored. We have previously shown that in experimental populations of Trinidadian guppies Poecilia reticulata the opportunity for sexual selection (i.e. the variance in male reproductive fitness) was higher following exposure to a simulated risk of predation than in a no-risk condition. We build upon this result by exploring whether imminent predation risk affects: 1) the relationship between the opportunity for sexual selection and the actual strength of selection on male traits and 2) the traits contributing to male fitness, and he shape of selection on these traits. While predation risk increased the variance in male fitness, realised selection on traits remained unaffected. Pre- and postcopulatory traits follow complex patterns of nonlinear and correlational selection in both treatments. Differences in selection gradients deviate from predictions based on evolutionary responses to predation, the most notable being stronger selection on courtship rate under predation risk. Our results demonstrate that the operation of sexual selection can be altered by perception of an imminent predation risk and reinforce the notion that both trait-based and variance-based metrics should be employed for an informative quantification.

sentences: Overcoming the effect of ASH1L haploinsufficiency on stem cells amenability to genome editing and differentiation into the neuronal lineage - a technical report Genome editing and neuronal differentiation protocols have proliferated in the last decade. Mutations in genes that control pluripotency could lead to a potential obstacle with regards to the survival and differentiation potential of the genome-edited cell lines. Here we describe a protocol for the generation, and differentiation, of cell lines containing CRISPR/Cas9 induced mutations in the histone methyl transferase ASH1L. This chromatin modifier was previously implicated in hematopoietic stem cell pluripotency and is a major genetic risk factor for autism spectrum disorders (ASD). We find that haploinsufficiency of ASH1L leads to decreased NANOG gene expression leading to reduce cell survival and increased spontaneous differentiation. We report a method that provides improved single-cell survival with higher colony formation efficiency in ASH1L mutant stem cells. Additionally, we describe a modified dual-SMAD inhibition neuronal induction methodology that permits the successful generation of human neurons with mutations in ASH1L, in a smaller scale than previously reported methods. With our modified CRISPR-genome editing and neuronal differentiation protocols, it is possible to generate genome-edited stem cells containing mutations in genes that impact pluripotency and could affect subsequent cell lineage specific differentiation. Our detailed technical report presents cost-effective strategies that will benefit researchers focusing on both translational and basic science using stem cell experimental systems.

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Kv3 channels contribute to the excitability of sub-populations of spinal cord neurons in lamina VII Autonomic parasympathetic preganglionic neurons (PGN) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be due to recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within lamina VII of the lumbosacral spinal cord, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphological analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.

Significance statementNeural circuits in the spinal cord and pons mediate the micturition reflex. The spinal cord drives bladder contraction during micturition through the activation of parasympathetic preganglionic neurons in lamina VII of the lumbosacral spinal cord. Despite the significant contribution of these neurons to a crucial physiological reflex, neurons in this region have been under-characterised. This study therefore elucidated and thoroughly characterised distinct neuronal populations in this lamina; we propose that these populations included a fast firing interneuron and subtypes of parasympathetic preganglionic neurons. Further investigation revealed the critical importance of Kv3 channels in the fast firing ability of the interneurons, as well as in synaptic release onto parasympathetic preganglionic neurons.

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Atherosis of trophoblast type: A specific form of decidual vasculopathy distinct from atherosis of macrophage type BackgroundThere are three types of decidual vasculopathy, namely, acute atherosis, fibrinoid medial necrosis and mural arterial hyerptrophy. Persistence of vascular trophoblasts is also known to be related to maternal vascular malperfusion, but detailed study is lacking.

Material and methodsA total 1017 placentas from 2021 were collected with clinical, neonatal and placental information, and routine placental pathology examination was performed. Decidual vasculopathy was classified based on the new classification scheme including atherosis of macrophage type atherosis of trophoblast type, fibrinoid medial necrosis, mural arterial hypertrophy and mixed type vasculopathy. The significance of these morphologic changes were examined based on the clinical, neonatal and placental pathology features.

ResultsDecidual vasculopathy is classified as classic type, mural hypertrophy and mixed type. Classic type vasculopathy is further separated as atherosis and fibrinoid medial necrosis. Atherosis is defined as atherosis of macrophage type and atherosis of trophoblast type. Each category of decidual vasculopathy was evaluated in association with maternal, neonatal and placental pathologic findings. Atherosis of macrophage type and mixed type vasculopathy showed statistically significant association with preeclampsia/pregnancy induced hypertension, low birth weight and low placental weight. Atherosis of trophoblast type was associated with lower placental weight but not with specific clinical features. There is no neonatal sex dimorphism in decidual vasculopathy.

ConclusionAtherosis of trophoblast type is a distinct pathologic feature in late pregnancy, and it is associated with lower placental weight. New classification of decidual vasculopathy helps with better stratification and categorization of placental maternal vascular abnormalities.

sentences: The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species A significant challenge today within protein lipidology is to understand the relationship between cell structure, lipid membrane integrity, ion homeostasis and the embedded membrane proteins. The bacterial magnesium transporter A (MgtA) is a specialized P-type ATPase important for Mg2+ import into the cytoplasm; disrupted magnesium homeostasis is linked to intrinsic ribosome instability and nitro-oxidative stress in Salmonella strains. MgtAs function is highly dependent on anionic lipids, particularly cardiolipin, and further co-localization of cardiolipin with MgtA at the E. coli cell poles has been revealed. Here, we show that MgtA has functional specificity for cardiolipin 18:1, but it reaches maximum activity only in combination with cardiolipin 16:0, equivalent to the major components of native cardiolipin found in E. coli membranes. This is the first time it has been shown experimentally that two different lipid species from the same class, individually promoting low activity, can enhance activity in combination. Native mass spectrometry verifies the presence of two binding sites for cardiolipin and kinetic studies reveal that a cooperative relationship likely exists between the two different cardiolipin variants (cardiolipin 16:0 and cardiolipin 18:1). This is the first experimental evidence of cooperative effects between lipids of the same class, with only minor variations in their acyl chain composition, acting on a membrane protein. In summary, our results reveal that MgtA exhibits a highly complex interaction with one cardiolipin 18:1 and one cardiolipin 16:0, affecting protein activity and stability, and contributing to our understanding of the particular interactions between lipid environment and membrane proteins. Further, a better understanding of Mg2+ homeostasis in bacteria, due to its role as a virulence regulator, will provide further insights into the regulation and mechanism of bacterial infections.

sentences: Biophysical Characterisation of Human LincRNA-p21 Sense and Antisense Alu Inverted Repeats Human Long Intergenic Noncoding RNA-p21 (LincRNA-p21) is a regulatory noncoding RNA that plays an important role in promoting apoptosis. LincRNA-p21 is also critical in down-regulating many p53 target genes through its interaction with a p53 repressive complex. The interaction between LincRNA-p21 and the repressive complex is likely dependent on the RNA tertiary structure. Previous studies have determined the two-dimensional secondary structures of the sense and antisense human LincRNA-p21 AluSx1 IRs using SHAPE. However, there were no insights into its three-dimensional structure. Therefore, we in vitro transcribed the sense and antisense regions of LincRNA-p21 AluSx1 Inverted Repeats (IRs) and performed analytical ultracentrifugation, size exclusion chromatography, light scattering, and small angle X-ray scattering (SAXS) studies. Based on these studies, we determined low-resolution, three-dimensional structures of sense and antisense LincRNA-p21. By adapting previously known two-dimensional information, we calculated their sense and antisense high-resolution models and determined that they agree with the low-resolution structures determined using SAXS. Thus, our integrated approach provides insights into the structure of LincRNA-p21 Alu IRs. Our study also offers a viable pipeline for combining the secondary structure information with biophysical and computational studies to obtain high-resolution atomistic models for long noncoding RNAs.

sentences: Variable autoinhibition among deafness-associated variants of Diaphanous 1 (DIAPH1) One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to the WT protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S and I530S mutations having an affinity similar to WT and the 1213x and {Delta}ag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to reduced inhibition of actin assembly.

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Fewer but higher quality of pollinator visits determines plant invasion success in simulated plant-pollinator networks O_LIInvasive plants often use mutualisms to establish and spread in their new habitats. These plants tend to be well-integrated into plant-pollinator networks in terms of being visited by resident pollinators similarly or more frequently than the native plants.
C_LIO_LIThe long-term persistence of non-native plants, however, not only depends on the quantity of visits they receive but also on the visit quality and the resulting reproductive success, which have been rarely studied in a network context.
C_LIO_LIWe evaluated the potential of non-native plants to invade and impact plant-pollinator networks based on their ability to attract pollinators via production of floral rewards, to produce and attach pollen on pollinators, and the consequent seed production and population growth.
C_LIO_LIWe simulated the introduction of different types of non-native plants into thousands of different networks using a model that includes population dynamics of plants and pollinators, quantity and quality of pollinator visits, the dynamics of floral rewards, and pollinators adaptive foraging.
C_LIO_LIWe found the counterintuitive result that introduced plants visited by fewer pollinators were more successful at securing the high-quality visits necessary to invade. These plants did not experience the depletion of floral rewards that plants visited by too many pollinators experienced, which caused pollinators to reassign their visits to other plants and, therefore, dilute the conspecific pollen they carried.
C_LIO_LINative pollinators increased their abundance with the plant invasions, but the reallocated foraging effort concentrated on invaders reduced the quantity and quality of visits to native plants and made the networks visitation structure more modular and nested. These effects were buffered by plant richness.
C_LIO_LIInterestingly, the significant changes in visitation structure only caused a minimal decline in native plant abundance and no extinctions.
C_LIO_LIOur results call for evaluating the impact of invasive plants not only on visitation rates and network structure, but also on the demographics of native plants, which depend on other processes beyond pollination including seed production and recruitment.
C_LI

sentences: Spread of new mutations through space The terms population size and population density are often used interchangeably, when in fact they are quite different. When viewed in a spatial landscape, density is defined as the number of individuals within a square unit of distance, while population size is simply the total count of a population. In discrete population genetics models, the effective population size is known to influence the interaction between selection and random drift with selection playing a larger role in large populations while random drift has more influence in smaller populations. Using a spatially explicit simulation software we investigate how population density affects the flow of new mutations through a geographical space. Using population density, selectional advantage, and dispersal distributions, a model is developed to predict the speed at which the new allele will travel, obtaining more accurate results than current diffusion approximations provide. We note that the rate at which a neutral mutation spreads begins to decay over time while the rate of spread of an advantageous allele remains constant. We also show that new advantageous mutations spread faster in dense populations.

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Homo-and heterofermentative lactobacilli are differently affected by lignocellulosic inhibitory compounds Second generation (2G) ethanol is produced through the use of lignocellulosic biomass. However, the pretreatment processes generates a variety of molecules (furan derivatives, phenolic compounds and organic acids) that act as inhibitors of microbial metabolism, and thus reduce the efficiency of the fermentation step in this process. In this context, the present study aimed to investigate the effect of furan derivatives on the physiology of lactic acid bacteria (LAB) strains that are potential contaminants of ethanol production. Homofermentative and heterofermentative strains of laboratory LAB and isolated from first generation ethanol fermentation were used. LAB strains were challenge to grow in the presence of furfural and hydroxymethyylfurfural (HMF). We found that the effect of HMF and furfural on the growth rate of LAB is dependent of the metabolic type, and growth kinetics in the presence of these compounds is enhanced for heterofermentative LAB, whereas is inhibitory to homofermentative LAB. Sugar consumption and product formation were also enhanced in the presence of furaldehydes in heterofermentative LAB, that displayed an effective detoxification kinetics when compared to the homofermentative LAB. This knowledge is important because LAB can be explored both within the scope of bio-detoxification, being applied before the fermentation.

Key points- Heterofermentative LAB presented the ability to decrease the concentrations of furfural and HMF
- LAB can be used in the bio-detoxification to remove the inhibitors before fermentations
- The presence of furan derivatives had a growth stimulus observed in heterofermentative LAB

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BiSulfite Bolt: A BiSulfite Sequencing Analysis Platform BackgroundBisulfite sequencing is commonly employed to measure DNA methylation. Processing bisulfite sequencing data is often challenging due to the computational demands of mapping a low complexity, asymmetrical library and the lack of a unified processing toolset to produce an analysis ready methylation matrix from read alignments. To address these shortcomings, we have developed BiSulfite Bolt (BSBolt); a fast and scalable bisulfite sequencing analysis platform. BSBolt performs a pre-alignment sequencing read assessment step to improve efficiency when handling asymmetrical bisulfite sequencing libraries.

FindingsWe evaluated BSBolt against simulated and real bisulfite sequencing libraries. We found that BSBolt provides accurate and fast bisulfite sequencing alignments and methylation calls. We also compared BSBolt to several existing bisulfite alignment tools and found BSBolt outperforms Bismark, BSSeeker2, BISCUIT, and BWA-Meth based on alignment accuracy and methylation calling accuracy.

ConclusionBSBolt offers streamlined processing of bisulfite sequencing data through an integrated toolset that offers support for simulation, alignment, methylation calling, and data aggregation. BSBolt is implemented as a python package and command line utility for flexibility when building informatics pipelines. BSBolt is available at https://github.com/NuttyLogic/BSBolt under an MIT license.

sentences: Method for Zero-Waste Circular Economy using worms for plastic agriculture: Augmenting polystyrene consumption and plant growth Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that meal-worms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food waste contamination and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these finding present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.

sentences: ModelMatcher: A scientist-centric online platform to facilitate collaborations between stakeholders of rare and undiagnosed disease research Next-generation sequencing is a prevalent diagnostic tool for undiagnosed diseases, and has played a significant role in rare disease gene discovery. While this technology resolves some cases, others are given a list of possibly damaging genetic variants necessitating functional studies. Productive collaborations between scientists, clinicians, and patients can help resolve such medical mysteries, and provide insights into in vivo function of human genes. Furthermore, facilitating interactions between scientists and research funders, including non-profit organizations or commercial entities, can dramatically reduce the time to translate discoveries from bench to bedside. Several systems designed to connect clinicians and researchers with a shared gene of interest have been successful. However, these platforms exclude some stakeholders based on their role or geography. Here we describe ModelMatcher, a global online matchmaking tool designed to facilitate cross-disciplinary collaborations, especially between scientists and other stakeholders of rare and undiagnosed disease research. ModelMatcher is integrated into the Rare Diseases Models and Mechanisms Network and Matchmaker Exchange, allowing users to identify potential collaborators in other registries. This living database decreases the time from when a scientist or clinician is making discoveries regarding their genes of interest, to when they identify collaborators and sponsors to facilitate translational and therapeutic research.

sentences: Marburg and Ebola virus infections elicit a muted inflammatory state in bats The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To understand how bats resist the disease caused by filoviruses, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, Principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction of coagulation and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. Many of the responsive genes were found to be evolutionarily divergent, providing a framework for understanding the differences in outcomes of filovirus infections between bats and humans. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

sentences: MaPPeRTrac: A Massively Parallel, Portable, and Reproducible Tractography Pipeline Large-scale diffusion MRI tractography remains a significant challenge. Users must orchestrate a complex sequence of instructions that requires many software packages with complex dependencies and high computational cost. We developed MaPPeRTrac, a probabilistic tractography pipeline that simplifies and vastly accelerates this process on a wide range of high performance computing (HPC) environments. It fully automates the entire tractography pipeline, from management of raw MRI machine data to edge density imaging (EDI) of the structural connectome. Dependencies are containerized with Docker or Singularity and de-coupled from code to enable rapid proto-typing and modification. Data artifacts are strictly organized with the Brain Imaging Data Structure (BIDS) to ensure that they are findable, accessible, interoperable, and reusable following FAIR principles. The pipeline takes full advantage of HPC resources using the Parsl parallel programming frame-work, resulting in the creation of connectome datasets of unprecedented size. MaPPeRTrac is publicly available and tested on commercial and scientific hardware, so that it may accelerate brain connectome research for a broader user community.

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Analysis of Aedes aegypti microRNAs in response to Wolbachia wAlbB infection and their potential role in mosquito longevity The mosquito Aedes aegypti is the primary vector of a range of medically important viruses including dengue, Zika, West Nile, yellow fever, and chikungunya viruses. The endosymbiotic bacterium Wolbachia pipientis wAlbB strain has emerged as a promising biocontrol agent for blocking viral transmission by Ae. aegypti. In order to predict and manage the long-term efficacy of its application in the field, a thorough understanding of the molecular interactions between symbiont, host, and pathogen is required. Wolbachia are known to influence host physiology in a variety of ways including reproduction, innate immunity, metabolism, and longevity. MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that are important regulators of gene expression in eukaryotes and viruses. A number of miRNAs have been functionally described in Drosophila and mosquitoes as having roles in regulating a broad range of biological processes, as well as facilitating Wolbachia maintenance. We generated the first chromosomal map of Ae. aegypti miRNAs, and compared the miRNA expression profiles between a wAlbB-transinfected Ae. aegypti mosquito line and a tetracycline cleared derivative using deep small RNA-sequencing. We found a number of miRNAs to be modulated by wAlbB infection. We also found that some miRNAs were upregulated with age in a manner that was affected by wAlbB. By selectively inhibiting some differentially expressed miRNAs, we identified miR-2946-3p and miR-317-3p as effecting mosquito longevity.

ImportanceWolbachia is an endosymbiotic bacterium found in about 65% of insect species. It is mostly known for reproductive manipulations of the host, and also blocking replication of negative sense RNA viruses. Transinfection of Wolbachia into Aedes aegypti mosquitoes, which transmit a variety of arboviruses, including dengue virus, has provided a novel biological approach in reducing transmission of arboviruses. To gain a better understanding of Wolbachia-mosquito interactions, we investigated the impact of Wolbachia on the microRNA profile of Ae. aegypti mosquitoes. Besides identifying several differentially expressed microRNAs and their putative target genes, we found alterations of microRNAs in mosquitoes due to age. Inhibition of two miRNAs led to reduced mosquito life span. The outcomes provide insights into underlying molecular mechanisms involved in Wolbachia-host interactions.

sentences: A novel Alex3/Gαq protein complex regulating mitochondrial dynamics, dendritic complexity, and neuronal survival In neurons, mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neuronal activity. Recent studies point to GPCR and G proteins as important regulators of mitochondrial dynamics and energy metabolism. Here we show that activation of Gq negatively regulates mitochondrial dynamics and trafficking in neurons. Gq interacts with the mitochondrial trafficking protein Alex3. By generating a CNS-specific armcx3 knock-out mouse line, we demonstrate that Alex3 is required for Gq effects on mitochondrial dynamics and trafficking, and dendritic growth. Armcx3-deficient mice present decreased OXPHOS complex and ER stress response protein levels, which correlate with increased neuronal death, motor neuron and neuromuscular synaptic loss, and severe motor alterations. Finally, we show that Alex3 disassembles from the Miro1/Gq complex upon calcium rise. These data uncover a novel Alex3/Gq complex that regulates neuronal mitochondrial dynamics and neuronal death and allows the control of mitochondrial functions by GPCRs.

sentences: Recalibrating vision-for-action requires years after sight restoration from congenital blindness Being able to perform goal-directed actions requires predictive, feed-forward control, including a mapping between the visually estimated target locations and the motor commands reaching for them. When the mapping is perturbed, e.g., due to muscle fatigue or optical distortions, we are quickly able to recalibrate the sensorimotor system to update this mapping. Here we investigated whether early visual and visuomotor experience is essential for developing the ability to recalibrate. To this end, we assessed young individuals deprived from vision due to dense congenital bilateral cataracts, who were surgically treated for sight restoration only years after birth. We compared their recalibration performance to such distortion to that of age-matched sighted controls. Their recalibration performance was impaired right after surgery. This finding cannot be explained by their still lower visual acuity, since blurring vision in controls to a matching degree did not lead to comparable behavior. Nevertheless, the recalibration ability of cataract-treated participants improved with experience, matching controls performance after around 2 years from surgery. Thus, the lack of early visual experience affects visuomotor recalibration. However, this ability is not lost but slowly develops after sight restoration, highlighting the importance of sensorimotor experience for brain plasticity late in life.

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Insulin-like peptide receptor mediated signaling pathways orchestrate regulation of energy homeostasis in the Pacific oyster, Crassostrea gigas The involvement of insulin/insulin-like growth factor (IIS) signaling pathway in growth regulation of marine invertebrates remains largely unexplored. In this study, we used a fast-growing Pacific oyster (Crassostrea gigas) variety "Haida No.1" as material to unravel the role of IIS system in growth regulation in oysters. Systematic bioinformatics analyses allowed to identify major components of IIS signaling pathway and insulin-like peptide receptor (ILPR) mediated signaling pathways, including PI3K-AKT, RAS-MAPK, and TOR, in C. gigas. Expression levels of the major genes in IIS and its downstream signaling pathways were significantly higher in "Haida No.1" than wild oysters, suggesting their involvement in growth regulation of C. gigas. Expression profiles of IIS and its downstream signaling pathway genes were significantly altered by nutrient abundance and culture temperature. These results suggested that IIS signaling pathway coupled with the ILPR mediated signaling pathways orchestrated energy homeostasis to regulate growth in the Pacific oyster.

Research HighlightsO_LIILPR, IRS, IGFBPRP, and IGFALS genes were characterized in the C. gigas.
C_LIO_LIMajor genes of IIS signaling pathway were highly expressed in fast-growing C. gigas.
C_LIO_LIIIS and downstream pathways participates in energy homeostasis of oysters.
C_LIO_LIILPR mediated signaling pathways orchestrate growth regulation in oysters.
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Eukaryotic initiation factor 2α kinases regulate virulence functions, stage conversion, and the stress response in Entamoeba invadens Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. Since infection is acquired by ingestion of cysts from contaminated food and water, this parasite is prevalent in underdeveloped countries. A reptilian pathogen, Entamoeba invadens, which can encyst in culture, has long-served as a surrogate to study stage conversion. In the host, the Entamoebae must manage stress including nutrient deprivation and host immune pressure. In many systems, the stress response is characterized by down-regulation of translation, which is initiated by the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2). In mammalian cells, this phosphorylation is carried out by a family of eIF2 kinases. A canonical eIF2 translational control system exists in the Entamoebae; however, no eIF2 kinases have been characterized. In this study, we identified two eIF2 kinases in E. invadens, EiIF2K-A and EiIF2K-B. Their identity as eIF2 kinases was validated using a heterologous yeast system. We used an RNAi Trigger-mediated silencing system to reduce expression of EiIF2K-A, which also reduced expression of EiIF2K-B. Parasites with decreased kinase expression exhibited decreased phosphorylation of eIF2 and increased sensitivity to oxidative stress. Diminished kinase expression also correlates with an increased rate of encystation, a decreased the rate of excystation, and an increase in several virulence functions, erythrophagocytosis and adhesion to host cells. Taken together, these data suggest that EiIF2K-A and EiIF2K-B are authentic eIF2 kinases that may regulate the Entamoeba stress response.

ImportanceEntamoeba histolytica is a human pathogen that causes dysentery and affects millions of people worldwide. This parasite possesses a two-stage life cycle: an environmentally stable cyst and the pathogenic trophozoite. Cysts are ingested from contaminated food and water; thus, this parasite in prevalent in underdeveloped countries. Current therapies commonly cause adverse side effects; therefore, new treatments are needed. In the host, Entamoeba experiences stress brought on, in part, by the host immune system. Understanding stage conversion and the stress response of this pathogen may lead to new drug therapies. Using the model organism, E. invadens, we identified two kinases, similar to those involved in stress and stage conversion in other systems. We determined that these kinases may regulate the oxidative stress response, stage conversion, and virulence. This work is significant as it will inform future studies on the life cycle and pathogenicity of the Entamoeba species.

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Hippocampal delta oscillations entrain neuronal activity, modulate gamma amplitude and convey information about running speed on a treadmill Locomotion has long been associated with rhythmic oscillations in the rat hippocampus. Running speed and acceleration affect the spectral density at the theta (6-10 Hz) and gamma (30-150 Hz) bands and the rhythmic entrainment of neuronal activity. However, less is known about other oscillatory rhythms. Recent studies have shown that oscillatory activity in the delta (1-4 Hz) band also relates to locomotion in stationary conditions, such as running on a treadmill or in a running wheel. To further investigate the effects of stationary running on hippocampal oscillations, we recorded CA1 local field potentials and neuronal activity while rats ran at different speed protocols on a treadmill. We found a remarkable oscillatory activity at 2 Hz that was strongly modulated by running speed. Delta power and peak frequency were highest at the fastest running speed, both in constant and progressively increasing speed protocols. Delta and theta oscillations co-occurred and showed independent relationships in their instantaneous power and frequency. Moreover, the delta phase modulated the amplitude of low-gamma (20-50 Hz) oscillations and the spiking activity of putative pyramidal neurons in a speed-dependent manner. Finally, spectral components in the delta frequency range, but not theta, predicted running speed using a naive Bayes classifier. In summary, our study shows that locomotion-related delta oscillations convey information about stationary running speed and coordinate hippocampal rhythmic activity in a speed-dependent manner.

Statement of significanceTheta and gamma oscillations are hallmarks of rhythmic activity in the rat hippocampus during locomotion and exploratory behaviors, but less is known about other oscillatory rhythms. This study shows the emergence of delta ([~]2 Hz) oscillations in the dorsal CA1 of rats running on a treadmill. Locomotion-related delta oscillations are independent of the concomitant theta activity and convey information about running speed. Delta waves modulate low-gamma oscillations and the spiking activity of pyramidal neurons in a speed-dependent way. These results suggest that oscillatory activity in the delta band aids rhythmic coordination during stationary locomotor behaviors.

sentences: PDAUG - a Galaxy based toolset for peptide library analysis, visualization, and machine learning modeling Computational methods based on initial screening and prediction of peptides for desired functions have been proven effective alternatives to the lengthy and expensive methods traditionally utilized in peptide research, thus saving time and effort. However, for many researchers, the lack of expertise in utilizing programming libraries and the lack of access to computational resources and flexible pipelines are big hurdles to adopting these advanced methods. To address these barriers, we have implemented the Peptide Design and Analysis Under Galaxy (PDAUG) package, a Galaxy based python powered collection of tools, workflows, and datasets for a rapid in-silico peptide library analysis. PDAUG offers tools for peptide library generation, data visualization, in-built and public database based peptide sequence retrieval, peptide feature calculation, and machine learning modeling. In contrast to the existing methods like standard programming libraries or rigid web-based tools, PDAUG offers a GUI based toolset thus providing flexibility to build and distribute reproducible pipelines and workflows without programming expertise. Additionally, this toolset facilitates researchers to combine PDAUG with hundreds of compatible existing Galaxy tools for limitless analytic strategies. Finally, we demonstrate the usability of PDAUG on predicting anticancer properties of peptides using four different feature sets and assess the suitability of various machine learning algorithms.

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Structural effects driven by rare point mutations in amylin hormone, the type II diabetes-associated peptide BackgroundAmylin is a 37-amino-acid peptide hormone co-secreted with insulin, which participates in glucose homeostasis. This hormone is able to aggregate in a {beta}-sheet conformation and deposit in islet amyloids, a hallmark in type II diabetes. Since amylin is a gene-encoded hormone, this peptide has variants caused by point mutations that can impact its functions.

MethodsHere, we analyzed the structural effects caused by S20G and G33R point mutations which, according to the 1000 Genomes Project, have frequency in East Asian and European populations, respectively. The analyses were performed by means of aggrescan server, SNP functional effect predictors, and molecular dynamics.

ResultsWe found that both mutations have aggregation potential and cause changes in the monomeric forms when compared with wild-type amylin. Furthermore, comparative analyses with pramlintide, an amylin drug analogue, allowed us to infer that second -helix maintenance may be related to the aggregation potential.

ConclusionsThe S20G mutation has been described as pathologically related, which is in agreement with our findings. In addition, our data suggest that the G33R mutation might have a deleterious effect. The data presented here also provide new therapy opportunities, whether for creating more effective drugs for diabetes or implementing specific treatment for patients with these mutations.

General SignificanceOur data could help to better understand the impact of mutations on the wild-type amylin sequence, as a starting point for the evaluation and characterization of other variations. Moreover, these findings could improve the health of patients with type II diabetes.

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Retraction ATPase motors are promiscuous among type IVa pilus systems Bacterial surface appendages called type IVa pili (T4aP) promote diverse activities including DNA uptake, twitching motility, and virulence. These activities rely on the ability of T4aP to dynamically extend and retract from the cell surface. Dynamic extension relies on a motor ATPase commonly called PilB. Most T4aP also rely on specific motor ATPases, commonly called PilT and PilU, to dynamically and forcefully retract. Here, we systematically assess whether motor ATPases from three orthologous T4aP can functionally complement Vibrio cholerae mutants that lack their endogenous motors. We found that the PilT and PilU retraction ATPases from the three T4aP systems tested are promiscuous and promote retraction of the V. cholerae competence T4aP despite a high degree of sequence divergence. In contrast, the orthologous extension ATPases from the same T4aP systems were not able to mediate extension of the V. cholerae competence T4aP despite exhibiting a similar degree of sequence divergence. Also, we show that one of the PilT orthologs characterized does not support PilU-dependent retraction and provide some data to indicate that the C-terminus of PilT is important for PilU-dependent retraction. Together, our data suggest that retraction ATPases may have maintained a high degree of promiscuity for promoting retraction of T4aP, while extension ATPases may have evolved to become specific for their cognate systems.

IMPORTANCEOne way that bacteria interact with their environments is via hair-like appendages called type IVa pili (T4aP). These appendages dynamically extend and retract from the cell surface via the action of distinct ATPase motors. T4aP are present in diverse bacterial species. Here, we demonstrate that retraction motors from three T4aP are promiscuous, and capable of promoting retraction of a heterologous T4aP system. By contrast, the extension ATPase motors from these same T4aP systems are specific and cannot promote extension of a heterologous T4aP. Thus, these results suggest that T4aP extension may be more tightly regulated compared to T4aP retraction.

sentences: Facilitation drives estrogen receptor positive (ER+) breast cancer growth and survival under drug pressure The interplay of positive and negative interactions between drug sensitive and resistant cells determines the efficacy of treatment in heterogeneous cancer cell populations. We developed isogenic estrogen receptor positive (ER+) breast cancer cell lineages sensitive and resistant to ribociclib-induced CDK4/6 inhibition to investigate cell interactions under selective pressure. During treatment with ribociclib alone or in combination with endocrine therapies, sensitive cells survive and proliferate better when grown together with resistant cells than when grown in monoculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays detected estrone, a less active metabolite, in medium taken from each lineage but detected highly active estradiol only from resistant cells. Because sensitive cells are more dependent on estrogen signaling than resistant cells, they benefit from the increase in local estradiol from resistant cells, leading to facilitated growth when grown under treatment in coculture. We developed a suite of mathematical models to quantify the strength of competition and facilitation between cell lines and evaluate the effects of treatments that modify these interactions. These analyses support the conclusion that resistant cell lineages facilitate growth of sensitive lineages during therapy through the increased production and sharing of local estradiol and themselves suffer from increased competition and suppression as a result. These models show that blocking facilitation has the potential to control both resistant and sensitive cell populations and inhibit the emergence of a refractory population. This combination of experiments and models shows that interactions within heterogeneous cancer cell populations can combine positive and negative interactions to shape the dynamics of resistance.

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Hippocampal GFAP-positive astrocyte responses to amyloid and tau pathologies IntroductionIn Alzheimers disease clinical research, glial fibrillary acidic protein (GFAP) released in the cerebrospinal fluid and blood is widely measured and perceived as a biomarker of reactive astrogliosis. However, it was recently demonstrated that plasma GFAP levels are associated with amyloid-{beta} (A{beta}) but not tau pathology. The molecular underpinnings behind this specificity are unexplored. Here we investigated biomarker and transcriptomic associations of GFAP-positive astrocytes with A{beta} and tau pathologies in humans and mouse models.

MethodsWe studied 90 individuals with plasma GFAP, A{beta}- and Tau-PET to investigate the association between biomarkers. Then, transcriptomic analysis in hippocampal GFAP-positive astrocytes isolated from mouse models presenting A{beta} (PS2APP) or tau (P301S) pathologies was applied to explore differentially expressed genes (DEGs), Gene Ontology processes, and protein-protein interaction networks associated with each phenotype.

FindingsIn humans, we found that plasma GFAP associates with A{beta} but not tau pathology. Supporting these results, mouse transcriptomics showed scarce overlap of DEGs between the A{beta} and tau mouse models, revealing the unique nature of GFAP-positive astrocytic responses to A{beta} or tau pathology. While A{beta} GFAP-positive astrocytes were overrepresented with genes associated with proteostasis and exocytosis-related processes, tau hippocampal GFAP-positive astrocytes presented greater abnormalities in functions related to DNA/RNA processing and cytoskeleton dynamics.

InterpretationOur results offer insights into A{beta}- and tau-driven specific signatures in GFAP-positive astrocytes. Characterizing how different underlying pathologies distinctly influence astrocyte responses is critical for the biological interpretation of astrocyte-related biomarker and suggests the need to develop context-specific astrocyte targets to study AD.

FundingThis study was supported by Instituto Serrapilheira, Alzheimers Association, CAPES, CNPq and FAPERGS.

Evidence before the studyThe glial fibrillary acidic protein (GFAP) is commonly used as a proxy of reactive astrogliosis in Alzheimers disease (AD) clinical studies. Recent evidence demonstrated that increases in GFAP levels are associated with a specific aspect of AD pathology, namely amyloid-{beta} (A{beta}). However, the biological interpretation behind it was not explored in depth molecularly. Uncovering the phenotypes acquired by GFAP-positive astrocytes under A{beta} or tau influence might directly impact clinical research by improving the biological interpretation of clinical glial biomarkers used in AD.

Added value of this studyThis study revealed molecular signatures driven by AD hallmarks in hippocampal GFAP-positive astrocytes, including 1) a scarce overlap of differentially expressed genes (DEG) driven by A{beta} and tau; 2) abnormalities in proteostasis and exocytosis-related processes in A{beta}-driven reactive astrocytes and 3) impaired DNA/RNA processing and cytoskeleton dynamics in tau-driven astrocytes. Additionally, this study revealed several astrocyte-related DEGs and biological pathways specific to each aspect of AD core pathology.

Implications of all the available evidenceThese results shed light on the existence of GFAP-positive astrocyte signatures driven by A{beta}- and tau pathologies in AD. Characterizing how upstream triggers influence GFAP-positive astrocyte molecular signals may contribute to the biological interpretation of GFAP as a biomarker of AD. Additionally, the signature genes and related biological processes identified in this study might guide other studies to identify context-specific astrocyte biomarkers for AD. Finally, it paves the way to unraveling novel mechanisms and therapeutic targets capable of potentiating the effects of emerging anti[Formula] and anti-tau therapies.

sentences: A comparison of feature selection methodologies and learning algorithms in the development of a DNA methylation-based telomere length estimator The field of epigenomics holds great promise in understanding and treating disease with advances in machine learning (ML) and artificial intelligence being vitally important in this pursuit. Increasingly, research now utilises DNA methylation measures at cytosine-guanine dinucleotides (CpG) to detect disease and estimate biological traits such as aging. Given the high dimensionality of DNA methylation data, feature-selection techniques are commonly employed to reduce dimensionality and identify the most important subset of features. In this study, we test and compare a range of feature-selection methods and ML algorithms in the development of a novel DNA methylation-based telomere length (TL) estimator. We found that principal component analysis in advance of elastic net regression led to the overall best performing estimator when evaluated using a nested cross-validation analysis and two independent test cohorts. In contrast, the baseline model of elastic net regression with no prior feature reduction stage performed worst - suggesting a prior feature-selection stage may have important utility. The variance in performance across tested approaches shows that estimators are sensitive to data set heterogeneity and the development of an optimal DNA methylation-based estimator should benefit from the robust methodological approach used in this study. Additionally, we observed that different DNA methylation-based TL estimators, which have few common CpGs, are associated with many of the same biological entities. Moreover, our methodology which utilises a range of feature-selection approaches and ML algorithms could be applied to other biological markers and disease phenotypes, to examine their relationship with DNA methylation and predictive value.

sentences: Single-cell RNA sequencing of conditional 	Pten knockout neurons in mice PTEN is a well-known tumor suppressor whose mutations are also strongly associated with Autism Spectrum Disorder (ASD). The activity and function of PTEN in neurons have been studied extensively in various settings, whereas animal models offer the best opportunity to experimentally modify and test the role of PTEN in neuronal development in vivo. On the molecular level, PTENs importance in the mTOR pathways suppression is well-known but many other interactions have been suggested and yet others likely remain undiscovered. Therefore, to systematically explore the regulatory landscape downstream of PTEN on the genetic level, we have set out to establish a single-cell RNA sequencing (scRNA-seq) workflow to measure gene expression changes as a result of knocking out the Pten gene in vivo. We were able to collect brain tissue from four conditionally knocked out mouse samples and dissociate them into single live cells, which were subsequently flow-sorted and a scRNA-seq library was prepared. All of these steps were completed in a single day, yielding sequencing data from hundreds of cells suitable for cell clustering, cell type identification, and differential expression measurements. Having demonstrated its feasibility, this approach promises to aid hypothesis generation and discovery in mapping the function of Pten as well other genes whose regulatory networks remain to be fully defined.

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IDENTIFICATION OF A NOVEL BDNF-SPECIFIC CORTICOSTRIATAL CIRCUITRY BDNF is released from axon terminals originating in the cerebral cortex onto striatal neurons. Here, we characterized BDNF in the corticostriatal circuitry. First, we utilized Bdnf-Cre and Ribotag transgenic mouse lines to label BDNF-positive cells in the cortex, and detected BDNF expression in the motor cortex, medial prefrontal cortex (mPFC) and the orbitofrontal cortex (OFC). Next, we used a retrograde viral tracing strategy, in combination with Bdnf-Cre knockin mice, to map the cortical outputs of BDNF neurons in the dorsal striatum. We found that the BDNF-positive prefrontal regions differentially project to the dorsal striatum. Specifically, BDNF-expressing neurons located in the mPFC project to both dorsolateral striatum (DLS) and dorsomedial striatum (DMS), and those located in the motor cortex project to the DLS. Surprisingly however, the BDNF-expressing OFC neurons differentially target the dorsal striatum depending on their mediolateral location. Specifically, the DMS is mainly innervated by the medial part of the OFC (mOFC) whereas, the DLS receives projections specifically from the ventrolateral region of the OFC (vlOFC). Next, using an anterograde viral tracing strategy, we confirmed the presence of a BDNF-specific vlOFC-DLS circuit. Finally, we show that overexpression of BDNF in the vlOFC activates TrkB signaling specifically in the DLS but not in the DMS demonstrating the functionality of this circuit. Our study uncovers a previously unknown neural circuit composed of BDNF-positive vlOFC neurons projecting to the DLS. These findings could have important implications for the role of BDNF signaling in the OFC as well as in other corticostriatal circuitries.

Significance StatementBDNF is released in axons upon neuronal depolarization. Surprisingly, careful mapping of BDNF projecting neurons in CNS has not been conducted. Using anterograde and retrograde viral strategies in combination with transgenic reporter mice, we mapped out corticostriatal BDNF circuits. We found that mPFC BDNF neurons project to both the DMS and DLS, and the motor cortex to the DLS only. In contrast, BDNF neurons in the OFC are anatomically segregated. Whereas BDNF in mOFC neurons project to the DMS, BDNF in vlOFC project specifically to the DLS. Our findings could be important to the study of BDNF in corticostriatal circuitries.

sentences: Comparative whole plastome and low copy number phylogenetics of the core Saccharinae and Sorghinae. AO_SCPLOWBSTRACTC_SCPLOWDespite over 60 years worth of taxonomic efforts, the relationships between sugarcane (Saccharum hybrid cultivars), Sorghum and their closest evolutionary relatives remain largely unresolved. Even relationships between generally accepted genera such as Miscanthus and Saccharum have not been examined in any large-scale molecular detail. Genera such as Erianthus, Miscanthidium and Narenga pose even greater taxonomic contention. Erianthus is not monophyletic and Erianthus sect. Ripidium (Valdes and Scholz 2006, Lloyd Evans et al. 2019a; Welker et al. 2019) represents a distinct and separate genus, Tripidium Scholz. Miscanthidium is placed within Miscanthus by many workers, whilst the New World Erianthus species and Narenga are currently placed within Saccharum. As these species represent a significant portion of the gene pool that sugarcane breeders use for introgression into sugarcane, their taxonomic placement and relationships to Saccharum are of significant economic import. Erianthus species from the Americas have not been significantly employed in sugarcane breeding and may represent an untapped genetic resource. In an attempt to resolve the taxonomic relationships of these genera, we have assembled three novel chloroplasts, from Miscanthidium capense, Miscanthidium junceum and Narenga porphyrocoma (this latter assembled from transcriptomic and long read data). In parallel, five low copy number loci have been assembled from species within Saccharum, Miscanthus, Sarga and Sorghum. Phylogenetic analyses were performed using both low copy number genes and whole chloroplasts. The phylogenetic results were compared with karyotype data to circumscribe the genera most closely related to sugarcane. We reveal that genera Miscanthus and Saccharum are monophyletic and have never undergone polyploidization outside their own genera. Genera Erianthus, Miscanthidium and Narenga are allopolyploids, which excludes them from being members of Saccharum and Miscanthus. Moreover, all three of these genera have divergent evolutionary histories. We therefore support the use of the genera Miscanthus, Miscanthidium, Erianthus (for the New World Species) and Narenga for those species and genera most closely allied to Saccharum. Our data demonstrate that all these genera should be excluded from Saccharum sensu lato.

sentences: Temporal characteristics of functional connectome reconfigurations are heritable and related to cognition. The brains functional connectome is dynamic, constantly reconfiguring in an individual-specific manner. However, which characteristics of such reconfigurations are subject to genetic effects, and to what extent, is largely unknown. Here, we identified heritable dynamic features, quantified their heritability, and determined their association with cognitive phenotypes. In resting-state fMRI, we obtained multivariate features, each describing a temporal or spatial characteristic of connectome dynamics jointly over a set of connectome states. We found strong evidence for heritability of temporal features, particularly fractional occupancy (FO) and transition probability (TP), describing the trajectory of state transitions. Genetic effects explained a substantial proportion of phenotypic variance of these features (h2=.39, 95% CI= [.24.54] for FO; h2=.43, 95% CI=[.29.57] for TP). Moreover, these temporal phenotypes were associated with cognitive performance. Contrarily, we found no robust evidence for heritability of spatial features of the dynamic states (i.e., states Modularity and connectivity pattern). Genetic effects may therefore primarily contribute to how the connectome transitions across states, rather than the precise spatial instantiation of the states in individuals. In sum, genetic effects impact the duration spent in each connectivity configuration and the frequency of shifting between configurations, and such temporal features may act as endophenotypes for cognitive abilities.

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Biologging of emperor penguins 	- attachment techniques and associated deployment performance An increasing number of marine animals are equipped with biologgers, to study their physiology, behaviour and ecology, often for conservation purposes. To minimise the impacts of biologgers on the animals welfare, the Refinement principle from the Three Rs framework (Replacement, Reduction, Refinement) urges to continuously test and evaluate new and updated biologging protocols.

Here, we propose alternative and promising techniques for emperor penguin (Aptenodytes forsteri) capture and on-site logger deployment that aim to mitigate the potential negative impacts of logger deployment on these birds. We equipped adult emperor penguins for short-term (GPS, Time-Depth Recorder (TDR)) and long-term (i.e. planned for one year) deployments (ARGOS platforms, TDR), as well as juvenile emperor penguins for long-term deployments (ARGOS platforms) in the Weddell Sea area where they had not yet been studied.

We describe and qualitatively evaluate our protocols for the attachment of biologgers on-site at the colony, the capture of the animals and the recovery of the devices after deployment. We report unprecedented recaptures of long-term equipped adult emperor penguins (50% of equipped individuals recaptured after 290 days). Our data demonstrate that the traditional technique of long-term attachment by gluing the biologgers directly to the back feathers is detrimental to the birds. It causes excessive feather breakage and the loss of the devices at an early stage. We therefore propose an alternative method of attachment for back-mounted devices. This technique led to successful year-round deployments on 37.5% of the equipped juveniles. Finally, we also disclose the first deployments of leg-bracelet mounted TDRs on emperor penguins.

Our findings highlight the importance of monitoring potential impacts of biologger deployments on the animals and the need to remain critical towards established and new protocols.

sentences: Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBDs activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.

sentences: Membrane interactions of mitochondrial lipid transfer proteins The mitochondrial inner membrane is an integral part of the cellular lipid biosynthesis network. Intramitochondrial lipid transfer shuttles specific lipid species between the two mitochondrial membranes. This pathway is facilitated by designated protein complexes in the intermembrane space. A hetero-dimeric complex of Ups1 and Mdm35 has been identified in yeast to transfer phosphatidic acid from the outer to the inner membrane. Here, we define pH and lipid charge dependency of Ups1 membrane interaction. We show that Ups1 interacts with membranes in a membrane curvature dependent manner. Ups1 predominantly binds to membrane domains of positive curvature. Desorption of phosphatidic acid from positively curved membranes is energetically favorable over flat or negatively curved membranes. Hence, our results demonstrate that Ups1 specifically binds to membrane regions where extraction and insertion of lipids is enhanced.

sentences: Bioluminescent zebrafish transplantation model for drug discovery In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

sentences: Emergence of distinct and heterogeneous strains of amyloid beta as Alzheimer s disease progresses in Down syndrome 0.Amyloid beta (A{beta}) is thought to play a critical role in the pathogenesis of Alzheimers disease (AD). Prion-like A{beta} polymorphs, or "strains", can have varying pathogenicity and may underlie the phenotypic heterogeneity of the disease. In order to develop effective AD therapies, it is critical to identify the strains of A{beta} that might arise prior to the onset of clinical symptoms and understand how they may change with progressing disease. Down syndrome (DS), as the most common genetic cause of AD, presents promising opportunities to compare such features between early and advanced AD. In this work, we evaluate the neuropathology and A{beta} strain profile in the post-mortem brain tissues of 210 DS, AD, and control individuals. We assayed the levels of various A{beta} and tau species and used conformation-sensitive fluorescent probes to detect differences in A{beta} strains among individuals and populations. We found that these cohorts have some common but also some distinct strains from one another, with the most heterogeneous populations of A{beta} emerging in subjects with high levels of AD pathology. The emergence of distinct strains in DS at these later stages of disease suggests that the confluence of aging, pathology, and other DS-linked factors may favor conditions that generate strains that are unique from sAD.

sentences: A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage Somatic cells age and die, but the germ-cell lineage is immortal. In C. elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte-maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in ER activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.

sentences: A rigorous framework for detecting SARS-CoV-2 spike protein mutational ensemble from genomic and structural features The recent release of SARS-CoV-2 genomic data from several countries has provided clues into the potential antigenic drift of the coronavirus population. In particular, the genomic instability observed in the spike protein necessitates immediate action and further exploration in the context of viralhost interactions. By temporally tracking 527,988 SARS-CoV-2 genomes, we identified invariant and hypervariable regions within the spike protein. We evaluated combination of mutations from SARS-CoV-2 lineages and found that maximum number of lineage-defining mutations were present in the N-terminal domain (NTD). Based on mutant 3D-structural models of known Variants of Concern (VOCs), we found that structural properties such as accessibility, secondary structural type, and intra-protein interactions at local mutation sites are greatly altered. Further, we observed significant differences between intra-protein networks of wild-type and Delta mutant, with the latter showing dense intra-protein contacts. Extensive molecular dynamics simulations of D614G mutant spike structure with hACE2 further revealed dynamic features with 47.7% of mutations mapping on flexible regions of spike protein. Thus, we propose that significant changes within spike protein structure have occurred that may impact SARS-CoV-2 pathogenesis, and repositioning of vaccine candidates is required to contain the spread of COVID-19 pathogen.

sentences: Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor-selective degradation of nucleolin PROteolysis TArgeting Chimeras (PROTACs) induce targeted protein degradation by hijacking the intracellular ubiquitin proteasome system, thus emerging as a new strategy for drug development. However, most PROTACs generated lack cell-type selectivity and have poorly soluble in water. To address this drawback, we developed a novel PROTAC ZL216 using aptamer AS1411 as a targeting ligand of nucleolin to conjugate with a small molecule ligand of E3 ligase VHL, which shows high aqueous solubility and serum stability. Based on the differential expression of nucleolin on the cell surface, ZL216 could bind to and internalize into breast cancer cells, but not normal breast cells. Furthermore, we revealed that ZL216 promoted the formation of a nucleolin-ZL216-VHL ternary complex in breast cancer cells and potently induced nucleolin degradation in vitro and in vivo. As a result, ZL216 inhibited the proliferation and migration of breast cancer cells. These studies demonstrate that in addition to peptides and small molecule compounds, nuclei acid aptamers can also be used to generate PROTACs, which broadens the toolbox constructing PROTACs and provides a promising strategy for development of tumor-selective PROTACs.

sentences: 4 SPECIES OF BACTERIA DETERMINISTICALLY FORM A STABLE BIOFILM IN A MILLIFLUIDIC CHANNEL: ASSEMBLY PRINCIPLES AO_SCPLOWBSTRACTC_SCPLOWMultispecies microbial adherent communities are widespread in nature and organisms but the principles of their assembly and development remain unclear. Yet, the demand to understand and predict the responses of such living communities to environmental changes is increasing, calling for new approaches. Here, we test the possibility to establish a simplified but relevant model of multispecies biofilm in a laboratory setup enabling in situ real-time monitoring of the community development and control of the environmental parameters in order to decipher the mechanisms underlying the formation of the community. Using video-microscopy and species combinatorial approach, we assess the global and individual species spatiotemporal development in millifluidic channels under constant flow of nutrients. Based on quantitative measurements of expansion kinetics, local dynamics and spatial distribution, we demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians and Rhodocyclus sp.) form a dynamical community that deterministically reaches its equilibrium after about 30 hours of growth. We evidence the emergence of complexity in this simplified community as reported by spatial heterogeneity rise and non-monotonic developmental kinetics. We find interspecies interactions consisting in competition for resources -- in particular oxygen -- and both direct and indirect physical interactions but no positive feedback. Thereby, we introduce a model of multispecies adherent community where effective couplings result from individual species quest for fitness optimization in a moving and heterogenous environment. This control and the understanding of this simplified experimental model shall open new avenues to apprehend adherent bacterial communities behavior in a context of rapid global change.

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RNA is required for the maintenance of multiple cytoplasmic and nuclear membrane-less organelles. Numerous membrane-less organelles composed of a combination of RNA and proteins, referred to as RNP granules, are observed in the nucleus and cytoplasm of eukaryotic cells, including stress granules, processing bodies, Cajal bodies, and nuclear speckles. An unresolved issue is how frequently RNA molecules are required for the maintenance of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. Strikingly, we find the majority of RNP granules, including stress granules, processing bodies, Cajal bodies, nuclear speckles and the nucleolus are altered by the degradation of their RNA components. In contrast, super-enhancer complexes and TIS granules were largely unaffected by widespread intracellular RNA degradation. This highlights a critical and widespread role of RNA in the organization of many, but not all, RNP granules.

SUMMARYDecker et al. examine the role of RNA in maintaining membrane-less organelles composed of RNA and protein by activating RNase L in the cytoplasm or nucleus. Degradation of RNA alters the majority of membrane-less organelles indicating that RNA plays a widespread role in their organization.

sentences: Site level factors that affect the rate of adaptive evolution in humans and chimpanzees; the effect of contracting population size. It has previously been shown in other species that the rate of adaptive evolution is higher at sites that are more exposed in a protein structure and lower between amino acid pairs that are more dissimilar. We have investigated whether these patterns are found in the divergence between humans and chimpanzees using an extension of the MacDonald-Kreitman test. We confirm previous findings and find that the rate of adaptive evolution, relative to the rate of mutation, is higher for more exposed amino acids, lower for amino acid pairs that are more dissimilar in terms of their polarity, volume and lower for amino acid pairs that are subject to stronger purifying selection, as measured by the ratio of the numbers of non-synonymous to synonymous polymorphisms (pN /pS). However, the slope of this latter relationship is significantly shallower than in Drosophila species. We suggest that this is due to the population contraction that has occurred since humans and chimpanzees diverged. We demonstrate theoretically that population size reduction can generate an artefactual positive correlation between the rate of adaptive evolution and any factor that is correlated to the mean strength of selection acting against deleterious mutations, even if there has been no adaptive evolution (the converse is also expected). Our measure of selective constraint, pN /pS, is negatively correlated to the mean strength of selection, and hence we would expect the correlation between the rate of adaptive evolution to also be negatively correlated to pN /pS, if there is no adaptive evolution. The fact that our rate of adaptive evolution is positively correlated to pN /pS suggests that the correlation does genuinely exist, but that is has been attenuated by population size contraction.

sentences: Discovery and Biosynthesis of Persiathiacins: Unusual Polyglycosylated Thiopeptides Active against Multi-drug resistant Tuberculosis Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, in particular their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinobacterial extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2475 and Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus (MRSA) and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 M. In vitro translation assays showed that, like other thiopeptide antibiotics, persiathiacin A targets protein biosynthesis. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster. A cytochrome P450 encoded by this gene cluster catalyses the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyse the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. The discovery of the persiathiacins and their biosynthetic gene cluster thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.

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Regulatory network-based imputation of dropouts in single-cell RNA sequencing data Single-cell RNA sequencing (scRNA-seq) methods are typically unable to quantify the expression levels of all genes in a cell, creating a need for the computational prediction of missing values ( dropout imputation). Most existing dropout imputation methods are limited in the sense that they exclusively use the scRNA-seq dataset at hand and do not exploit external gene-gene relationship information. Further, it is unknown if all genes equally benefit from imputation or which imputation method works best for a given gene.

Here, we show that a transcriptional regulatory network learned from external, independent gene expression data improves dropout imputation. Using a variety of human scRNA-seq datasets we demonstrate that our network-based approach outperforms published state-of-the-art methods. The network-based approach performs particularly well for lowly expressed genes, including cell-type-specific transcriptional regulators. Further, the cell-to-cell variation of 12.6% to 48.2% of the genes could not be adequately imputed by any of the methods that we tested. In those cases gene expression levels were best predicted by the mean expression across all cells, i.e. assuming no measurable expression variation between cells. These findings suggest that different imputation methods are optimal for different genes. We thus implemented an R-package called ADImpute (available via Bioconductor https://bioconductor.org/packages/release/bioc/html/ADImpute.html) that automatically determines the best imputation method for each gene in a dataset.

Our work represents a paradigm shift by demonstrating that there is no single best imputation method. Instead, we propose that imputation should maximally exploit external information and be adapted to gene-specific features, such as expression level and expression variation across cells.

Author summarySingle-cell RNA-sequencing (scRNA-seq) allows for gene expression to be quantified in individual cells and thus plays a critical role in revealing differences between cells within tissues and characterizing them in healthy and pathological conditions. Because scRNA-seq captures the RNA content of individual cells, lowly expressed genes, for which few RNA molecules are present in the cell, are easily missed. These events are called  dropouts and considerably hinder analysis of the resulting data. In this work, we propose to make use of gene-gene relationships, learnt from external and more complete datasets, to estimate the true expression of genes that could not be quantified in a given cell. We show that this approach generally outperforms previously published methods, but also that different genes are better estimated with different methods. To allow the community to use our proposed method and combine it with existing ones, we created the R package ADImpute, available through Bioconductor.

sentences: How increased cognitive load affects the dual-task cost in healthy young adults? A randomized, double-blind sham-controlled study Our ability to interact flexibly with the surrounding environment and achieve an adaptive goal-directed response is one of the necessities of balance control. This study aimed to examine the interaction between cognitive demand and the necessity for keeping balance in unstable conditions. We examined the effects of performing two cognitive tasks, namely the Stroop test and Wisconsin Card Sorting Test (WCST), on postural balance in healthy young adults. Stroop and the WCST test assess selective attention and cognitive flexibility in shifting between rules, respectively. Thirty-two healthy adults were included in two experimental conditions (control and treatment) in random order, separated by at least seven days. Standing balance was evaluated by the Sway Medical Mobile application in eyes open (EO) and eyes closed (EC) in different stance positions: feet apart, feet together, semi-tandem, tandem, and single-leg stance (SLS). Balance was evaluated before and after the cognitive test in each experimental condition. Our findings indicate that performing cognitively demanding tasks adversely affected the balance ability in more demanding balance tests such as the SLS with EC (P {square} 0.05). However, no significant changes were seen in other balance tests (P {square} 0.05). Additionally, no significant changes were seen in balance ability after the Stroop or Wisconsin card sorting test alone. These results confirm that performing cognitively demanding tasks significantly reduced the ability to keep balance in less stable conditions. These findings have significant implications in understanding and preventing falls and incidents resulting from an impaired balance in complex and cognitively demanding conditions.

sentences: Rvb1/Rvb2 proteins couple transcription and translation during glucose starvation During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly represses translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester the mRNAs into phase-separated granules and repress their translation. Additionally, this Rvb-tethering to the mRNA drove further transcriptional upregulation of the target genes. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

sentences: The YcnI protein from Bacillus subtilis contains a copper-binding domain Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through its ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized DUF1775 domain-containing YcnI. DUF1775 domains are found across bacterial phylogeny and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance (EPR) and inductively-coupled plasma-mass spectrometry (ICP-MS) find that it can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper binding site featuring a unique mono-histidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

sentences: Alpha variant versus D614G strain in the Syrian hamster model Late 2020, SARS-CoV-2 Alpha variant from lineage B.1.1.7 emerged in United Kingdom and gradually replaced the G614 strains initially involved in the global spread of the pandemic. In this study, we used a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeeded to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicated to almost the same level and induced a comparable disease and immune response. A slight fitness advantage was noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium.

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Genome sequencing and functional genes comparison between Sphingopyxis USTB-05 and Sphingomonas morindae NBD5 Sphingomonadaceae has a large number of strains that can biodegrade hepatotoxins or environmental pollutants. The latest research reported that certain strains can also produce lutein. Based on the third-generation sequencing technology, we analyzed the whole genome sequence and compared related functional genes of two strains of Sphingomonadaceae isolated from different habitats. The genome of Sphingopyxis USTB-05 was 4,679,489 bp and contained 4312 protein coding genes. The 4,239,716 bp nuclear genome of Sphingomonas morindae NBD5, harboring 3882 protein coding genes, has two sets of chromosomes. Both strains had lutein synthesis metabolism pathway sharing identical synthetic genes of crtB, crtE, crtI, crtQ, crtL, crtR, atoB, dxs, dxr, ispD, ispE, ispDF, gcpE, ispG, ispH, ispA, ispB and ispU. Sphingopyxis USTB-05 had hepatotoxins microcystins and nodularin metabolic pathways related to 16 genes (ald, ansA, gdhA, crnA, phy, ocd, hypdh, spuC, nspC, speE, murI, murD, murC, hmgL, bioA and glsA), while these genes were not found in Sphingomonas morindae NBD5. The unique protein sequences of strain NBD5 and strain USTB-05 were 155 and 199, respectively. The analysis of whole genome of the two Sphingomonadaceae strains provides insights into prokaryote evolution, the new pathway for lutein production and the new genes for environmental pollutant biodegradation.

IMPORTANCEUnderstanding the functional genes related to the special functions of strains is essential for humans to utilize microbial resources. The ability of Sphingopyxis USTB-05 to degrade hepatotoxins microcystins and nodularin has been studied in depth, however the complete metabolic process still needs further elucidation. Sphingomonas morindae NBD5 can produce lutein, and it is necessary to determine whether there is a new pathway of lutein. In this study, the whole genome sequencing of Sphingopyxis USTB-05 and Sphingomonas morindae NBD5 were performed for the first time. Lutein synthesis metabolic pathways and synthetic genes were discovered in Sphingomonadaceae. We predicted the existence of new lutein synthesis pathways and revealed most of the genes of the new synthesis pathways. A comparative analysis of the functional genes of the two strains revealed that Sphingopyxis USTB-05 contains a large number of functional genes related to the biodegradation of hepatotoxins or hexachlorocyclohexane. Among them, the functional genes related to the biodegradation and metabolism of hexachlorocyclohexane had not been previously reported. These findings lay the foundation for the biosynthesis of lutein using Sphingomonas morindae NBD5 or Sphingopyxis USTB-05 and the application of Sphingopyxis USTB-05 for the biodegradation of hepatotoxins microcystins and nodularin or environmental pollutants.

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Young APPKI NL-G-F/NL-G-F mice display high-fat diet-induced metabolic disturbances and specific disorders associated with brain energy homeostasis. AimType 2 diabetes mellitus (T2DM) is an increased risk factor for Alzheimers disease (AD); however, the relationship between the two conditions is controversial. High-fat diet (HFD) causes cognitive impairment with/without A{beta} accumulation in middle-aged or aged transgenic (Tg) and knock-in (KI) AD mouse models, except for metabolic disorders, which commonly occur in all mice types. Alternatively, whether HFD in early life impacts energy metabolism and neurological phenotypes in young AD mouse models remains unknown. In the present study, we examined the effects of HFD on young APPKI NL-G-F/NL-G-F mice, one of the novel knock-in (KI)-AD mouse models.

MethodsThe mice were categorized by diet into two experimental groups, normal diet (ND) and HFD. Four-week-old WT and APPKI NL-G-F/NL-G-F mice were fed ND or HFD for nine weeks. Both types of mice on ND and HFD were examined during young adulthood.

ResultsHFD causes T2DM-related metabolic disturbances in young WT and APPKI NL-G-F/NL-G-F mice and specific impairment of brain energy homeostasis only in young APPKI NL-G-F/NL-G-F mice. However, HFD-induced metabolic dysfunctions had no impact on behaviors, A{beta} levels, and specific IRS1 modifications in both young APPKI NL-G-F/NL- G-F mice and young WT mice.

ConclusionHFD in early life is effective in causing metabolic disturbances in young WT and APPKI NL-G-F/NL-G-F mice but is ineffective in inducing neurological disorders in young mice, which suggests that the aging effects along with long-term HFD cause neurological alterations.

sentences: SCARF1-induced efferocytosis plays an immunomodulatory role in humans, and autoantibodies targeting SCARF1 are produced in patients with systemic lupus erythematosus Deficiency in the clearance of cellular debris is a major pathogenic factor in the emergence of autoimmune diseases. We previously demonstrated that mice deficient for scavenger receptor class F member 1 (SCARF1) develop a lupus-like autoimmune disease with symptoms similar to human systemic lupus erythematosus (SLE), including a pronounced accumulation of apoptotic cells (ACs). Therefore, we hypothesized that SCARF1 will be important for clearance of ACs and maintenance of self-tolerance in humans, and that dysregulation of this process could contribute to SLE. Here, we show that SCARF1 is highly expressed on phagocytic cells, where it functions as an efferocytosis receptor. In healthy individuals, we discovered that engagement of SCARF1 by ACs on BDCA1+ dendritic cells (DCs) initiates an interleukin-10 (IL-10) anti-inflammatory response mediated by the phosphorylation of signal transducer and activator of transcription 1 (STAT1). Unexpectedly, there was no significant difference in SCARF1 expression in SLE patient samples compared to healthy donor samples. However, we detected anti-SCARF1 autoantibodies in 26% of SLE patients, which was associated with dsDNA antibody positivity. Furthermore, our data shows a direct correlation of the levels of anti-SCARF1 in the serum and defects in the removal of ACs. Depletion of immunoglobulin restores efferocytosis in SLE serum, suggesting that defects in the removal of ACs is partially mediated by SCARF1 pathogenic autoantibodies. Our data demonstrate that human SCARF1 is an AC receptor in DCs and plays a role in maintaining tolerance and homeostasis.

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

sentences: Unique Characterization of Spatiotemporal Neuronal Network Activity We prove that three-spike motifs and phase relationships between frequency bands are essential to characterizing spatiotemporal neural activity and, in fact, sufficient. This follows from applying a theorem from optical science to show that all finite neural data have both unique triple (third-order) correlation and unique bispectrum. We simplify triple correlation by classifying three-spike motifs into motif-classes according to their sequencing, which can embody well-studied neural properties such as synchrony, feedback, feedforward, convergence, and divergence. Summing triple correlations within these classes yields a summary revealing structure underlying the neural network data. This simple approach demonstrates the power of complete statistical representations of neural activity. Since these representations generalize across recording modalities, we present our analysis as a potential avenue for investigation throughout the field.

sentences: Assessing the efficacy of antibiotic treatment for the creation of axenic earthworms Earthworms are an integral part of soil ecosystems, especially for their role in soil functions such as organic matter (OM) decomposition and nutrient cycling. Earthworms and microorganisms are interdependent, and a considerable portion of the contribution earthworms make to influencing OM fate is through interactions with microorganisms. However, the importance of the earthworm-associated microbiome is not fully understood, because it is difficult to separate the direct influence of the earthworms from the indirect influence of their microbiome. Here, we evaluated an antibiotic-based procedure for producing axenic ecologically-contrasting earthworm species (E. fetida, L. terrestris, A. chlorotica) as the first step towards soil studies aimed at understanding the importance of the earthworm microbiome for host health and function. Individual earthworms were exposed to antibiotics: cycloheximide (150 g ml-1), ampicillin (100 g ml-1), ciprofloxacin (50 g ml-1), nalidixic acid (50 g ml-1), and gentamicin (50 g ml-1) either singly or in a cocktail via culture (96 h) in a semi-solid agar carrier. Compared to the non-antibiotic treated control, the cocktail (for all three species) and ciprofloxacin (for E. fetida and A. chlorotica) treatments significantly reduced (P<0.05) culturable microbial abundance on nutrient agar and potato dextrose agar. The microbial counts were reduced to below detection (<50 CFU individual-1) for E. fetida and A. chlorotica receiving the cocktail. Illumina 16S rDNA amplicon sequence analysis of culturable L. terrestris -associated bacteria showed that antibiotic treatment influenced community composition revealing putative sensitive (Comomonas, Kosakonia and Sphingobacterium) and insensitive (Aeromonas, Pseudochrobactrum) taxa. Overall, we report a rapid, with minimal earthworm-handling, process of creating  axenic E. fetida and A. chlorotica individuals or L. terrestris with a suppressed microbiome as a tool to be used in future ecological studies of earthworm microbial interactions affecting host health and function.

sentences: Global phylogeography of marine Synechococcus in coastal areas reveals strikingly different communities than in the open ocean Marine Synechococcus comprise a numerically and ecologically prominent phytoplankton group, playing a major role in both carbon cycling and trophic networks in all oceanic regions except in the polar oceans. Despite their high abundance in coastal areas, our knowledge of Synechococcus communities in these environments is based on only a few local studies. Here, we use the global metagenome dataset of the Ocean Sampling Day (June 21st 2014) to get a snapshot of the taxonomic composition of coastal Synechococcus communities worldwide, by recruitment on a reference database of 141 picocyanobacterial genomes, representative of the whole Prochlorococcus, Synechococcus and Cyanobium diversity. This allowed us to unravel drastic community shifts over small to medium scale gradients of environmental factors, in particular along European coasts. The combined analysis of the phylogeography of natural populations and the thermophysiological characterization of eight strains, representative of the four major Synechococcus lineages (clades I to IV), also brought novel insights about the differential niche partitioning of clades I and IV, which most often co-dominate the Synechococcus community in cold and temperate coastal areas. Altogether, this study tackles the main differences between open-ocean and coastal communities worldwide.

sentences: A functional bacterial-derived restriction modification system in the mitochondrion of a heterotrophic protist The overarching trend in mitochondrial evolution is functional streamlining coupled with gene loss; therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in organellar genomes, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification system originating from a horizontal gene transfer event involving bacteria related to flavobacteria. This restriction modification system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase. We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that toxin-antitoxin selfish elements, such as restriction modification systems, could be co-opted by eukaryotic genomes to drive uniparental organellar inheritance.

sentences: mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines1,2. These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known3-6. Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers3,5,6. Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection7,8. However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.

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Effect of TSS on anti-CVB3 through TLR3 pathway Toll-like receptors (TLRs) can recognize molecular patterns associated with microorganisms and induce immune responses. TLR3 is responsible for recognizing viruses and making hosts resistant to Coxsackie B3 (CVB3) infection. In this study, we found that Tectorigenin Sodium Sulfonate (TSS) has a TLR3 inhibitory effect and exerts strong anti-CVB3 activity through the TLR3 pathway both in vivo and in vitro. Balb/c mice were infected by ip injection of CVB3 virus, and subcutaneously injected with TSS at 264mg{middle dot}Kg-1{middle dot}d-1 for 7 d. The life extension rate of CVB3-infected mice was up to 46.67%, and the mean time to death was prolonged from 6.00 {+/-} 0.47 d to 8.80 {+/-} 2.78 d (P < 0.01). TSS was also able to reduce the level of weight loss in mice, significantly reduce the symptoms of myocarditis caused by CVB3, reduce the virus titer in the heart, improve the spleen and thymus index, and enhance immune function. We further explored the underlying mechanism of TSS in CVB3-infected HT-29 cells and mice: TSS reduced TLR3 expression and downregulated its downstream TRIF, TRAF6, IRF3, NF-{kappa} B. MAPK, AP1 expression, reduced expression of IFN-{beta}IL-6TNF-. TSS has strong anti-CVB3 activity in vitro and in vivo and has the effect of inhibiting the activation of TLR3 pathway.

IMPORTANCETSS is expected to be an antiviral drug for the treatment of CVB3 infection. Its regulatory effect on the immune system provides a new approach for antiviral research.

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Active Sinking Particles: Sessile Suspension Feeders significantly alter the Flow and Transport to Sinking Aggregates AO_SCPLOWBSTRACTC_SCPLOWSinking or sedimentation of biological aggregates plays a critical role in carbon sequestration in the ocean and in vertical material fluxes in waste-water treatment plants. In both these contexts, the sinking aggregates are "active," since they are biological hot-spots and are densely colonized by microorganisms including bacteria and sessile protists, some of which generate feeding currents. However, the effect of these feeding currents on the sinking rates, trajectories, and mass transfer to these "active sinking particles," has not previously been studied. Here we use a novel scale-free vertical-tracking microscope (a.k.a. Gravity Machine, Krishnamurthy et al. "Scale-free vertical tracking microscopy." Nature Methods (2020)) to follow model sinking aggregates (agar spheres) with attached protists (Vorticella convallaria), sinking over long distances while simultaneously measuring local flows. We find that activity due to attached Vorticella cause substantial changes to the flow around aggregates in a dynamic manner and reshape mass transport boundary layers. Further, we find that activity-mediated local flows along with sinking significantly changes how aggregates interact with the water-column at larger scales by modifying the encounter and plume cross-sections and by inducing sustained aggregate rotations. In this way our work suggests an important role of biological activity in understanding the growth, degradation, composition and sinking speeds of aggregates with consequences for predicting vertical material fluxes in marine, freshwater and man-made environments.

1 Significance StatementSinking aggregates are a critical part of aquatic ecosystems. Plentiful sinking aggregates account for the majority of carbon sequestration in the oceans. These aggregates are densely colonized by microorganisms, including many that generate feeding currents. Utilizing a novel instrument for high resolution imaging of sinking particles, we demonstrate that these feeding currents significantly change how water flows near the aggregates. We show that these changes in flow are likely to affect aquatic system processes, including aggregation rates, degradation rates, sinking speeds, and aggregate composition. Our work provides a starting point for exploring the larger-scale implications of attached organisms on these system processes, which, in turn, are critical for understanding carbon sequestration in the oceans or efficiency in waste-water treatment plants.

sentences: Assortative Mating Biases Marker-based Heritability Estimators Many complex traits are subject to assortative mating (AM), with recent molecular genetic findings confirming longstanding theoretical predictions that AM alters genetic architecture by inducing long range dependence across causal variants. However, all marker-based heritability estimators assume mating is random. We provide mathematical and simulation-based evidence demonstrating that both method-of-moments estimators and likelihood-based estimators produce biased estimates in the presence of AM and that common approaches to account for population structure fail to mitigate this bias. Then, examining height and educational attainment in the UK Biobank, we demonstrate that these biases affect real world traits. Finally, we derive corrected heritability estimators for traits under equilibrium AM.

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Astrocyte nanoscale morphology controls Ca2+ signals at tripartite synapses Much of the Ca2+ activity in astrocytes is spatially restricted to microdomains and occurs in fine processes that form a complex anatomical meshwork, the so-called spongiform domain. A growing body of literature indicates that those astrocytic Ca2+ signals can influence the activity of neuronal synapses and thus tune the flow of information through neuronal circuits. Because of technical difficulties in accessing the small spatial scale involved, the role of astrocyte morphology on Ca2+ microdomain activity remains poorly understood. Here, we use computational tools and idealized 3D geometries of fine processes based on recent super-resolution microscopy data to investigate the mechanistic link between astrocytic nanoscale morphology and local Ca2+ activity. Simulations demonstrate that the nano-morphology of astrocytic processes powerfully shapes the spatio-temporal properties of Ca2+ signals and promotes local Ca2+ activity. The model predicts that this effect is attenuated upon astrocytic swelling, hallmark of brain diseases, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neurotransmitter release events, the model predicts that swelling hinders astrocytic signal propagation. Overall, this study highlights the influence of the complex morphology of astrocytes at the nanoscale and its remodeling in pathological conditions on neuron-astrocyte communication at so-called tripartite synapses, where astrocytic processes come into close contact with pre- and postsynaptic structures.

Table of ContentsO_ST_ABSMain PointsC_ST_ABSO_LIAstrocyte nano-morphology favors the compartmentalization of biochemical signals
C_LIO_LIThis compartmentalization promotes local Ca2+ activity and signal propagation robustness
C_LIO_LIIn contrast, its pathological remodeling upon swelling attenuates Ca2+ activity
C_LI

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org.highwire.dtl.DTLVardef@22b80forg.highwire.dtl.DTLVardef@1621218org.highwire.dtl.DTLVardef@191e971org.highwire.dtl.DTLVardef@751637_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1:C_FLOATNO Proposed mechanisms that regulate astrocytic Ca2+ activity in perisynaptic astrocytic processes.

Our simulation results demonstrate that the nano-morphology of astrocytic processes, consisting in the alternation of nodes and shafts, favors the compartmentalization of biochemical signals. This compartmentalization promotes local Ca2+ activity and signal propagation robustness. Astrocyte swelling, observed in pathological conditions such as brain injury, stroke and epilepsy, results in an increased shaft width without altering node size. Our results suggest that such pathological alterations of the nanoscale morphology of astrocytes result in a decreased local Ca2+ activity, which we confirm experimentally in hypo-osmotic conditions. Upon repeated neuronal stimuli, we predict that swelling hinders astrocytic signal propagation. Overall, this study highlights the impact of astrocyte nano-morphology on astrocyte activity at tripartite synapses, in health and disease.

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PyRodentTracks: flexible computer vision and RFID based system for multiple rodent tracking and behavioral assessment. PyRodentTracks (PRT) is a scalable and customizable computer vision and RFID- based system for multiple rodent tracking and behavior assessment that can be set up within minutes in any user-defined arena at minimal cost. PRT is composed of the online Raspberry Pi-based video and RFID acquisition and the subsequent offline analysis tools. The system is capable of tracking up to 6 mice in experiments ranging from minutes to days. PRT maintained a minimum of 88% detections tracked with an overall accuracy >85% when compared to manual validation of videos containing 1-4 mice in a modified home-cage. As expected, chronic recording in home-cage revealed diurnal activity patterns. Moreover, it was observed that novel non-cagemate mice pairs exhibit more similarity in travel trajectory patterns over a 10-minute period in the openfield than cagemates. Therefore, shared features within travel trajectories between animals may be a measure of sociability that has not been previously reported. Moreover, PRT can interface with open-source packages such as Deeplabcut and Traja for pose estimation and travel trajectory analysis, respectively. In combination with Traja, PRT resolved motor deficits exhibited in stroke animals. Overall, we present an affordable, open-sourced, and customizable/scalable rodent-specific behavior recording and analysis system.

Statement of SignificanceAn affordable, customizable, and easy-to-use open-source rodent tracking system is described. To tackle the increasingly complex questions in neuroscience, researchers need a flexible system to track rodents of different coat colors in various complex experimental paradigms. The majority of current tools, commercial or otherwise, can only be fully automated to track multiple animals of the same type in a single defined environment and are not easily setup within custom arenas or cages. Moreover, many tools are not only expensive but are also difficult to set up and use, often requiring users to have extensive hardware and software knowledge. In contrast, PRT is easy to install and can be adapted to track rodents of any coat color in any user-defined environment with few restrictions. We believe that PRT will be an invaluable tool for researchers that are quantifying behavior in identified animals.

sentences: An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity. The analysis of single-cell proteomes has recently become a viable complement to transcriptomics and genomics studies. Proteins are the primary driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have lately attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lag. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE(R), reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1,500 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,600 proteins across 170 multiplexed single cells from two highly similar human cell types. This dedicated loss-less workflow allows distinguishing in vitro co-differentiated cell types of self-organizing cardiac organoids based on indicative markers across 150 single cells. In-depth characterization revealed enhanced cellular motility of cardiac endothelial cells and sarcomere organization in cardiomyocytes. Our versatile and automated sample preparation has not only proven to be easily adaptable but is also sufficiently sensitive to drive biological applications of single-cell proteomics.

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CcLBD25 functions as a key regulator of haustorium development in Cuscuta campestris Parasitic plants reduce yield of crops worldwide. Cuscuta campestris is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, CcLBD25, as highly expressed in prehaustoria and haustoria. Our gene co-expression networks (GCN) from different tissue types and laser-capture microdissection (LCM) RNA-Seq data indicate that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing (HIGS) by generating transgenic tomato hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had less biomass compared with C. campestris growing on wild type host. This suggests that the parasites growing on the transgenic plants were stressed due to insufficient nutrient acquisition. Anatomy of C. campestris haustoria growing on CcLBD25 RNAi tomatoes showed reduced pectin digestion and lack of searching hyphae, which interfered with haustorium penetration and the formation of vascular connections. We developed an in vitro haustorium (IVH) system to assay the number of prehaustoria produced on strands from C. campestris. When C. campestris was grown on CcLBD25 RNAi tomatoes or wild type tomatoes, the former produce fewer prehaustoria than the latter, indicating that down-regulating CcLBD25 may affect haustorium initiation. The results of this study shed light on the role of CcLBD25 in haustorium development and might help to develop a parasite-resistant system in crops.

One-sentence summaryCcLBD25 plays a pivotal role in haustorium initiation, regulating pectin digestion, and searching hyphae development during the haustorium penetration process.

sentences: Couching the brain's multiple-demand and language-specific systems within a macroscale gradient of cortical connectivity The cerebrum comprises a set of specialised systems that tile across the cortical sheet, forming a tapestry-like configuration. For example, the multiple-demand and language-specific systems occupy largely separate neural estates and exhibit disparate functional profiles. Although delimiting the boundary between systems informs where cortical sheet functionally fractionates, it remains unclear why different systems topographical placements are spatially configured in typical manners and how a macroscale architecture arises from this topography. Novel approaches have tackled this challenge by condensing the topography into a principal gradient, which represents the workflow of information processing from sensory-motoric to abstract-cognitive. To understand how the multiple-demand and language-specific systems are accommodated in the gradient framework, here we used fMRI to probe cognitive operations in semantic vs. visuospatial domains and projected functional activities onto the principal gradient. We found that the two systems showed distinct trajectories of distribution along gradient tiers, suggesting different roles in the transition from sensation to cognition. Critically, when semantic processing became difficult, the brain recruited a specialised  semantic-control system that was a functional and anatomical  hybrid juxtaposed between the multi-demand and language systems. We discuss how the brains modular division can be better understood through the lens of a dimensionality-reduced gradient-like architecture.

sentences: FebRNA: an automated fragment-ensemble-based model for building RNA 3D structures Knowledge of RNA 3-dimensional (3D) structures is critical to understanding the important biological functions of RNAs. Although various structure prediction models have been developed, high accuracy of predicted RNA 3D structures is still limited to the RNAs with short length or with simple topology. In this work, we proposed a new model, namely FebRNA, for building RNA 3D structures through fragment assembly based on coarse-grained (CG) fragment ensembles. Specifically, FebRNA is composed of four processes: establishing the library of different types of CG fragment ensembles, building CG 3D structure ensemble through fragment assembly, identifying top-1 CG structure through a CG scoring function, and rebuilding the all-atom structure from the top-1 CG one. Extensive examination on different types of RNA structures indicates that FebRNA gives consistently reliable predictions on RNA 3D structures including pseudoknots, 3-way junction, 4-way and 5-way junctions, and RNAs in the RNA-Puzzles. FebRNA is available at website: https://github.com/Tan-group/FebRNA.

sentences: Nav1.1 in mammalian sensory neurons is required for normal motor behaviors AO_SCPLOWBSTRACTC_SCPLOWThe mammalian voltage-gated sodium channel (NaV), NaV1.1, has been well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we report a new role for peripherally expressed NaV1.1 in murine motor behaviors. RNAscope analysis found 100% of proprioceptors express NaV1.1 transcript, consistent with in vitro patch clamp recordings showing this channel is required for repetitive firing in proprioceptors. Notably, genetic deletion of NaV1.1 in all sensory neurons caused profound motor coordination deficits in homozygous conditional knockout animals of both sexes, a phenotype similar to conditional Piezo2-knockout animals. Movement deficits were also observed in heterozygotes, demonstrating that NaV1.1 haploinsufficiency in sensory neurons leads to motor deficiencies. This behavioral phenotype was not due to reduced proprioceptor numbers or abnormal muscle spindle formation; however, we observed decreased proprioceptor innervation of motor neurons in the spinal cord in conditional knockouts, indicating loss of NaV1.1 in sensory neurons alters spinal cord circuitry. Ex vivo muscle afferent recordings also support the notion that loss of NaV1.1 leads to aberrant proprioceptor function. Collectively, these data provide the first evidence that NaV1.1 in mammalian sensory neurons is essential for motor coordination. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia. Thus, our data suggest sensory neuron dysfunction may contribute to the clinical manifestations and co-morbidities of neurological disorders in which NaV1.1 function is compromised.

sentences: Vertebrate hemicentin-1 interacts physically and genetically with nidogen-2 Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mammalian hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 for basement membrane integrity. HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately ten-fold lower affinity and in a competitive manner. Immunofluorescence and immunogold labellings reveal that HMCN1/Hmcn1 is localized closely attached to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as loss of Laminin-5 (Lama5) or Hmcn1. Combined partial loss-of-function studies further indicate that nid2a genetically interacts with both hmcn1 and lama5. Together, this suggests that despite their mutually exclusive physical binding, hemicentins, nidogens and laminins tightly cooperate and support each other during formation, maintenance and function of basement membranes to confer tissue linkage.

sentences: Impulsivity and Emotional Dysregulation Predict Choice Behavior During a Competitive Multiplayer Game in Adolescents with Borderline Personality Disorder Impulsivity and emotional dysregulation are two core features of borderline personality disorder (BPD), and the neural mechanisms recruited during mixed-strategy interactions overlap with frontolimbic networks that have been implicated in BPD. We investigated strategic choice patterns during the classic two-player game, Matching Pennies, where the most efficient strategy is to choose each option randomly from trial-to-trial to avoid exploitation by one's opponent. Twenty-seven female adolescents with BPD (mean age: 16 years) and twenty-seven age-matched female controls (mean age: 16 years) participated in an experiment that explored the relationship between strategic choice behavior and impulsivity in both groups and emotional dysregulation in BPD. Relative to controls, BPD participants showed fewer reinforcement learning biases, increased coefficient of variation in reaction times (CV), and more anticipatory decisions. A subset of BPD participants characterized by high levels of impulsivity and emotional dysregulation showed increased reward rate, increased entropy in choice patterns, decreased CV, and fewer anticipatory decisions relative to participants with lower indices, and emotion dysregulation mediated the relationship between impulsivity and CV in BPD. Finally, exploratory analyses revealed that increased vigilance to outcome was associated with higher reward rates, decreased variability in SRT, and fewer anticipatory decisions. In BPD, higher levels of emotion dysregulation corresponded to increased vigilance to outcome, and mediated its relationship with choice behavior. Together, our results suggest that impulsivity and emotional dysregulation contribute to variability in mixed-strategy decision-making in BPD, the latter of which may influence choice behavior by increasing attention to outcome information during the task.

sentences: Impact of K-Pg Mass Extinction Event on Crocodylomorpha Inferred from Phylogeny of Extinct and Extant Taxa Crocodilians and their allies have survived several mass extinction events. However, the impact of the K-Pg mass extinction event on crocodylomorphs is considered as minor or non-existent although other clades of archosaurs, e.g., non-avian dinosaurs and pterosaurs, went extinct completely. Previous approaches using fossil occurrence data alone have proven inconclusive. In this paper, we take a phylogenetic approach using extant and extinct species. The time-calibrated phylogeny of extant crocodilians provides insights into the pattern of recent biodiversity changes whereas fossil occurrence data provide insights about the more ancient past. The two data sources combined into a single phylogeny with extinct and extant taxa provide a holistic view of the historical biodiversity. To utilize this combined data and to infer the impact of the K-Pg mass extinction event, we derive the likelihood function for a time-varying (episodic) serially sampled birth-death model that additionally incorporates mass extinctions and bursts of births. We implemented the likelihood function in a Bayesian framework with recently developed smoothing priors to accommodate for both abrupt and gradual changes in speciation, extinction and fossilization rates. Contrary to previous research, we find strong evidence for the K-Pg extinction event in crocodiles and their allies. This signal is robust to uncertainty in the phylogeny and the prior on the mass extinctions. Through simulated data analyses, we show that there is high power to detect this mass extinction and little risk of false positives.

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Ancient migrations - The first complete genome assembly, annotation and variants of the Zoroastrian-Parsi community of India With the advent of Next Generation Sequencing, many population specific whole genome sequences published thus far, predominantly represent individuals of European ancestry. While sequencing efforts of underrepresented communities in genomes datasets, like the Yoruba West-African, Han Chinese, Tibetan, South Korean, Egyptian and Japanese have recently added to the public genomic repositories, a comprehensive understanding of human genomic diversity and discovery of trait-associated variants necessitates the need for additional population specific analysis. In this context, the genomics of the population from the Indian sub-continent, given its genetic heterogeneity needs further elucidation.

In this context, the endogamous Zoroastrian-Parsi community of India, offer an exceptional insight into a homogenous population that has culturally, socially, and genetically remained intact, for 13 centuries amidst the genomic, social and cultural Indian landscape, consequent to their migration from the ancient Persian plateau.

Notwithstanding longevity as a trait, this endangered community is highly susceptible to cancers, rare genetic disorders, and display a documented high incidence of neurodegenerative and autoimmune conditions. The community as a matter of cultural practice abstains from smoking.

Here, we describe the assembly and annotation of the genome of an adult female, Zoroastrian-Parsi individual sequenced at a high depth of 173X using a combination of short Illumina reads (160X) and long nanopore reads (13X). Using a combination of hybrid assemblers, we created a new, population-specific human reference genome, The Zoroastrian-Parsi Genome Reference Female, AGENOME-ZPGRF, contains 2,778,216,114 nucleotides as compared to 3,096,649,726 in GRCh38 constituting 93.235% of the total genomic fraction. Annotation identified 20833 genomic features, of which 14996 are almost identical to their counterparts on GRCh38 while 5837 genomic features were covered in partial. AGENOME-ZPGRF contained 5,426,310 variants of which the majority were SNPs (4,291,601) and 960,867 SNPs were AGENOME-ZPGRF specific personal variants not listed in dbSNP.

We present, AGENOME-ZPGRF as a whole reference for any genetic studies involving Zoroastrian-Parsi individuals extending their application to identify disease relevant prognostic biomarkers and variants in global population genomics studies.

sentences: A Two-Component System FleS/FleR Regulates Multiple Virulence-Related Traits in Pseudomonas aeruginosa Microorganisms commonly use two-component systems (TCSs) to detect specific environmental changes and respond accordingly for their own benefit. However, the regulatory mechanisms and physiological roles of a majority of TCSs are still elusive. In this study, we focused on a previously predicted TCS FleS/FleR in Pseudomonas aeruginosa to systematically investigate its regulation and physiological roles. Loss of fleS or fleR or both genes led to decreased biofilm formation and attenuated motility in PAO1, which could be restored by heterologously complementation of FleR but not FleS, confirming that the sensor kinase FleS and the response regulator FleR constitute a TCS pair. To determine the regulatory spectrum of this TCS, we conducted transcriptome sequencing and comparison between the wild-type strain and the fleR deletion mutant. The result showed that the TCS regulates about 440 genes including most of them are involved in the virulence-related pathways, e.g. siderophore biosynthesis, pyocyanin biosynthesis, type III/VI secretion systems, c-di-GMP metabolism, flagellar assembly etc. In addition to its roles in controlling biofilm formation and motility we have already shown, FleR was demonstrated to regulate the production of virulence factors such as pyocyanin and elastase, mediate stress response to SDS, and autoregulate its own expression. Moreover, EMSA assays revealed that FleR regulates flagellum biosynthesis genes flgBCDE, flgFGHIJKL, filC, which are essential for the bacterial motility, by directly interacting with their promoters. Taken together, these results expanded our understanding on the biological roles of FleS/FleR and provided new insights on its regulatory mechanisms.

sentences: Nitrate-regulated growth processes involve activation of gibberellin pathway Nitrate, one of the main nitrogen (N) sources for crops, acts as a nutrient and key signaling molecule coordinating gene expression, metabolism and various growth processes throughout the plant life cycle. It is widely accepted that nitrate-triggered developmental programs cooperate with hormone synthesis and transport, to finely adapt plant architecture to N availability. Here, we report that nitrate, acting through its signaling pathway, promotes growth in Arabidopsis and wheat, in part by modulating the accumulation of gibberellin (GA)-regulated DELLA growth repressors. We show that nitrate reduces the abundance of DELLAs by increasing GA contents through activation of GA metabolism gene expression. Consistently, the growth restraint conferred by nitrate deficiency is partially rescued in global-DELLA mutant that lacks all DELLAs. At the cellular level, we show that nitrate enhances both cell proliferation and elongation in a DELLA-dependent and -independent manner, respectively. Our findings establish a connection between nitrate and GA signaling pathways that allow plants to adapt their growth to nitrate availability.

sentences: The mechanism underlying the organization of Borna disease virus inclusion bodies is unique among mononegaviruses Inclusion bodies (IBs) are characteristic biomolecular condensates organized by mononegaviruses. Here, we characterize the IBs of Borna disease virus 1 (BoDV-1), a unique mononegavirus that forms IBs in the nucleus, in terms of liquid-liquid phase separation (LLPS). The BoDV-1 phosphoprotein (P) alone induces LLPS and the nucleoprotein (N) is incorporated into the P droplet in vitro. In contrast, co-expression of N and P is required for the formation of IB-like structure in cells. Furthermore, while BoDV-1 P binds to RNA, an excess amount of RNA dissolves the liquid droplets formed by N and P. Notably, the N-terminal intrinsically disordered region of BoDV-1 P is essential to drive LLPS and bind to RNA, suggesting that both abilities could compete with one another. These features are unique among mononegaviruses, and thus this study will contribute to a deeper understanding of LLPS-driven organization and RNA-mediated regulation of biomolecular condensates.

sentences: Lin28a rejuvenates muscle stem cells via mitochondrial optimization The well-conserved correlation between juvenility and tissue regeneration was first discussed by Charles Darwin. Ectopic Lin28 is known to play an important role in somatic reprogramming and tissue regeneration, but endogenous Lin28s role in tissue homeostasis and juvenility had remained unclear. Through lineage tracing, we found that a rare subset of muscle stem cells (MuSCs) expressing Lin28a can respond to acute injury by proliferating as Pax3+ or Pax7+ MuSCs, and contribute to all types of myofibers during muscle regeneration. Compared with conventional Pax7+ MuSCs, Lin28a+ MuSCs express more Pax3 and show enhanced myogenicity in vitro. In terms of the epigenetic clock, adult Lin28a+ MuSCs lie between adult Pax7+ MuSCs and embryonic Pax7+ myoblasts according to their DNA methylation profiles. We found that Lin28a+ MuSCs upregulate several embryonic limb bud mesoderm transcription factors and could maintain a juvenile state with enhanced stem cell self-renewal and stress-responsiveness in vitro and in vivo. When combined with telomerase and TP53 inhibition to biomimic mouse Lin28a+ MuSCs, we found that Lin28a can rejuvenate and dedifferentiate aged human primary myoblasts into engraftable, self-renewing MuSCs. Mechanistic studies revealed that Lin28a activated the HIF1A pathway by optimizing mitochondrial ROS (mtROS), thereby rejuvenating MuSC self-renewal and muscle regeneration. Our findings connect the stem cell factor Lin28, mtROS metabolism and stress response pathways to the process of stem cell rejuvenation and tissue regeneration.

sentences: Differential bioactivity of four BMP-family members as function of biomaterial stiffness Whereas soft biomaterial is not able to induce cell spreading, BMP-2 presented by a soft film has been described to be sufficient to trigger cell spreading, migration and downstream BMP-2 signaling. Based on thin polyelectrolyte films of controlled stiffness, we investigated whether the presentation of four BMP members (2, 4, 7, 9) in a matrix-bound manner may differentially impact cell adhesion and bone differentiation of skeletal progenitors. We performed high content and automated screening of cellular responses, including cell number, cell spreading area, SMAD phosphorylation and alkaline phosphatase activity. The basolateral presentation of the different BMPs allowed us to discriminate the specificity of cellular response and the role of BMP receptors type I, type II, as well as three {beta} integrins, in a BMP type and stiffness-dependent manner.

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The Expression Pattern of miR-17, -24, -124 and -145 as Diagnostic Factor for Metastatic Gastric Cancer; a Lesson from Gastric Cancer Stem cells BackgroundDistant metastasis of Gastric Cancer (GC) causes more than 700 000 deaths worldwide. Cancer Stem Cells (CSCs) are a subpopulation of cancer cells responsible for aggressiveness and chemoresistance in clinical settings. MicroRNAs (miRNAs) emerge as important players in regulating self-renewal and metastasis in CSCs. Understanding the role of miRNAs in CSCs offer a potential diagnostic tool for GC patients. This study is aimed to identify miRNAs that target both stemness and metastasis in gastric cancer stem cells (GCSCs) and differentially expressed in metastatic GC patients as diagnostic biomarkers for GC metastasis.

MethodsWe investigate the gene expression profile of patients using the GEO database and Rstudio software. To obtain the regulatory networks and miRNAs, the STRING and miRwalk database used. The gastric cancer tissues were obtained from Iranian National Tumor Bank (INTB) to validate the results.

ResultsOur results indicated three important regulatory cores affecting the immune systems regulation, tumor progress, and metastasis. Based on the bioinformatics results, four miRNAs miR-17-5p, miR-24-3p, miR-124-3p, and miR-145-5p, were selected, and their expression pattern was evaluated in 10 patients metastatic tumors compared to 10 nonmetastatic tumors by real-time PCR. The expression level of mir-17, -24, and -124 was upregulated about 8, 10, 60 folds, respectively, and miR-145 was down-regulated 4.5 folds in metastatic tumors compared to nonmetastatic tumors.

Conclusionthe high expression level of miR-17, -24, -124, and low level of miR-145 in GC patients samples could be a potential biomarker for the presence of GCSCs and the diagnosis of metastasis.

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Possible Regulation of Toll-Like Receptor 4 By Lysine Acetylation Through LPCAT2 Activity in RAW264.7 Cells. 1Inflammation is central to several diseases. TLR4 mediates inflammatory signals, however, there are gaps in the understanding of its mechanisms. Recently, TLR4 was found to co-localise with LPCAT2, a lysophospholipid acetyltransferase. This interaction influenced TLR4 subcellular localisation through an unknown mechanism.

In this study, we have combined computational analysis, RNA interference technology, and biochemical analysis to investigate the possibility of TLR4 lysine acetylation and the influence of LPCAT2 on the detected lysine acetylation.

The results suggest for the first time that TLR4 can undergo lysine acetylation and LPCAT2 can influence TLR4 lysine acetylation. This lays a foundation for further research on the role of lysine acetylation on TLR4 and characterisation of LPCAT2 as a protein acetyltransferase.

sentences: Scalable and model-free detection of spatial patterns and colocalization The expeditious growth in spatial omics technologies enable profiling genome-wide molecular events at molecular and single-cell resolution, highlighting a need for fast and reliable methods to characterize spatial patterns. We developed SpaGene, a model-free method to discover any spatial patterns rapidly in large scale spatial omics studies. Analyzing simulation and a variety of spatial resolved transcriptomics data demonstrated that SpaGene is more powerful and scalable than existing methods. Spatial expression patterns by SpaGene reconstructed unobserved tissue structures. SpaGene also successfully discovered ligand-receptor interactions through their colocalization.

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RTX-KG2: a system for building a semantically standardized knowledge graph for translational biomedicine BackgroundBiomedical translational science is increasingly leveraging computational reasoning on large repositories of structured knowledge (such as the Unified Medical Language System (UMLS), the Semantic Medline Database (SemMedDB), ChEMBL, DrugBank, and the Small Molecule Pathway Database (SMPDB)) and data in order to facilitate discovery of new therapeutic targets and modalities. Since 2016, the NCATS Biomedical Data Translator project has been working to federate autonomous reasoning agents and knowledge providers within a distributed system for answering translational questions. Within that project and within the field more broadly, there is an urgent need for an open-source framework that can efficiently and reproducibly build an integrated, standards-compliant, and comprehensive biomedical knowledge graph that can be either downloaded in standard serialized form or queried via a public application programming interface (API) that accords with the FAIR data principles.

ResultsTo create a knowledge provider system within the Translator project, we have developed RTX-KG2, an open-source software system for building--and hosting a web API for querying--a biomedical knowledge graph that uses an Extract-Transform-Load (ETL) approach to integrate 70 knowledge sources (including the aforementioned sources) into a single knowledge graph. The semantic layer and schema for RTX-KG2 follow the standard Biolink metamodel to maximize interoperability within Translator. RTX-KG2 is currently being used by multiple Translator reasoning agents, both in its downloadable form and via its SmartAPI-registered web interface. Serializations of RTX-KG2 are available for download in both the pre-canonicalized form and in canonicalized form (in which synonym concepts are merged). The current canonicalized version (KG2.7.3) of RTX-KG2 contains 6.4M concept nodes and 39.3M relationship edges with a rich set of 77 relationship types.

ConclusionRTX-KG2 is the first open-source knowledge graph of which we are aware that integrates UMLS, SemMedDB, ChEMBL, DrugBank, SMPDB, and 65 additional knowledge sources within a knowledge graph that conforms to the Biolink standard for its semantic layer and schema at the intersections of these databases. RTX-KG2 is publicly available for querying via its API at arax.rtx.ai/api/rtxkg2/v1.2/openapi.json. The code to build RTX-KG2 is publicly available at github:RTXteam/RTX-KG2.

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Associative memory of structured knowledge A long standing challenge in biological and artificial intelligence is to understand how new knowledge can be constructed from known building blocks in a way that is amenable for computation by neuronal circuits. Here we focus on the task of storage and recall of structured knowledge in longterm memory. Specifically, we ask how recurrent neuronal networks can store and retrieve multiple knowledge structures. We model each structure as a set of binary relations between events and attributes (attributes may represent e.g., temporal order, spatial location, role in semantic structure), and map each structure to a distributed neuronal activity pattern using a vector symbolic architecture (VSA) scheme.

We then use associative memory plasticity rules to store the binarized patterns as fixed points in a recurrent network. By a combination of signal-to-noise analysis and numerical simulations, we demonstrate that our model allows for efficient storage of these knowledge structures, such that the memorized structures as well as their individual building blocks (e.g., events and attributes) can be subsequently retrieved from partial retrieving cues. We show that long-term memory of structured knowledge relies on a new principle of computation beyond the memory basins. Finally, we show that our model can be extended to store sequences of memories as single attractors.

sentences: All-trans retinoic acid induces GADD34 gene expression via transcriptional regulation by Six1-TLE3 and post-transcriptional regulation by p38-TTP in skeletal muscle All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response (UPR) in differentiating leukemic blasts. The downstream transcriptional factors of PERK, a major arm of UPR, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of it via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in myoblasts. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in myoblasts changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation in myoblasts, which changes muscle fiber type in myotubes.

sentences: Parallel Evaluation of Nucleophilic and Electrophilic Chemical Probes for Sulfenic Acid: Reactivity, Selectivity and Biocompatibility S-sulfenylation of cysteine thiols (Cys-SOH) is a regulatory posttranslational modification in redox signaling and an important intermediate to other cysteine chemotypes. Owing to the dual chemical nature of the sulfur in sulfenic acid, both nucleophilic and electrophilic chemical probes have been developed to react with and detect Cys-SOH; however, the efficiency of existing probes has not been evaluated in a side-by-side comparison. Here, we employ small-molecule and protein models of Cys-SOH and compare the chemical probe reactivity. These data clearly show that 1,3-diketone-based nucleophilic probes react more efficiently with sulfenic acid as compared to strained alkene/alkyne electrophilic probes. Kinetic experiments that rigorously address the selectivity of the 1,3-diketone-based probes are also reported. Consideration of these data alongside relative cellular abundance, indicates that biological electrophiles, including cyclic sulfenamides, aldehydes, disulfides and hydrogen peroxide, are not meaningful targets of 1,3-diketone-based nucleophilic probes, which still remain the most viable tools for the bioorthogonal detection of Cys-SOH.

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Genome-wide analysis reveals PhoP regulates pathogenicity in Riemerella anatipestifer Duck infectious serositis, also known as the Riemerella anatipestifer disease infects domestic ducks, geese, turkeys, and wild birds. However, the regulatory mechanism of its pathogenicity remains unclear. The phoP/phoR two-component system was first reported in gram-negative bacteria in our previous research and was demonstrated to be involved in virulence and gene expression. Here, the DNA-affinity-purified sequencing (DAP-seq) was applied to further explore the regulation of phoP/phoR to pathogenicity in R. anatipestifer. A conserved motif was identified in the upstream of 583 candidate target genes which were directly regulated by phoP. To further confirm the genes which are regulated by phoP/phoR, phoR and phoP, the single-gene deletion strains were constructed. The results of transcriptome analysis using next-generation RNA sequencing showed 136 differential expression genes (DEGs) between {Delta}phoP and RA-YM, and 183 DEGs between {Delta}phoR and WT. The candidate target genes of PhoP were further identified by combining transcriptome analysis and DAP-seq. The results of DAP-seq and RNA-seq of {Delta}phoP in combination revealed that the main direct regulons of PhoP are located on the membrane and PhoP is involved in regulating aerotolerance. Using the in vivo duck model, the pathogenicity of {Delta}phoP or {Delta}phoR was significantly lower than that of the WT. Together, our findings provided a perception about the direct regulation of PhoP and suggested that phoP/phoR is essential for the pathogenicity of R. anatipestifer, and phoP/phoR is related to the aerotolerance of R. anatipestifer. The gene deletion strains are expected to be the candidate live vaccine strains of R. anatipestifer which can be used as ideal genetic engineering vector strains for the expression of foreign antigens.

Author summaryRiemerella anatipestifer is a severe pathogen in the poultry industry with high mortality in ducks and geese mainly due to acute septicemia and infectious polyserositis. A two-component system phoP/phoR was previously characterized in R. anatipestifer, and the phoP/phoR TCS was reported for the first time in Gram-negative bacteria. A deleted phoP/phoR in R. anatipestifer has been reported to almost lose its pathogenicity in ducklings. However, the mechanism of phoP/phoR regulating the virulence of R. anatipestifer had not been explored in detail. This study has utilized DAP-seq to explore the DNA-binding sites of PhoP as a response regulator in the global genome. Furthermore, the phoP and phoR were deleted separately and the transcriptomics of the corresponding gene-deleted strains was analyzed. A series of directly regulated genes of phoP/phoR TCS were determined in combination. The duckling model revealed both PhoP and PhoR as essential virulence-related factors of R. anatipestifer.

sentences: Assessment of local potato cultivars found in Cis-Himalayan region of West Bengal through morphology and biochemical profiling Potato is a major global food crop grown for tubers (underground storage stems) that are high in carbohydrates, minerals, and vitamins. The presence of anthocyanins in tuber skin and flesh can have a significant impact on customer preferences. Potatoes are also high in resistant starches, which have a variety of health benefits, including enhanced fullness, cholesterol reduction, and a reduced risk of colon cancer etc. In West Bengal, diabetes is prevalent. Similarly, according to ICMR studies, colon cancer affects 8.9% of all cancer patients in West Bengal cancer which is caused by ill eating habits or the consumption of foods that are low in antioxidants. 5-10% of colon cancers are genetically caused, while the remainder are caused by poor eating habits or the consumption of foods that are low in antioxidants. To address these issues, one strategy is to eat foods with a low glycemic index and high antioxidant content as a staple food. The tuber tissues of the potato (S. tuberosum L.) accumulate various quantities of anthocyanins, which are commonly consumed around the world. Anthocyanins are pigments that range in colour from red to purple and are found throughout the plant kingdom. Anthocyanins are powerful antioxidants that are water soluble. The red skin potato is well-known among the general public. The epidermal layer contains a high quantity of red anthocyanins, which causes the skin to be red. With this background the present study has been undertaken to address the following objectives morphological (quantitative and qualitative traits) and biochemical characterization of local potato cultivars and identification of most stable genotypes based on the anthocyanin content and tuber yield of local potato cultivars. For our recent research 14 local potato cultivar from North Bengal were collected and evaluated for DUS characterization showed wide range of variability with respect to different phenotypic variants of ten characters. Overall predominant DUS characters of local potato cultivars found in North Bengal are medium sized apical length of sprout, short stem height, compact foliage structure, open leaf structure, ovate lanceolate type leaflet shape, purple leaf sprout predominant colour, spherical leaf sprout shape, medium intensity of anthicyanin coloraton at the base, light intensity of anthicyanin coloraton at the tip and weak nature of light sprout pubescence base. The results of the present investigation suggest that local potato cultivars collected from the northern part of West Bengal showed a high level of genetic variation. The differences between genotypes were highly significant at the 1% and 5% alpha level for all characters like tuber yield, length and weight of tuber, length of sprout, chlorophyll content, canopy temperature, and number of tuber per plant, according to the analysis of variance. Three quantitative traits namely tuber yield, tuber breadth, tuber length played major role in the genetic variance. Anthocyanin content had more contribution to diversify the local potato cultures according to biochemical characterization AMMI analysis suggested that Jalpai from CoochBehar is the most stable potato cultivars in respect to anthocyanin content and tuber yield per plant.

sentences: Sharks, Lies, and Videotape: A content analysis of 32 years of Shark Week documentaries Despite evidence of their importance to marine ecosystems, at least 25% of all chondrichthyan species are estimated or assessed as threatened with extinction. In addition to the logistical difficulties of effectively conserving wide-ranging marine species, shark conservation is believed to have been hindered in the past by public perceptions of sharks as dangerous to humans. Shark Week is a high-profile, international programming event that has potentially enormous influence on public perceptions of sharks, shark research, shark researchers, and shark conservation. However, Shark Week has received regular criticism for poor factual accuracy, fearmongering, bias, and inaccurate representations of science and scientists. This research analyzes the content and titles of Shark Week episodes across its entire 32 years of programming to determine if there are trends in species covered, research techniques featured, expert identity, conservation messaging, type of programming, and portrayal of sharks. We analyzed titles from 272 episodes (100%) of Shark Week programming and the content of all available (201; 73.9%) episodes. Our data demonstrate that the majority of episodes are not focused on shark bites, although such shows are common and many Shark Week programs frame sharks around fear, risk, and adrenaline. While anecdotal descriptions of disproportionate attention to particular charismatic species (e.g. great whites, bull sharks, and tiger sharks) are accurate and supported by data, 79 shark species have been featured briefly at least once. Shark Weeks depictions of research and of scientists are biased towards a small set of (typically visual and expensive) research methodologies and (mostly white, mostly male) scientists, including presentation of many white male non-scientists as experts. While sharks are more often portrayed negatively than positively, limited conservation messaging does appear in 53% of episodes analyzed. Results suggest that as a whole, while Shark Week is likely contributing to the collective perception of sharks as monsters, even relatively small alterations to programming decisions could substantially improve the presentation of sharks and shark science and conservation issues.

sentences: Synergistic regulation of bZIP53 and dimerizing partners results in abnormal seed phenotype in Arabidopsis: Use of a designed dominant negative protein A-ZIP53 In Arabidopsis, basic leucine zipper (bZIP) family of transcription factors (TFs) are key proteins to regulate the expression of seed maturation (MAT) genes. bZIPs are functionally redundant and their DNA-binding activity is dependent on dimerization partner. The intervention of loss of function mutation is inadequate to understand and regulate the redundant behavior of TFs and one such example is bZIP53, which is known as a key regulator of seed maturation phenomena. Here, to examine the consequences of hindering the function of bZIP53 and its known and unknown heterodimerizing partners, a transgenic Arabidopsis constitutively expressing a novel dominant negative (DN) protein A-ZIP53 was raised. Transgenic plants demonstrated a delayed growth and retarded seed phenotype. The in vivo inhibition of DNA binding of bZIP53, bZIP10, and bZIP25 to the G-box demonstrated the efficacy of A-ZIP53 protein. In first generation, majority of plants failed to survive beyond four weeks suggesting a pleiotropic nature of bZIP53. Plants expressing A-ZIP53 have small flower, shorter siliques, and small-seeded phenotype. RNA seq analysis of the transgenic lines revealed the reduced expression of target genes of bZIP53 and its heterodimerizing partners. Furthermore, immunoprecipitation followed by mass spectrometry (IP-MS/MS) of transgenic plants helped to identify the additional heterodimerizing partners of the A-ZIP53. The interactions were subsequently confirmed with the transient transfection experiments. Unlike other gene knock out technologies, DN protein can inhibit the function of members of the same group of bZIP TFs.

sentences: pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopes dipolespread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Greens tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14{degrees} orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

sentences: Chimeric chromosome landscapes of human somatic cell cultures show dependence on stress and regulation of genomic repeats by CGGBP1. Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help us understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long read as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.

sentences: Reverse plasticity underlies rapid evolution by clonal selection within populations of fibroblastspropagated on a novel soft substrate Mechanical properties such as substrate stiffness are a ubiquitous feature of a cells environment. Many types of animal cells exhibit canonical phenotypic plasticity when grown on substrates of differing stiffness, in vitro and in vivo. Whether such plasticity is a multivariate optimum due to hundreds of millions of years of animal evolution, or instead is a compromise between conflicting selective demands, is unknown. We addressed these questions by means of experimental evolution of populations of mouse fibroblasts propagated for ~90 cell generations on soft or stiff substrates. The ancestral cells grow twice as fast on stiff substrate as on soft substrate and exhibit the canonical phenotypic plasticity. Soft-selected lines derived from a genetically diverse ancestral population increased growth rate on soft substrate to the ancestral level on stiff substrate and evolved the same multivariate phenotype. The pattern of plasticity in the soft-selected lines was opposite of the ancestral pattern, suggesting that reverse plasticity underlies the observed rapid evolution. Conversely, growth rate and phenotypes did not change in selected lines derived from clonal cells. Overall, our results suggest that the changes were the result of genetic evolution and not phenotypic plasticity per se. Whole-transcriptome analysis revealed consistent differentiation between ancestral and soft-selected populations, and that both emergent phenotypes and gene expression tended to revert in the soft-selected lines. However, the selected populations appear to have achieved the same phenotypic outcome by means of at least two distinct transcriptional architectures related to mechano-transduction and proliferation.

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Fewer non-native insects in freshwater than in terrestrial habitats across continents AimBiological invasions are a major threat to biodiversity in both aquatic and terrestrial habitats. Insects represent an important group of species in freshwater and terrestrial habitats, and they constitute a large proportion of non-native species. However, while many non-native insects are known from terrestrial ecosystems, it remains unclear how they are represented in freshwater habitats. Comparisons of the richness of invaders relative to the richness of native species between freshwater and terrestrial habitats are scarce, which hinders syntheses of invasion processes. Here, we used data from three regions on different continents to determine whether non-native insects are under- or overrepresented in freshwater compared to terrestrial assemblages.

LocationEurope, North America, New Zealand

MethodsWe compiled a comprehensive inventory of the native and non-native insect species established in freshwater and terrestrial habitats of the three study regions. We then contrasted the richness of non-native and native species among freshwater and terrestrial insects for all insect orders in each region. Using binomial regression, we analysed the proportions of non-native species in freshwater and terrestrial habitats across the three regions.

ResultsIn most insect orders living in freshwater, non-native species were under-represented, while they were over-represented in a number of terrestrial orders. This pattern occurred in purely aquatic orders as well as in orders with both freshwater and terrestrial species. Overall, the proportion of non-native species was significantly lower in freshwater than in terrestrial species.

Main conclusionsDespite the numerical and ecological importance of insects among all non-native species, non-native insect species are surprisingly rare in freshwater habitats, and this pattern is consistent across the three investigated study regions. We briefly review hypotheses concerning species traits and invasion pathways that are most likely to explain these patterns. Our findings contribute to a growing appreciation of drivers and impacts of biological invasions.

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Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a. Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1. While IRE1 can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1 complexes as well as the targeting mechanism that recruits XBP1 to IRE1 oligomers, remain unknown.

Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1 clusters, which only assemble upon overexpression of fluorescently-tagged IRE1 during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1 assemblies that are homogenously distributed throughout the ER membrane.

sentences: Improved estimation of cell type-specific gene expression through deconvolution of bulk tissues with matrix completion Single cell RNA-seq (scRNA-seq) has been widely used to uncover cellular heterogeneity, however, the constraints of cost make it impractical as a routine on large patient cohorts. Here we present ENIGMA, a method that accurately deconvolute bulk tissue RNA-seq into single cell-type resolution given the knowledge gained from scRNA-seq. ENIGMA applies a matrix completion strategy to minimize the distance between mixture transcriptome and weighted combination of cell type-specific expression, allowing quantification of cell type proportions and reconstruction of cell type-specific transcriptome. The superior performance of ENIGMA was validated in simulated and realistic datasets, including disease-related tissues, demonstrating its ability in novel biological findings.

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

sentences: NAD+ repletion by the PARP inhibitor PJ34 prevents Sarm1 activation and rotenone-induced cell death Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+, it is also allosterically inhibited by NAD+. However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP1 inhibitor, PJ34 restored mitochondrial homeostasis and prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncovers a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders mediated by Sarm1.

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Qualitative and quantitative top-down proteomics of human colorectal cancer cell lines identified 23000 proteoforms and revealed drastic proteoform-level differences between metastatic and non-metastatic cancer cells Understanding cancer metastasis at the proteoform level is crucial for discovering new protein biomarkers for cancer diagnosis and drug development. Proteins are the primary effectors of function in biology and proteoforms from the same gene can have drastically different biological functions. Here, we present the first qualitative and quantitative top-down proteomics (TDP) study of a pair of isogenic human metastatic and non-metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). This study pursues a global view of human CRC proteome before and after metastasis in a proteoform specific manner. We identified 23,319 proteoforms of 2,297 genes from the CRC cell lines using capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS), representing nearly one order of magnitude improvement in the number of proteoform identifications from human cell lines compared to literature data. We identified 111 proteoforms containing single amino acid variants (SAAVs) using a proteogenomic approach and revealed drastic differences between the metastatic and non-metastatic cell lines regarding SAAVs profiles. Quantitative TDP analysis unveiled statistically significant differences in proteoform abundance between the SW480 and SW620 cell lines on a proteome scale for the first time. Ingenuity Pathway Analysis (IPA) disclosed that many differentially expressed genes at the proteoform level had diversified functions and were closely related to cancer. Our study represents a milestone in TDP towards the definition of human proteome in a proteoform specific manner, which will transform basic and translational biomedical research.

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sentences: Soluble ECM promotes organotypic formation in lung alveolar model Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

sentences: The neural code for 'face cells' is not face specific 1 Face cells are visual neurons that selectively respond more to faces than other objects. Clustered together in inferotemporal cortex, they are thought to form a network of modules specialized in face processing by encoding face-specific features. Here we reveal that their category selectivity is instead captured by domain-general attributes. Analyzing neural responses in and around macaque face patches to hundreds of objects, we discovered graded tuning for non-face objects that was more predictive of face preference than was tuning for faces themselves. The relationship between category-level face selectivity and image-level non-face tuning was not predicted by color and simple shape properties, but by domain-general information encoded in deep neural networks trained on object classification. These findings contradict the long-standing assumption that face cells owe their category selectivity to face-specific features, challenging the prevailing idea that visual processing is carried out by discrete modules, each specialized in a semantically distinct domain.

sentences: Acoustic regularities in infant-directed speech and song across cultures The forms of many species vocal signals are shaped by their functions1-15. In humans, a salient context of vocal signaling is infant care, as human infants are altricial16, 17. Humans often alter their vocalizations to produce "parentese", speech and song produced for infants that differ acoustically from ordinary speech and song18-35 in fashions that have been proposed to support parent-infant communication and infant language learning36-39; modulate infant affect33, 40-45; and/or coordinate communicative interactions with infants46-48. These theories predict a form-function link in infant-directed vocalizations, with consistent acoustic differences between infant-directed and adult-directed vocalizations across cultures. Some evidence supports this prediction23, 27, 28, 32, 49-52, but the limited generalizability of individual ethnographic reports and laboratory experiments53 and small stimulus sets54, along with intriguing reports of counterexamples55-62, leave the question open. Here, we show that people alter the acoustic forms of their vocalizations in a consistent fashion across cultures when speaking or singing to infants. We collected 1,615 recordings of infant- and adult-directed singing and speech produced by 410 people living in 21 urban, rural, and small-scale societies, and analyzed their acoustic forms. We found cross-culturally robust regularities in the acoustics of infant-directed vocalizations, such that infant-directed speech and song were reliably classified from acoustic features found across the 21 societies studied. The acoustic profiles of infant-directedness differed across language and music, but in a consistent fashion worldwide. In a secondary analysis, we studied whether listeners are sensitive to these acoustic features, playing the recordings to 51,065 people recruited online, from many countries, who guessed whether each vocalization was infant-directed. Their intuitions were largely accurate, predictable in part by acoustic features of the recordings, and robust to the effects of linguistic relatedness between vocalizer and listener. By uniting rich cross-cultural data with computational methods, we show links between the production of vocalizations and cross-species principles of bioacoustics, informing hypotheses of the psychological functions and evolution of human communication.

sentences: Designing multi-epitope based peptide vaccine targeting spike protein SARS-CoV-2 B1.1.529 (Omicron) variant using computational approaches. Since the SARS-CoV-2 outbreak in 2019, millions of people have been infected with the virus, and due to its high human-to-human transmission rate, there is a need for a vaccine to protect people. Although some vaccines are in use, due to the high mutation rate in the SARS-CoV-2 multiple variants, the current vaccines may not be sufficient to immunize people against new variant threats. One of the emerging variants of concern is B1.1.529 (Omicron), which carries[~]30 mutations in the Spike protein of SARS-CoV-2 is predicted to evade antibodies recognition even from vaccinated people. We used a structure-based approach along with an epitope prediction server to develop a Multi-Epitope based Subunit Vaccine (MESV) involving SARS-CoV-2 B1.1.529 variant spike glycoprotein. The predicted epitope with better antigenicity and non-toxicity were used for designing and predicting vaccine construct features and structure models. The MESV construct In-silico cloning in pET28a expression vector predicted the construct to be highly translational. The proposed MESV vaccine construct was also subjected to immune simulation prediction and was found to be highly antigenic and elicit a cell-mediated immune response. The proposed MESV in the present study has the potential to be evaluated further for vaccine production against the newly identified B1.1.529 (Omicron) variant of concern.

sentences: A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness Our ability to understand and modulate mammalian physiology and disease requires knowing how all genes contribute to any given phenotype in the organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for the genetic dissection of cellular processes1,2, but the need to stably deliver single guide RNAs to millions of cells has restricted its implementation to ex vivo systems. These ex vivo systems cannot reproduce all of the cellular phenotypes observed in vivo nor can they recapitulate all of the factors that influence these phenotypes. There thus remains a pressing need for high-throughput functional genomics in a living organism. Here, we establish accessible genome-wide screening in the mouse liver and use this approach to uncover the complete regulation of cellular fitness in a living organism. We discover novel sex-specific and cell non-autonomous regulation of cell growth and viability. In particular, we find that the class I major histocompatibility complex is essential for preventing immune-mediated clearance of hepatocytes. Our approach provides the first comprehensive picture of cell fitness in a living organism and highlights the importance of investigating cellular phenomena in their native context. Our screening method is robust, scalable, and easily adapted to examine diverse cellular processes using any CRISPR application. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into mammalian physiology and disease.

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

sentences: High-speed multifocus phase imaging in thick tissue Phase microscopy is widely used to image unstained biological samples. However, most phase imaging techniques require transmission geometries, making them unsuited for thick sample applications. Moreover, when applied to volumetric imaging, phase imaging generally requires large numbers of measurements, often making it too slow to capture live biological processes with fast 3D index-of-refraction variations. By combining oblique back-illumination microscopy and a z-splitter prism, we perform phase imaging that is both epi-mode and multifocus, enabling high-speed 3D phase imaging in thick, scattering tissues with a single camera. We demonstrate here 3D qualitative phase imaging of blood flow in chick embryos over a field of view of 546 x 546 x 137 m3 at speeds up to 47 Hz.

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SNHG16 promotes cell proliferation and inhibits cell apoptosis via regulation of the miR-1303p/STARD9 axis in renal cell carcinoma BackgroundRenal cell carcinoma (RCC) is a fatal malignant tumor with high morbidity. Numerous medical studies have suggested that long noncoding RNAs (lncRNAs) exert their biological function on various cancerous progresses. Herein, functions of lncRNA SNHG16 in RCC cells and the mechanism medicated by SNHG16 were investigated.

MethodsThe expression levels of SNHG16 and its downstream genes in RCC cells and tissues were examined utilizing reverse transcription quantitative polymerase chain reaction analyses. Cell counting kit-8 and 5-Ethynyl-2-deoxyuridine assays were carried out to evaluate the proliferation of RCC cells, and flow cytometry analyses were employed to determine the apoptosis of RCC cells. Western blot analysis was applied to examine protein levels associated with cell proliferation and apoptosis. The combination between SNHG16 and miRNA as well as miRNA and its target gene were explored by luciferase reporter, RNA pull down, and RNA immunoprecipitation assays.

ResultsThe significant upregulation of SNHG16 was observed in RCC tissues and cells. SNHG16 downregulation inhibited the proliferation and promoted the apoptosis of RCC cells. In addition, SNHG16 served as a competing endogenous RNA for miR-1301-3p, and STARD9 was a target gene of miR-1301-3p in RCC cells. SNHG16 upregulated STARD9 expression by binding with miR-1301-3p in RCC cells. Rescue assays validated that SNHG16 promoted RCC cell promotion and induced RCC cell apoptosis by upregulating STARD9 expression.

ConclusionsSNHG16 promotes RCC cell proliferation and suppresses RCC cell apoptosis via interaction with miR-1301-3p to upregulate STARD9 expression in RCC cells.

sentences: Physical model of electrical synapses in a neural network A theoretical model of electrical synapses is proposed, in which connexons play the role of "nails" that hold unmyelinated areas of neurons at a distance of about 3.5 nm, and the electrical connection between them is provided by charging the membrane of an inactive neuron with currents generated in the intercellular electrolyte (saline) by the action potential in the active neuron. This mechanism is similar to the salutatory conduction of the action potential between the nodes of Ranvier in myelinated axons and the ephaptic coupling of sufficiently close spaced neurons.

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Human mesenchymal stem cells and derived extracellular vesicles reduce sensory neuron hyperexcitability and pain-related behaviors in a mouse model of osteoarthritis Osteoarthritis (OA) is a common degenerative joint disease characterized by joint pain and stiffness. In humans, mesenchymal stem cells (MSCs) and derived extracellular vesicles (MSC-EVs) have been reported to alleviate pain in knee OA. Here, we used the destabilization of the medial meniscus (DMM) mouse model of OA to investigate mechanisms by which MSCs and MSC-EVs influence pain-related behavior. We found that MSC and MSC-EV treated DMM mice displayed improved OA pain-related behavior (i.e. locomotion, digging and sleep) compared to untreated DMM mice. Improved behavior was not the result of reduced joint damage, but rather knee-innervating sensory neurons from MSC and MSC-EV treated mice did not display the hyperexcitability observed in untreated DMM mice. Furthermore, we found that MSC-EVs normalize sensory neuron hyperexcitability induced by nerve growth factor in vitro. Our study suggests that MSCs and MSC-EVs may reduce pain in OA by direct action on peripheral sensory neurons.

TeaserMesenchymal stem cells and secreted extracellular vesicles normalize sensory neuron excitability to reduce pain.

sentences: Deep brain stimulation in the subthalamic nucleus for Parkinson's disease can restore dynamics of striatal networks Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is highly effective in alleviating movement disability in patients with Parkinsons disease (PD). However, its therapeutic mechanism of action is unknown. The healthy striatum exhibits rich dynamics resulting from an interaction of beta, gamma and theta oscillations. These rhythms are at the heart of selection, initiation and execution of motor programs, and their loss or exaggeration due to dopamine (DA) depletion in PD is a major source of the behavioral deficits observed in PD patients. Interrupting abnormal rhythms and restoring the interaction of rhythms as observed in the healthy striatum may then be instrumental in the therapeutic action of DBS. We develop a biophysical networked model of a BG pathway to study how abnormal beta oscillations can emerge throughout the BG in PD, and how DBS can restore normal beta, gamma and theta striatal rhythms. Our model incorporates STN projections to the striatum, long known but understudied, that were recently shown to preferentially target fast spiking interneurons (FSI) in the striatum. We find that DBS in STN is able to normalize striatal medium spiny neuron (MSN) activity by recruiting FSI dynamics, and restoring the inhibitory potency of FSIs observed in normal condition. We also find that DBS allows the re-expression of gamma and theta rhythms, thought to be dependent on high DA levels and thus lost in PD, through cortical noise control. Our study shows how BG connectivity can amplify beta oscillations, and delineates the role of DBS in disrupting beta oscillations and providing corrective input to STN efferents to restore healthy striatal dynamics. It also suggests how gamma oscillations can be leveraged to enhance or supplement DBS treatment and improve its effectiveness.

sentences: Visualizing reactive astrogliosis extends survival in glioblastoma patients Glioblastoma multiforme (GBM) is a devastating brain tumor with dismal prognosis of only 15-month survival regardless of surgical resection. Here, we report an advanced neuroimaging technique combining 11C-acetate PET and MRI (AcePET), visualizing the boundary beyond the MRI-defined tumor. Targeted biopsy of the regions with increased 11C-acetate uptake revealed the presence of reactive astrocytes with enhanced acetate-transporter MCT1, along with cancer stem cells. Reactive astrogliosis and MCT1-dependent 11C-acetate-uptake were recapitulated in U87MG-orthotopic models. Mechanistically, glycolytic tumor cells release excessive acetate causing reactive astrogliosis, leading to the release of aberrant astrocytic GABA and H2O2, which further down-regulate the neuronal glucose uptake through GLUT3. Clincally, AcePET-guided surgery allows complete tumor resection of infiltrating cancer stem cells and extends the overall survival of patients by 5.25 months compared to conventional MRI-guided surgery. We established a new concept of the metabolic interactions between GBM cells and neighboring neurons through reactive astrocytes and developed AcePET-guided surgery to fight against GBM.

sentences: Western Amazon was a center of Neotropical fish dispersal, as evidenced by the continental-wide time-stratified biogeographic analysis of the hyper-diverse Hypostomus catfish The Amazon is probably the most diverse realm on Earth, and is considered to be the primary source of diversity and a center of dispersal for Neotropical terrestrial organisms. Yet, the assumption that the Amazon basin is a primordial place of fish species origination and dispersal into other drainages still need to be tested. We addressed this issue by inferring a time-stratified biogeographic history and reconstructing the ancestral habitat preference of Hypostomus, a continentally widespread and species-rich Neotropical genus. We found that Hypostomus emerged in the Western Amazon ([~]14.7 Ma), when the Western Amazon River was flowing northwards and disconnected from the Eastern Amazon. We show that dispersal events in the first half of Hypostomus evolution occurred from the Western Amazon into adjacent basins, initiating its Neotropical radiation. The ancestral preferred habitat consisted in small rivers with running waters, a predominant habitat in river headwaters. Because of strong niche conservatism in the early evolution of Hypostomus, we suggest that most of the out-of-Western-Amazon dispersal occurred via headwater captures. The radiation of Hypostomus was further promoted by major reconfigurations of river basins, which opened dispersal opportunities into new drainages. Diversification in habitat preference coincided with colonization of basins already occupied by congenerics, indicative of niche shifts triggered by inter-specific competition and species coexistence. By analyzing the evolutionary history of Hypostomus, we show that Western Amazon was the main center of fish dispersal in the Neotropical Region from Middle Miocene to the present, supporting the cradle hypothesis of fish origination and dispersal.

sentences: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. In recent years, the signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. Here, we set out to systematically compare the effect of two major ECM classes, fibrillar collagen type I and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all differentiated and undifferentiated cell types present in both cultures, albeit at different ratios. Specific to the collagen culture was a subset of cells with a fetal-like gene expression program. In contrast, laminin, but not collagen IV, increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture, resembles the gut development in vivo. Here, the ECM is dramatically remodelled by mesenchymal cells, which is accompanied by a specific and local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

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Distinct Protofilament-scale Dynamics Define the Differential Remodeling of Microtubule Arrays The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes, implicated in both the complete demolition and fine size control of microtubule-based arrays, are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here we use Atomic Force Microscopy (AFM) to image depolymerizing arrays at single microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-sized cytoskeletal structures.

SIGNIFICANCE STATEMENTOne cannot help but marvel at the precise organization of microtubule polymers in cellular structures such as the axoneme and the spindle. However, our understanding of the biochemical mechanisms that sculpt these arrays comes largely from in vitro experiments with a small number (one or two) of microtubules. This is somewhat akin to studying the architecture of multilane highways by studying one-lane streets. Here we directly visualize depolymerizing microtubule arrays at individual microtubule and protofilament resolution using Atomic Force Microscopy. Our results reveal differences in microtubule depolymerase activity and provide insights into how these differences in enzymatic activity on the nanometer scale can result in the differential remodeling of multi-microtubule arrays on the micron-length scale.

sentences: Single-cell Chromatin Accessibility and Lipid Profiling Reveals a Metabolic Shift in Adipocytes Induced by Bariatric Surgery Obesity promotes type 2 diabetes and cardiometabolic pathologies. Vertical sleeve gastrectomy (VSG) is used to treat obesity resulting in long-term weight loss and health improvements that precede weight loss; however, the mechanisms underlying the immediate benefits remain incompletely understood. Because adipose plays a crucial role in energy homeostasis and utilization, we hypothesized that VSG exerts its influences, in part, by modulating adipose functional states. We applied single-cell ATAC sequencing and lipid profiling to inguinal and epididymal adipose depots from mice that received sham surgery or VSG. We observed depot-specific cellular composition and chromatin accessibility patterns that were altered by VSG. Specifically, accessibility at Scd1, a fatty acid desaturase, was substantially reduced after VSG in mature adipocytes of inguinal but not epididymal depots. This was accompanied by reduced accumulation of SCD1-produced unsaturated fatty acids. Given these findings and reports that reductions in Scd1 attenuate obesity and insulin resistance and that unsaturated fatty acids stimulate glucose uptake, storage, and oxidation, our results suggest VSG exerts its beneficial effects through modifications of fatty acid profiles mediated by Scd1.

sentences: Comprehensive transcriptional profiling and mouse phenotyping reveals dispensable role for adipose tissue selective long noncoding RNA Gm15551 1Cold and nutrient activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via uncoupled respiration and secretion of endocrine factors thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to inter-scapular brown adipose tissue (iBAT) function in high fat diet (HFD) and cold stressed mice. We focused on Gm15551 which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis and downregulated by {beta}-adrenergic activation in mature adipocytes. Albeit we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

sentences: High-altitude adaptation and incipient speciation in geladas Survival at high altitude requires adapting to extreme conditions such as environmental hypoxia. To understand high-altitude adaptations in a primate, we assembled the genome of the gelada (Theropithecus gelada), an endemic Ethiopian monkey, and complemented it with population resequencing, hematological, and morphometric data. Unexpectedly, we identified a novel karyotype that may contribute to reproductive isolation between gelada populations. We also identified genomic elements including protein-coding sequences and gene families that exhibit accelerated changes in geladas and may contribute to high-altitude adaptation. Our findings lend insight into mechanisms of speciation and adaptation while providing promising avenues for functional hypoxia research.

sentences: Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. Tissue- specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

sentences: A CRISPR-Cas9-based system for the dose-dependent study of DNA double strand breaks sensing and repair The integrity of DNA is put at risk by different lesions, among which double strand breaks (DSBs) occur at low frequency, yet remain one of the most life-threatening harms. The study of DSB repair requests tools provoking their accumulation, and include the use of chemical genotoxins, ionizing radiations or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nucleases expression permits assessments at precise locations. In this work, we have exploited the repetitiveness of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled creation of 0, 1, 15 or 59 cuts in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1. By comparing different strategies to create DNA breaks, we find that Tel1 is capable of forming multiple foci, in striking contrast with other DSB-related proteins. Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.

sentences: Assessment of Neurovascular Coupling & Cortical Spreading Depression in Mixed Models of Atherosclerosis & Alzheimer's Disease Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in preparations that modelled Alzheimers disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9-12m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, levels of oxyhaemoglobin & deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3x fold increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimers-related amyloid-plaques.

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Tropical origin, global diversification and dispersal in the pond damselflies (Coenagrionoidea) revealed by a new molecular phylogeny AimThe processes responsible for the formation of Earths most conspicuous diversity pattern, the latitudinal diversity gradient (LDG), remain unexplored for many clades in the Tree of Life. Here, we present a densely-sampled and dated molecular phylogeny for the most speciose clade of damselflies worldwide (Odonata: Coenagrionoidea), and investigate the role of time, macroevolutionary processes and dispersal dynamics in shaping the LDG in this ancient insect superfamily.

Locationglobal

Time period240-0 mya

Major taxa studiedpond damselflies (Coenagrionidae) and featherlegs (Platycnemididae)

MethodsWe used a process-based biogeographic model to jointly infer ancestral ranges and speciation times and to characterise dispersal dynamics between latitudinal regions. We then used this phylogenetic inference to investigate temporal and latitudinal variation in diversification rates.

ResultsOur results uncover a tropical origin of pond damselflies and featherlegs [~] 105 mya, with their common ancestor arising in either Northern South America or Western Africa. While diversification rates have declined since the origin of this clade, the tropics have acted as a net source of lineages dispersing into temperate areas, where both speciation and extinction rates have been relatively higher.

Main conclusionsThis study underscores the importance of biogeographic origin and time to diversify as important drivers of the LDG in pond damselflies. Diversification dynamics may have instead resulted in the formation of ephemeral species in temperate regions. Dispersal has played a role reducing the steepness of the LDG, rather than amplifying it, which would have been expected under strong tropical niche conservatism. With ongoing climate change and increasing northward range expansions of many damselfly taxa, the LDG may continue to shallow. Our results support recent calls to unify biogeographic and macroevolutionary approaches to increase our understanding of how latitudinal diversity gradients are formed and why they vary across time and among taxa.

sentences: Environmentally-induced mdig is a major contributor to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism The novel {beta}-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 101 million people and resulted in 2.2 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provide evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is a master regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. These data, accordingly, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.

sentences: The HIV-1 ribonucleoprotein dynamically regulates its condensate behavior and drives acceleration of protease activity through membraneless granular phase separation A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase separated molecular  sponges, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ~2400 Gag and ~120 GagPol by viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak-strong-moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

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Augmenting Whole Recombinant Antibody Production in HEK293E Cells: Optimizing Transfection and Culture Parameters BackgroundOptimising recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialisation, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterisations and investigations.

MethodsInvestigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analysed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day and common nutrient supplements.

ResultsUsing 2 mL adherent HEK293E cell culture transfections with 25 kDa linear Polyethylenimine in the most optimised parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 g respectively in a cost-effective manner. With the addition of peptone, {kappa} light chain increased by ~4-fold to 1032 g while whole antibody increased to a lesser extent by ~2.5-fold to 51 g, with benefits potentially for antibodies limited by their light chains in production.

ConclusionsOur optimised findings show promise for a more efficient and convenient antibody production method through transfection and culture optimisations that can be incorporated to scale up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E cells.

Statement of SignificanceRecombinant antibody production is crucial for antibody research and development. Systematically investigating transfection and culture parameters such as PEI/DNA concentrations, complexation time, volume, and temperature, supplements, etc., we demonstrated a ~4-fold light chain alone production increase to 1032 g and a 2.5-fold whole antibody production increase to 51 g from 2 mL transfections.

sentences: Allosteric modulation of the SARS-CoV-2 spike conformation Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Forster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

sentences: The potential of aquatic haematophagous, liquidosomatophagous and macrophagous leeches as a tool for iDNA characterisation Leeches play important roles in food webs due to their abundance, diversity and feeding habits. Studies using invertebrate-derived DNA (iDNA) extracted from leech gut contents to target vertebrate DNA have focused on the Indo-Pacific region and mainly leveraged the leech family Haemadipsidae, composed of haematophagous terrestrial leeches, while the aquatic haematophagous, liquidosomatophagous and macrophagous counterparts have largely been disregarded. While there is general knowledge regarding the taxonomic groups that leeches prefer to feed on, detailed taxonomic resolution is still missing and therefore, their potential use for monitoring animals is not known. In this study, 116 non-haemadipsid leeches belonging to 12 species and spanning the three feeding habits were collected in Mexico and Canada. We used DNA metabarcoding to investigate their diet and assess their potential use for vertebrate monitoring. We detected vertebrate taxa from five orders including fish, turtles and birds in the diet of the aquatic haematophagous leeches; eight invertebrate orders of annelids, arthropods and molluscs in the liquidosomatophagous leeches; and ten orders of invertebrates belonging to Arthropoda and Annelida, as well as one vertebrate and one parasitic nematode, in the macrophagous leeches. These results show the potential use of iDNA from the gut content of aquatic haematophagous leeches for retrieving vertebrate taxa, and from macrophagous and liquidosomatophagous counterparts for invertebrates. Our study provides information about the dietary range of the freshwater leeches and the non-haemadipsid terrestrial leech and proof-of-concept for the use of non-haemadipsid leeches for animal monitoring, expanding our knowledge of the use of iDNA from leech gut contents to North America.

sentences: Trophic ecology of deep-sea megafauna in the ultra-oligotrophic Southeastern Mediterranean Sea The trophic ecology of fourteen species of bathybenthic and bathypelagic fishes and six species of bathybenthic decapod crustaceans from the continental slope and rise of the Southeastern Mediterranean Sea (SEMS) was examined using stable isotope analysis. Mean {delta}13C values among fish species varied by more than 4.0{per thousand}, from -20.85{per thousand} (Macroramphosus scolopax) to -16.57{per thousand} (Conger conger and Centrophorus granulosus), and increased as a function of depth (200 - 1400 m). Mean {delta}13C values of the crustaceans showed smaller variation, between -16.38{per thousand} (Polycheles typhlops) and -18.50{per thousand} (Aristeus antennatus). This suggests a shift from pelagic to regenerated benthic carbon sources with depth. Benthic carbon regeneration is further supported by the low benthic-pelagic POM-{delta}13C values, averaging -24.7 {+/-} 1.2{per thousand}, and the mixing model results, presenting very low contribution of epipelagic POM to the bathyal fauna. Mean {delta}15N values of fish and crustacean species ranged 7.91 {+/-} 0.36{per thousand} to 11.36 {+/-} 0.39{per thousand} and 6.15 {+/-} 0.31{per thousand} to 7.69 {+/-} 0.37{per thousand}, respectively, resulting in trophic position estimates, occupying the third and the fourth trophic levels. Thus, despite the proximity to the more productive areas of the shallow shelf, low number of trophic levels (TL~1.0) and narrow isotopic niche breadths (SEAc <1) were observed for bathybenthic crustaceans (TL = 3.62 {+/-} 0.22) and bathypelagic fishes (TL = 4.33 {+/-} 0.34) in the study area - probably due to the ultra-oligotrophic state of the SEMS resulting in limited carbon sources. Our results, which provide the first trophic description of deep-sea megafauna in the SEMS, offer insight into the carbon sources and food web structure of deep-sea ecosystems in oligotrophic marginal seas, and can be further used in ecological modeling and support the sustainable management of marine resources in the deep Levantine Sea.

sentences: Discovery of a carbonic anhydrase-Rubisco supercomplex within the alpha-carboxysome Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation, Rubisco and carbonic anhydrase, and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the -carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how CsoSCA, the carbonic anhydrase, is incorporated into the -carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into -carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 [A] single-particle cryo-electron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCAs binding site overlaps with that of CsoS2 but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of biogenesis of carboxysomes and highlights the importance of Rubisco, not only as an enzyme, but also as a hub protein central for assembling supercomplexes.

sentences: Assessment and Optimization of the Interpretability of Machine Learning Models Applied to Transcriptomic Data Explainable artificial intelligence aims to interpret how the machine learning models make decisions, and many model explainers have been developed in the computer vision field. However, the understandings of the applicability of these model explainers to biological data are still lacking. In this study, we comprehensively evaluated multiple explainers by interpreting pretrained models of predicting tissue types from transcriptomic data, and by identifying top contributing genes from each sample with the greatest impacts on model prediction. To improve the reproducibility and interpretability of results generated by model explainers, we proposed a series of optimization strategies for each explainer on two different model architectures of Multilayer Perceptron (MLP) and Convolutional Neural Network (CNN). We observed three groups of explainer and model architecture combinations with high reproducibility. Group II, which contains three model explainers on aggregated MLP models, identified top contributing genes in different tissues that exhibited tissue-specific manifestation and were potential cancer biomarkers. In summary, our work provides novel insights and guidance for exploring biological mechanisms using explainable machine learning models.

sentences: Oncogenic role of a developmentally regulated NTRK2 splice variant Temporally-regulated alternative splicing choices are vital for proper development yet the wrong splice choice may be detrimental. Here we highlight a novel role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in both humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis and forced over-expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring TRK fusions. Affinity purification-mass spectrometry (AP-MS) proteomic analysis reveals TrkB.T1 has distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, TGF-{beta}, Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

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Sex differences and similarities in the neuroimmune response to central administration of poly I:C BackgroundThe neuroimmune system is required for normal neural processes, including modulation of cognition, emotion, and adaptive behaviors. Aberrant neuroimmune activation is associated with dysregulation of memory and emotion, though the precise mechanisms at play are complex and highly context dependent. Sex differences neuroimmune activation and function further complicate our understanding of its roles in cognitive and affective regulation.

MethodsHere, we characterized the physiological sickness and inflammatory response of the hippocampus following intracerebroventricular (ICV) administration of a synthetic viral mimic, polyinosinic:polycytidylic acid (poly I:C), in both male and female C57Bl/6 mice.

ResultsWe observed that poly I:C induced weight loss, fever, and elevations of cytokine and chemokines in the hippocampus of both sexes. Specifically, we found transient increases in gene expression and protein levels of IL-1, IL-1{beta}, IL-4, IL-6, TNF, CCL2, and CXCL10, where males showed a greater magnitude of response compared with females. Only males showed increased IFN and IFN{gamma} in response to poly I:C, whereas both males and females exhibited elevations of IFN{beta}, demonstrating a specific sex difference in the anti-viral response in the hippocampus.

ConclusionOur data suggest that type I interferons are one potential node mediating sex-specific cytokine responses and neuroimmune effects on cognition. Together, these findings highlight the importance of using both males and females and analyzing a broad set of inflammatory markers in order to identify the precise, sex-specific roles for neuroimmune dysregulation in neurological diseases and disorders.

sentences: Effects of Intravenous Magnesium Sulfate on Calcium-Regulating Hormones, Plasma and Urinary Electrolytes in Healthy Horses Intravenous magnesium sulfate (MgSO4) is used in equine practice to treat hypomagnesemia, dysrhythmias, neurological disorders, and calcium dysregulation. MgSO4 is also used as a calming agent in equestrian events. Hypercalcemia affects calcium-regulating hormones, as well as plasma and urinary electrolytes; however, the effect of hypermagnesemia on these variables is unknown. The goal of this study was to investigate the effect of hypermagnesemia on blood parathyroid hormone (PTH), calcitonin (CT), ionized calcium (Ca2+), ionized magnesium (Mg2+), sodium (Na+), potassium (K+), chloride (Cl-) and their urinary fractional excretion (F) after intravenous administration of MgSO4 in healthy horses. Twelve healthy female horses of 4-18 years of age and 432-600 kg of body weight received a single intravenous dose of MgSO4 (60 mg/kg) over 5 minutes, and blood and urine samples were collected at different time points over 360 minutes. Plasma Mg2+ concentrations increased 3.7-fold over baseline values at 5 minutes and remained elevated for 120 minutes (P < 0.05), Ca2+ concentrations decreased from 30-60 minutes (P < 0.05), but Na+, K+ and Cl- concentrations did not change. Serum PTH concentrations dropped initially to rebound and remain elevated from 30 to 60 minutes, while CT concentrations increased at 5 minutes to return to baseline by 10 minutes (P < 0.05). The FMg, FCa, FNa, FK, and FCl increased, while urine osmolality decreased from 30-60 minutes compared baseline (P < 0.05). Experimental hypermagnesemia alters calcium-regulating hormones (PTH, CT), reduces plasma Ca2+ concentrations, and increases the urinary excretion of Mg2+, Ca2+, K+, Na+ and Cl- in healthy horses. This information has clinical implications on the short-term effects of hypermagnesemia on calcium-regulating hormones as well as plasma and urine electrolytes.

sentences: Identification of Hsp90 inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

sentences: Developmental changes in story-evoked responses in the neocortex and hippocampus How does the representation of naturalistic life events change with age? Here we analyzed fMRI data from 415 children and adolescents (5 - 19 years) as they watched a narrative movie. In addition to changes in the degree of inter-subject correlation (ISC) with age in sensory and medial parietal regions, we used a novel measure (between-groups ISC) to reveal age-related shifts in the responses across the majority of the neocortex. Over the course of development, brain responses became more discretized into stable and coherent events, and shifted earlier in time to anticipate upcoming event transitions. However, hippocampal responses to event boundaries actually decreased with age, suggesting a shifting division of labor between episodic encoding processes and schematic event representations between the ages of 5 and 19.

sentences: The Euler Characteristic and Topological Phase Transitions in Complex Systems In this work, we use methods and concepts of applied algebraic topology to comprehensively explore the recent idea of topological phase transitions (TPT) in complex systems. TPTs are characterized by the emergence of nontrivial homology groups as a function of a threshold parameter. Under certain conditions, one can identify TPTs via the zeros of the Euler characteristic or by singularities of the Euler entropy. Recent works provide strong evidence that TPTs can be interpreted as a complex networks intrinsic fingerprint. This work illustrates this possibility by investigating some classic network and empirical protein interaction networks under a topological perspective. We first investigate TPT in protein-protein interaction networks (PPIN) using methods of topological data analysis for two variants of the Duplication-Divergence model, namely, the totally asymmetric model and the heterodimerization model. We compare our theoretical and computational results to experimental data freely available for gene co-expression networks (GCN) of Saccharomyces cerevisiae, also known as bakers yeast, as well as of the nematode Caenorhabditis elegans. Supporting our theoretical expectations, we can detect topological phase transitions in both networks obtained according to different similarity measures. Later, we perform numerical simulations of TPTs in four classical network models: the Erd[o]s-Renyi model, the Watts-Strogatz model, the Random Geometric model, and the Barabasi-Albert. Finally, we discuss some perspectives and insights on the topic. Given the universality and wide use of those models across disciplines, our work indicates that TPT permeates a wide range of theoretical and empirical networks.

sentences: AP-4 regulates neuronal lysosome composition, function and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimers disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP.

sentences: Rostrocaudal Patterning and Neural Crest Differentiation of Human Pre-Neural Spinal Cord Progenitors in vitro The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by Wnt/FGF signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which complicates the full range production of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity by co-expressing the transcription factors SOX2 and CDX2, and lose mesodermal potential by downregulating TBXT. For 7 to 10 passages PNPs divide to self-renew and to make trunk neural crest (NC), while gradually adopting a more posterior identity by activating colinear HOX gene expression. This HOX clock can be halted at the thoracic level for up to 30 passages by blocking the trunk-to-tail transition through GDF11-mediated signal inhibition.

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Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy BackgroundInnovative cancer immunotherapy approaches aim at combining immune checkpoint inhibitors with other immunomodulatory agents. Epigenetic regulators can control immune-related genes, therefore targeting them with specific inhibitors may be a potential way forward. Here we identified immune-related signatures induced by four classes of epigenetic drugs in human melanoma cells to define the most promising agent and to understand its biological activity in-vitro, in-vivo and in clinical samples.

MethodsHuman melanoma cell lines were characterized for mutational and differentiation profile and treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), bromodomain and extraterminal domain proteins (JQ1 and OTX-015) and enhancer of zeste homolog 2 (GSK126). Drug-specific gene signatures were identified by Clariom S and Nanostring platforms. Modulation of 14 proteins was determined by quantitative western blot. Ingenuity Pathway Analysis (IPA) identified Upstream Regulator (UR) molecules explaining changes in gene expression and biological activity of drugs. Gene set enrichment and IPA were used to test modulation of guadecitabine-specific gene and UR signatures, respectively, in on-treatment tumor biopsies from melanoma patients enrolled in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial.

ResultsDrug-specific gene and UR signatures were identified for each of the four inhibitors. Immune-related genes were frequently upregulated by guadecitabine, to a lesser extent by givinostat, but downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Treatment of melanoma cells with combination of two epigenetic drugs revealed a dominant effect of guadecitabine and JQ1 on immune-related gene modulation. Drug-specific modulatory profiles were confirmed at the protein level. The guadecitabine-specific UR signature was characterized by activated molecules of the TLR, NF-kB, and IFN innate immunity pathways and was induced in drug-treated melanoma, mesothelioma, hepatocarcinoma cell lines and human melanoma xenografts. Most of the guadecitabine-specific signature genes (n>160) were upregulated in on-treatment tumor biopsies from NIBIT-M4 trial. Progressive activation of guadecitabine UR signature molecules was observed in on-treatment tumor biopsies from responding compared to non-responding patients.

ConclusionsGuadecitabine was the most promising immunomodulatory agent among those investigated. This DNA methyltransferases inhibitor emerged as a strong inducer of innate immunity pathways, supporting the rationale for its use in combinatorial immunotherapy approaches.

sentences: An environmentally ultrasensitive fluorine-19 NMR probe for monitoring protein conformational equilibria Limited chemical shift dispersion represents a major barrier to studying multi-state equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe with a dramatically larger chemical shift dispersion than existing probes. The improved conformational sensitivity and line shape enabled the detection of hidden states of a critical membrane transporter, guiding subsequent structural elucidation by single-particle cryo-electron microscopy.

sentences: Pak1 and PP2A antagonize aPKC function to support cortical tension induced by the Crumbs-Yurt complex The Drosophila polarity protein Crumbs is essential for the establishment and growth of the apical domain in epithelial cells. The protein Yurt limits the ability of Crumbs to promote apical membrane growth, thereby defining proper apical/lateral membrane ratio that is crucial for forming and maintaining complex epithelial structures such as tubes or acini. Here, we show that Yurt also increases Myosin-dependent cortical tension downstream of Crumbs. Yurt overexpression thus induces apical constriction in epithelial cells. The kinase aPKC phosphorylates Yurt, thereby dislodging the latter from the apical domain and releasing apical tension. In contrast, the kinase Pak1 promotes Yurt dephosphorylation through activation of the phosphatase PP2A. The Pak1- PP2A module thus opposes aPKC function and supports Yurt-induced apical constriction. Hence, the complex interplay between Yurt, aPKC, Pak1 and PP2A contributes to the functional plasticity of Crumbs. Overall, our data increase our understanding of how proteins sustaining epithelial cell polarization and Myosin-dependent cell contractility interact with one another to control epithelial tissue architecture.

sentences: Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an -helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin {beta} subunit. Moreover, a PN2-3 N-terminal stretch lies in a {beta}-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecule inhibitors of microtubule assembly.

sentences: Maintenance of community function through compensation breaks down over time in a desert rodent community Understanding the ecological processes that maintain community function in systems experiencing species loss, and how these processes change over time, is key to understanding the relationship between community structure and function and predicting how communities may respond to perturbations in the Anthropocene. Using a 30-year experiment on desert rodents, we show that the impact of species loss on community-level energy use has changed repeatedly and dramatically over time, due to 1) the addition of new species to the community, and 2) a reduction in functional redundancy among the same set of species. Although strong compensation, initially driven by the dispersal of functionally redundant species to the local community, occurred in this system from 1997-2010, since 2010, compensation has broken down due to decreasing functional overlap within the same set of species. Simultaneously, long-term changes in sitewide community composition due to niche complementarity have decoupled the dynamics of compensation from the overall impact of species loss on community-level energy use. Shifting, context-dependent compensatory dynamics, such as those demonstrated here, highlight the importance of explicitly long-term, metacommunity, and eco-evolutionary perspectives on the link between species-level fluctuations and community function in a changing world.

sentences: The VersaLive platform enables pipette-compatible microfluidic mammalian cell culture for versatile applications Microfluidic-based cell culture allows for precise spatio-temporal regulation of microenvironment, live cell imaging and better recapitulation of physiological conditions, while minimizing reagents consumption. Despite their usefulness, most microfluidic systems are designed with one specific application in mind and require specialized equipment and expertise for their operation. All these requirements prevent microfluidic-based cell culture to be widely adopted. Here, we designed and implemented a versatile and easy-to-use perfusion cell culture microfluidic platform for multiple application (VersaLive) requiring only standard pipettes. Here, we showcase the multiple uses of VersaLive (e.g., time-lapse live cell imaging, immunostaining, cell recovery, cell lysis) on mammalian cell lines and primary cells. VersaLive can replace standard cell culture formats in several applications, thus decreasing costs and increasing reproducibility across laboratories. The layout, documentation and protocols are open-source and available online at https://versalive.tigem.it/.

sentences: Chromatin accessibility determines cell-type specific intron retention Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including cancer. Despite consistent reports demonstrating intrinsic sequence features that predispose introns to become retained, conflicting findings about cell type-specific IR regulation demand a systematic analysis in a controlled experimental setting. We integrated matched transcriptomics and epigenetics data (including DNA methylation, nucleosome occupancy, histone modifications) from primary human myeloid and lymphoid cells. Using machine learning we trained two complementary models to determine the role of epigenetic factors in the regulation of IR. We show that increased chromatin accessibility contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. With mounting reports linking pathogenic alterations to RNA processing, our findings may have profound implications for the design of therapeutic approaches targeting aberrant splicing.

sentences: The MIF SNP rs755622 is a germline determinant of tumor immune activation in Glioblastoma While immunotherapies have shown durable responses for multiple tumors, their efficacy remains limited in some advanced cancers, including glioblastoma. This may be due to differences in the immune landscape, as the glioblastoma microenvironment strongly favors immunosuppressive myeloid cells, which are linked to an elevation in immune-suppressive cytokines, including macrophage migration inhibitory factor (MIF). We now find that a single-nucleotide polymorphism (SNP) rs755622 in the MIF promoter associates with increased leukocyte infiltration in glioblastoma. Furthermore, we identified lactotransferrin expression as being associated with the rs755622 SNP, which could also be used as a biomarker for immune infiltrated tumors. These findings provide the first example in glioblastoma of a germline SNP that underlies differences in the immune microenvironment and identifies high lactotransferrin as a potential factor promoting immune activation.

sentences: Alzheimer's disease-associated TM2D genes regulate Notch signaling and neuronal function in Drosophila TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each species. Rare variants in TM2D3 are associated with Alzheimers disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. We knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the {gamma}-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD.

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Comparison of heat-inactivated and infectious SARS-CoV-2 across indoor surface materials shows comparable RT-qPCR viral signal intensity and persistence Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with COVID-19 and inform appropriate infection mitigation responses. Research groups have reported detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2 positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over seven days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle (Cq)) can be correlated to surface viral load using only one linear regression model per material category. The same experiment was performed with infectious viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods.

ImportanceEnvironmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made towards containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) [1], a large-scale environmental monitoring effort in elementary school and child care settings, has processed > 13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT - qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.

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CREB regulates the expression of Type 1 Inositol 1,4,5-trisphosphate receptors Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a central role in regulating intracellular calcium signals in response to a variety of internal/external cues. Dysregulation of IP3R signaling is the underlying cause for numerous pathological conditions. It is also well established that the activity of IP3Rs is governed by several post-translational modifications including phosphorylation by protein kinase A (PKA). However, the long-term effects of PKA activation on expression of IP3R sub-types, remains largely unexplored. In this report, we investigate the effect of chronic activation of PKA on expression of IP3R sub-types. We demonstrate that expression of IP3R1 is augmented upon prolonged activation of PKA or upon ectopic over-expression of CREB but does not alter IP3R2 and IP3R3 sub-type abundance. Conversely, inhibition of PKA or blocking endogenous CREB diminished IP3R1 expression. We also demonstrate that agonist-induced Ca2+-release mediated by IP3R1 is significantly attenuated upon blocking endogenous CREB. Moreover, CREB by regulating the expression of KRAS-induced actin-interacting protein (KRAP) ensures proper localization and licensing of IP3R1. Overall, we report a crucial role for CREB in governing both the expression and proper localization of IP3R1.

Summary statementWe report a critical role of CREB in regulating the expression and proper localization of IP3R1. Agonist-induced Ca2+ release and Ca2+ puffs generated by IP3R1 are diminished upon blocking the function of endogenous CREB.

sentences: The extracellular loops of Salmonella Typhimurium outer membrane protein A (OmpA) maintain the stability of Salmonella containing vacuole (SCV) in murine macrophages and protect the bacteria from autophagy-dependent lysosomal degradation After entering the host cells, Salmonella Typhimurium (STM) stays inside a modified membrane-bound compartment called Salmonella containing vacuole (SCV). The biogenesis and stability of SCV are crucial for the intracellular proliferation of Salmonella. Our research has provided a novel mechanistic view on the role of a bacterial porin OmpA in maintaining the stability of SCV. We found that the deletion of OmpA forces the bacteria to escape from the SCV during the immediate early stage of infection. In the absence of OmpA, the bacteria failed to retain the LAMP-1 and came into the host cells cytosol. Subsequently, the cytosolic population of STM{Delta} ompA activated the host autophagy machinery after colocalizing with syntaxin 17 and LC3B. The autophagosomes carrying STM{Delta} ompA were targeted to the lysosomes for degradation. Inhibition of autophagy pathway using bafilomycin A1 restored the intracellular proliferation of STM{Delta} ompA. We further showed that the four extracellular loops of OmpA played a crucial role in holding the LAMP-1 pool around the SCV. We have altered the extracellular loop sequences of Salmonella OmpA by site-directed mutagenesis and observed that the bacteria failed to maintain the LAMP-1 pool around the SCV, which finally resulted in their release into the cytosol of the host macrophages. Surprisingly, the cytosolic population of Salmonella having mutations in the extracellular loops of OmpA didnt activate the lysosomal degradation pathway like STM{Delta} ompA, which helped them to survive within the murine macrophages. In summary, our study revealed an OmpA dependent novel strategy utilized by Salmonella to combat host autophagy by promoting the stability of SCV.

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Cargo genes of Tn7-like transposons comprise an enormous diversity of defense systems, mobile genetic elements and antibiotic resistance genes Transposition is a major mechanism of horizontal gene mobility in prokaryotes. However, exploration of the genes mobilized by transposons (cargo) is hampered by the difficulty in delineating integrated transposons from their surrounding genetic context. Here, we present a computational approach that allowed us to identify the boundaries of 6,549 Tn7-like transposons at base pair resolution. We found that 96% of these transposons carry at least one cargo gene. Delineation of distinct communities in a gene-sharing network demonstrates how transposons function as a conduit of genes between phylogenetically distant hosts. Comparative analysis of the cargo genes reveals significant enrichment of mobile genetic elements (MGEs) nested within Tn7-like transposons, such as insertion sequences and toxin-antitoxin modules, genes involved in recombination and anti-MGE defense, and in antibiotic resistance. More unexpectedly, cargo also includes genes encoding central carbon metabolism enzymes. Twenty- two Tn7-like transposons carry both an anti-MGE defense system and antibiotic resistance genes, illustrating how bacteria can overcome these combined pressures upon acquisition of a single transposon. This work substantially expands the distribution of Tn7-like transposons, defines their evolutionary relationships and provides a large-scale functional classification of prokaryotic genes mobilized by transposition.

SignificanceTransposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries. We developed a computational approach for detecting transposon ends and applied it to perform a comprehensive census of the cargo genes of Tn7-like transposons, a large class of bacterial mobile genetic elements (MGE), many of which employ a unique, CRISPR-mediated mechanism of site- specific transposition. The cargo genes encompass a striking diversity of MGE, defense and antibiotic resistance systems. Unexpectedly, we also identified cargo genes encoding metabolic enzymes. Thus, Tn7-like transposons mobilize a vast repertoire of genes that can have multiple effects on the host bacteria.

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VirusTaxo: Taxonomic classification of virus genome using multi-class hierarchical classification by k-mer enrichment Taxonomic classification of viruses is a multi-class hierarchical classification problem, as taxonomic ranks (e.g., order, family and genus) of viruses are hierarchically structured and have multiple classes in each rank. Classification of biological sequences which are hierarchically structured with multiple classes is challenging. Here we developed a machine learning architecture, VirusTaxo, using a multi-class hierarchical classification by k-mer enrichment. VirusTaxo classifies DNA and RNA viruses to their taxonomic ranks using genome sequence. To assign taxonomic ranks, VirusTaxo extracts k-mers from genome sequence and creates bag-of-k-mers for each class in a rank. VirusTaxo uses a top-down hierarchical classification approach and accurately assigns the order, family and genus of a virus from the genome sequence. The average accuracies of VirusTaxo for DNA viruses are 99% (order), 98% (family) and 95% (genus) and for RNA viruses 97% (order), 96% (family) and 82% (genus). VirusTaxo can be used to detect taxonomy of novel viruses using full length genome or contig sequences.

AvailabilityOnline version of VirusTaxo is available at https://omics-lab.com/virustaxo/.

sentences: Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media Three web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of E. coli that is commonly used in molecular biology and biochemistry research, for example in the production of recombinant proteins. Metal availabilities have been examined for cells grown in LB medium aerobically, anaerobically and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by qPCR. The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterised in related bacterial cells enabling gene expression to be read-out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial which discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

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Modelling the impact of urban and hospital wastewaters eco-exposomes on the antibiotic-resistance dynamics. Antibiotic-resistance emergence and selection have become major public health issues globally. The presence of antibiotic resistant bacteria (ARB) in natural and anthroposophical environments threatens to compromise the sustainability of care in human and animal populations. This study was undertaken to develop a simple model formalizing the selective impact of antibiotics and pollutants on the dynamics of bacterial resistance in water and use the model to analyze longitudinal spatiotemporal data collected in hospital and urban wastewaters. Longitudinal-sampling data were collected between 2012 and 2015 in four different locations in Haute-Savoie, France: hospital and urban wastewaters, before and after water-treatment plants. Concentration in three different types of compounds: 1) heavy metals 2) antibiotics and 3) surfactants; and abundance of 88 individual genes and mobile genetic elements, mostly conferring resistance to antibiotics, were simultaneously collected. A simple hypothesis-driven model describing the weekly ARB dynamics was proposed to fit available data by assuming normalized gene abundance to be proportional to ARB populations in water. Compounds impacts on the dynamics of 17 genes found in multiple sites were estimated. We found that while mercury and vancomycin had relevant effects on ARB dynamics, respectively positively affecting the dynamics of 10 and 12 identified genes, surfactants antagonistically affected genes dynamics (identified for three genes). This simple model enables analyzing the relationship between resistance-gene persistence in aquatic environments and specific compounds inherent to human activities. Applying our model to longitudinal data, we identified compounds that act as co-selectors for antibiotic resistance.

HighlightsO_LIWe analyzed longitudinal wastewater resistance genes and environmental data
C_LIO_LIWe developed a simple hypothesis-driven model to assess resistance selection
C_LIO_LIMercury and vancomycin were key drivers of antibiotic resistance in wastewater
C_LI

sentences: Neuroimaging signature associated with symptom exacerbation in early-stage psychosis Recent reports have indicated that the occurrence of symptom exacerbation in early-stage psychosis could result in brain changes, which are likely to underlie the poorer disease outcome. Thus, it is important to identify neuroimaging signature associated with symptom exacerbation in early-stage psychosis. We studied 85 patients with psychosis within two years after onset and 94 healthy controls (HC). The patient group was subdivided into two groups: 54 patients who did not experience major symptom exacerbation between the onset and study enrollment (P1), and 31 patients who experienced major symptom exacerbation (P2). We analyzed three brain imaging measures derived from resting-state functional MRI, such as global efficiency, nodal efficiency, and resting-state functional connectivity (rs-FC). After excluding some brain imaging measures that were potentially affected by clinical variables, we conducted a comparison between overall patient group and HC group as well as comparsions between HC, P1, and P2 groups for these three types of brain imaging measures, respectively. By integrating the information, we pinned down the dorsal anterior cingulate cortex and thalamus as key hubs in the context of several large-scale brain networks associated with symptom exacerbations in early-stage psychosis. Our study implies the importance of considering neural mechanism associated with symptom exacerbations in early stages of psychotic disorders.

sentences: Visualizing sequential compound fusion and kiss-and-run in live excitable cells Vesicle fusion is assumed to occur at flat membrane of excitable cells. In live neuroendocrine cells, we visualized vesicle fusion at {Omega}-shape membrane generated by preceding fusion, termed sequential compound fusion, which may be followed by fusion pore closure, termed compound kiss-and-run. These novel fusion modes contribute to vesicle docking, multi-vesicular release, asynchronous release, and endocytosis. We suggest modifying current models of exo-endocytosis to include these new fusion modes.

sentences: LAVA: An integrated framework for local genetic correlation analysis Genetic correlation (rg) analysis is commonly used to identify traits that may have a shared genetic basis. Traditionally, rg is studied on a global scale, considering only the average of the shared signal across the genome; though this approach may fail to detect scenarios where the rg is confined to particular genomic regions, or show opposing directions at different loci. Tools dedicated to local rg analysis have started to emerge, but are currently restricted to analysis of two phenotypes. For this reason, we have developed LAVA, an integrated framework for local rg analysis which, in addition to testing the standard bivariate local rgs between two traits, can evaluate the local heritability for all traits of interest, and analyse conditional genetic relations between several traits using partial correlation or multiple regression. Applied to 20 behavioural and health phenotypes, we show considerable heterogeneity in the bivariate local rgs across the genome, which is often masked by the global rg patterns, and demonstrate how our conditional approaches can elucidate more complex, multivariate genetic relations between traits.

sentences: Genomic analyses of wild and cultivated bacanora agave (Agave angustifolia var. pacifica) reveal inbreeding, few signs of cultivation history and shallow population structure Due to the recent increase in demand for agave-based beverages, many wild agave populations have experienced rapid decline and fragmentation; whereas cultivated plants are now managed at monocultural plantations, in some cases involving clonal propagation. We examined the relative effect of migration, genetic drift, natural selection and human activities on the genetic repertoire of Agave angustifolia var. pacifica, an agave used for bacanora (an alcoholic spirit similar to tequila) production in northwestern Mexico. We sampled 34 wild and cultivated sites and used over eleven thousand genome-wide SNPs. We found shallow genetic structure among wild samples, although, detected differentiation between coastal and inland sites. Surprisingly, no differentiation was found between cultivated and wild populations. Moreover, we detected moderate inbreeding (FIS [~] 0.13) and similar levels of genomic diversity in wild and cultivated agaves. Nevertheless, the cultivated plants had almost no private alleles and presented evidence of clonality. The overall low genetic structure in A. angustifolia var. pacifica is apparently the result of high dispersibility promoted by pollinators and possibility of clonal reproduction. Incipient cultivation history and reliance on wild seeds and plants are probably responsible for the observed patterns of high genetic connectivity and considerable diversity in cultivated samples.

sentences: Categorical working memory codes in human visual cortex Working memory contents are represented in neural activity patterns across multiple regions of the cortical hierarchy. It has remained unclear to which degree this reflects a specialization for different levels of abstraction. Here, we demonstrate that for color stimuli categorical codes are already present at the level of extrastriate visual cortex (V4 and VO1). Interestingly, this categorical coding was observed during working memory, but not during perception.

sentences: An Omics Analysis Search and Information System (OASIS) for Enabling Biological Discovery in the Old Order Amish The "Omics Analysis Search and Information System" (OASIS), developed at the University of Maryland School of Medicine, enables discovery by allowing researchers to mine results from genome wide association studies (GWAS). When interesting signals are found, the research can immediately ask follow-up questions and get answers in real-time. OASIS provides this unique capability with a web-based, scientist-friendly search system and a variety of real-time analysis tools (linkage disequilibrium calculations, conditional analysis, and direct variant comparison) plus on-demand visualizations (boxplots, histograms, LocusZoom & Haploview plots, and pedigree charts). Because OASIS uses a web-based user interface, an understanding of programming or the UNIX operating system is not required. The OASIS application has been used to enable discovery from whole-exome, whole-genome, metabolome, transcriptome and methylome association results for Old Order Amish studies at the University of Maryland School of Medicine.

sentences: Agrochemical control of gene expression using evolved split RNA polymerase Chemical inducible gene expression systems are valuable tools for rational control of gene expression both for basic research and biotechnology. However, most chemical inducers are confined to certain groups of organisms. Therefore, dissecting interaction between different organisms could be challenging using existing chemical inducible systems. We engineered a mandipropamid induced gene expression system (Mandi-T7) based on evolved split T7 RNAP system. As a proof-of-principle, we induced GFP expression in E. coli cells grown inside plant tissue.

sentences: Description and comparison of PIMS-TS innate cell signature and immunophenotype with a cohort of healthy children, severe viral and bacterial infections and Kawasaki Disease. A new clinical syndrome associated to SARS-CoV-2 has been described in children. It has been named as Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). This new disease is a main cause of hospital and pediatric intensive care unit (PICU). In this work we describe the innate cell signature and immunophenotype of children admitted to PICU because of PIMS-TS. Also, we compare it with healthy controls and children admitted to PICU because bacterial infection, viral infection and Kawasaki disease. We made a prospective-retrospective observational study in a tertiary pediatric hospital. Children admitted to PICU because of PIMS-TS from March 2020 to September 2020 were consecutively included. They were compare with previous cohorts from our center. A total of 247 children were included: 183 healthy controls, 25 viral infections, 20 bacterial infections, 6 Kawasaki disease and 13 PIMS-TS. PIMT-TS showed the lowest percentage of lymphocytes and monocytes with higher relative numbers of CD4+ (p =0,000). At the same time, we describe a differential expression of CD64, CD11a and CD11b. Monocytes and neutrophils in PIMS-TS showed higher levels of CD64 expression compared to all groups (p = 0,000). Also, proteins involved in leukocyte tissue migration, like CD11a and CD11b were highly expressed compare to other severe viral or bacterial infections (p = 0,000). In PIMS-TS this increased CD11a expression could be a sign of the activation and trafficking of these leukocytes. These findings are congruent with an inflammatory process and the trend of these cells to leave the bloodstream. In conclusion, we compare for the first time the innate cellular response of children with PIMS-TS with other severe forms of viral or bacterial infection and Kawasaki disease. Our findings define a differential cell innate signature. These data should be further studied and may facilitate the diagnosis and management of these patients.

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Injury-Free In Vivo Delivery and Engraftment into the Cornea Endothelium Using Extracellular Matrix Shrink-Wrapped Cells Cell injection is a common clinical approach for therapeutic delivery of cells into diseased and damaged tissues in order to achieve regeneration. However, cell retention, viability, and engraftment at the injection site has generally been poor, driving the need for improved approaches. Here, we developed a technique to shrink-wrap micropatterned islands of corneal endothelial cells in a basement membrane-like layer of extracellular matrix (ECM) that enables the cells to maintain their cell-cell junctions and cytoskeletal structure while in suspension. These Monolayers exhibited the ability to rapidly engraft into intact, high-density corneal endothelial monolayers in both in vitro and in vivo model systems. Importantly, the engrafted Monolayers increased local cell density, something that the clinical-standard single cells in suspension failed to do. These results show that shrink-wrapping cells in ECM dramatically improves engraftment and provides a potential alternative to cornea transplant when low endothelial cell density is the cause of corneal blindness.

One Sentence SummaryShrink-wrapped patches of endothelial cells can rapidly attach and integrate into an intact cornea endothelium when injected into the anterior chamber, increasing cell density.

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Detecting ice artefacts in processed macromolecular diffraction data with machine learning Contamination with diffraction from ice crystals can negatively affect, or even impede macromolecular structure determination and therefore, detecting the resulting artefacts in diffraction data is crucial. However, once the data have been processed, it can be very difficult to automatically recognize this problem. To address this, a set of convolutional neural networks named Helcaraxe has been developed which can detect ice diffraction artefacts in processed diffraction data from macromolecular crystals. The networks outperform previous algorithms and will be available as part of the AUSPEX webserver and CCP4-distributed software.

SynopsisA program utilizing artificial learning and convolutional neural networks, named Helcaraxe, has been developed which can detect ice crystal artefacts in processed macromolecular diffraction data with unprecedented accuracy.

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Rhinovirus reduces the severity of subsequent respiratory viral infections by interferon-dependent and -independent mechanisms Coinfection by unrelated viruses in the respiratory tract is common and can result in changes in disease severity compared to infection by individual virus strains. We have previously shown that inoculation of mice with rhinovirus (RV) two days prior to inoculation with a lethal dose of influenza A virus (PR8), provides complete protection against mortality and reduces morbidity. In this study, we extended that finding to a second lethal respiratory virus, pneumonia virus of mice (PVM) and analyzed potential mechanisms whereby RV reduces lethal viral pneumonia caused by PR8 and PVM. RV prevented mortality and weight loss associated with PVM infection, suggesting that RV-mediated protection is more effective against PVM than PR8. Major changes in host gene expression upon PVM infection were delayed compared to PR8, which likely provides a larger time frame for RV-induced gene expression to alter the course of disease. Overall, RV induced earlier recruitment of inflammatory cells, while these populations were reduced at later times in RV-inoculated mice. Findings common to both virus pairs included upregulated expression of mucin-associated genes and dampening of inflammation-related genes in mice that were inoculated with RV prior to lethal virus infection. However, type I IFN signaling was required for RV-mediated protection against PR8, but not PVM. IFN signaling had minor effects on PR8 replication and contributed to controlling neutrophilic inflammation and subsequent hemorrhagic lung pathology in RV/PR8 infected mice. These findings, combined with differences in virus replication levels and disease severity, suggest that the suppression of inflammation in RV/PVM infected mice may be due to early, IFN-independent suppression of viral replication, while in RV/PR8 infected mice may be due to IFN-dependent modulation of immune responses. Thus, a mild upper respiratory viral infection can reduce the severity of a subsequent severe viral infection in the lungs through virus-dependent mechanisms.

Author SummaryRespiratory viruses from diverse families co-circulate in human populations and are frequently detected within the same host. Though clinical studies suggest that infection by more than one unrelated respiratory virus may alter disease severity, animal models in which we can control the doses, timing, and strains of coinfecting viruses are critical to understand how coinfection affects disease severity. In this study, we compared gene expression and immune cell recruitment between two pairs of viruses (RV/PR8 and RV/PVM) inoculated sequentially in mice that both result in reduced severity compared to lethal infection by PR8 or PVM alone. Reduced disease severity was associated with suppression of inflammatory responses in the lungs. However, differences in disease kinetics and host and viral gene expression suggest that protection by coinfection with RV may be due to distinct molecular mechanisms. Indeed, we found that antiviral cytokine signaling was required for RV-mediated protection against lethal infection by PR8, but not PVM.

sentences: Robustness of organ morphology is associated with modules of co-expressed genes related to plant cell wall Reproducibility in organ size and shape is a fundamental trait of living organisms. The mechanisms underlying such robustness remain, however, to be elucidated. Taking the sepal of Arabidopsis as a model, we investigated whether variability of gene expression plays a role in variation of organ morphology. To address this question, we produced a dataset composed of both transcriptomic and morphological information obtained from 27 individual sepals from wild-type plants. Although nearly identical in their genetic background, environment, and developmental stage, these sepals exhibited appreciable variability in both morphology and transcriptome. We identified modules of co-expressed genes in sepals, three of which correlated significantly with morphology, revealing biologically relevant gene regulatory networks. Interestingly, cell-wall related genes were overrepresented in two of these three modules. Additionally, we found that highly variable genes were unexpectedly enriched in cell-wall related processes. We then analyzed sepal morphology from 16 cell-wall mutants and found that the more a gene is expressed in wild-type, the more variable the morphology of the corresponding mutant. Altogether, our work unravels the contribution of cell-wall related genes to the robustness of sepal morphology. More generally, we propose that canalizing traits during development could rely on the modulation of highly expressed genes.

sentences: Epstein-Barr virus infection promotes Th1-mediated disease in a humanized immune mouse model of multiple sclerosis Infection with the human-tropic Epstein-Barr virus (EBV) is a strong risk factor for multiple sclerosis (MS), though the underlying mechanisms remain unclear. To investigate EBV infection directly, we induced experimental autoimmune encephalomyelitis (EAE) in immunocompromised mice humanized with peripheral blood mononuclear cells (PBMCs) from individuals with or without a history of EBV infection and/or diagnosis of relapsing MS. HuPBMC EAE mice generated from EBV seronegative healthy donors (HD) were less susceptible to developing severe clinical symptoms than EBV seropositive cohorts. Donor EBV seropositivity and RRMS diagnosis led to a significant incremental increase in the human Th1:Treg CD4+ T cell ratio in the brain and spinal cord, as well as increased human cytotoxic CD8+ T cell and murine macrophage infiltration and demyelination. The data indicate that a history of EBV infection, further compounded by a diagnosis of RRMS, promotes Th1-mediated disease in a novel humanized mouse model of MS.

sentences: Direct tissue sensing reprograms TLR4+ Tfh-like cells inflammatory profile in the joints of rheumatoid arthritis patients CD4+ T cells mediate rheumatoid arthritis (RA) pathogenesis through both antibody-dependent and independent mechanisms. It remains unclear how synovial microenvironment impinges on CD4+ T cells pathogenic functions. Here, we identified a TLR4+ follicular helper T (Tfh) cell-like population present in the blood and expanded in synovial fluid. Mechanistically, we unveiled that homotypic T-T cell interactions through non-cognate HLA-DR:TCR contacts regulate TLR4 expression on T cells. TLR4+ T cells possess a two-pronged pathogenic activity. Upon TCR and ICOS engagement, TLR4+ T cells produce IL-21, a cytokine known to sponsor antibody production. However, direct TLR4+ engagement on T cells, by endogenous ligands in the arthritic joint, reprograms them towards an IL-17 inflammatory profile compatible with tissue damage program. Blocking TLR4 signaling with a specific inhibitor impaired IL-17 production in response to synovial fluid recognition. Ex vivo, synovial fluid TLR4+ T cells produced IL-17, but not IL-21. TLR4+ T cells appear to uniquely reconcile an ability to promote systemic antibody production with a local synovial driven tissue damage program. TLR4+ T cells could constitute an attractive cellular target and predictive biomarker for erosive arthritis.

sentences: The Temporal Dynamics of Willed Attention in Vision Most models of attention distinguish between voluntary and involuntary attention, the latter being driven in a bottom-up fashion by salient sensory signals. Studies of voluntary visual-spatial attention have used informational or instructional cues, such as arrows, to induce or instruct observers to direct selective attention to relevant locations in visual space in order to detect or discriminate subsequent target stimuli. In everyday vision, however, voluntary attention is influenced by a host of factors, most of which are quite different from the laboratory paradigms that utilize attention-directing cues. These factors include priming, experience, reward, meaning, motivations, and high-level behavioral goals. Attention that is endogenously directed in the absence of external cues has been referred to as self-initiated attention, or in our prior work as "willed attention". Such studies typically replace attention-directing cues with a "prompt" that signals the subject when to choose where they will attend in preparation for the upcoming target stimulus. We used a novel paradigm that was designed to minimize external influences (i.e., cues or prompts) as to where, as well as when, spatial attention would be shifted and focused. Participants were asked to view bilateral dynamic dot motion displays, and to shift their covert spatial attention to either the left or right visual field patch at a time of their own choosing, thus allowing the participants to control both when and where they attended on each trial. The task was to discriminate and respond to a pattern in the attended dot motion patch. Our goal was to identify patterns of neural activity in the scalp-recorded EEG that revealed when and where attention was focused. Using machine learning methods to decode attention-related EEG alpha band activity, we were able to identify the onset of voluntary (willed) shifts of visual-spatial attention, and to determine where attention was focused. This work contributes to our understanding of the neural antecedents of voluntary attention, opening the door for improved models of attentional control, and providing steps toward development of brain-computer interfaces using non-invasive electrical recordings of brain activity.

sentences: YAP/TAZ Maintain the Proliferative Capacity and Structural Organization of Radial Glial Cells During Brain Development The Hippo pathway regulates the development and homeostasis of many tissues and in many species. It controls the activity of two paralogous transcriptional coactivators, YAP and TAZ (YAP/TAZ). Although previous studies have established that aberrant YAP/TAZ activation is detrimental to mammalian brain development, whether and how endogenous levels of YAP/TAZ activity regulate brain development remain unclear. Here, we show that during mammalian cortical development, YAP/TAZ are specifically expressed in apical neural progenitor cells known as radial glial cells (RGCs). The subcellular localization of YAP/TAZ undergoes dynamic changes as corticogenesis proceeds. YAP/TAZ are required for maintaining the proliferative potential and structural organization of RGCs, and their ablation during cortical development reduces the numbers of cortical projection neurons and causes the loss of ependymal cells, resulting in hydrocephaly. Transcriptomic analysis using sorted RGCs reveals gene expression changes in YAP/TAZ-depleted cells that correlate with mutant phenotypes. Thus, our study has uncovered essential functions of YAP/TAZ during mammalian brain development and revealed the transcriptional mechanism of their action.

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The Alternative Sigma Factor SigL Influences Clostridioides difficile Toxin Production, Sporulation, and Cell Surface Properties The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages - the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain, but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficiles virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.

Contribution to the FieldAlternative sigma factors modulate bacterial gene expression in response to environmental and metabolic cues. C. difficile encodes multiple alternative sigma factors which mediate critical cellular processes. A Sigma-54 ortholog, SigL, is conserved across all sequenced strains, yet its function remains to be elucidated. We demonstrate that SigL dynamically influences C. difficile biology in fundamental, but distinct, ways in two strains representing two clinically-relevant C. difficile phylogenetic lineages (ribotypes). Loss of SigL results in changes in sporulation kinetics, biofilm formation, alterations in cell surface properties, and secreted toxin levels in a strain-specific manner. SigL overexpression, however, uniformly impedes sporulation. Thus, SigL plays a fundamental role in regulating the expression of C. difficile virulence factors and likely profoundly impacts pathogenesis in a strain-specific manner.

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Optimizing Oscillators for Specific Tasks Predicts Preferred Biochemical Implementations Oscillatory processes are used throughout cell biology to control time-varying physiology including the cell cycle, circadian rhythms, and developmental patterning. It has long been understood that free-running oscillations require feedback loops where the activity of one component depends on the concentration of another. Oscillator motifs have been classified by the positive or negative net logic of these loops. However, each feedback loop can be implemented by regulation of either the production step or the removal step. These possibilities are not equivalent because of the underlying structure of biochemical kinetics. By computationally searching over these possibilities, we find that certain molecular implementations are much more likely to produce stable oscillations. These preferred molecular implementations are found in many natural systems, but not typically in artificial oscillators, suggesting a design principle for future synthetic biology. Finally, we develop an approach to oscillator function across different reaction networks by evaluating the biosynthetic cost needed to achieve a given phase coherence. This analysis predicts that phase drift is most efficiently suppressed by delayed negative feedback loop architectures that operate without positive feedback.

PACS numbers47.15.-x

sentences: Size Matters: A Mechanistic Model of Nanoparticle Curvature Effects on Amyloid Fibril Formation The aggregation of peptides into amyloid fibrils is linked to ageing-related diseases, such as Alzheimers disease and type 2 diabetes. Interfaces, particularly those with large nanostructured surface areas, can affect the kinetics of peptide aggregation, ranging from a complete inhibition to strong acceleration. While a number of physiochemical parameters determine interface effects, we here focus on the role of nanoparticle curvature for the aggregation of the amyloidogenic peptides A{beta}40, NNFGAIL, GNNQQNY and VQIYVK. Nanoparticles (NPs) provided a surface for peptide monomers to adsorb, enabling the nucleation into oligomers and fibril formation. High surface curvature, however, destabilized prefibrillar structures, providing an explanation for inhibitory effects on fibril growth. Thioflavin T (ThT) fluorescence assays as well as dynamic light scattering (DLS), atomic force microscopy (AFM) and electron microscopy experiments revealed NP size-dependent effects on amyloid fibril formation, with differences between the peptides. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of most peptides, larger 20 nm gold NPs (AuNP-20) tended to accelerate peptide aggregation. Molecular dynamics (MD) studies demonstrated that NPs ability to catalyze or inhibit oligomer formation was influenced by the oligomer stability at curved interfaces which was lower at more highly curved surfaces. Differences in the NP effects for the peptides resulted from the peptide properties (size, aggregation propensity) and concomitant surface binding affinities. The results can be applied to the design of future nanostructured materials for defined applications.

sentences: HYPK scaffolds the Nedd8 and LC3 proteins to initiate formation of autophagosome around polyneddylated huntingtin exon1 aggregates Selective degradation of protein aggregates by autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like modifier proteins, Nedd8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3 interacting region which bind to Nedd8 and LC3 respectively. Both Nedd8 and HYPK are positive modulators of basal and induced-autophagy, leading to desensitizing cells from protein aggregates, such as aggregates of mutant huntingtin-exon1. Thus, we propose an additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress.

sentences: Wide Diversity of Recombinant Noroviruses Circulating in Spain, 2016 to 2020 Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years most norovirus infections have been caused by the pandemic genotype GII.4, yet recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia (Spain) from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus with a positive rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup GII and 49 to genogroup GI. Using dual genotyping based on sequencing the ORF1/ORF2 junction region we detected 25 different capsid-polymerase type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6 and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 Sydney[P16] was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harboring within six different capsid genes. A new subcluster of GII.4 Sydney associated with the P31 polymerase was identified by phylogenetic analysis. GI.4 and GI.3 were the predominant genotypes in genogroup GI, in which recombinant strains were also found, such as GI.3[P10], GI.3[P13] and GI.5[P4]. Interestingly, the GI.3[P10] strain could represent a new capsid genotype. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses.

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C2cd6-encoded CatSper{tau} Targets Sperm Calcium Channel to Ca2+ Signaling Domains in the Flagellar Membrane In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSper{tau}, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSper{tau} loss-of-function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSper{tau} targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSper{tau} interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting.

HighlightsO_LICatSper{tau} encoded by C2cd6 is a C2 membrane-associating domain containing protein
C_LIO_LICatSper{tau} loss-of-function impairs sperm hyperactivation and male fertility
C_LIO_LICatSper{tau} adopts ciliary trafficking machineries for flagellar targeting via C2 domain
C_LIO_LICatSper{tau} targets the CatSper channel into nanodomains of developing sperm flagella
C_LI

sentences: Enhanced food motivation in obese mice is controlled by D1R expressing spiny projection neurons in the nucleus accumbens. Obesity is a chronic relapsing disorder that is caused by an excess of caloric intake relative to energy expenditure. In addition to homeostatic feeding mechanisms, there is growing recognition of the involvement of food reward and motivation in the development of obesity. However, it remains unclear how brain circuits that control food reward and motivation are altered in obese animals. Here, we tested the hypothesis that signaling through pro-motivational circuits in the core of the nucleus accumbens (NAc) is enhanced in the obese state, leading to invigoration of food seeking. Using a novel behavioral assay that quantifies physical work during food seeking, we confirmed that obese mice work harder than lean mice to obtain food, consistent with an increase in the relative reinforcing value of food in the obese state. To explain this behavioral finding, we recorded neural activity in the NAc core with both in vivo electrophysiology and cell-type specific calcium fiber photometry. Here we observed greater activation of D1-receptor expressing NAc spiny projection neurons (NAc D1SPNs) during food seeking in obese mice relative to lean mice. With ex vivo slice physiology we identified both pre- and post-synaptic mechanisms that contribute to this enhancement in NAc D1SPN activity in obese mice. Finally, blocking synaptic transmission from D1SPNs decreased physical work during food seeking and attenuated high-fat diet-induced weight gain. These experiments demonstrate that obesity is associated with a selective increase in the activity of D1SPNs during food seeking, which enhances the vigor of food seeking. This work also establishes the necessity of D1SPNs in the development of diet-induced obesity, establishing these neurons as a potential therapeutic target for preventing obesity.

sentences: Testing for heterogeneous rates of discrete character evolution on phylogenies Many hypotheses in the field of phylogenetic comparative biology involve specific changes in the rate or process of trait evolution. We present a method designed to test whether the rate of evolution of a discrete character has changed in one or more clades, lineages, or time periods. This method differs from other related approaches (such as the  covarion model) in that the  regimes in which the rate or process is postulated to have changed are specified a priori by the user, rather than inferred from the data. Similarly, it differs from methods designed to model a correlation between two binary traits in that the regimes mapped onto the tree are fixed. We apply our method to investigate the rate of dewlap color and/or caudal vertebra number evolution in Caribbean and mainland clades of the diverse lizard genus Anolis. We find little evidence to support any difference between mainland and island evolution in either character. We also examine the statistical properties of the method more generally and show that it has acceptable type I error, parameter estimation, and power. Finally, we discuss the relationship of our method to existing models of heterogeneity in the rate of discrete character evolution on phylogenies.

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Detection and characterization of replication origins defined by DNA polymerase epsilon BackgroundAlthough the process of DNA replication is highly conserved the location of origins of replication (ORI) may vary from one tissue to the next or one round of replication to the next in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location and there are no methods available to compare them.

ResultsHere we report the genome-wide localization of ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high-fidelity between individuals and tumor types. Using a Smith-Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods.

ConclusionsMany DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

sentences: Cortico-subcortical β burst dynamics underlying movement cancellation in humans Dominant neuroanatomical models hold that humans regulate their movements via loop-like cortico-subcortical networks, including the subthalamic nucleus (STN), thalamus, and sensorimotor cortices (SMC). Inhibitory commands across these networks are purportedly sent via transient, burst-like signals in the {beta} frequency (15-29Hz). However, since human depth-recording studies are typically limited to one recording site, direct evidence for this proposition is hitherto lacking. Here, we present simultaneous multi-site depth-recordings from SMC and either STN or thalamus in humans performing the stop-signal task. In line with their purported function as inhibitory signals, subcortical {beta}-bursts were increased on successful stop-trials and were followed within 50ms by increased {beta}-bursting over SMC. Moreover, between-site comparisons (including in a patient with simultaneous recordings from all three sites) confirmed that {beta}-bursts in STN precede thalamic {beta}-bursts. This provides first empirical evidence for the role of {beta}-bursts in conveying inhibitory commands along long-proposed cortico-subcortical networks underlying movement regulation in humans.

sentences: Language processing in brains and deep neural networks: computational convergence and its limits AO_SCPLOWBSTRACTC_SCPLOWDeep Learning has recently led to major advances in natural language processing. Do these models process sentences similarly to humans, and is this similarity driven by specific principles? Using a variety of artificial neural networks, trained on image classification, word embedding, or language modeling, we evaluate whether their architectural and functional properties lead them to generate activations linearly comparable to those of 102 human brains measured with functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG). We show that image, word and contextualized word embeddings separate the hierarchical levels of language processing in the brain. Critically, we compare 3,600 embeddings in their ability to linearly map onto these brain responses. The results show that (1) the position of the layer in the network and (2) the ability of the network to accurately predict words from context are the main factors responsible for the emergence of brain-like representations in artificial neural networks. Together, these results show how perceptual, lexical and compositional representations precisely unfold within each cortical region and contribute to uncovering the governing principles of language processing in brains and algorithms.

sentences: AGES: An auxin-inducible, GAL4-compatible, gene expression system for Drosophila The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here we describe the Auxin-inducible Gene Expression System (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.

sentences: Condensin DC spreads linearly and bidirectionally from recruitment sites to create loop-anchored TADs in C. elegans Condensins are molecular motors that compact DNA for chromosome segregation and gene regulation. In vitro experiments have begun to elucidate the mechanics of condensin function but how condensin loading and translocation along DNA controls eukaryotic chromosome structure in vivo remains poorly understood. To address this question, we took advantage of a specialized condensin, which organizes the 3D conformation of X chromosomes to mediate dosage compensation (DC) in C. elegans. Condensin DC is recruited and spreads from a small number of recruitment elements on the X chromosome (rex). We found that ectopic insertion of rex sites on an autosome leads to bidirectional spreading of the complex over hundreds of kilobases. On the X chromosome, strong rex sites contain multiple copies of a 12-bp sequence motif and act as TAD borders. Inserting a strong rex and ectopically recruiting the complex on the X chromosome or an autosome creates a loop-anchored TAD. Unlike the CTCF system, which controls TAD formation by cohesin, direction of the 12-bp motif does not control the specificity of loops. In an X;V fusion chromosome, condensin DC linearly spreads into V and increases 3D DNA contacts, but fails to form TADs in the absence of rex sites. Finally, we provide in vivo evidence for the loop extrusion hypothesis by targeting multiple dCas9-Suntag complexes to an X chromosome repeat region. Consistent with linear translocation along DNA, condensin DC accumulates at the block site. Together, our results support a model whereby strong rex sites act as insulation elements through recruitment and bidirectional spreading of condensin DC molecules and form loop-anchored TADs.

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Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen The discovery and characterization of tumor associated antigens is increasingly important to advance the field of immuno-oncology. Data on the, 1) topology, 2) amino acid (a.a.) homology analyses and 3) cell surface localization are presented as evidence in support of labyrinthin as a pan-adenocarcinoma marker. Multiple bioinformatics tools were used to collectively predict calcium binding domain(s), N-myristoylation sites, kinase II phosphorylation sites, and transmembrane domain(s) with predicted N- and C-termini orientations. Sequence homology comparisons were found and alignments made for labyrinthin (255 a.a.) vs. the intracellular aspartyl/asparaginyl beta-hydroxylase (ASPH; 758 a.a.) and the ASPH-gene related protein junctate (299 a.a.), which are both type II proteins. The epitopes for commonly used MCA 44-3A6 anti-labyrinthin and FB-50 anti-ASPH/junctate antibodies were present in all three proteins. The selective expression of labyrinthin was investigated by third party fluorescent activated cell sorter (FACS) analyses of MCA 44-3A6 binding to non-permeabilized adenocarcinoma and normal cell line models. Labyrinthin was detected on non-permeabilized A549 human lung adenocarcinoma cells, but not on normal WI-38 human lung fibroblasts nor primary cultures of normal human glandular-related cells. Microscopic images of immunofluorescent labelled MCA 44-3A6 binding to A549 cells at random cell cycle stages complement the FACS results by further showing that labyrinthin persisted on the cell surfaces along with some cell internalization within 20 minutes. The dataset presented herein is related to research article: "Labyrinthin: A distinct pan-adenocarcinoma diagnostic and immunotherapeutic tumor specific antigen" by Babich et al., Heliyon, 2022 [1].

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@19222bborg.highwire.dtl.DTLVardef@1264bcdorg.highwire.dtl.DTLVardef@13a8158org.highwire.dtl.DTLVardef@1dcf0org.highwire.dtl.DTLVardef@503a9f_HPS_FORMAT_FIGEXP  M_TBL C_TBL Value of the data O_LIBecause identification and characterization of neoantigens [3, 4, 5] are rare yet increasingly important for diagnostics and therapeutics, the identification of labyrinthin as a pan-adenocarcinoma target is significant for further study by clinicians and cancer researchers.
C_LIO_LIThe data provide a basis to determine to what extent labyrinthin is a distinguishing marker for adenocarcinomas vs. conventional methods.
C_LIO_LIThe results indicate that therapeutic strategies can be employed that take advantage of labyrinthins convenient presence on adenocarcinoma cell surfaces.
C_LIO_LIThe discovery of a calcium binding sequence in labyrinthin provides a basis to research if it is key to explaining the long-known phenomena of elevated calcium and irregular signaling in cancer cells [8, 9].
C_LIO_LIThe data reveal a need to explore distinctions between ASPH and labyrinthin, in particular because ASPH [10, 11] and labyrinthin-based treatments have already been FDA approved for clinical trials.
C_LI

sentences: Virtue as the mean: Pan-human consensus genome significantly improves the accuracy of RNA-seq analyses The Human Reference Genome serves as the foundation for modern genomic analyses. However, in its present form, it does not adequately represent the vast genetic diversity of the human population. In this study, we explored the consensus genome as a potential successor of the current Reference genome and assessed its effect on the accuracy of RNA-seq read alignment. In order to find the best haploid genome representation, we constructed consensus genomes at the Pan-human, Super-population and Population levels, utilizing variant information from the 1000 Genomes project. Using personal haploid genomes as the ground truth, we compared mapping errors for real RNA-seq reads aligned to the consensus genomes versus the Reference genome. For reads overlapping homozygous variants, we found that the mapping error decreased by a factor of ~2-3 when the Reference was replaced with the Pan-human consensus genome. Interestingly, we also found that using more population-specific consensuses resulted in little to no increase over using the Pan-human consensus, suggesting a limit in the utility of incorporating more specific genomic variation. To assess the functional impact, we compared splice junction expression in the different genomes and found that the Pan-human consensus increases accuracy of splice junction quantification for hundreds of splice junctions.

sentences: A Kink in DWORF Helical Structure Controls the Activation of the Sarco-plasmic Reticulum Ca2+-ATPase The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase embedded in the sarcoplasmic reticulum. For each enzymatic cycle, SERCA transports 2 Ca2+ ions per ATP hydrolyzed in exchange for 2 to 3 H+ ions. SERCA is responsible for approximately 70% of Ca2+ transport and plays a central role in muscle relaxation. SERCAs function is regulated by endogenous regulins, single-pass membrane proteins that bind the ATPase within the membrane. While most of the regulins, such as phospholamban and sarcolipin, inhibit SERCAs activity, a newly discovered protein DWarf Open Reading Frame (DWORF) has a unique activating effect. DWORF is a 3.8 kDa bitopic membrane protein expressed in cardiac muscle. In this work, we determine the structure, topology, and per-residue lipid interactions of DWORF in lipid bilayers using a combination of high-resolution oriented sample solid-state NMR (OS-ssNMR) spectroscopy and refinement by replica-averaged orientationally-restrained molecular dynamics (RAOR-MD). We found that DWORFs structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64{degrees} and a transmembrane (TM) C-terminal helix with an angle of 32{degrees}. A kink induced by Pro15, unique to DWORF, separated the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORFs activating effect on SERCA. Overall, our findings directly link DWORFs structural topology to its unique activating effect on SERCA.

sentences: A polyyne toxin produced by an antagonistic bacterium blinds and lyses a green microalga Microalgae are key contributors to global carbon fixation and the basis of many food webs. In nature, their growth is often supported or suppressed by other microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology. Using a combination of Raman microspectroscopy, genome mining and mutational analysis, we discovered a novel polyyne toxin we name protegencin that is secreted by P. protegens and penetrates algal cells to destroy their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin prevents the phototactic behavior of C. reinhardtii needed to perform optimal photosynthesis. A protegencin-deficient biosynthetic mutant of P. protegens does not affect growth or eyespot carotenoids of C. reinhardtii. Thus, protegencin acts in a direct and destructive way, and reveals at least a two-pronged molecular strategy used by algicidal bacteria.

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Sequence based prediction of protein phase separation into disordered condensates using machine learning Several proteins which are responsible for neuro-degenrerative disorders (Alzheimers, Parkinsons etc) are shown to undergo a mechanism known as liquid liquid phase separation (LLPS). We in this research build a predictor which would answer whether a protein molecule would undergo LLPS or not. For this we used some protein sequences for which we already knew the answer. The ones who undergo LLPS were considered as the positive set and the ones who do not, were taken as the negative set. Depending on the knowledge of amino-acid sequences we identified some relevant variables in the context of LLPS e.g. number of amino acids, length of the best pairings, average register shifts. Using these variables we built a number of scoring functions which were basically analytic functions involving these variables and we also combined some scores already existing in the literature. We considered a total of 43636 protein sequences, among them only 121 were positive. We applied logistic regression and performed cross validation, where 25% of the data were used as the training set and the performance of the obtained results were tested on the remaining 75% of the data. In the training process, we used Simplex algorithm to maximize area under the curve (AUC) in receiver operator characteristics (ROC) space for each of the scores we defined. The optimised parameters were then used to evaluate AUC on the test set to check the accuracy. The best performing score was identified as the predicting model to answer the question whether a protein chain would undergo phase separating behavior or not.

Author summaryLiquid-liquid phase separation (LLPS) is a classic subject in polymer physics. The essen- tial physics is aptly captured within the Flory-Huggins (FH) approach, a simple lattice mean-field theory. Evidence has been mounting in the last decade that protein LLPS underlies the formation of membrane-less organelles (MLOs) in living cells, explaining why proteins and other bio-molecules can remain in a dense liquid condensate without diffusing away. Intrinsically disordered regions (IDRs), with a sequence-intrinsic prefer- ence for conformational heterogeneity or disorder under native conditions, are enriched in proteins that have the ability of switching on LLPS. The detailed understanding of the biological function of disordered bio-molecular condensates, whose formation is driven by LLPS, is currently the focus of a major effort in cell biology. Several key proteins in neuro-degenerative disorders are components of MLOs, and a further liquid-to-solid transition to unsoluble amyloid aggregates may be triggered by pathogenic mutants. Non-equilibrium active processes are also known to drive non trivial spatio-temporal organization patterns in MLOs. In this research we aim to predict which proteins can undergo LLPS in physiological conditions in living cells, and the corresponding phase behavior, based on theoretical tools and on the knowledge of the amino-acid sequence alone. We derive specific knowledge-based potentials for the different kind of short-range interactions that are believed to drive protein LLPS and use them to build a simple yes/no predictor of phase separation in physiological conditions.

sentences: Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl- and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 [A] resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl-) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

sentences: Ultraviolet light-induced collagen degradation inhibits melanoma invasion Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma1-4. The ageing, sun-exposed dermis accumulates UVR damage5, and older patients develop more melanomas at UVR-exposed sites4,6,7. As fibroblasts are functionally heterogeneous and play key roles in the stromal contribution to cancer8,9, we asked whether UVR modifies dermal fibroblast function. Here we confirmed the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) by UVR-damaged fibroblasts was persistently upregulated to reduce local levels of collagen 1 (COL1A1), and found dermal COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, we show inhibiting extracellular matrix degradation and MMP1 expression restored melanoma invasion to UVR damaged dermis. We confirmed in vitro findings in a cohort of primary cutaneous melanomas of aged humans, showing more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen. We found collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. These data indicate melanomas arising over UVR-damaged, collagen-poor skin of the elderly are less invasive, and this reduced invasion improves survival. Consequently, although UVR increases tumour incidence, it delays primary melanoma invasion by degrading collagen. However, we show melanoma-associated fibroblasts can restore invasion in low-collagen primary tumours by increasing collagen synthesis. Finally, we demonstrate high COL1A1 gene expression is a biomarker of poor outcome across a broad range of primary cancers.

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Conserved Untranslated Regions of Multipartite Viruses: Natural Markers of Novel Viral Genomic Components and Tags of Viral Evolution Viruses with split genomes are categorized as being either segmented or multipartite according to whether their genomic segments occur in single or different virions. Some complexity will exist, in that inherited "core" vital segments viruses may renew the others once host and environmental alterations keep driving viral evolution. Despite this uncertainty, empirical observations have been made across the split genomes in the untranslated regions (UTRs) on the short or long stretches of conserved or identical sequences. In this study, we describe a methodology that combines RNA and small RNA sequencing, conventional BLASTx, and iterative BLASTn of UTRs to detect viral genomic components even if they encode orphan genes (ORFans). Within the phylum Kitrinoviricota, novel putative multipartite viruses and viral genomic components were annotated using data obtained from our sampling or publicly available sources. The novel viruses, as extensions or intermediate nodes, enriched the information of the evolutionary networks. Furthermore, the diversity of novel genomic components emphasized the evolutionary roles of reassortment and recombination, as well as genetic deletion, strongly supporting the genomic complexity. These data also suggest insufficient knowledge of these genomic components for categorizing some extant viral taxa. The relative conservation of UTRs at the genome level may explain the relationships between monopartite and multipartite viruses and how the multipartite viruses can have a life strategy involving multiple host cells.

Author summaryThe current workflows for virus identification are largely based on high-throughput sequencing and coupled protein sequence homology-dependent analysis methods and tools. However, for viruses with split genomes, the identification of genomic components whose deduced protein sequences are not homologous to known sequences is inadequate. Furthermore, many plant-infecting multipartite viruses contain conserved UTRs across their genomic components. Based on this, we propose a practical method of UTR-backed iterative BLASTn (UTR-iBLASTn) to explore the components with ORFans and study virus evolution using the UTRs as signals. These shed light on viral "dark matter"--unknown/omitted genomic components of segmented/multipartite viruses from different kingdoms and hosts, and the origins of these components.

sentences: Antagonistic inhibitory subnetworks control cooperation and competition across cortical space The cortical microcircuit can dynamically adjust to dramatic changes in the strength, scale, and complexity of its input. In the primary visual cortex (V1), pyramidal cells (PCs) integrate widely across space when signals are weak, but narrowly when signals are strong, a phenomenon known as contrast-dependent surround suppression. Theoretical work has proposed that local interneurons could mediate a shift from cooperation to competition of PCs across cortical space, underlying this computation. We combined calcium imaging and electrophysiology to constrain a stabilized supralinear network model that explains how the four principal cell types in layer 2/3 (L2/3) of mouse V1- somatostatin (SST), parvalbumin (PV), and vasoactive intestinal peptide (VIP) interneurons, and PCs- transform inputs from layer 4 (L4) PCs to encode drifting gratings of varying size and contrast. Using bidirectional optogenetic perturbations, we confirmed key predictions of the model. Our data and modeling showed that recurrent amplification drives a transition from a positive PC[->]VIP{dashv}SST{dashv}PC feedback loop at small size and low contrast to a negative PC[->]SST{dashv}PC feedback loop at large size and high contrast to contribute to this flexible computation. This may represent a widespread mechanism for gating competition across cortical space to optimally meet task demands.

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The ecology and quantitative genetics of seed and seedling traits in upland and lowland ecotypes of a perennial grass Plants have evolved diverse reproductive allocation strategies and seed traits to aid in dispersal, persistence in the seed bank, and establishment. In particular, seed size, dormancy, and early seedling vigor are thought to be key functional traits with important recruitment and fitness consequences across abiotic stress gradients. Selection for favored seed-trait combinations, or against maladaptive combinations, is likely an important driver shaping recruitment strategies. Here, we test for seed-trait plasticity and local adaptation in contrasting upland and lowland ecotypes of Panicum hallii with field experiments in native versus foreign habitats. Furthermore, we test whether seed traits have been under directional selection in P. hallii using the v-test (Fraser 2020) based on trait variance in a genetic cross. Finally, we evaluate the genetic architecture of ecotypic divergence for these traits with Quantitative Trait Locus (QTL) mapping. Field experiments reveal little plasticity but support a hypothesis of local adaptation among ecotypes based on recruitment. Patterns of segregation within recombinant hybrids provides strong support for directional selection driving ecotypic divergence in seeds traits. Genetic mapping revealed a polygenic architecture with evidence of genetic correlation between seed mass, dormancy, and seedling vigor. Our results suggest that the evolution of these traits may involve constraints that affect the direction of adaptive divergence. For example, seed size and germination percentage shared two colocalized QTL with antagonistic additive effects. This supports the hypothesis of a functional genetic relationship between these traits, resulting in either large seed/strong dormancy or small seed/weak dormancy trait combinations. Overall, our study provides insights into the factors facilitating and potentially constraining ecotypic differentiation in seed traits.

Impact SummarySeed size and dormancy are key functional traits with important recruitment and fitness consequences. Theory suggests tradeoffs and plasticity in offspring quantity and quality are important in seed evolution. The genetics of seed size and dormancy traits have been studied extensively, but these studies are mostly limited to model system or domesticated crops. We also know very little about the genetic architecture and evolution of seed-based life history traits especially considering adaptation to xeric and mesic habitats. Here, we explored the genetic basis of trade-offs between seed size and dormancy in a C4 perennial grass, Panicum hallii, endemic to North America. We planted seeds of recombinant inbred lines from a cross between a xeric and mesic ecotype of P.hallii and mapped quantitative trait (QTL) loci for seed size, dormancy and seedling vigor traits. We detected a genetic basis of trade-offs between seed size and dormancy, suggesting that natural selection strongly favored specific trait combinations in ecotype formation. We further explored the role of seed size variation on seedling and adult recruitment in contrasting habitats. Our data showed that seed size was under strong selection through recruitment. Overall, our results demonstrate that adaptive differentiation for seed size and early life stages are important factors in adaptation to contrasting habitats.

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ACCUMULATION OF MICROPLASTIC AND MICRORUBBER PARTICLES IN STORMWATER POND FISH AND INVERTEBRATES Stormwater ponds serve as best management practices to trap or remove both physical and chemical pollutants before further discharge of stormwater into receiving natural waterbodies. Stormwater ponds can therefore accumulate high levels of pollutants, including microplastics (MP) and microrubber (MR) from road runoff. It was hypothesized that biota in stormwater ponds that receive large amounts of road runoff, or are in proximity to roadways and major developments, would contain significant amounts of MP per individual, with high abundances of tire particles (TP). Fish and invertebrates were collected from five stormwater ponds and their adjacent tidal creeks in Mt. Pleasant, SC, USA. Whole organisms were digested using KOH and digested contents were filtered and analyzed by visual microscopy to identify and count MPs.

The majority (>80%) of MP recovered from biota across all sites were suspected TP. The average number of MP per individual ranged from 0.3 to 71 MP and the average number of suspected tire particles per individual ranged from 0 to 57.7 tire particles.

There were significant differences in MP per individual observed between sites and between species. A combination of factors such as availability of MPs based on surrounding land use, pond hydrodynamics, organism size, and species feeding habitat likely influenced the total MP observed among the different sites and species analyzed. These data provide preliminary examination into the fate and transport of MP and MR in stormwater ponds and an evaluation of MP and MR abundance in organisms from stormwater ponds in the coastal zone.

sentences: Osteoarthritis Early-, Mid- and Late-Stage Progression in the Rat Medial Meniscus Transection Model Osteoarthritis is a degenerative disease of synovial joints affecting all tissues, including the articular cartilage and underlying subchondral bone. Osteoarthritis animal models can recapitulate aspects of human disease progression and are commonly used to test the development of drugs, biomaterials, and cell therapies for treatment. The rat medial meniscus transection (MMT) model is a surgically induced post-traumatic osteoarthritis model and is one of the most commonly used models for therapeutic development; however, it is typically used to evaluate the efficacy of therapies to prevent disease development rather than testing the treatment of disease progression in already established disease. We describe herein, the qualitative and quantitative changes to articular cartilage, subchondral bone, and formation of osteophytes in rats at early-(3-weeks post-surgery), mid-(6-weeks post-surgery) and late-(12-weeks post-surgery) stages of osteoarthritis progression. Tibiae of MMT-operated animals showed loss of proteoglycan and fibrillation formation on articular cartilage surfaces as early as 3-weeks post-surgery. Using a contrast-enhanced CT technique, quantitative, 3-dimensional analysis of the tibiae showed that the articular cartilage initially thickened at 3- and 6-weeks post-surgery and then decreased at 12-weeks post-surgery. This decrease in cartilage thickness corresponded with increased lesions in the articular cartilage, including fully degraded surfaces down to the subchondral bone layer. In this rat MMT model, subchondral bone thickening was significant at 6-weeks post-surgery and seem to follow cartilage damage. Osteophytes were found at 3-weeks post-surgery, which coincided with articular cartilage degradation. Cartilaginous osteophytes preceded mineralization suggesting that these marginal tissue growths most likely occurred through endochondral ossification. The use of the rat MMT model has predominantly been used out to 3-weeks, and most studies determine the effect of therapies to delay or prevent the onset of osteoarthritis. We provide evidence that an extension of the rat MMT model out to 6 and 12 weeks resembled more severe phenotypes of human osteoarthritis. The mid- to late-stages of rat MMT model can be used to evaluate the therapeutic efficacy of novel treatments to treat the progression of established disease -- since patients typically present in the clinic when the disease is established and becomes symptomatic, thus evaluating the efficacy of new treatments at the late stage will be important for eventual clinical translation.

sentences: Structural basis for the ARF GAP activity and specificity of the C9orf72 complex Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required [~]10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP.

sentences: Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

sentences: Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A locus Cellular oxygen-sensing pathways are primarily regulated by hypoxia inducible factor-1 (HIF-1) in chronic hypoxia and are well studied. Intermittent hypoxia also occurs in many pathological conditions, yet little is known about its biological effects. In this study, we investigated how two proposed cellular oxygen sensing systems, HIF-1 and KDM4A-C, respond to cells exposed to intermittent hypoxia and compared to chronic hypoxia. We found that intermittent hypoxia increases HIF-1 activity through a pathway distinct from chronic hypoxia, involving the KDM4A, -B and -C histone lysine demethylases. Intermittent hypoxia increases the quantity and activity of KDM4A-C resulting in a decrease in H3K9 methylation. This contrasts with chronic hypoxia, which decreases KDM4A-C activity, leading to hypermethylation of H3K9. Demethylation of histones bound to the HIF1A gene in intermittent hypoxia increases HIF1A mRNA expression, which has the downstream effect of increasing overall HIF-1 activity and expression of HIF target genes. This study highlights how multiple oxygen-sensing pathways can interact to regulate and fine tune the cellular hypoxic response depending on the period and length of hypoxia.

sentences: GWAS and functional studies implicate a role for altered DNA repair in the evolution of drug resistance in Mycobacterium tuberculosis The emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is alarming and demands in-depth knowledge for timely diagnosis. We performed genome-wide association analysis (GWAS) using 2237 clinical strains of Mtb to identify novel genetic factors that evoke drug resistance. In addition to the known direct targets, for the first time, we identified a strong association between the mutations in the DNA repair genes and the multidrug-resistant phenotype. To evaluate the impact of variants identified in the clinical samples in the evolution of drug resistance, we utilized knockouts and complemented strains in Mycobacterium smegmatis (Msm) and Mtb. Results show that variant mutations abrogated the function of MutY and UvrB. MutY variant showed enhanced survival compared with wild-type (Rv) when the Mtb strains were subjected to multiple rounds of ex vivo antibiotic stress. Notably, in an in vivo Guinea pig infection model, the MutY variant outcompeted the wild-type strain. Collectively, we show that novel variant mutations in the DNA repair genes abrogate their function and contribute to better survival under antibiotic/host stress conditions.

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Philympics 2021: Prophage Predictions Perplex Programs Most bacterial genomes contain integrated bacteriophages--prophages--in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms.

Here, we present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses.

We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. We discuss caveats and concerns in this analysis and how those concerns may be mitigated.

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Epitope-Based Peptide prediction of vaccine against European bat lyssaviruses type 2 Glycoprotein G with Immunoinformatic Approach ObjectiveEuropean bat lyssaviruses (EBLV) type 2 is present in many European countries. Infection is usually seen in bats, the primary reservoirs of the viruses. Human deaths have been documented within few days following bat exposures. So, it is very useful to design an insilco peptide vaccine for European bat lyssaviruses type 2 virus using glycoprotein G as an immunogen to stimulate protective immune response

ResultsB cell tests were conducted for Bepipred with 15 conserved epitopes, Emini surface accessibility prediction with 7 conserved epitopes in the surface and Kolaskar and Tongaonkar antigenicity tested with three conserved epitopes being antigenic. 357 conserved epitopes were predicted to interact with different MHC-1 alleles with (IC50) [≤]500 while 282 conserved epitopes found to interact with MHC-II alleles with IC50[≤] 1000. Among all tested epitopes for world population coverage the epitope VFSYMELKV binding to MHC11 alleles was 97.94% and it found to bind 10 different alleles that indicate strong potential to formulate peptide vaccine for lyssaviruses type 2 virus. To the best of our knowledge this is the first study to propose peptide vaccine for European bat lyssavirus type 2.

sentences: Complex regulatory role of DNA methylation in caste- and age-specific expression of a termite. The reproductive castes of eusocial insects are often characterised by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite, Macrotermes natalensis. We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens.

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Reduced Dimension, Biophysical Neuron Models Constructed From Observed Data Using methods from nonlinear dynamics and interpolation techniques from applied mathematics, we show how to use data alone to construct discrete time dynamical rules that forecast observed neuron properties. These data may come from from simulations of a Hodgkin-Huxley (HH) neuron model or from laboratory current clamp experiments. In each case the reduced dimension data driven forecasting (DDF) models are shown to predict accurately for times after the training period.

When the available observations for neuron preparations are, for example, membrane voltage V(t) only, we use the technique of time delay embedding from nonlinear dynamics to generate an appropriate space in which the full dynamics can be realized.

The DDF constructions are reduced dimension models relative to HH models as they are built on and forecast only observables such as V(t). They do not require detailed specification of ion channels, their gating variables, and the many parameters that accompany an HH model for laboratory measurements, yet all of this important information is encoded in the DDF model.

As the DDF models use only voltage data and forecast only voltage data, they can be used in building networks with biophysical connections. Both gap junction connections and ligand gated synaptic connections among neurons involve presynaptic voltages and induce postsynaptic voltage response. Biophysically based DDF neuron models can replace other reduced dimension neuron models, say of the integrate-and-fire type, in developing and analyzing large networks of neurons.

When one does have detailed HH model neurons for network components, a reduced dimension DDF realization of the HH voltage dynamics may be used in network computations to achieve computational efficiency and the exploration of larger biological networks.

sentences: An amino residue that guides the correct photoassembly the water-oxidation complex but is not required for high affinity Mn2+ binding The assembly of the Mn4O5Ca cluster of the photosystem II (PSII) starts from the initial binding and photooxidation of the first Mn2+ at a high affinity site (HAS). Recent cryo-EM apo-PSII structures reveal an altered geometry of amino ligands in this region and suggest the involvement of D1-Glu189 ligand in the formation of the HAS. We now find that Gln and Lys substitution mutants photoactivate with reduced quantum efficiency compared to the wild-type. However, the affinity of Mn2+ at the HAS in D1-E189K was very similar to the wild-type (~2.2 M). Thus, we conclude that D1-E189 does not form the HAS (~2.9 M) and that the reduced quantum efficiency of photoactivation in D1-E189K cannot be ascribed to the initial photooxidation of Mn2+ at the HAS. Besides reduced quantum efficiency, the D1-E189K mutant exhibits a large fraction of centers that fail to recover activity during photoactivation starting early in the assembly phase, becoming recalcitrant to further assembly. Fluorescence relaxation kinetics indicate on the presence of an alternative route for the charge recombination in Mn-depleted samples in all studied mutants and exclude damage to the photochemical reaction center as the cause for the recalcitrant centers failing to assemble and show that dark incubation of cells reverses some of the inactivation. This reversibility would explain the ability of these mutants to accumulate a significant fraction of active PSII during extended periods of cell growth. The failed recovery in the fraction of inactive centers appears to a reversible mis-assembly involving the accumulation of photooxidized, but non-catalytic high valence Mn at the donor side of photosystem II, and that a reductive mechanism exists for restoration of assembly capacity at sites incurring mis-assembly. Given the established role of Ca2+ in preventing misassembled Mn, we conclude that D1-E189K mutant impairs the ligation of Ca2+ at its effector site in all PSII centers that consequently leads to the mis-assembly resulting in accumulation of non-catalytic Mn at the donor side of PSII. Our data indicate that D1-E189 is not functionally involved in Mn2+ oxidation\binding at the HAS but rather involved in Ca2+ ligation and steps following the initial Mn2+ photooxidation.

sentences: Identifying targetable metabolic dependencies across colorectal cancer progression Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system - comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

sentences: Parameterization of regulatory nodes for engineering broad host range heterologous gene expression By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacIq-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of cross-regulation offer a panoply of choices to potential users and help interoperability of the specific constructs.

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Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly known. Here we show that NRC4, from the  NRC solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Significance statementPlant NLR immune receptors function as intracellular sensors of pathogen virulence factors known as effectors. In resting state, NLRs localize to subcellular sites where the effectors they sense operate. However, the extent to which NLRs alter their subcellular distribution during infection remains elusive. We describe dynamic changes in spatiotemporal localization of an NLR protein in infected plant cells. Specifically, the NLR protein accumulates at the newly synthesized plant-pathogen interface membrane, where the corresponding effectors are deployed. Following immune recognition, the activated receptor re-organizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary immune receptors, but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.

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A small molecule, ACAi-028, with anti-HIV-1 activity targets a novel hydrophobic pocket on HIV-1 capsid The human immunodeficiency virus type 1 (HIV-1) capsid (CA) is an essential viral component of HIV-1 infection, and an attractive therapeutic target for antivirals. We report that a small molecule, ACAi-028, inhibits HIV-1 replication by targeting a hydrophobic pocket in the N-terminal domain of CA (CA-NTD). ACAi-028 is one of more than 40 candidate anti-HIV-1 compounds identified by in silico screening and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Our binding model showed that ACAi-028 interacts with the Q13, S16, and T19 amino acid residues, via hydrogen bonds, in the targeting pocket of CA-NTD. Using recombinant fusion methods, TZM-bl, time-of-addition, and colorimetric reverse transcriptase (RT) assays, the compound was found to exert anti-HIV-1 activity in the early stage between a reverse transcriptase inhibitor, azidothymidine (AZT), and an integrase inhibitor, raltegravir (RAL), without any effect on RT activity, suggesting that this compound may affect HIV-1 core disassembly (uncoating). Moreover, electrospray ionization mass spectrometry (ESI-MS) also showed that the compound binds directly and non-covalently to the CA monomer. CA multimerization and thermal stability assays showed that ACAi-028 decreased CA multimerization and thermal stability via S16 or T19 residues.

ImportanceThese results indicate that ACAi-028 is a novel CA inhibitor that binds to the novel hydrophobic pocket of CA-NTD. This study demonstrates that a compound targeting the new hydrophobic pocket is a promising anti-HIV-1 inhibitor. The findings presented here may offer the development of a novel class of anti-viral agents that can be used, providing HIV-1 patients with more options for Anti-retroviral therapy (ART) treatment. Despite many years of successful pharmaceutical developments in the area of anti-retroviral therapy, the prevalence of drug-resistant mutations in HIV-1, necessitates the continued development of novel agents, such as ACAi-028.

sentences: Sensory evidence accumulation using optic flow in a naturalistic navigation task Sensory evidence accumulation is considered a hallmark of decision-making in noisy environments. Integration of sensory inputs has been traditionally studied using passive stimuli, segregating perception from action. Lessons learned from this approach, however, may not generalize to ethological behaviors like navigation, where there is an active interplay between perception and action. We designed a sensory-based sequential decision task in virtual reality in which humans and monkeys navigated to a memorized location by integrating optic flow generated by their own joystick movements. A major challenge in such closed-loop tasks is that subjects actions will determine future sensory input, causing ambiguity about whether they rely on sensory input rather than expectations based solely on a learned model of the dynamics. To test whether subjects performed sensory integration, we used three independent experimental manipulations: unpredictable optic flow perturbations, which pushed subjects off their trajectory; gain manipulation of the joystick controller, which changed the consequences of actions; and manipulation of the optic flow density, which changed the reliability of sensory evidence. Our results suggest that both macaques and humans relied heavily on optic flow, thereby demonstrating a critical role for sensory evidence accumulation during naturalistic action-perception closed-loop tasks.

sentences: Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the feces of a wild blackbuck antelope The anaerobic gut fungi (AGF, phylum Neocallimastigomycota) reside in the alimentary tracts of herbivores. Multiple novel, yet-uncultured AGF taxa have recently been identified in culture-independent diversity surveys. Here, we report on the isolation and characterization of the first representative of the RH5 lineage from fecal samples of a wild blackbuck (Indian Antelope) from Sutton County, Texas, USA. The isolates displayed medium sized (2-4 mm) compact circular colonies on agar roll tubes and thin loose biofilm-like growth in liquid medium. Microscopic examination revealed monoflagellated zoospores and polycentric thalli with highly branched nucleated filamentous rhizomycelium, a growth pattern encountered in a minority of described AGF genera so far. The obtained isolates are characterized by formation of spherical vesicles at the hyphal tips from which multiple sporangia formed either directly on the spherical vesicles or at the end of sporangiophores. Phylogenetic analysis using the D1/D2 regions of the large ribosomal subunit (D/D2 LSU) and the ribosomal internal transcribed spacer 1 (ITS1) revealed sequence similarities of 93.5%, and 81.3%, respectively, to the closest cultured relatives (Orpinomyces joyonii strain D3A (D1/D2 LSU), and Joblinomyces apicalis strain GFH681 (ITS1). Substrate utilization experiments using the type strain (BB-3) demonstrated growth capabilities on a wide range of mono-, oligo-, and polysaccharides, including glucose, xylose, mannose, fructose, cellobiose, sucrose, maltose, trehalose, lactose, cellulose, xylan, starch, and raffinose. We propose accommodating these novel isolates in a new genus and species, for which the name Paucimyces polynucleatus is proposed. The type species is strain BB-3.

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SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development.

HIGHLIGHTSO_LIIterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogens
C_LIO_LISpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1
C_LIO_LIPassively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge
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sentences: A Numerical Representation and Classification of Codons to Investigate Codon Alternation Patterns during Genetic Mutations on Disease Pathogenesis Alteration of amino acid is possible due to mutation in codons that could have potential reasons to occur disease. Effective mutation analysis can help to predict the fate of the diseased individual which can be validated later by in-vitro experiments. It may also help an individual who is asymptomatic but having a particular genetic change for early detection and diagnosis during any terminal diseases. We try to investigate the codon alteration patterns and its impact during mutation for the genes known to be responsible for a particular disease. For our current study, we consider two neurodegenerative diseases, Parkinsons disease and Glaucoma. We use numerical representation of four nucleotides based on the number of hydrogen bonds in their chemical structures and make classification of 64 codons as well as amino acids into three different classes (Strong, Weak and Transition). The entire analysis has been carried out based on these classifications. Our analysis reveals that the strong class codons are highly mutated followed by weak and transition class. We observe that most of the mutations occur in the first or second positions in the codon rather than the third. While looking into the chemical properties of amino acid, we observe that amino acids belong to the aliphatic group are affected most during missense mutations. Our investigation further emphasises that in most of the cases the change in the determinative degree of codon due to mutation is directly proportional to the physical density property. Further calculation of determinative degree of 20 amino acids and getting determinative degree of any arbitrary set of amino acid sequences from them may help biologists to understand the evolutionary information of amino acid occurrence frequencies of in proteins. In addition, our scheme gives a more microscopic and alternative representation of the existing codon table that helps in deciphering interesting codon alteration patterns during mutations in disease pathogenesis.

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Heme overdrive rewires pan-cancer cell metabolism All cancers reprogram their metabolism. In this study, we report that all cancer cells employ imbalanced and dynamic heme metabolic pathways essential for their oncogenic growth and coined the term  heme overdrive. Three key characteristics define heme overdrive, i.e., cancer universality, cancer essentiality and cancer specificity. While heme overdrive is absent in diverse differentiated cells or somatic stem cells, it is present in hundreds of cancers, and is delineated in patient-derived tumor progenitor cells by single cell RNAseq. The only exception to heme overdrive in normal tissues is identified in preimplantation human embryos, where heme overdrive likely links to embryonic omnipotency. Among the major drivers are proteins involved in biosynthesis of heme intermediates (hydroxymethylbilane synthase (HMBS) and uroporphyrinogen decarboxylase (UROD)) and heme trafficking (FLVCR1). CRISPR/Cas9 editing to engineer leukemia cells with impaired heme biosynthesis steps confirmed our whole genomic data analyses that heme overdrive is linked to oncogenic states and cellular differentiation. Finally, we devised a novel "bait- and-kill" strategy to target this cancer metabolic vulnerability.

One-Sentence SummaryHeme overdrive re-programs cellular metabolism across diverse types of cancer.

sentences: On stability of Canonical Correlation Analysis and Partial Least Squares with application to brain-behavior associations Associations between datasets, each comprising many features, can be discovered through multivariate methods like Canonical Correlation Analysis (CCA) or Partial Least Squares (PLS). Application of CCA/PLS to high-dimensional datasets raises critical questions about reliability and interpretability. To study this, we developed a generative modeling framework to simulate synthetic datasets, parameterized by dimensionality, variance structure, and association strength. We found that CCA/PLS associations could be highly inaccurate when the number of samples per feature is relatively small. For PLS, profiles of feature weights exhibit detrimental bias toward leading principal component axes. We confirmed these trends in state-of-the-art neuroimaging datasets, Human Connectome Project (n{approx}1000) and UK Biobank (n=20000), finding that only the latter comprised sufficient samples for stable estimates. Analysis of the neuroimaging literature using CCA to map brain-behavior relationships revealed also that the commonly employed sample sizes yield unstable CCA solutions. Finally, we provide a calculator of dataset properties required for CCA/PLS stability. Collectively, we characterize how to limit detrimental effects of overfitting on CCA/PLS stability, and provide recommendations for future studies.

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Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST.

SYNOPSISWe track cow, pig, and human fecal pollution in lake water over time with metagenomics and benchmark these novel protocols against standard culture-based and qPCR tests for water quality monitoring.

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REVOLVER: a low-cost automated protein purifier based on parallel preparative gravity column workflows Protein purification is a ubiquitous operation in biochemistry and life sciences and represents a key step to producing purified proteins for research (understanding how proteins work) and various applications. The need for scalable and parallel protein purification systems keeps growing due to the increase in throughput in the production of recombinant proteins and in the ever-growing scale of biochemistry research. Therefore, automating the process to handle multiple samples in parallel with minimal human intervention is highly desirable; yet only a handful of such tools have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable and automatic protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER completes a full protein purification process with almost no human intervention and yields results equivalent to those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems will be useful to accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.

Specifications Table

O_TBL View this table:
org.highwire.dtl.DTLVardef@13fff66org.highwire.dtl.DTLVardef@98aa2forg.highwire.dtl.DTLVardef@cd2e70org.highwire.dtl.DTLVardef@a3bd86org.highwire.dtl.DTLVardef@1efc67d_HPS_FORMAT_FIGEXP  M_TBL C_TBL

sentences: Food or physics: plankton communities structured across Gulf of Alaska eddies Oceanic features, such as mesoscale eddies that entrap and transport water masses, create heterogeneous seascapes to which biological communities may respond. To date, however, our understanding of how internal eddy dynamics influence plankton community structuring is limited by sparse sampling of eddies and their associated biotic communities. In this paper, we used 10 years of archived Continuous Plankton Recorder (CPR) data (2002-2013) associated with 9 mesoscale eddies in the Northeast Pacific/Gulf of Alaska to test the hypothesis that eddy origin and rotational direction determines the structure and dynamics of entrained plankton communities. Using generalized additive models and accounting for confounding factors (e.g., timing of sampling), we found peak diatom abundance within both cyclonic and anticyclonic eddies near the eddy edge. Zooplankton abundances, however, varied with distance to the eddy center/edge by rotational type and eddy life stage, and differed by taxonomic group. For example, the greatest abundance of small copepods was found near the center of anticyclonic eddies during eddy maturation and decay, but near the edge of cyclonic eddies during eddy formation and intensification. Distributions of copepod abundances across eddy surfaces were not mediated by phytoplankton distribution. Our results therefore suggest that physical mechanisms such as internal eddy dynamics exert a direct impact on the structure of zooplankton communities rather than indirect mechanisms involving potential food resources.

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Re-evaluation of the phylogenetic diversity and global distribution of the genus Candidatus Accumulibacter Candidatus Accumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO), important for phosphorus removal from wastewater. This genus is diverse, and the current phylogeny and taxonomic framework appears complicated, with the majority of publicly available genomes classified as "Candidatus Accumulibacter phosphatis", despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer scale differentiation into different "types" and "clades", nevertheless taxonomic assignments remain confusing and inconsistent across studies. Therefore, a comprehensive re-evaluation is needed to establish a common understanding of this genus, both in terms of naming and basic conserved physiological traits. Here, we provide this re-assessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive datasets. We identified 15 novel species, along with the well-known Ca. A. phosphatis, Ca. A. deltensis and Ca. A. aalborgensis. To compare the species in situ, we designed new species-specific FISH probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, Ca. Accumulibacter species were widespread in WWTPs with phosphorus removal, indicating the process design as a major driver for their abundance. Genome mining for PAO related pathways and FISH-Raman microspectroscopy confirmed the potential for the PAO metabolism in all Ca. Accumulibacter species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed fine-scale differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal.

ImportanceCandidatus Accumulibacter is the most studied PAO organism, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to 16S rRNA gene- and other phylogenetic markers phylogeny, we obtained a higher resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of gene and pathways of interest, validated in situ by application of a new set of FISH probes and Raman micromicrospectroscopy, provided additional high-resolution metabolic insights into these organisms.

sentences: Nitroxoline as a promising alternative drug for the treatment of Lyme disease based on an in-vitro study Lyme disease (LD) is the most common vector-borne disease in USA and Europe and is caused by Borrelia burgdorferi. Despite proper treatment, approximately one fifth of patients will develop post-treatment LD syndrome (PTLDS), a condition which is poorly understood. One of the possible causes is thought to be due to persister forms of B. burgdorferi that are not effectively killed by the current Lyme antibiotics. In this study, we evaluated nitroxoline, an antibiotic used to treat urinary tract infections, for its activity against a stationary-phase culture enriched with persister forms of B. burgdorferi. Nitroxoline was found to be equivalent in activity against B. burgdorferi to cefuroxime (standard Lyme antibiotic) in different experiments. Moreover, we found that the three-drug combination cefuroxime + nitroxoline + clarithromycin eradicated 98.3% of stationary phase bacteria in the drug-exposure experiment and prevented the regrowth in the subculture study after drug exposure, as well as two-drug combinations cefuroxime + nitroxoline and clarithromycin + nitroxoline. These drug combinations should be further evaluated in a LD mouse model to assess if eradication of persister forms of B. burgdorferi in-vivo is possible and if so, whether nitroxoline could be repurposed as an alternative drug for the treatment of LD.

sentences: Liquid-Liquid Phase Separation Protects Amyloidogenic and Aggregation-Prone Peptides in Heterologous Expression Systems Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid-liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in E. coli and linked it to highly amyloidogenic peptides, including A{beta} amyloid. We find that the fusion proteins form membraneless organelles during overexpression and remain soluble. We also developed a facile purification method of functional A{beta} peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.

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Temperature drives variation in flying insect biomass across a German malaise trap network O_LIAmong the many concerns for biodiversity in the Anthropocene, recent reports of flying insect loss are particularly alarming, given their importance as pollinators and as a food source for many predators. Few insect monitoring programs cover large spatial scales required to provide more generalizable estimates of insect responses to global change drivers.
C_LIO_LIWe ask how climate and surrounding habitat affect flying insect biomass and day of peak biomass using data from the first year of a new standardized distributed monitoring network at 84 locations across Germany comprising spatial gradient of land-cover types from protected to urban areas.
C_LIO_LIFlying insect biomass increased linearly with monthly temperature across Germany. However, the effect of temperature on flying insect biomass flipped to negative in the hot months of June and July when local temperatures most exceeded long-term averages.
C_LIO_LILand-cover explained little variation in insect biomass, but biomass was lowest in forested sites. Grasslands, pastures and orchards harbored the highest insect biomass. The date of peak biomass was primarily driven by surrounding land-cover type, with grasslands especially having earlier insect biomass phenologies.
C_LIO_LIStandardized, large-scale monitoring is pivotal to uncover underlying processes of insect decline and to develop climate-adapted strategies to promote insect diversity. In a temperate climate region, we find that the benefits of temperature on flying insect biomass diminish in a German summer at locations where temperatures most exceeded long-term averages. These results highlighting the importance of local adaptation in climate change-driven impacts on insect communities.
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sentences: Antimicrobial Effects of Ibuprofen Combined with Standard of Care Antimicrobials against Cystic Fibrosis Pathogens Cystic Fibrosis (CF) is a common fatal genetic disease caused by mutations happened to cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lungs of CF patients are often colonized or infected with microorganisms. Drug resistant bacterial infection has been problematic in cystic fibrosis patient. The chronic bacterial infections and concomitant airway inflammation could damage the lung and lead to respiratory failure. Several clinical trials have demonstrated that high-dose ibuprofen reduces the rate of pulmonary function decline in CF patients. This beneficial effect has been attributed to the anti-inflammatory properties of ibuprofen. Previously, we have confirmed that high-dose ibuprofen demonstrated antimicrobial activity against P. aeruginosa in in vitro and in vivo. However, no study has examined the antimicrobial effect of combining ibuprofen with standard-of-care (SoC) antimicrobials. Here, we evaluated possible synergistic activity of combinations of common nonsteroidal anti-inflammatory drugs (NSAIDs), namely, aspirin, naproxen, and ibuprofen, with commonly used antibiotics for CF patients. The drug combinations were screened against different CF clinical isolates. Drugs that demonstrated efficacy in the presence of ibuprofen were further verified synergistic effects between these antimicrobials and NSAIDs. Finally, the survival analysis of an P. aeruginosa murine infection model was used to demonstrate the efficacy of synergistic combination. Our results suggest that combinations of ibuprofen with commonly used antibiotics demonstrate synergistic antimicrobial activity against drug resistant, clinical bacterial strains in in vitro. The efficacy of combination ceftazidime and ibuprofen was demonstrated in in vivo.

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Naked mole-rat hematopoietic stem and progenitors are highly quiescent with an inherent myeloid bias Naked mole-rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which is a neotenic trait. A myeloid bias towards granulopoiesis in concert with decreased B-lymphopoiesis defines the marrow composition, resembling fetal leukopoiesis. Very similar to primates, the primitive stem cell compartment is marked by CD34 and THY1. Remarkably, stem and progenitor respiration rates are as low as in human cells, while NMR cells show a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. Our work provides a platform to study immunology and stem cell biology in an animal model of exceptional longevity.

TeaserJuvenile features of hematopoiesis shape the blood system of the longest-lived rodent.

sentences: Polycomb repressive complex 2 coordinates with Sin3 histone deacetylase complex to epigenetically reprogram genome-wide expression of effectors and regulate pathogenicity in Magnaporthe oryzae The strict suppression and reprogramming of gene expression are necessary at different development stages and/or in response to environment stimuli in eukaryotes. In Rice Magnaporthe oryzae pathosystem, effectors from pathogen are kept transcriptionally silenced in the vegetative growth stage and are highly expressed during invasive growth stage to adapt to the host environment. However, the mechanism of how such effectors are stably repressed in the vegetative stage and its roles during rice blast infection remain unclear so far. Here, we showed that all subunits of Polycomb Repressive Complex 2 are required for such repression by direct H3K27me3 occupancy and pathogenic process in M. oryzae. Suppression of polycomb-mediated H3K27me3 causes an improper induction of effectors during vegetative growth thus simulating a host environment. Notably, the addition subunit P55 not only acts as the bridge to connect with core subunits to form a complex in M. oryzae, but also recruits Sin3 histone deacetylase complex to prompt H3K27me3 occupancy for stable maintenance of transcriptional silencing of the target genes in the absence of PRC1. In contrast, during invasive growth stage, the repressed state of effectors chromatin can be partially erased during pathogenic development resulting in transcriptional activation of effectors therein. Overall, Polycomb repressive complex 2 coordinates with Sin3 histone deacetylase complex to epigenetically reprogram genome-wide expression of effectors, which act as molecular switch to memorize the host environment from vegetative to invasive growth, thus contributing to the infection of rice blast.

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Detection of SARS-CoV-2 Omicron variant (B.1.1.529) infection of white-tailed deer White-tailed deer (Odocoileus virginianus) are highly susceptible to infection by SARS-CoV-2, with multiple reports of widespread spillover of virus from humans to free-living deer. While the recently emerged SARS-CoV-2 B.1.1.529 Omicron variant of concern (VoC) has been shown to be notably more transmissible amongst humans, its ability to cause infection and spillover to non-human animals remains a challenge of concern. We found that 19 of the 131 (14.5%; 95% CI: 0.10-0.22) white-tailed deer opportunistically sampled on Staten Island, New York, between December 12, 2021, and January 31, 2022, were positive for SARS-CoV-2 specific serum antibodies using a surrogate virus neutralization assay, indicating prior exposure. The results also revealed strong evidence of age-dependence in antibody prevalence. A significantly ({chi}2, p < 0.001) greater proportion of yearling deer possessed neutralizing antibodies as compared with fawns (OR=12.7; 95% CI 4-37.5). Importantly, SARS-CoV-2 nucleic acid was detected in nasal swabs from seven of 68 (10.29%; 95% CI: 0.0-0.20) of the sampled deer, and whole-genome sequencing identified the SARS-CoV-2 Omicron VoC (B.1.1.529) is circulating amongst the white-tailed deer on Staten Island. Phylogenetic analyses revealed the deer Omicron sequences clustered closely with other, recently reported Omicron sequences recovered from infected humans in New York City and elsewhere, consistent with human to deer spillover. Interestingly, one individual deer was positive for viral RNA and had a high level of neutralizing antibodies, suggesting either rapid serological conversion during an ongoing infection or a "breakthrough" infection in a previously exposed animal. Together, our findings show that the SARS-CoV-2 B.1.1.529 Omicron VoC can infect white-tailed deer and highlights an urgent need for comprehensive surveillance of susceptible animal species to identify ecological transmission networks and better assess the potential risks of spillback to humans.

Key FindingsThese studies provide strong evidence of infection of free-living white-tailed deer with the SARS-CoV-2 B.1.1.529 Omicron variant of concern on Staten Island, New York, and highlight an urgent need for investigations on human-to-animal-to-human spillovers/spillbacks as well as on better defining the expanding host-range of SARS-CoV-2 in non-human animals and the environment.

sentences: Quantifying habitat and landscape effects on composition and structure of plant-pollinator networks in the US Northern Great Plains Community structure contributes to ecosystem persistence and stability. To understand the mechanisms underlying pollination and community stability of natural areas in a human influenced landscape, a better understanding of the interaction patterns between plants and pollinators in disturbed landscapes is needed. The Northern Great Plains still retain extensive tracts of remnant temperate grassland habitat within a matrix of varying land-uses. We used a network-based approach to quantify how temperate grassland attributes and landscape heterogeneity influence plant-pollinator community structure in natural habitats. We also quantified pollinator diversity and floral diversity to assess the functional role of temperate grassland attributes and the surrounding landscape on the composition of the plant-pollinator communities in natural habitats. We found that the amount of local nectar sugar and increased proportions of certain land-uses contribute to pollinator diversity that in turn influences the structure of interactions between plants and pollinators. Understanding the factors contributing to plant-pollinator network structure can guide management decisions to support resilient plant-pollinator communities and conserve the stability of pollination services.

sentences: Polar lipids are linked to nanoparticles in xylem sap of temperate angiosperm species Xylem sap of angiosperm species has been found to include low concentrations of polar lipids and nanoparticles, including surfactant-coated nanobubbles. Although the nanoparticles have been suggested to consist of polar lipids, no attempt has been made to determine if nanoparticle and lipid concentrations are related. Here, we examined concentrations of nanoparticles and lipids in xylem sap and contamination control samples of six temperate angiosperm species with a NanoSight device and based on mass spectrometry. We found (1) that the concentration of nanoparticles and lipids were both diluted when an increasing amount of sap was extracted, (2) that their concentrations were significantly correlated in three species, (3) that their concentrations were affected by vessel anatomy, and (4) that concentrations of nanoparticles and lipids were very low in contamination-control samples. Moreover, there was little seasonal difference, no freezing-thawing effect on nanoparticles, and little seasonal variation in lipid composition. These findings indicate that lipids and nanoparticles are related to each other, and largely do not pass interconduit pit membranes. Further research is needed to examine the formation and stability of nanoparticles in xylem sap in relation to lipid composition, and the complicated interactions among the gas, liquid, and solid phases in xylem conduits.

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Fis connects two sensory pathways, quorum sensing and surface sensing, to control motility in Vibrio parahaemolyticus Fis (Factor for Inversion Stimulation) is a global regulator that is highly expressed during exponential growth and undetectable in stationary growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to cell density and growth phase. In V. parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation required for biofilm formation and a repressor of swarming motility. In Vibrio parahaemolyticus, we showed, using genetics and DNA binding assays, that Fis bound directly to the regulatory regions of the qrr genes and was a positive regulator of these genes. In the {Delta}fis mutant, opaR expression was induced and a robust CPS and biofilm was produced, while swarming motility was abolished. Expression analysis and promoter binding assays showed that Fis was a direct activator of both the lateral flagellum laf operon and the surface sensing scrABC operon, both required for swarming motility. In in vitro growth competition assays, {Delta}fis was outcompeted by wild type in minimal media supplemented with intestinal mucus, and we showed that Fis directly modulated catabolism gene expression. In in vivo colonization competition assays, {Delta}fis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a direct role for Fis in QS, motility, and metabolism in V. parahaemolyticus.

IMPORTANCEIn this study, we examined the role of Fis in modulating expression of the five-quorum sensing regulatory sRNAs, qrr1 to qrr5, and showed that Fis is a direct positive regulator of QS, which oppositely controls CPS and swarming motility in V. parahaemolyticus. The {Delta}fis deletion mutant was swarming defective due to a requirement for Fis in lateral flagella and surface sensing gene expression. Thus, Fis links QS and surface sensing to control swarming motility and, indirectly, CPS production. Fis was also required for cell metabolism, acting as a direct regulator of several carbon catabolism loci. Both in vitro and in vivo competition assays showed that the {Delta}fis mutant had a significant defect compared to wild type. Overall, our data demonstrates that Fis plays a critical role in V. parahaemolyticus physiology that was previously unexamined.

sentences: Prisoner of War dynamics explains the time-dependent pattern of substitution rates in viruses Molecular clock dating is widely used to estimate timescales of phylogenetic histories and to infer rates at which species evolve. One of the major challenges with inferring rates of molecular evolution is the observation of a strong correlation between estimated rates and the timeframe of their measurements. Recent empirical analysis of virus evolutionary rates suggest that a power-law rate decay best explains the time-dependent pattern of substitution rates and that the same pattern is observed regardless of virus type (e.g. groups I-VII in the Baltimore classification). However there exists no explanation for this trend based on molecular evolutionary mechanisms. We provide a simple predictive mechanistic model of the time-dependent rate phenomenon, incorporating saturation and host constraints on the evolution of some sites. Our model recapitulates the ubiquitous power-law rate decay with a slope of -0.65 (95% HPD: -0.72, -0.52) and can satisfactorily account for the variation in inferred molecular evolutionary rates over a wide range of timeframes. We show that once the saturation of sites starts - typically after hundreds of years in RNA viruses and thousands of years in DNA viruses - standard substitution models fail to correctly estimate divergence times among species, while our model successfully re-creates the observed pattern of rate decay. We apply our model to re-date the diversification of genotypes of hepatitis C virus (HCV) to 396,000 (95% HPD: 326,000 - 425,000) years before present, a time preceding the dispersal of modern humans out of Africa, and also showed that the most recent common ancestor of sarbecoviruses dates back to 23,500 (95% HPD: 21,100 - 25,300) years ago, nearly thirty times older than previous estimates. This not only creates a radical new perspective for our understanding the origins of HCV but also suggests a substantial revision of evolutionary timescales of other viruses can be similarly achieved.

sentences: Characterization of peptide-protein relationships in protein ambiguity groups via bipartite graphs In bottom-up proteomics, proteins are enzymatically digested into peptides before measurement with mass spectrometry. The relationship between proteins and their corresponding peptides can be represented by bipartite graphs. We conduct a comprehensive analysis of bipartite graphs using quantified peptides from measured data sets as well as theoretical peptides from an in silico digestion of the corresponding complete taxonomic protein sequence databases. The aim of this study is to characterize and structure the different types of graphs that occur and to compare them between data sets. We observed a large influence of the accepted minimum peptide length during in silico digestion. When changing from theoretical peptides to measured ones, the graph structures are subject to two opposite effects. On the one hand, the graphs based on measured peptides are on average smaller and less complex compared to graphs using theoretical peptides. On the other hand, the proportion of protein nodes without unique peptides, which are a complicated case for protein inference and quantification, is considerably larger for measured data. Additionally, the proportion of graphs containing at least one protein node without unique peptides rises when going from database to quantitative level. The fraction of shared peptides and proteins without unique peptides as well as the complexity and size of the graphs highly depends on the data set and organism. Large differences between the structures of bipartite peptide-protein graphs have been observed between database and quantitative level as well as between analyzed species. In the analyzed measured data sets, the proportion of protein nodes without unique peptides ranged from 6.4% to 55.0%. This highlights the need for novel methods that can quantify proteins without unique peptides. The knowledge about the structure of the bipartite peptide-protein graphs gained in this study will be useful for the development of such algorithms.

sentences: Diffusion approximations in population genetics and the rate of Muller's ratchet Diffusion theory is a central tool of modern population genetics, yielding simple expressions for fixation probabilities and other quantities that are not easily derived from the underlying Wright-Fisher model. Unfortunately, the textbook derivation of diffusion equations as scaling limits requires evolutionary parameters (selection coefficients, mutation rates) to scale like the inverse population size--a severe restriction that does not always reflect biological reality. Here we note that the Wright-Fisher model can be approximated by diffusion equations under more general conditions, including in regimes where selection and/or mutation are strong compared to genetic drift. As an illustration, we use a diffusion approximation of the Wright-Fisher model to improve estimates for the expected time to fixation of a strongly deleterious allele, i.e. the rate of Mullers ratchet.

sentences: Inferring population structure in biobank-scale genomic data Inferring the structure of human populations from genetic variation data is a key task in population and medical genomic studies. While a number of methods for population structure inference have been proposed, current methods are impractical to run on biobank-scale genomic datasets containing millions of individuals and genetic variants. We introduce SCOPE, a method for population structure inference that is orders of magnitude faster than existing methods while achieving comparable accuracy. SCOPE infers population structure in about a day on a dataset containing one million individuals and variants as well as on the UK Biobank dataset containing 488,363 individuals and 569,346 variants. Furthermore, SCOPE can leverage allele frequencies from previous studies to improve the interpretability of population structure estimates.

sentences: Genomic insights into metabolic flux in ruby-throated hummingbirds Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome level de novo genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long and short read sequencing and scaffolding using other existing assemblies. We then used hybrid long and short-read RNA-sequencing for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and a deletion of critical genes (GLUT4, GCK) involved in glucostasis in other vertebrates. We found expression of fructose-specific GLUT5 putatively in place of insulin-sensitive GLUT4, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds suggesting key pathways for the rapid metabolic switch hummingbirds undergo.

sentences: High-Throughput Translational Profiling with riboPLATE-seq Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages rRNA immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.

sentences: Lumican regulates cervical corticospinal axon collateralization via non-autonomous crosstalk between distinct corticospinal neuron subpopulations Corticospinal neurons (CSN) are the cortical projection neurons that innervate the spinal cord and some brainstem targets with segmental precision to control voluntary movement of specific functional motor groups, limb sections, or individual digits. CSN subpopulations exhibit striking axon targeting specificity from development into maturity: Evolutionarily newer rostrolateral CSN exclusively innervate bulbar-cervical targets (CSNBC-lat), while evolutionarily older caudomedial CSN (CSNmedial) are more heterogeneous, with distinct subpopulations extending axons to either bulbar-cervical or thoraco-lumbar segments. However, molecular regulation over specificity of CSN segmental target innervation is essentially unknown. The cervical cord, with its evolutionarily enhanced precision of forelimb movement, is innervated by multiple CSN subpopulations, suggesting inter-neuronal interactions in establishing cervical corticospinal circuitry. Here, we identify that Lumican, previously unrecognized in axon development, controls the balance of innervation between CSNBC-lat and CSNmedial within the cervical spinal cord. Remarkably, Lumican, an extracellular matrix protein expressed by CSNBC-lat, non-cell-autonomously suppresses axon collateralization in the cervical cord by CSNmedial. Intersectional viral labeling and mouse genetics further identify that Lumican controls axon collateralization by multiple CSN subpopulations in caudomedial sensorimotor cortex. These results identify inter-axonal molecular crosstalk between CSN subpopulations as a novel mechanism controlling corticospinal circuitry, target density, and competitive specificity. Further, this mechanism has potential implications for evolutionary diversification of corticospinal circuitry with finer scale precision.

sentences: Single cell transcriptome analysis of decidua macrophages in normal and recurrent spontaneous abortion patients Due to the heterogeneity and different polarization state of decidual macrophages (dM{Phi}), they play an important role during the pregnancy, but their definition and exact function remain elusive. We isolated CD14+CD45+ dM{Phi} from the normal or RSA decidua by flow cytometry, followed by single cell RNA sequencing (scRNA-seq). In total, 23,062 single-cell transcriptomes of macrophage were profiled (12,470 Normal and 10,592 RSA), which were divided into 13 major clusters via T-distributed stochastic neighbor embedding (t-SNE) visualization. We observed that there is higher percentage composition of M1 cells (70.6%) in the normal decidua, and higher percentage composition of M2 cells (68.3%) in the RSA decidua. We identified new markers (M1: S100A8, S100A9, M2: SELENOP, FOLR2, RNASE1) and secreted cytokines (M1: IL1{beta}, TNFSF13B and MMP9; M2: CCL3, CCL4) of the dM{Phi}. We found that cluster 10 as the specific cluster of dM{Phi} highly expressing BAG3 in normal group and cluster 7 specific highly expressing CXCL9/10/11 chemokine. After pseudo-time trajectory analysis, we found that the dM{Phi} formed a continuous "V-shaped" trajectory, with M1 and M2 type cells mainly occupying the two heads. We found that NF{kappa}B1, MYC and TCF12 acted as the key transcription factors of dM{Phi}. Our study redefined the polarization state and physiological characteristic of dM{Phi} in early normal and RSA pregnancy, which suggests a novel view and therapeutic target for spontaneous abortion prevention.

sentences: Progenitor cell integration into a barrier epithelium during adult organ turnover Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. These replacement cells--the progeny of basal stem cells--are born without apical, barrier-forming structures such as a protective, lumen-facing membrane and occluding junctions. How stem cell progeny acquire these structures to become part of the barrier is unknown. Here we use Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), Correlative Light-Electron Microscopy (CLEM), and volumetric imaging of live and fixed organs to investigate progenitor integration in the intestinal epithelium of adult Drosophila. We find that stem cell daughters gestate their future lumenal-apical membrane beneath a transient, basal niche formed by an umbrella-shaped occluding junction that shelters the growing cell and adheres it to mature neighbor cells. The umbrella junction both targets formation of a deep, microvilli-lined, apical invagination and closes it off from the contents of the gut lumen. When the growing cell is sufficiently mature, the umbrella junction retracts to expose this Pre-Assembled Apical Compartment (PAAC) to the gut lumen, thus incorporating the new cell into the intestinal barrier. When we block umbrella junctions, stem cell daughters grow and attempt to differentiate but fail to integrate; when we block cell growth, no umbrella junctions form and daughters arrest in early differentiation. Thus, stem cell progeny build new barrier structures in the shelter of a transient niche, where they are protected from lumenal insults until they are prepared to withstand them. By coordinating this dynamic junctional niche with progenitor cell differentiation, a physiologically active epithelial organ incorporates new cells while upholding integrity of its barrier.

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Yield Maintenance Under Drought is Orchestrated by the qDTY12.1-Encoded DECUSSATE Gene of Rice Through a Network With Other Flowering-Associated Genes Across the Genetic Background Introgression of major-effect QTLs is an important component of rice breeding for yield-retention under drought. While largely effective, the maximum potentials of such QTLs have not been consistent across genetic backgrounds. We hypothesized that synergism or antagonism with additive-effect peripheral genes across the background could either enhance or undermine the QTL effects. To elucidate the molecular underpinnings of such interaction, we dissected qDTY12.1 synergy with numerous peripheral genes in context of network rewiring effects. By integrative transcriptome profiling and network modeling, we identified the DECUSSATE (OsDEC) within qDTY12.1 as the core of the synergy and shared by two sibling introgression lines in IR64 genetic background, i.e., LPB (low-yield penalty) and HPB (high-yield penalty). OsDEC is expressed in flag leaves and induced by progressive drought at booting stage in LPB but not in HPB. The unique OsDEC signature in LPB is coordinated with 35 upstream and downstream peripheral genes involved in floral development through the cytokinin signaling pathway, which are lacking in HPB. Results further support the differential network rewiring effects through genetic coupling-uncoupling between qDTY12.1 and other upstream and downstream peripheral genes across the distinct genetic backgrounds of LPB and HPB. We propose that the functional DEC-network in LPB defines a mechanism for early flowering as a means for avoiding the depletion of photosyntate needed for reproductive growth due to drought. Its impact on yield-retention is likely through the timely establishment of stronger source-sink dynamics that sustains a robust reproductive transition under drought.

Author summaryWhile the Green Revolution of the 1960s significantly increased rice grain yields through the creation of high-yielding varieties for high input systems, current marginal climates pose a significant challenge for providing consistent yield. In rice growing regions of the world, drought affects the livelihood of small-scale and subsistence farmers by inflicting significant yield penalties to their production systems. Breeding of next-generation rice varieties with optimal balance of survivability and productivity traits will be key to providing consistent yields year to year. Within this paradigm, the use of large effect QTLs such as qDTY12.1 to improve yield retention under drought have been largely successful. By integrating the use of high resolution transcriptome datasets with a focused biological interrogation of agronomic results from this and previous studies, we uncovered a putative functional genetic network, anchored by the DECUSSATE gene (OsDEC) within qDTY12.1, that effectively minimizes drought penalties to yield by driving cellular processes that culminate in timely flowering that maximizes the use of photosynthetic sources for efficient reproduductive transition and ultimately seed development. Our study further illuminates the qDTY12.1 function and speaks to the misconception that qDTY introgression alone is sufficient for providing consistently large positive effects to yield retention under reproductive stage drought.

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Queuine, a bacterial derived hypermodified nucleobase, shows protection in in vitro models of neurodegeneration Growing evidence suggests that human gut bacteria, comprising the microbiome that communicates with the brain through the so-called  gut-brain-axis, are linked to neurodegenerative disorders. Imbalances in the microbiome of Parkinsons disease (PD) and Alzheimers disease (AD) patients have been detected in several studies. Queuine is a hypermodified nucleobase enriched in the brain and exclusively produced by bacteria and salvaged by humans through their gut epithelium. Queuine replaces guanine at the wobble position of tRNAs with GUN anticodons and promotes efficient cytoplasmic and mitochondrial mRNA translation.

To elucidate whether queuine could facilitate protein folding and prevent aggregation and mitochondrial defects, hallmarks of neurodegenerative disorders, we tested the effect of chemically synthesized queuine, STL-101, in several in vitro models of neurodegeneration. Treatment with STL-101 led to increased neuronal survival as well as a significant decrease in hyper-phosphorylated alpha-synuclein, a marker of alpha-synuclein aggregation in a PD model and a decrease in tau hyperphosphorylation in an AD model. Our work has identified a new role for queuine in neuroprotection uncovering a therapeutic potential for STL-101 in neurological disorders.

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Jumper Enables Discontinuous Transcript Assembly in Coronaviruses Genes in SARS-CoV-2 and, more generally, in viruses in the order of Nidovirales are expressed by a process of discontinuous transcription mediated by the viral RNA-dependent RNA polymerase. This process is distinct from alternative splicing in eukaryotes, rendering current transcript assembly methods unsuitable to Nidovirales sequencing samples. Here, we introduce the DO_SCPLOWISCONTINUOUSC_SCPLOW TO_SCPLOWRANSCRIPTC_SCPLOW AO_SCPLOWSSEMBLYC_SCPLOW problem of finding transcripts [Formula] and their abundances c given an alignment [Formula] under a maximum likelihood model that accounts for varying transcript lengths. Underpinning our approach is the concept of a segment graph, a directed acyclic graph that, distinct from the splice graph used to characterize alternative splicing, has a unique Hamiltonian path. We provide a compact characterization of solutions as subsets of non-overlapping edges in this graph, enabling the formulation of an efficient mixed integer linear program. We show using simulations that our method, JO_SCPLOWUMPERC_SCPLOW, drastically outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1 and SARS-CoV-2 samples, we find that JO_SCPLOWUMPERC_SCPLOW not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are well supported by direct evidence from long-read data, presence in multiple, independent samples or a conserved core sequence. JO_SCPLOWUMPERC_SCPLOW enables detailed analyses of Nidovirales transcriptomes.

Code availabilitySoftware is available at https://github.com/elkebir-group/Jumper

sentences: Change in protein, oil and fatty acid contents in soybeans (Glycine max (L.) Merr.) of different seed coat colors and seed weight Soybean seeds are one of the best sources of plant-based high-quality proteins and oils. The contents of these metabolites are affected by both environmental and genetic factors. In this study, 49 soybean germplasms were cultivated in Korea, the contents of total protein, total oil and five fatty acids were determined, and the influences of seed coat color and seed weight on each were assessed. The total protein and total oil contents were evaluated using Kjeldahl and Soxhlet methods and were in the ranges of 36.28-44.19% and 13.45-19.20%, respectively. Moreover, the contents of individual fatty acids were determined as area percentage from acquired gas-chromatography peaks. The contents of palmitic, stearic, oleic, linoleic and linolenic acids were in the ranges of 9.90-12.55, 2.45-4.00, 14.97-38.74, 43.22-60.26, and 5.37-12.33%, respectively and each significantly varied between the soybean germplasms. Unlike total oil and fatty acid contents, total protein content was not significantly affected by both seed coat color and seed weight. Cluster analysis grouped the soybeans into two classes with notable content differences. Fatty acids were the main factors for the variabilities seen between the soybean germplasms as observed in the principal component analysis. Correlation analysis revealed a significant but negative association between total oil and total protein contents (r = -0.714, p < 0.0001). Besides, a trade-off relationship was observed between oleic acid and linoleic acid (r = -0.936, p < 0.0001) which was reflected with respect to both seed coat color and seed weight. Among all colored soybeans, pale-yellow soybeans had the highest and the lowest levels of oleic acid and linoleic acid, respectively each being significantly different from the rest of colored soybeans (p < 0.05). Likewise, oleic acid content increased with seed weight while that of linoleic acid decreased with seed weight (p < 0.05). In general, this study showed the significance of seed coat color and seed weight to discriminate soybean genotypes, mainly in terms of their fatty acid contents. Moreover, the soybean germplasms with distinct characters and fatty acid contents identified in this study could be important genetic resources for cultivar development.

sentences: Self-contained and modular structured illumination microscope We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our instrument, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions. We illustrate the performance of the instrument by imaging optically large samples containing sequence-specifically stained DNA fragments.

sentences: Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. In trypanosomes, gene expression regulation depends heavily on post-transcriptional mechanisms. Both the RNA-binding protein RBP10 and glycosomal phosphoglycerate kinase PGKC are expressed only in mammalian-infective forms. RBP10 targets procyclic-specific mRNAs for destruction, while PGKC is required for bloodstream-form glycolysis. Developmental regulation of both is essential: expression of either RBP10 or PGKC in procyclic forms inhibits their proliferation. We show that the 3'-untranslated region of the RBP10 mRNA is extraordinarily long - 7.3kb - and were able to identify six different sequences, scattered across the untranslated region, which can independently cause bloodstream-form-specific expression. The 3'-untranslated region of the PGKC mRNA, although much shorter, still contains two different regions, of 125 and 153nt, that independently gave developmental regulation. No short consensus sequences were identified that were enriched either within these regulatory regions, or when compared with other mRNAs with similar regulation, suggesting that more than one regulatory RNA-binding protein is important for repression of mRNAs in procyclic forms. We also identified regions, including an AT repeat, that increased expression in bloodstream forms, or suppressed it in both forms. Trypanosome mRNAs that encode RNA-binding proteins often have extremely extended 3'-untranslated regions. We suggest that one function of this might be to act as a fail-safe mechanism to ensure correct regulation even if mRNA processing or expression of trans regulators is defective.

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Ontology-Aware Biomedical Relation Extraction MotivationAutomatically extracting relationships from biomedical texts among multiple sorts of entities is an essential task in biomedical natural language processing with numerous applications, such as drug development or repurposing, precision medicine, and other biomedical tasks requiring knowledge discovery. Current Relation Extraction (RE) systems mostly use one set of features, either as text, or more recently, as graph structures. The state-of-the-art systems often use resource-intensive hence slow algorithms and largely work for a particular type of relationship. However, a simple yet agile system that learns from different sets of features has the advantage of adaptability over different relationship types without an extra burden required for system re-design.

ResultsWe model RE as a classification task and propose a new multi-channel deep neural network designed to process textual and graph structures in separate input channels. We extend a Recurrent Neural Network (RNN) with a Convolutional Neural Network (CNN) to process three sets of features, namely, tokens, types, and graphs. We demonstrate that entity type and ontology graph structure provide better representations than simple token-based representations for RE. We also experiment with various sources of knowledge, including data resources in the Unified Medical Language System (UMLS) to test our hypothesis. Extensive experiments on four well-studied biomedical benchmarks with different relationship types show that our system outperforms earlier ones. Thus, our system has state-of-the-art performance and allows processing millions of full-text scientific articles in a few days on one typical machine.

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Time series analysis of trial-to-trial variability of MEG power spectrum during rest state, unattented listening and frequency-modulated tones classification The nonstationarity of EEG/MEG signals is important for understanding the functioning of human brain. From the previous research we know that even very short, i.e. 250--500ms MEG signals are variance-nonstationary. The covariance of stochastic process is mathematically associated with its spectral density, therefore we investigate how the spectrum of such nonstationary signals varies in time.

We analyze the data from 148-channel MEG, that represent rest state, unattented listening and frequency-modulated tones classification. We transform short-time MEG signals to the frequency domain using the FFT algorithm and for the dominant frequencies 8--12 Hz we prepare the time series representing their trial-to-trial variability. Then, we test them for level- and trend-stationarity, unit root, heteroscedasticity and gaussianity and based on their properties we propose the ARMA-modelling for their description.

The analyzed time series have the weakly stationary properties independently of the functional state of brain and localization. Only their small percentage, mostly related to the cognitive task, still presents nonstationarity. The obtained mathematical models show that the spectral density of analyzed signals depends on only 2--3 previous trials.

The presented method has limitations related to FFT resolution and univariate models, but it is not computationally complicated and allows to obtain a low-complex stochastic models of the EEG/MEG spectrum variability.

Although the physiological short-time MEG signals are in principle nonstationary in time domain, its power spectrum at the dominant frequencies varies as weakly stationary stochastic process. Described technique has the possible applications in prediction of the EEG/MEG spectral properties in theoretical and clinical neuroscience.

sentences: Assortative mate choice and epistatic mating-trait architecture induce complex movement of the crow hybrid zone Hybrid zones provide a window into the evolutionary processes governing species divergence. While the role of postzygotic isolation has been extensively characterized in the context of hybrid zones, the contribution of prezygotic isolation is less well explored. Here, we investigate the effects of assortative mate choice, the underlying preference function and mating-trait architecture, and the strength of sexual selection on hybrid zone dynamics. We explore this question by means of a mathematical model parameterized with phenotype and genotype data from the hybrid zone between all-black carrion and grey-coated hooded crows. The best-fit model resulted in narrow clines for two mating-trait loci coding for colour phenotype maintained by a moderate degree of assortative mating. Epistasis between the two loci induced hybrid-zone movement in favor of alleles conveying dark plumage followed by a shift in the opposite direction favouring grey-coated phenotypes [~]1,200 generations after secondary contact. Unlinked neutral loci diffused near-unimpeded across the zone. These results were generally robust to the choice of matching rule (self-referencing or parental imprinting) and effects of genetic drift. Overall, this study illustrates under which conditions assortative mating can maintain steep clines in mating-trait loci without generalizing to genome-wide reproductive isolation. It further emphasizes the importance of mating-trait architecture for spatio-temporal hybrid-zone dynamics.

sentences: Strain-dependent induction of primary bile acid 7-dehydroxylation by cholic acid Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism and immunomodulation. Primary BAs are synthesised by the host and their concentrations tightly regulated, while secondary BAs are produced by the gut microbiome through transformation of the former. Regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or the mouse ursodeoxycholic acid, UDCA) and it is unknown whether they induce bai gene expression. Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by Clostridium scindens is induced by CA suggesting that CA-dependent transcriptional regulation of 7-dehydroxylation is generalisable to CDCA and UDCA. Nevertheless, this effect is strain-specific and the murine isolate Extibacter muris did not respond to CA exposure, suggesting that bai genes are regulated differently in this strain. Moreover, the accessory gene baiJ was only upregulated in the Clostridium scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the C. scindens strains were also capable of 7-DH-ing muricholic acids (MCAs) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro as a result of CA-driven induction and suggests that it is likely that a different BA plays the same role in the murine gut.

sentences: The probability of joint monophyly of all species in an arbitrary species tree. Monophyly is a feature of a set of genetic lineages in which every lineage in the set is more closely related to all other members of the set than it is to any lineage outside the set. Multiple sets of lineages that are separately monophyletic are said to be reciprocally monophyletic, or jointly monophyletic. The prevalence of reciprocal monophyly, or joint monophyly, has been used to evaluate phylogenetic and phylogeographic hypotheses, as well as to delimit species. These applications often make use of a probability of joint monophyly under models of gene lineage evolution. Studies in coalescent theory have computed this joint monophyly probability for small numbers of separate groups in arbitrary species trees, and for arbitrary numbers of separate groups in trivial species trees. Here, generalizing existing results on monophyly probabilities under the multispecies coalescent, we derive the probability of joint monophyly for arbitrary numbers of separate groups in arbitrary species trees. We illustrate how our result collapses to previously examined cases. We also study the effect of tree height, sample size, and number of species on the probability of joint monophyly. The result also enables computation of relatively simple lower and upper bounds on the joint monophyly probability. Our results expand the scope of joint monophyly calculations beyond small numbers of species, subsuming past formulas that have been used in simpler cases.

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TET1 controls Cxcl1 induction by DNA demethylation and promotes neutrophil recruitment during acute lung injury Neutrophils are rapidly recruited from the peripheral blood to the inflammatory site to initiate inflammatory response against pathogenic infections. The process to recruit neutrophils must be properly regulated since the abnormal accumulation of neutrophils can cause organ damage and dysfunction. The acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a common cause of respiratory failure that is characterized by the infiltration of neutrophils and epithelial integrity disruption. Indeed, recent studies suggest a pathogenic role of neutrophils in the clinic severity of the coronavirus disease 2019 (COVID-19) ARDS. The chemokine CXCL1, which is rapidly induced by inflammatory stimuli, plays a key role in neutrophil influx during lung inflammation. The molecular basis of Cxcl1 induction is not fully understood. Here we report that TET1, a member of the ten eleven translocation (TET) methylcytosine dioxygenase protein family, displays a striking specificity in the regulation of gene expression in macrophages. RNA sequencing (RNA-seq) analysis showed that Tet1 disruption significantly altered the expression of only 48 genes that include Cxcl1 and several other genes known to be important for cell migration and trafficking in bone marrow derived macrophages (BMDMs) in response to LPS stimulation. TET1 regulates the induction of Cxcl1 by facilitating the DNA demethylation of the Cxcl1 promoter. In Tet1-/- mice, the induction of Cxcl1 was suppressed, resulting in defective neutrophil recruitment to the lung during LPS-induced acute lung injury. Our results identify a novel epigenetic mechanism that selectively controls Cxcl1 induction and neutrophil recruitment during acute lung injury.

Key PointsO_LITET1 has a striking specificity in macrophage gene regulation and controls Cxcl1 induction by inflammatory stimuli via DNA demethylation
C_LIO_LINeutrophil recruitment is defective in Tet1 deficient mice during acute lung injury
C_LI

sentences: Three-dimensional structure of the single domain cupredoxin AcoP Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

sentences: Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well-documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 sgRNAs. We used this toolkit to explore the limits of multiplexing in two major model species, and report on isolation of transgene-free octuple Nicotiana benthamiana and duodecuple (12x) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on production of toxic 5-fluorouracyl in presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (~112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher order multiplexing applications. We identify a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

sentences: Differences in the genetic structure between and within two landlocked Ayu groups with different migration patterns in Lake Biwa revealed by environmental DNA analysis Ayu, Plecoglossus altivelis, is largely an annual amphidromous fish, although a landlocked population lives in Lake Biwa, the largest lake in Japan. The landlocked population comprises two migrant groups, spring migrants and autumn migrants, which run to inlet rivers from the lake at different seasons. We used environmental DNA (eDNA) analysis, which is reported to be more sensitive and cost-effective than capture surveys, to clarify the genetic structure of this landlocked Ayu population with different migration patterns in Lake Biwa. We took water samples in 11 inlet rivers in the spring and autumn for two years in a row and quantitatively detected a total of 265 haplotypes of the mitochondrial D-loop region. The pairwise fixation index (FST) value and haplotype diversity indicated that there were genetic differences between the two migrant groups in their respective rivers, and the FST values were negatively related to latitude and the presence of artificial fish release. Additionally, the isolation by distance within each migrant group was observed when the lake was divided into the east and west sides. These findings show that the landlocked Ayu population in Lake Biwa has the genetic structure associated with migration patterns and geographical distance. This study demonstrates that the eDNA approach will be effective for conducting a large-scale investigation of genetic structure beyond simple presence/absence tests.

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The volatile cedrene from plant beneficial Trichoderma guizhouense modulates Arabidopsis root development through auxin transport and signaling Rhizosphere microorganisms interact with plant roots by producing chemical signals to regulate root development. However, the involved distinct bioactive compounds and the signal transduction pathways are remaining to be identified. Here, we show that sesquiterpenes (SQTs) are the main volatile compounds produced by plant beneficial Trichoderma guizhouense NJAU 4742, inhibition of SQTs synthesis in this strain indicated their involvement in plant-fungus cross-kingdom signaling. SQTs component analysis further identified the cedrene, a high abundant SQT in strain NJAU 4742, could stimulate plant growth and root development. Genetic analysis and auxin transport inhibition showed that auxin receptor TIR1, AFB2, auxin-responsive protein IAA14, and transcription factor ARF7, ARF19 affect the response of lateral roots to cedrene. Moreover, auxin influx carrier AUX1, efflux carrier PIN2 were also indispensable for cedrene-induced lateral root formation. Confocal imaging showed that cedrene affected the expression of pPIN2:PIN2:GFP and pPIN3:PIN3:GFP, which may be related to the effect of cedrene on root morphology. These results suggest that a novel SQT molecule from plant beneficial T. guizhouense can regulate plant root development through auxin transport and signaling.

One-sentence SummaryCedrene, a high- abundance sesquiterpenes produced by plant beneficial Trichoderma guizhouense NJAU 4742, stimulates Arabidopsis lateral root formation and primary root elongation by relying on auxin signaling pathway and auxin transporter PIN2 and AUX1.

sentences: A sensitive and specific genetically encodable biosensor for potassium ions Potassium ions (K+) play a critical role as an essential electrolyte in all biological systems. Here we report the crystal structure-guided optimization and directed evolution of an improved genetically encoded fluorescent K+ biosensor, GINKO2. GINKO2 is highly sensitive and specific for K+ and enables in vivo detection of K+ dynamics in multiple species.

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Uncertainty-guided learning with scaled prediction errors in the basal ganglia To accurately predict rewards associated with states or actions, the variability of observations has to be taken into account. In particular, when the observations are noisy, the individual rewards should have less influence on tracking of average reward, and the estimate of the mean reward should be updated to a smaller extent after each observation. However, it is not known how the magnitude of the observation noise might be tracked and used to control prediction updates in the brain reward system. Here, we introduce a new model that uses simple, tractable learning rules that track the mean and standard deviation of reward, and leverages prediction errors scaled by uncertainty as the central feedback signal. We provide a normative analysis, comparing the performance of the new model with that of conventional models in a value tracking task. We find that the new model has an advantage over conventional models when tested across various levels of observation noise. Further, we propose a possible biological implementation of the model in the basal ganglia circuit. The scaled prediction error feedback signal is consistent with experimental findings concerning dopamine prediction error scaling relative to reward magnitude, and the update rules are found to be consistent with many features of striatal plasticity. Our results span across the levels of implementation, algorithm, and computation, and might have important implications for understanding the dopaminergic prediction error signal and its relation to adaptive and effective learning.

Author SummaryThe basal ganglia system is a collection of subcortical nuclei in the mammalian brain. This system and its dopaminergic inputs are associated with learning from rewards. Here, dopamine is thought to signal errors in reward prediction. The structure and function of the basal ganglia system are not fully understood yet--for example, the basal ganglia are split into two antagonistic pathways, but the reason for this split and the role of the two pathways are unknown. Further, it has been found that under some circumstances, rewards of different sizes lead to dopamine responses of similar size, which cannot be explained with the reward prediction error theory. Here, we propose a new model of learning in the basal ganglia--the scaled prediction error model. According to our model, both reward average and reward uncertainty are tracked and represented in the two basal ganglia pathways. The learned reward uncertainty is then used to scale dopaminergic reward prediction errors, which effectively renders learning adaptive to reward noise. We show that such learning is more robust than learning from unscaled prediction errors and that it explains several physiological features of the basal ganglia system.

sentences: Comparative analysis of zooplankton diversity in freshwaters: What can we gain from metagenomic analysis? Molecular genetic approaches applied to environmental DNA have great potential for biodiversity research and ecosystem monitoring. A metagenome contains genetic information from all organisms captured in an environmental sample. It has been primarily used to study bacteria and archaea, but promising reports focusing on metazoan diversity are emerging. However, methodological uncertainties remain, and studies are required to validate the power and the limitations of such an approach when applied to macro-eukaryotes. Here, we analyzed water sample metagenomes to estimate zooplankton diversity in 22 freshwater lakes across Eastern Canada. We tested the coherence of data based on morphologically identified zooplankton taxa and molecular genetic data derived from shotgun sequencing of environmental DNA collected at the same time. RV coefficients showed a significant correlation between the relative abundance of zooplankton families derived from small subunit rRNA genes extracted from the metagenomes and morphologically identified zooplankton. However, differences in congruence with morphological counts were detected when varied bioinformatic approaches were applied to presence-absence data. This study presents one of the first diversity assessments of a group of aquatic metazoans using metagenomes and validates the coherence of the community composition derived from genetic and classical species surveys. Overall, our results suggest that metagenomics has the potential to be further developed to describe metazoan biodiversity in aquatic ecosystems, and to advance this area we provide key recommendations for workflow improvement.

sentences: Establishing regulated unconventional secretion for production of heterologous proteins Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

sentences: Pose Estimation of Free-Flying Fruit Flies Insect flight is a complex interdisciplinary phenomenon. Understanding its multiple aspects, such as flight control, sensory integration and genetics, often requires the analysis of large amounts of free flight kinematic data. Yet, one of the main bottlenecks in this field is automatically and accurately extracting such data from multi-view videos. Here, we present a model-based method for pose-estimation of free-flying fruit flies from multi-view high-speed videos. To obtain a faithful representation of the fly with minimum free parameters, our method uses a 3D model that mimics two new aspects of wing deformation: a non-fixed wing hinge and a twisting wing surface. The method is demonstrated for free and perturbed flight. Our method does not use prior assumptions on the kinematics apart from the continuity of one wing angle. Hence, this method can be readily adjusted for other insect species.

sentences: ILC homeostasis phenotyping in various tissues, aging, and sex differences Innate lymphoid cells are the innate counterpart to CD4+ T cells that are mediated by the same transcription factors and produce similar cytokines. ILCs are being investigated in many different disease states, but the field current lacks foundational information on ILC representation whether it be in tissues, between males and females, or in aging as these are all vital components in disease etiology and severity. Our descriptive study used flow cytometry to characterize ILCs compared to the entire CD45+ (e.g., lymphocyte) and lineage negative (e.g., ILC) compartments to understand their homeostatic balance and plasticity. Moreover, we defined ILC2 expression and subsets based on their cytokine production and created several mathematical models to elucidate the correlation of extra- and intra-cellular ILC2 markers from least to most complex. ILC studies would benefit from more unbiased, holistic experiments including RNA-seq and mass spectroscopy to further define ILCs in steady state before adding more complex pathways like different disease states to enhance translational value and therapeutic targeting of these cells.

sentences: Whole animal modelling reveals neuronal mechanisms of decision-making and reproduces unpredictable swimming in frog tadpoles Animal behaviour is based on interaction between nervous, musculoskeletal and environmental systems. How does an animal process sensory stimuli, use it to decide whether and how to respond, and initiate the locomotor behaviour? We build the whole body computer models of a simple vertebrate with a complete chain of neural circuits and body units for sensory information processing, decision-making, generation of spiking activities, muscle innervation, body flexion, body-water interaction, and movement. Our Central Nervous System (CNS) model generates biologically-realistic spiking and reveals that sensory memory populations on two hindbrain sides compete for swimming initiation and first body flexion. Biomechanical 3-dimensional "Virtual Tadpole" (VT) model is constructed to evaluate if motor outputs of CNS model can produce swimming-like movements in a volume of "water". We find that whole animal modelling generates reliable and realistic swimming. The combination of CNS and VT models opens a new perspective for experiments with immobilised tadpoles.

sentences: The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3 In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5 exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.

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Acquisition of ionic copper by a bacterial outer membrane protein Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how Gram-negative bacteria acquire ionic copper. Here we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed "methionine track" leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

SignificanceCopper is an essential metal in biology due to its role in the structure and function of many proteins. Despite this, it is not very clear how bacteria acquire copper, especially for Gram-negative organisms. In this study we show that the outer membrane protein OprC has an unusual metal binding site that allows OprC to bind both reduced and oxidised ionic copper near-irreversibly. Given the versatility of OprC, its presence in many Proteobacteria and its abundance during lung infection in mice, our study shows that OprC is an important component of prokaryote copper biology that warrants further study to uncover its regulation and to assess its role in bacterial virulence.

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Impact of stimulation location relative to grey and white matter on single pulse electrical stimulation responses in the human brain BackgroundElectrical neuromodulation is an increasingly common therapy for a wide variety of neuropsychiatric diseases. Unfortunately, therapeutic efficacy is inconsistent, possibly a result of our limited understanding of the mechanisms and the massive stimulation parameter space.

Objective/HypothesisTo better understand the role different parameters play in inducing a response, we systematically examined single pulse-induced cortico-cortico evoked potentials (CCEP) as a function of stimulation amplitude, duration and location in the brain and relative to grey and white matter.

MethodsWe measured voltage peak amplitudes and area under the curve of intracranially recorded stimulation responses as a function of distance from the stimulation site, pulse width, current injected, location relative to grey and white matter, and brain region stimulated (N=52, n=719 stimulation sites).

ResultsIncreasing stimulation pulse width increased response values near the stimulation location. Increasing stimulation amplitude (current) increased responses nonlinearly. Locally (<15 mm from the stimulation site), stimulation closer to the grey matter-white matter boundary induced larger responses. In contrast, for distant sites (>15 mm), white matter stimulation consistently produced larger responses than stimulation in or near grey matter. These relationships were different between cingulate, lateral prefrontal, and lateral temporal cortical stimulation.

ConclusionThese results demonstrate the importance of location and stimulation parameters in inducing a specific output and indicate that a stronger local response may require stimulation in the grey-white boundary while stimulation in the white matter may be needed for network activation, suggesting that stimulation location can be tailored for a specific outcome, key to informed neuromodulatory therapy.

HighlightsO_LIIntracranial single pulse electrical stimulation (SPES) response increases with increased pulse duration mostly near the stimulation site
C_LIO_LISPES response varies nonlinearly with injected current with an effect of distance from the stimulation site.
C_LIO_LISPES near the grey-white boundary and 90{degrees} to the nearest cortical axis induces larger local responses, but white matter stimulation produces larger distant responses.
C_LIO_LIThe relationship between SPES location and responses depends on brain region stimulated
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sentences: SMaSH: A scalable, general marker gene identification framework for single-cell RNA sequencing and Spatial Transcriptomics Spatial transcriptomics is revolutionising the study of single-cell RNA and tissue-wide cell heterogeneity, but few robust methods connecting spatially resolved cells to so-called marker genes from single-cell RNA sequencing, which generate significant insight gleaned from spatial methods, exist. Here we present SMaSH, a general computational framework for extracting key marker genes from single-cell RNA sequencing data for spatial transcriptomics approaches. SMaSH extracts robust and biologically well-motivated marker genes, which characterise the given data-set better than existing and limited computational approaches for global marker gene calculation.

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The memory of airway epithelium damage in smokers and COPD patients BackgroundChronic obstructive pulmonary disease (COPD) is a devastating lung disease, representing the third cause of mortality worldwide. In COPD, the bronchial epithelium displays several structural and functional abnormalities affecting barrier integrity, cell polarity, and differentiation, as well as epithelial-to-mesenchymal transition (EMT) and inflammation. Although COPD is currently considered an irreversible disease, the (ir)reversible nature of epithelium changes ex vivo remains poorly known.

MethodsThe persistence of COPD epithelial abnormalities was addressed in very long-term (10 weeks) primary cultures of air/liquid interface (ALI)-reconstituted airway epithelium from non-smoker controls, smoker controls, and COPD patients. The role of inflammation was also explored by stimulating ALI cultures with a cytokine mix consisting of TNF-, IL-6 and IL-1{beta}. Finally, the cellular niche holding epithelial memory was studied by exploiting a single cell RNA-sequencing database.

ResultsAlmost all epithelial defects (barrier dysfunction, impaired polarity, lineage abnormalities) observed in smokers and COPD patients persisted in vitro up to week 10, except IL-8/CXCL-8 release and EMT which declined over time. Cytokine treatment induced COPD-like changes and reactivated EMT in COPD cells. Progenitor cells of large and small airways, namely basal and club cells, exhibited EMT-related signatures reminiscent of features observed in situ.

ConclusionsThe airway epithelium from smokers and COPD patients displays a memory of its native state and previous injuries by cigarette smoking, which is multidimensional and sustained for years. This memory probably resides in progenitor cells of the airway epithelium.

sentences: Long-term quality assessment and monitoring of light microscope performance through accessible and reliable protocols, tools and metrics Reliable, reproducible and comparable results are what biology requires from microscopy. To achieve that level of confidence, monitoring the stability of the microscope performance over time with standardized quality testing routines is essential for mining quantitative data. Three levels of microscope quality control procedures should be considered: i) usage of accessible and affordable tools and samples, ii) execution of easy and fast, preferably automatized, acquisition protocols, iii) analysis of data in the most automated way possible with adequate metrics for long-term monitoring. In this paper, we test the acquisition protocols on the mainly used microscope techniques (wide-field, spinning disk and confocal microscopy) with simple quality control tools. Seven protocols specify metrics on measuring the lateral and axial resolution (Point-Spread Function) of the system, field flatness, chromatic aberrations and co-registration, illumination power monitoring and stability, stage drift and positioning repeatability and finally temporal and spatial noise sources of camera detectors. We designed an ImageJ/FiJi java plugin named MetroloJ_QC to incorporate the identified metrics and automatize the data processing for the analysis. After processing and comparing the data of microscopes from more than ten imaging facilities, we test the robustness of the metrics and the protocols by determining experimental limit values. Our results give a first extensive characterization of the quality control procedures of a light microscope, with an automated data processing and experimental limit values that can be used by core facility staff and researchers to monitor the microscope performance over time.

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Reward-specific satiety affects subjective value signals in orbitofrontal cortex during multi-component economic choice Sensitivity to satiety constitutes a basic requirement for neuronal coding of subjective reward value. Satiety from natural on-going consumption affects reward functions in learning and approach behavior. More specifically, satiety reduces the subjective economic value of individual rewards during choice between options that typically contain multiple reward components. The unconfounded assessment of economic reward value requires tests at choice indifference between two options, which is difficult to achieve with sated rewards. By conceptualizing choices between options with multiple reward components ( bundles), Revealed Preference Theory may offer a solution. Despite satiety, choices against an unaltered reference bundle may remain indifferent when the reduced value of a sated bundle reward is compensated by larger amounts of an unsated reward of the same bundle; then the value loss of the sated reward is indicated by the amount of the added unsated reward. Here we show psychophysically titrated choice indifference in monkeys between bundles of differently sated rewards. Neuronal chosen value signals in orbitofrontal cortex (OFC) followed closely the subjective value change within recording periods of individual neurons. A neuronal classifier distinguishing the bundles and predicting choice substantiated the subjective value change. Choice between conventional single rewards confirmed the neuronal changes seen with two-reward bundles. Thus, reward-specific satiety reduces subjective reward value signals in OFC. With satiety being an important factor of subjective reward value, these results extend the notion of subjective economic reward value coding in OFC neurons.

SignificanceOn-going consumption reduces the subjective value of rewards to different degrees depending on their individual properties, a phenomenon referred to as sensory-specific satiety. Such value change should be manifested in economic choices, and neuronal signals for subjective economic reward value should be sensitive to reward-specific satiety. We tested monkeys during choice between two options that each contained two different rewards ( bundles); the two rewards were prone to different degrees of satiety. On-going reward consumption affected choices in a way that indicated satiety-induced reward-specific change of subjective economic value. Neuronal responses in the monkey orbitofrontal cortex (OFC) followed the differential reduction of subjective economic value. These results satisfy a crucial requirement for subjective reward value coding in OFC neurons.

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Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, 1,3-fucosyltransferase and fucosyl 4- O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface- exposed lipid virulence factors.

IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

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N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus Several compounds have been tested against SARS-CoV-2; at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor- (PPAR-). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR- activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs.

Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF- production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.

sentences: Transcriptome analysis of alcohol dependence and stress interactions in the nucleus of the solitary tract Stress exposure contributes to the development of drug and alcohol use disorders. In animal models, stress exacerbates escalations in alcohol consumption in alcohol-dependent animals. The nucleus of the solitary tract (NTS) is a critical brainstem region for integrating and relaying peripheral signals to regulate stress responses. To define the molecular adaptions within this brain region that may contribute to stress-induced alcohol drinking, we exposed animals to chronic intermittent bouts of ethanol vapor (CIE), forced swim stress (FSS), or both (CIE + FSS) and then transcriptionally profiled the NTS at three different timepoints after the last vapor exposure (0-hr, 72-hr, and 186-hr). We identified interferon (IFN) signaling as a critical gene network correlated with alcohol consumption levels. Using a likelihood ratio test, we identified genes that were differentially expressed across time and between groups. Clustering analysis of these genes to identify unique expression patterns identified a subset of genes that fail to normalize in the CIE + FSS group, but not the others. These genes were enriched for cell-to-cell interaction and cellular movement pointing to long-term structural and functional changes in this brain region caused by the unique interaction of alcohol dependence and stress. Specific genes of interest identified in this group include Aqp4, Il16, Reln, Grm4, Gabrd, and Gabra6. We also compared gene expression changes in the NTS to the PFC and found a significant overlap of genes between the two brain regions. Overlapping NTS/PFC genes in the CIE + FSS group were enriched for type I IFN signaling. Finally, we tested the hypothesis that activation of type I IFN signaling increases alcohol consumption based on the three lines of evidence identifying type I IFN signaling as critical for escalations in alcohol intake. Mice treated with recombinant IFN{beta} showed significantly elevated levels of alcohol intake in a two-bottle choice procedure compared to saline-treated controls. Overall, these results define the transcriptomic changes across time in the NTS that may be critical to the development of stress-induced increases in alcohol consumption and alcohol dependence.

sentences: Reproducibility of in-vivo electrophysiological measurements in mice Understanding whole-brain-scale electrophysiological recordings will rely on the collective work of multiple labs. Because two labs recording from the same brain area often reach different conclusions, it is critical to quantify and control for features that decrease reproducibility. To address these issues, we formed a multi-lab collaboration using a shared, open-source behavioral task and experimental apparatus. We repeatedly inserted Neuropixels multi-electrode probes targeting the same brain locations (including posterior parietal cortex, hippocampus, and thalamus) in mice performing the behavioral task. We gathered data across 9 labs and developed a common histological and data processing pipeline to analyze the resulting large datasets. After applying stringent behavioral, histological, and electrophysiological quality-control criteria, we found that neuronal yield, firing rates, spike amplitudes, and task-modulated neuronal activity were reproducible across laboratories. To quantify variance in neural activity explained by task variables (e.g., stimulus onset time), behavioral variables (timing of licks/paw movements), and other variables (e.g., spatial location in the brain or the lab ID), we developed a multi-task neural network encoding model that extends common, simpler regression approaches by allowing nonlinear interactions between variables. We found that within-lab random effects captured by this model were comparable to between-lab random effects. Taken together, these results demonstrate that across-lab standardization of electrophysiological procedures can lead to reproducible results across labs. Moreover, our protocols to achieve reproducibility, along with our analyses to evaluate it are openly accessible to the scientific community, along with our extensive electrophysiological dataset with corresponding behavior and open-source analysis code.

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Development of a new militarily-relevant whole-body low-intensity blast model for mild and subconcussive traumatic brain injury: Examination of acute neurological and multi-organ pathological outcomes This work describes a newly developed experimental mouse model reproducing features of blast-induced neurotrauma (BINT), induced in operationally relevant manner using a compressed air-driven shock tube. Mild BINT (smBINT) was induced by one exposure to a low-intensity blast (LIB), whereas subconcussive BINT (rscBINT) was caused by repeated exposures to LIB.

To mimic an operational scenario when a soldier is standing when exposed to blast using a quadruped experimental animal (mouse), a whole-body holder was developed to position mice in a bipedal stance, face-on toward the pressure wave generated in a shock tube. This restraint avoids  bobble head movement, thus prevents tertiary blast effects, and allows administration of fast-acting inhaled anesthetics via nose cone.

Using this model, we established and validated paradigms for primary blast-induced mild and repetitive traumatic brain injuries Our results showed that a single exposure to 69 kPa (10 psi) was capable of inducing smBINT, whereas three-rounds of exposure to 41 kPa (6 psi) caused rscBINT.

Mice recovered rapidly from both types of BINT without prolonged neurological dysfunction. Mild superficial pathology was found predominantly in the lungs 24h after injury, with equivalent pathology after smBINT or repetitive rscBINT. The Purkinje layer of the cerebellum exhibited neuronal damage persisting up to 7d. Similar to some other models as well as clinical findings, this model reproduces blast-induced cerebellar pathology. In conclusion, this model positioning mice in a bipedal stance and facing front-on toward the shockwave provides realistic representation of operational scenarios and reproduces militarily-relevant smBINT and rscBINT in the laboratory.

sentences: Exposure to fruit-flavoring during adolescence increases nicotine consumption and promotes dose escalation The rise of e-cigarette popularity has sparked interest in the role of palatable flavors on nicotine use. Despite growing evidence that sweet flavorants enhance nicotine reward, their influence on nicotine consumption has not been studied extensively. In addition, the impact that flavored nicotine use in adolescence could have on nicotine reward and dependence in adulthood remains unclear. This study examined the role of flavored nicotine access on nicotine preference and consumption longitudinally, from adolescence to adulthood. Male and female adolescent mice preferred a fruit-flavored nicotine solution. However, only adolescent female mice with access to flavored nicotine consumed higher doses. Furthermore, while adolescent male mice escalated consumption of both flavored and unflavored nicotine, female mice only escalated when given access to flavored nicotine. As mice matured into adulthood, there was no evidence that a history of flavored-nicotine access altered preference for unflavored nicotine. However, when the nicotine concentration was reduced, mice that had consumed strawberry-flavored nicotine in adolescence maintained baseline nicotine consumption levels longer than mice that initiated nicotine use without flavor in adolescence. Finally, addition of fruit-flavorants into the nicotine solution during adulthood led to increased levels of nicotine consumption, regardless of previous flavored-nicotine access or of familiarity with the selected flavorant. These results indicate that flavorants increase nicotine consumption independent of life stage, possibly posing a disproportionate risk to adolescent females. Our results also point to an effect of adolescent flavored-nicotine use on nicotine dose maintenance in adulthood, which could have implications for the success of future quit attempts.

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Biome-scale woody encroachment threatens conservation potential and sustainability of U.S. rangelands O_LIRangelands of the United States provide ecosystem services that sustain biodiversity and rural economies. Native tree encroachment is a long-standing conservation challenge to these landscapes. Still, its impact is often overlooked due to the slow pace of tree invasions and the positive public perception of trees.
C_LIO_LIRecent innovations in satellite remote sensing permit the tracking of tree encroachment and the corresponding impact on herbaceous production. We analyzed tree cover change and herbaceous production across 17 western states from 1990 to 2019.
C_LIO_LIWe show that tree encroachment is a dominant change agent in western U.S. rangelands; tree cover has increased by 50% (77,323 km2) over 30 years, with more than 25% of U.S. rangelands experiencing sustained tree cover expansion. Since 1990, roughly 300 Tg of herbaceous biomass has been lost, totaling $5 billion in foregone revenue to small agricultural producers.
C_LIO_LISynthesis and applications: The impact of tree encroachment to rangeland loss is similar in magnitude to row-crop conversion, another primary threat to U.S. rangelands. Prioritizing conservation efforts to prevent tree encroachment can bolster ecosystem and economic sustainability, particularly among privately-owned lands threatened by land-use conversion.
C_LI

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Hierarchy-guided Neural Networks for Species Classification O_LISpecies classification is an important task that is the foundation of industrial, commercial, ecological, and scientific applications involving the study of species distributions, dynamics, and evolution.
C_LIO_LIWhile conventional approaches for this task use off-the-shelf machine learning (ML) methods such as existing Convolutional Neural Network (ConvNet) architectures, there is an opportunity to inform the ConvNet architecture using our knowledge of biological hierarchies among taxonomic classes.
C_LIO_LIIn this work, we propose a new approach for species classification termed Hierarchy-Guided Neural Network (HGNN), which infuses hierarchical taxonomic information into the neural networks training to guide the structure and relationships among the extracted features. We perform extensive experiments on an illustrative use-case of classifying fish species to demonstrate that HGNN outperforms conventional ConvNet models in terms of classification accuracy, especially under scarce training data conditions.
C_LIO_LIWe also observe that HGNN shows better resilience to adversarial occlusions, when some of the most informative patch regions of the image are intentionally blocked and their effect on classification accuracy is studied.
C_LI

sentences: SpeciesRax: A tool for maximum likelihood species tree inference from gene family trees under duplication, transfer, and loss. Species tree inference from gene family trees is becoming increasingly popular because it can account for discordance between the species tree and the corresponding gene family trees. In particular, methods that can account for multiple-copy gene families exhibit potential to leverage paralogy as informative signal. At present, there does not exist any widely adopted inference method for this purpose. Here, we present SpeciesRax, the first maximum likelihood method that can infer a rooted species tree from a set of gene family trees and can account for gene duplication, loss, and transfer events. By explicitly modelling events by which gene trees can depart from the species tree, SpeciesRax leverages the phylogenetic rooting signal in gene trees. SpeciesRax infers species tree branch lengths in units of expected substitutions per site and branch support values via paralogy-aware quartets extracted from the gene family trees. Using both empirical and simulated datasets we show that SpeciesRax is at least as accurate as the best competing methods while being one order of magnitude faster on large datasets at the same time. We used SpeciesRax to infer a biologically plausible rooted phylogeny of the vertebrates comprising 188 species from 31612 gene families in one hour using 40 cores. SpeciesRax is available under GNU GPL at https://github.com/BenoitMorel/GeneRax and on BioConda.

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A naturally arising broad and potent CD4-binding site antibody with low somatic mutation The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs exhibit extraordinary breadth and potency, but also rank among the most highly somatically-mutated bNAbs. Here we describe a VRC01-class antibody isolated from a viremic controller, BG24, that has less than half the mutations of most other relatives of its class, while achieving comparable breadth and potency. A 3.8 [A] X-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately-mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.

TeaserAn anti-HIV-1 antibody with comparable neutralization breadth and potency to similarly-classed antibodies, with half as many mutations.

sentences: Genome sequence of the aurodox-producing bacterium Streptomyces goldiniensis ATCC 21386 We report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71 %. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141.

sentences: Taurine or N-acetylcysteine treatments prevent memory impairment and metabolite profile alterations in the hippocampus of high-fat diet-fed female mice Obesity impacts brain metabolism and function, and constitutes a risk factor for dementia development. Long-term exposure to obesogenic diets leads to taurine accumulation in the hippocampus of rodents. Taurine has putative beneficial effects on cell homeostasis, and is reported to improve cognition. Therefore, hippocampal taurine accumulation in obese and diabetic models might constitute a counteracting response to metabolic stress. This study tested the hypothesis that treatment with taurine or with N-acetylcysteine (NAC), which provides cysteine for the synthesis of taurine and the antioxidant glutathione, prevent obesity-associated hippocampal alterations and memory impairment. Female mice were fed either a regular diet or a high-fat diet (HFD). For the same period, some mice had access to 3%(w/v) taurine or 3%(w/v) NAC in the drinking water. After 2 months, magnetic resonance spectroscopy was used to measure metabolite profiles. Then, memory was assessed in novel object and novel location recognition tests. HFD feeding caused memory impairment in both tests, and reduced concentration of lactate, phosphocreatine-to-creatine ratio, and the neuronal marker N-acetylaspartate in the hippocampus. Both taurine and NAC prevented HFD-induced memory impairment and N-acetylaspartate reduction. NAC, but not taurine, prevented the HFD-induced reduction of lactate and phosphocreatine-to-creatine ratio. Analysis of the metabolite profile further revealed prominent NAC/taurine-induced increase of glutamate and GABA levels in the hippocampus. We conclude that NAC and taurine prevent of neuronal dysfunction, while only NAC prevents energy metabolism dysregulation in HFD-induced obesity in female mice.

sentences: Experimental Control of a Reaction Occurring during the Interaction between Chicken Anemia Virus (CAV) and Its Corresponding Antibodies In our ongoing investigations, we have studied a specific interaction between electromagnetic fields and matter - the so-called Electromagnetic echo effect (EMEE). It enables rapid and contactless investigations of gases, liquids and solids to be performed, since the signal generated as a result of the effect is quite sensitive to all kinds of changes occurring within the studied samples. The effect can be considered universal for all matter and provides analysis in real time. We use this phenomenon to demonstrate the practical possibility to control reactions, occurring between Chicken anemia virus (CAV) and the corresponding antibodies. This methodology can be used for simple but reliable control of similar, otherwise hard to detect, antigen-antibody reactions, in order to confirm the presence of a certain viral species. The approach offers a high level of safety, since it enables measurements to be taken remotely, thus limiting exposure to contagion. We further discuss the possibility to register the presence of SARS-nCoV-2 in an attempt to address current global pandemic.

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AF-vapeR: A multivariate genome scan for detecting parallel evolution using allele frequency change vectors O_LIThe repeatability of evolution at the genetic level has been demonstrated to vary along a continuum from complete parallelism to divergence. In order to better understand why this continuum exists within and among systems, hypotheses must be tested using high confidence sets of candidate loci for repeatability. Despite this, few methods have been developed to scan SNP data for signatures specifically associated with repeatability, as opposed to local adaptation.
C_LIO_LIHere we present AF-vapeR (Allele Frequency Vector Analysis of Parallel Evolutionary Responses), an approach designed to identify genome regions exhibiting highly correlated allele frequency changes within haplotypes and among replicated allele frequency change vectors. The method divides the genome into windows of an equivalent number of SNPs, and within each window performs eigen decomposition over normalised allele frequency change vectors (AFV), each derived from a replicated pair of populations/species. Properties of the resulting eigenvalue distribution can be used to compare regions of the genome for those exhibiting strong parallelism, and can also be compared against a null distribution derived from randomly permuted AFV. Further, the shape of the eigenvalue distribution can reveal multiple axes of parallelism within datasets.
C_LIO_LIWe demonstrate the utility of this approach to detect different modes of parallel evolution using simulations, and also demonstrate a reduction in error rate compared with intersecting FST outliers. Lastly, we apply AF-vapeR to three previously published datasets (stickleback, guppies, and Galapagos finches) which comprise a range of sampling and sequencing strategies, and lineage ages. We detect known parallel regions whilst also identifying novel candidates.
C_LIO_LIThe main benefits of this approach include a reduced false-negative rate under many conditions, an emphasis on signals associated specifically with repeatable evolution as opposed to local adaptation, and an opportunity to identify different modes of parallel evolution at the first instance.
C_LI

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Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis.

We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from SSc patients deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This can be mimicked in healthy fibroblast stimulated by soluble factors that drive inflammation and fibrosis in SSc and is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling.

In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

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sentences: A metagenomic analysis of the wrackbed microbiome indicates a phylogeographic break along the North Sea - Baltic Sea transition zone Sandy beaches are biogeochemical hotspots that bridge marine and terrestrial ecosystems via the transfer of marine organic matter, such as seaweed (termed wrack). A keystone of this unique ecosystem is the microbial community, which helps to degrade wrack and re-mineralize nutrients. However, little is known about the wrackbed microbiome, its composition, trophic ecology, or how it varies over time and space. Here we characterize the wrackbed microbiome as well as the microbiome of a primary consumer, the seaweed fly Coelopa frigida, and examine how they change along one of the most studied ecological gradients in the world, the transition from the marine North Sea to the brackish Baltic Sea. We found that polysaccharide degraders dominated both the wrackbed and seaweed fly microbiomes but there were still consistent differences between wrackbed and fly samples. Furthermore, we observed a shift in both microbial communities and functionality between the North and Baltic Sea. These shifts were mostly due to changes in the frequency of different groups of known polysaccharide degraders (Proteobacteria and Bacteroidota). We hypothesize that microbes were selected for their abilities to degrade different polysaccharides corresponding to a shift in polysaccharide content in the seaweed communities of the North vs. Baltic Sea. Our results reveal the complexities of both the wracked microbial community, with different groups specialized to different roles, and the cascading trophic consequences of shifts in the near shore algal community.

sentences: Polygenic enrichment distinguishes disease associations of individual cells in single-cell RNA-seq data Gene expression at the individual cell-level resolution, as quantified by single-cell RNA-sequencing (scRNA-seq), can provide unique insights into the pathology and cellular origin of diseases and complex traits. Here, we introduce single-cell Disease Relevance Score (scDRS), an approach that links scRNA-seq with polygenic risk of disease at individual cell resolution without the need for annotation of individual cells to cell types; scDRS identifies individual cells that show excess expression levels for genes in a disease-specific gene set constructed from GWAS data. We determined via simulations that scDRS is well-calibrated and powerful in identifying individual cells associated to disease. We applied scDRS to GWAS data from 74 diseases and complex traits (average N =346K) in conjunction with 16 scRNA-seq data sets spanning 1.3 million cells from 31 tissues and organs. At the cell type level, scDRS broadly recapitulated known links between classical cell types and disease, and also produced novel biologically plausible findings. At the individual cell level, scDRS identified subpopulations of disease-associated cells that are not captured by existing cell type labels, including subpopulations of CD4+ T cells associated with inflammatory bowel disease, partially characterized by their effector-like states; subpopulations of hippocampal CA1 pyramidal neurons associated with schizophrenia, partially characterized by their spatial location at the proximal part of the hippocampal CA1 region; and subpopulations of hepatocytes associated with triglyceride levels, partially characterized by their higher ploidy levels. At the gene level, we determined that genes whose expression across individual cells was correlated with the scDRS score (thus reflecting co-expression with GWAS disease genes) were strongly enriched for gold-standard drug target and Mendelian disease genes.

sentences: Single Serine on TSC2 Exerts Biased Control over mTORC1 Activation by ERK1/2 but Not Akt The mammalian target of rapamycin complex 1 (mTORC1) is tightly controlled by tuberous sclerosis complex-2 (TSC2) that is regulated by phosphorylation from kinases responding to environmental cues. Protein kinase G specifically modifies serine-1365 (S1364, human), and its phosphorylation (or phosphomimetic SE mutant) potently blocks mTORC1 co-activation by pathological stress, while a phospho-silenced (SA) mutation does the opposite. Neither alter basal mTORC1 activity. Here we show S1365 exerts biased control over mTORC1 activity (S6K phosphorylation) modifying ERK1/2 but not Akt-dependent stimulation. Whereas mTORC1 activation by endothelin-1 is potently modified by S1365 status, insulin or PDGF stimulation are unaltered. TSC2-S1365 is also phosphorylated upon ET-1 but not insulin stimulation in a PKG-dependent manner, revealing intrinsic bias. Neither energy or nutrient modulation of mTORC1 are impacted by S1365. Consistent with these results, knock-in mice with either TSC2 SA or SE mutations develop identical obesity, glucose intolerance, and fatty liver disease from a high fat diet. Thus, S1365 provides an ERK1/2-selective mTORC1 control mechanism and a genetic means to modify pathological versus physiological mTOR stimuli.

sentences: Managing beach access and vehicle impacts following reconfiguration of the landscape by a natural hazard event After New Zealands 7.8 Mw Kaik[o]ura earthquake in late 2016 an unexpected anthropogenic effect involved increased motorised vehicle access to beaches. We show how these effects were generated by landscape reconfiguration associated with coastal uplift and widening of high-tide beaches, and present analyses of the distribution of natural environment values in relation to vehicle movements and impacts. Access changes led to extensive vehicle tracking in remote areas that had previously been protected by natural barriers. New dunes formed seaward of old dunes and have statutory protection as threatened ecosystems, yet are affected by vehicle traffic. Nesting grounds of nationally vulnerable banded dotterel (Charadrius bicinctus bicinctus) co-occur with vehicle tracking. An artificial nest experiment showed that vehicle strikes pose risks to nesting success, with 91% and 83% of nests destroyed in high and moderate-traffic areas, respectively, despite an increase in suitable habitat. Despite gains for recreational vehicle users there are serious trade-offs with environmental values subject to legal protection and associated responsibilities for management authorities. In theory, a combination of low-impact vehicle access and environmental protection could generate win-win outcomes from the landscape changes, but is difficult to achieve in practice. Detailed information on sensitive areas would be required to inform designated vehicle routes as a potential solution, and such sensitivities are widespread. Alternatively, vehicle access areas that accommodate longstanding activities such as boat launching could be formally established using identified boundaries to control impacts further afield. Difficulties for the enforcement of regulatory measures in remote areas also suggest a need for motivational strategies that incentivise low-impact behaviours. We discuss options for user groups to voluntarily reduce their impacts, the importance of interactions at the recreation-conservation nexus, and need for timely impact assessments across the social-ecological spectrum after physical environment changes -- all highly transferable principles for other natural hazard and disaster recovery settings worldwide.

sentences: TOR represses stress responses through global regulation of H3K27 trimethylation in plants Target of Rapamycin (TOR) functions as a central sensory hub to link a wide range of external stimuli to gene expression. However, the mechanisms underlying stimulus-specific transcriptional reprogramming by TOR remains elusive. Our in silico analysis in Arabidopsis demonstrates that TOR-repressed genes are associated with either bistable or silent chromatin states. Both states regulated by TOR signaling pathway are associated with high level of H3K27me3 deposited by CURLY LEAF (CLF) in specific context with LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Combinations of epigenetic modifications H3K4me3 and H3K27me3 implicate bistable feature which alternates between on and off state allowing rapid transcriptional changes upon external stimuli. Chromatin remodeler SWI2/SNF2 ATPase BRAHMA (BRM) activates TOR-repressed genes only at bistable chromatin domains to rapidly induce biotic stress responses. Here we demonstrated both in silico and in vivo that TOR represses transcriptional stress responses through global maintenance of H3K27me3.

sentences: TopoStats - a program for automated tracing of biomolecules from AFM images We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, 90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required.

sentences: A genetically-defined population in the lateral and ventrolateral periaqueductal gray selectively promotes flight to safety When encountering external threats, survival depends on the engagement of appropriate defensive reactions to minimize harm. There are major clinical implications for identifying the neural circuitry and activation patterns that produce such defensive reactions, as maladaptive overactivation of these circuits underlies pathological human anxiety and fear responses. A compelling body of work has linked activation of large glutamatergic neuronal populations in the midbrain periaqueductal gray (PAG) to defensive reactions such as freezing, flight and threat-induced analgesia. These pioneering data have firmly established that the overarching functional organization axis of the PAG is along anatomically-defined columnar boundaries. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains a diverse arrangement of cell types that vary in neurochemical profile and location. How these cell types contribute to defensive responses remains largely unknown, indicating that targeting sparse, genetically-defined populations can lead to a deeper understanding of how the PAG generates a wide array of behaviors. Though several prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (cck) cells selectively causes flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-cck cells reduces avoidance of a predatory threat without altering other defensive behaviors like freezing. Lastly, l/vlPAG-cck activity increases away from threat and during movements towards safer locations. In contrast, activating l/vlPAG cells pan-neuronally promoted freezing and these cells were activated near threat. These data underscore the importance of investigating genetically-identified PAG cells. Using this approach, we found a sparse population of cck-expressing l/vlPAG cells that have distinct and opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar anatomical boundaries. Thus, in addition to the anatomical columnar architecture of the APG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.

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Structural Covariance Networks in Post-Traumatic Stress Disorder: A Multisite ENIGMA-PGC Study IntroductionCortical thickness (CT) and surface area (SA) are established biomarkers of brain pathology in posttraumatic stress disorder (PTSD). Structural covariance networks (SCN) constructed from CT and SA may represent developmental associations, or unique interactions between brain regions, possibly influenced by a common causal antecedent. The ENIGMA-PGC PTSD Working Group aggregated PTSD and control subjects data from 29 cohorts in five countries (n=3439).

MethodsUsing Destrieux Atlas, we built SCNs and compared centrality measures between PTSD subjects and controls. Centrality is a graph theory measure derived using SCN.

ResultsNotable nodes with higher CT-based centrality in PTSD compared to controls were left fusiform gyrus, left superior temporal gyrus, and right inferior temporal gyrus. We found sex-based centrality differences in bilateral frontal lobe regions, left anterior cingulate, left superior occipital cortex and right ventromedial prefrontal cortex (vmPFC). Comorbid PTSD and MDD showed higher CT-based centrality in the right anterior cingulate gyrus, right parahippocampal gyrus and lower SA-based centrality in left insular gyrus.

ConclusionUnlike previous studies with smaller sample sizes ([≤]318), our study found differences in centrality measures using a sample size of 3439 subjects. This is the first cross-sectional study to examine SCN interactions with age, sex, and comorbid MDD. Although limited to group level inferences, centrality measures offer insights into a nodes relationship to the entire functional connectome unlike approaches like seed-based connectivity or independent component analysis. Nodes having higher centrality have greater structural or functional connections, lending them invaluable for translational treatments like neuromodulation.

sentences: High-throughput Oligopaint screen identifies druggable regulators of genome folding Although the molecular rules governing genome organization are being quickly elucidated, relatively few proteins regulating this process have been identified. To address this gap, we developed a fully automated imaging pipeline, called HiDRO (high-throughput DNA or RNA labeling with optimized Oligopaints), that permits quantitative measurement of chromatin interactions across a large number of samples. Using HiDRO, we screened the human druggable genome and identified >300 factors that regulate chromatin folding during interphase, including 43 validated hits that either increase or decrease interactions between topological associating domains (TADs). We discovered that genetic or chemical inhibition of the ubiquitous kinase GSK3A enhances long-range interactions by dysregulating cohesin-mediated chromatin looping. Collectively, these results highlight a noncanonical role for GSK3A signaling in nuclear architecture and underscore the broader utility of HiDRO-based screening to identify novel mechanisms that drive the spatial organization of the genome.

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Serum and sputum MMP-9/TIMP-1 in winter sports athletes and swimmers: relationships with airway function IntroductionCross-country skiers and swimmers present characteristics of airway inflammation and remodeling of the extracellular matrix similar to what is observed in mild asthma. We aimed to compare serum and sputum MMP-9/TIMP-1 levels, to assess the balance between airway fibrogenesis and inflammation process in both categories of athletes, and to observe its seasonal variations in winter sports athletes.

MethodsCompetitive winter sports athletes (n=41), swimmers (n=25) and healthy nonathletes (n=10) had blood sampling, lung function measurement, skin prick tests, eucapnic voluntary hyperpnea challenge, methacholine inhalation test (MIT), and induced sputum analysis. Twelve winter sport athletes performed the test during both summer and winter. Serum and sputum MMP-9 and TIMP-1 were measured by ELISA.

ResultsNo significant difference in serum or sputum MMP-9/TIMP-1 ratio was observed between groups, nor relationship with airway function or responsiveness. Serum MMP-9/TIMP-1 ratio was significantly higher during the summer in winter sport athletes compared with winter season (median [Interquartile range]: 3.65 [2.47-4.03] ng.ml-1 and 1.27 [0.97-1.62] ng.ml-1, respectively, p=0.005). Sputum MMP-9 correlated with Methacholine PC20 and serum CC16/SP-D ratio.

ConclusionMMP-9/TIMP-1 ratio in sputum or serum may fluctuate with training or environment but does not correlate with airway lung function or responsiveness in competitive athletes.

sentences: Spontaneous neural synchrony links intrinsic spinal sensory and motor networks during unconsciousness Non-random functional connectivity during unconsciousness is a defining feature of supraspinal networks. However, its generalizability to intrinsic spinal networks remains incompletely understood. Previously, Barry et al. (2014) used fMRI to reveal bilateral resting state functional connectivity within sensory-dominant and, separately, motor-dominant regions of the spinal cord. Here, we record spike trains from large populations of spinal interneurons in vivo in rats and demonstrate that spontaneous functional connectivity also links sensory- and motor-dominant regions during unconsciousness. The spatiotemporal patterns of connectivity could not be explained by latent afferent activity or by populations of interconnected neurons spiking randomly. We also document connection latencies compatible with mono- and di-synaptic interactions and putative excitatory and inhibitory connections. The observed activity is consistent with the hypothesis that salient, experience-dependent patterns of neural transmission introduced during behavior or by injury/disease are reactivated during unconsciousness. Such a spinal replay mechanism could shape circuit-level connectivity and ultimately behavior.

sentences: Discovery and synthesis of hydroxy-L-proline based blockers of the neutral amino acid transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2). The conformationally restricted heterocycle hydroxy-L-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened a series of hydroxy-L-proline derivatives or  prolinols using electrophysiological and radio-labeled uptake assays on amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We identified a number of synthetic prolinols that act as selective high-affinity inhibitors of the SLC1 functional subfamily comprising the neutral amino acid transporters SLC1A4 and SLC1A5. The active and inactive prolinols were computationally docked into a threaded homology model and analyzed with respect to predicted molecular orientation and observed pharmacological activity. The series of hydroxy-L-proline derivatives identified here represents a new class of potential agents to pharmacologically modulate SLC1A4 and SLC1A5, amino acid exchangers that play important roles in a wide range of physiological and pathophysiological processes.

sentences: Machine learning-based library design improves packaging and diversity of adeno-associated virus (AAV) libraries AAVs hold tremendous promise as delivery vectors for clinical gene therapy. Yet the ability to design libraries comprising novel and diverse AAV capsids, while retaining the ability of the library to package DNA payloads, has remained challenging. Deep sequencing technologies allow millions of sequences to be assayed in parallel, enabling large-scale probing of fitness landscapes. Such data can be used to train supervised machine learning (ML) models that predict viral properties from sequence, without mechanistic knowledge. Herein, we leverage such models to rationally trade-off library diversity with packaging capability. In particular, we show a proof-of-principle application of a general approach for ML-guided library design that allows the experimenter to rationally navigate the trade-off between sequence diversity and fitness of the library. Consequently, this approach, instantiated with an AAV capsid library designed for packaging, enables the selection of starting libraries that are more likely to yield success in downstream selections for therapeutics and beyond. We demonstrated this increased success by showing that the designed libraries are able to more easily infect primary human brain tissue. We expect that such ML-guided design of AAV libraries will have broad utility for the development of novel variants for therapeutic applications in the near future.

sentences: Genome assemblies of three closely related leaf beetle species (Galerucella spp). Galerucella (Coleoptera: Chrysomelidae) is a leaf beetle genus that has been extensively used for ecological and evolutionary studies. It has also been used as biological control agent against invading purple loosestrife in North America, with large effects on biodiversity. Here we report genome assembly and annotation of three closely related Galerucella species: G. calmariensis, G. pusilla and G. tenella. The three assemblies have a genome size ranging from 460Mb to 588Mb, with N50 from 31,588kb to 79.674kb, containing 29,202 to 40,929 scaffolds. Using an ab initio evidence-driven approach, 30,302 to 33,794 protein-coding genes were identified and functionally annotated. These draft genomes will contribute to the understanding of host-parasitoid interactions, evolutionary comparisons of leaf beetle species and future population genomics studies.

sentences: Applying And Promoting Open Science In Ecology - Surveyed Drivers And Challenges Open Science (OS) comprises a variety of practices and principles that are broadly intended to improve the quality and transparency of research, and the concept is gaining traction. Since OS has multiple facets and still lacks a unifying definition, it may be interpreted quite differently among practitioners. Moreover, successfully implementing OS broadly throughout science requires a better understanding of the conditions that facilitate or hinder OS engagement, and in particular, how practitioners learn OS in the first place. We addressed these issues by surveying OS practitioners that attended a workshop hosted by the Living Norway Ecological Data Network in 2020. The survey contained scaled-response and open-ended questions, allowing for a mixed-methods approach. Out of 128 registered participants we obtained survey responses from 60 individuals. Responses indicated usage and sharing of data and code, as well as open access publications, as the OS aspects most frequently engaged with. Men and those affiliated with academic institutions reported more frequent engagement with OS than women and those with other affiliations. When it came to learning OS practices, only a minority of respondents reported having encountered OS in their own formal education. Consistent with this, a majority of respondents viewed OS as less important in their teaching than in their research and supervision. Even so, many of the respondents suggestions for what would help or hinder individual OS engagement included more knowledge, guidelines, resource availability and social and structural support; indicating that formal instruction can facilitate individual OS engagement. We suggest that the time is ripe to incorporate OS in teaching and learning, as this can yield substantial benefits to OS practitioners, student learning, and ultimately, the objectives advanced by the OS movement.

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Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

Significance StatementCellulases are multi-modular enzymes produced by numerous microbes that catalyze cellulose hydrolysis into glucose. These enzymes play an important role in global carbon cycling as well as cellulosic biofuels production. CBMs are essential components of cellulolytic enzymes involved in facilitating hydrolysis of polysaccharides by tethered catalytic domains (CD). The subtle interplay between CBM binding and CD activity is poorly understood particularly for heterogeneous reactions at solid-liquid interfaces. Here, we report a highly multiplexed single-molecule force spectroscopy method to study CBM dissociation from cellulose to infer the molecular mechanism governing substrate recognition and dissociation. This approach can be broadly applied to study multivalent protein-polysaccharide binding interactions relevant to other carbohydrates such as starch, chitin, or hyaluronan to engineer efficient biocatalysts.

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Landscape management for grassland multifunctionality Land-use intensification has contrasting effects on different ecosystem services, often leading to land-use conflicts. While multiple studies have demonstrated how landscape-scale strategies can minimise the trade-off between agricultural production and biodiversity conservation, little is known about which land-use strategies maximise the landscape-level supply of multiple ecosystem services (landscape multifunctionality), a common goal of stakeholder communities.

We combine comprehensive data collected from 150 German grassland sites with a simulation approach to identify landscape compositions, with differing proportions of low-, medium-, and high-intensity grasslands, that minimise trade-offs between the six main grassland ecosystem services prioritised by local stakeholders: biodiversity conservation, aesthetic value, productivity, carbon storage, foraging, and regional identity. Results are made accessible through an online tool that provides information on which compositions best meet any combination of user-defined priorities (https://neyret.shinyapps.io/landscape_composition_for_multifunctionality/).

Results show that an optimal landscape composition can be identified for any pattern of ecosystem service priorities. However, multifunctionality was similar and low for all landscape compositions in cases where there are strong trade-offs between services (e.g. aesthetic value and fodder production), where many services were prioritised, and where drivers other than land use played an important role. We also found that if moderate service levels are deemed acceptable, then strategies in which both high and low intensity grasslands are present can deliver landscape multifunctionality. The tool presented can aid informed decision-making by predicting the impact of future changes in landscape composition, and by allowing for the relative roles of stakeholder priorities and biophysical trade-offs to be understood by scientists and practitioners alike.

HighlightsO_LIAn online tool identifies optimal landscape compositions for desired ecosystem services
C_LIO_LIWhen the desired services are synergic, the optimum is their common best landscape composition
C_LIO_LIWhen the desired services trade-off, a mix of grassland intensity is most multifunctional
C_LIO_LISuch tools could support decision-making processes and aid conflict resolution
C_LI

Graphical abstract

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sentences: Macrophage-produced peroxynitrite induces antibiotic tolerance and supersedes intrinsic mechanisms of persister formation Staphylococcus aureus is a leading human pathogen that frequently causes chronic and relapsing infections. Antibiotic tolerant persister cells contribute to frequent antibiotic failure in patients. Macrophages represent an important niche during S. aureus bacteremia and recent work has identified a role for oxidative burst in the formation of antibiotic tolerant S. aureus. We find that host-derived peroxynitrite, the reaction product of superoxide and nitric oxide, is the main mediator of antibiotic tolerance in macrophages. Using a collection of S. aureus clinical isolates, we find that, despite significant variation in persister formation in pure culture, all strains were similarly enriched for antibiotic tolerance following internalization by activated macrophages. Our findings suggest that host interaction strongly induces antibiotic tolerance and may negate bacterial mechanisms of persister formation, established in pure culture. These findings emphasize the importance of studying antibiotic tolerance in the context of bacterial interaction with the host suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.

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Increasing age is independently associated with higher free water in non-active MS brain - A multi-compartment analysis using FAST-T2 PurposeTo explore the relationship between the cerebral cortical perivascular space (PVS) and aging in non-active MS subjects by using the multi-echo T2 relaxometry based cerebrospinal fluid fraction (CSFF) map.

MethodsMulti-echo spiral T2 data from 111 subjects with non-active multiple sclerosis (MS) were retrospectively investigated by fitting the T2 data into a three-compartment model, the three water compartments including myelin water, intra-extracellular water, and cerebrospinal fluid. Segmentation of T1w image was performed to get the region of interest (ROI) in cerebral cortical regions. The white matter lesion segmentation was conducted using a convolutional neural network (CNN) based segmentation tool. The CSFF in the ROIs were correlated with age by controlling the gender, white matter hyperintensity lesion burden, and MS disease duration. Multiple linear models were created for the analysis of aging effect on the CSFF.

ResultsThe ROI analysis shows that the CSFF in the cerebral cortical regions (temporal, occipital, parietal, front, hippo, and mtl) are significantly linear increasing with age (p<0.01). The intra-extracellular water fraction (IEWF) in the ROIs are significantly linear decreasing (p<0.01).

ConclusionThe multi-echo T2 based three-compartment model can be used to quantify the CSFF. The linear increase of CSF water contents in the cerebral cortical regions indicates increased perivascular space load in cortex with aging. The quantification of CSFF may provide a way to understand the glymphatic clearance function in aging and neurodegenerations.

HighlightsO_LIMR T2 relaxometry is a valid method to quantify the cerebrospinal fluid fraction (CSFF) in cerebral cortical regions
C_LIO_LIThe CSFF in the cerebral cortical regions are positively correlated with age by controlling the white matter lesion load in non-active MS subjects.
C_LIO_LIQuantification of cerebral CSFF may reflect the perivascular space load in cortex and better interpret the disease progression in neurodegenerative disease, such as MS.
C_LI

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The genes controlling normal function of citrate and spermine secretion is lost in aggressive prostate cancer and prostate model systems BackgroundSecretion of the metabolites citrate and spermine into prostate lumen is a unique hallmark for normal prostate epithelial cells. However, the identity of the genes controlling citrate and spermine secretion remains mostly unknown despite their obvious relevance for progression to aggressive prostate cancer.

Materials & MethodsIn this study, we have correlated simultaneous measurement of citrate/spermine and transcriptomics data. We have refined these gene correlations in 12 prostate cancer cohorts containing 2915 tissue samples to create a novel gene signature of 150 genes connected with citrate and spermine secretion. We further explored the signature in public data, interrogating over 18 000 samples from various tissues and model systems, including 3826 samples from prostate and prostate cancer.

ResultsIn prostate cancer, the expression of this gene signature is gradually lost in tissue from normal epithelial cells through PIN, low grade (Gleason <= 7), high grade cancer (Gleason >= 8) and metastatic lesions. The accuracy of the signature is validated by its unique enrichment in prostate compared to other tissues, and its strong enrichment in epithelial tissue compartments compared to stroma. Several zinc-binding proteins that are not previously investigated in the prostate are present in the gene signature, suggesting new mechanisms for controlling zinc homeostasis in citrate/spermine secretion. However, the absence of the gene signature in all common prostate normal and cancer cell-lines, as well as prostate organoids, underlines the challenge to study the role of these genes during prostate cancer progression in model systems.

ConclusionsA large collection of transcriptomics data integrated with metabolomics identifies the genes related to citrate and spermine secretion in the prostate, and show that the expression of these genes gradually decreases on the path towards aggressive prostate cancer. In addition, the study questions the relevance of currently available model systems to study metabolism in prostate cancer development.

sentences: Internal oligo(dT) priming in bulk and single cell RNA sequencing Significant advances in RNA sequencing have been recently made possible by the use of oligo(dT) primers for simultaneous mRNA enrichment and reverse transcription priming. The associated increase in efficiency has enabled more economical bulk RNA sequencing methods as well as the advent of high throughput single cell RNA sequencing, now already one of the most widely adopted new methods in the study of transcriptomics. However, the effects of off-target oligo(dT) priming on gene expression quantification have not been fully appreciated. In the present study, we describe the extent, the possible causes, and the consequences of internal oligo(dT) priming across multiple publicly available datasets obtained from a variety of bulk and single cell RNA sequencing platforms. In order to explore and address this issue, we developed a computational algorithm for identification of sequencing read alignments that likely resulted from internal oligo(dT) priming and their subsequent removal from the data. Directly comparing filtered datasets to those obtained by an alternative method reveals significant improvements in gene expression measurement. Finally, we infer a list of genes whose expression quantification is most likely to be affected by internal oligo(dT) priming.

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VSTseed: periodic spaced seeds for reads with substitutions MotivationTechnical progress in computer hardware made it possible to access and process large amounts of data even on budget workstations. Therefore new or existing alignment algorithms may use large index files to increase performance. Spaced seeds with large weights reduce the number of possible locations of a read within a reference sequence. Optimal patterns for spaced seeds may guarantee to align reads even with several substitutions.

ResultsFor reads of 64-200 bp periodic spaced seeds of 32, 40, 48, 56, 64 weights are found that guarantee to locate all positions within a reference sequence for a specified number of point mutations. SIMD instructions to convert masked reads into 64, 80, 96, 112, 128-bit numbers are provided.

AvailabilityC codes to generate spaced seeds and find optimal SIMD instructions for them are freely available under MIT license at https://github.com/vtman/VSTseed

sentences: LmTag: functional-enrichment and imputation-aware tag SNP selection for population-specific genotyping arrays Despite the rapid development of sequencing technology, single-nucleotide polymorphism (SNP) array is still the most cost-effective genotyping solutions for large-scale genomic research and applications. Recent years have witnessed the rapid development of numerous genotyping platforms of different sizes and designs, but population-specific platforms are still lacking, especially for those in developing countries. We aim to develop methods to design SNP arrays for thse countries, so the arrays should be cost-effective (small size), yet can still generate key information needed to associate genotypes with traits. A key design principle for most current platforms is to improve genome-wide imputation so that more SNPs (imputed tag SNPs) not included in the array can be predicted. However, current tag SNP selection methods mostly focus on imputation accuracy and coverage, but not the functional content of the measured and imputed SNPs. It is those functional SNPs that are most likely associated to traits. Here, we propose LmTag, a novel method for tag SNP selection that not only improves imputation performance but also prioritizes highly functional SNP markers. We apply LmTag on a wide range of populations using both public and in-house whole genome sequencing databases. Our results showed that LmTag improved both functional marker prioritization and genome-wide imputation accuracy compared to existing methods. This novel approach could contribute to the next generation genotyping arrays that provide excellent imputation capability as well as facilitate array-based functional genetic studies. Such arrays are particularly suitable for under-represented populations in developing countries or non-model species, where little genomics data are available while investment in genome sequencing or high-density SNP arrays is limited.

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

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Disentangling heterogeneity of Malignant Pleural Mesothelioma through deep integrative omics analyses Malignant Pleural Mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Using the largest series of whole-genome sequencing data integrated with transcriptomic and epigenomic data using multi-omic factor analysis, we demonstrate that MPM heterogeneity arises from four sources of variation: tumor cell morphology, ploidy, adaptive immune response, and CpG island methylator phenotype. Previous genomic studies focused on describing only the tumor cell morphology factor, although we robustly find the three other sources in all publicly available cohorts. We prove how these sources of variation explain the biological functions performed by the cancer cells, and how genomic events shape MPM molecular profiles. We show how these new sources of variation help understand the heterogeneity of the clinical behavior of MPM and drug responses measured in cell lines. These findings unearth the interplay between MPM functional biology and its genomic history, and ultimately, inform classification, prognostication and treatment.

Graphical abstract

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sentences: Chromomycin A5 induces bonafide immunogenic cell death in metastatic melanoma Some first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA5-8). B16-F10, a metastatic melanoma cell line, treated with CA5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA5-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2 and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bonafide ICD induction by CA5 was confirmed by a C57BL/6 mice vaccination assay with CA5-treated cells. These findings support a high potential of CA5 as an anticancer candidate against AMM.

sentences: SmMIP-tools: a computational toolset for processing and analysis of single-molecule molecular inversion probes derived data Single-molecule molecular inversion probes (smMIPs) provides a modular and cost-effective platform for high-multiplex targeted next-generation sequencing (NGS). Nevertheless, translating the raw smMIP-derived sequencing data into accurate and meaningful information currently requires proficient computational skills and a large amount of computational work, prohibiting wide-scale adoption of smMIP-based technologies. To enable easy, efficient, and accurate interrogation of smMIP-derived data, we developed SmMIP-tools, a computational toolset that combines the critical analytic steps for smMIP data interpretation into a single computational pipeline. Here, we describe in detail two of the softwares major components. The first is a read processing tool that performs quality control steps, generates read-smMIP linkages and retrieves molecular tags. The second is an error-aware variant caller capable of detecting single nucleotide variants (SNVs) and short insertions and deletions (indels). Using a cell-line DNA dilution series and a cohort of blood cancer patients, we benchmarked SmMIP-tools and evaluated its performance against clinical sequencing reports. We anticipate that SmMIP-tools will increase accessibility to smMIP-technology, enabling cost-effective genetic research to push personalized medicine forward.

sentences: Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve novel dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling-edge pulse-detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile-signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.

sentences: Metabolomics of aging in primary fibroblasts from small and large breed dogs Among several animal groups (eutherian mammals, birds, reptiles) lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in co-enzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small versus large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

sentences: Evolution and connectivity influence the persistence and recovery of coral reefs under climate change in the Caribbean, Southwest Pacific, and Coral Triangle Corals are experiencing unprecedented decline from climate change-induced mass bleaching events. Dispersal not only contributes to coral reef persistence through demographic rescue but can also hinder or facilitate evolutionary adaptation. Locations of reefs that are likely to survive future warming therefore remain largely unknown, particularly within the context of both ecological and evolutionary processes across complex seascapes that differ in temperature range, strength of connectivity, network size, and other characteristics. Here, we used eco-evolutionary simulations to examine coral adaptation to warming across reef networks in the Caribbean, the Southwest Pacific, and the Coral Triangle. We assessed the factors associated with coral persistence in multiple reef systems to understand which results are general and which are sensitive to particular geographic contexts. We found that evolution can be critical in preventing extinction and facilitating the long-term recovery of coral communities in all regions. Furthermore, the strength of immigration to a reef (destination strength) and current sea surface temperature robustly predicted reef persistence across all reef networks and across temperature projections. However, we found higher initial coral cover, slower recovery, and more evolutionary lag in the Coral Triangle, which has a greater number of reefs and more larval settlement than the other regions. We also found the lowest projected future coral cover in the Caribbean. These findings suggest that coral reef persistence depends on ecology, evolution, and habitat network characteristics, and that, under an emissions stabilization scenario (RCP 4.5), recovery may be possible over multiple centuries.

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Personalized single-cell networks: a framework to predict the response of any gene to any drug for any patient BackgroundThe last decade has seen a major increase in the availability of genomic data. This includes expert-curated databases that describe the biological activity of genes, as well as high-throughput assays that measure gene expression in bulk tissue and single cells. Integrating these heterogeneous data sources can generate new hypotheses about biological systems. Our primary objective is to combine population-level drug-response data with patient-level single-cell expression data to predict how any gene will respond to any drug for any patient.

MethodsWe take 2 approaches to benchmarking a "dual-channel" random walk with restart (RWR) for data integration. First, we evaluate how well RWR can predict known gene functions from single-cell gene co-expression networks. Second, we evaluate how well RWR can predict known drug responses from individual cell networks. We then present two exploratory applications. In the first application, we combine the Gene Ontology database with glioblastoma single cells from 5 individual patients to identify genes whose functions differ between cancers. In the second application, we combine the LINCS drug-response database with the same glioblastoma data to identify genes that may exhibit patient-specific drug responses.

ConclusionsOur manuscript introduces two innovations to the integration of heterogeneous biological data. First, we use a "dual-channel" method to predict up-regulation and down-regulation separately. Second, we use individualized single-cell gene co-expression networks to make personalized predictions. These innovations let us predict gene function and drug response for individual patients. Taken together, our work shows promise that single-cell co-expression data could be combined in heterogeneous information networks to facilitate precision medicine.

sentences: MHC-I binding affinity derived metrics fail to predict tumor specific neoantigen immunogenicity Tumor-specific antigens emerging through somatic genomic rearrangements, known as neoantigens, play a critical role in current anticancer immunotherapy. They may or may not elicit an immune response when presented on the tumor cell surface bound to the MHC-I molecule, whose strength has been assumed as an indicator of immunogenicity. Several in silico peptide-MHC-I binding affinity predictors are used to prioritize putative immunogenic neoantigens to be experimentally and clinically explored either as biomarkers or targets for anticancer vaccines. This claims for a fair evaluation of such predictors, making essential the development of appropriate databases with experimentally validated, immunogenic/non-immunogenic neoantigens. Thus far, such a database is lacking. We herein present ITSNdb, a new and curated immunogenic neoantigen database and use it to benchmark current neoantigen immunogenicity predictors. Benchmark results failed to support the application of the predicted peptide- MHC-I binding affinity or its derived metrics as a tool to estimate neoantigen immunogenicity and the tumor neoantigen burden as an immunotherapy response biomarker. Moreover, binding affinity based immunogenicity definition leads to identifying wild-type peptide counterparts as predictors of immunotherapy response. We demonstrate that MHC-I binding affinity is insufficient to define neoantigen immunogenicity, despite being necessary for neoantigen tumor cell presentation suggesting that a paradigm shift for the emergence of new rules to identify immunogenic neoantigens is required.

sentences: Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals Neurons draw on alternative splicing for their increased transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. We found that in Drosophila, this splicing program is restricted to neurons by the post-transcriptional processing of the enhancer of microexons (eMIC) domain in Srrm234 by Elav and Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell-type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. Remarkably, we found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient post-transcriptional programs can evolve independently in different phyla to impact distinct cellular modules while maintaining cell-type specificity.

sentences: Rhes Deletion Prevents Age-Dependent Selective Motor Deficits and Reduces Phosphorylation of S6K in Huntington Disease Hdh150Q(CAG) Knock-In Mice. Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (mHTT) protein. This expansion is thought to promote striatal atrophy by a combination of cell- and non-cell-autonomous processes, but the mechanisms are unclear. Previous evidence suggests that the striatal-enriched SUMO E3-like protein Rhes could play a pathological role in HD. Rhes interacts with, and SUMOylates, mHTT and promotes toxicity and Rhes deletion ameliorates the HD phenotype in cell and severe mouse models of HD. However, the effect of Rhes on less severe knock-in models of HD remains obscure. Here, we report that a Hdh(CAG)150 knock-in murine model of HD showed diminished body weight but no changes in locomotor coordination or activity at 80 and 100 weeks of age. Conversely, Rhes deletion did not impact the body weight or behaviors but caused a significant reduction of gait, clasping, and tremor behaviors in Hdh150Q/150Q mice. Rhes deletion did not affect the loss of striatal DARPP-32 protein levels but abrogated the hyper ribosomal protein S6 kinase beta-1 (S6K) phosphorylation, which is a substrate for a mechanistic target of rapamycin complex 1 (mTORC1) signaling, in Hdh(CAG)150 mice. Interestingly, striatal Rhes protein levels were downregulated in the striatum of Hdh(CAG)150 mice, indicating a potential compensatory mechanisms at work. Thus, Rhes deletion prevents age-dependent behavioral deficits and diminishes hyperactive mTORC1-S6K signaling in Hdh(CAG)150 knock-in mice HD striatum.

sentences: Hypothermia Suppresses Uncoupling of Oxidative-Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia Hypothermia is the best available therapy for neonatal hypoxia-ischemia (HI) brain injury, but its primary mechanisms remain uncertain1. We hypothesize that HI induces, whereas hypothermia represses, uncoupling of oxidative-phosphorylation (OXPHOS)--an increase of the cerebral metabolic rate of oxygen (CMRO2) despite reduction of the mitochondrial energy output2,3. We used a multi-parametric photoacoustic microscopy (PAM) system to compare the effects of HI and post-HI hypothermic treatment on CMRO2 in awake 10-day-old (P10) mice4-9. Here we show that hypoxia (10% O2) elevated CMRO2, but the addition of unilateral carotid artery ligation suppressed CMRO2 and sparked a rapid overshoot of post-HI CMRO2 in the ipsilateral cerebral cortex for at least 2 hours. The post-HI surge of CMRO2 was linked to an increase of mitochondrial oxygen consumption and superoxide outburst, despite reduction of the mitochondrial membrane potential. Notably, post-HI hypothermia blocked the surge of superoxide and CMRO2--primarily by limiting oxygen extraction fraction (OEF)--leading to better preservation of adenosine triphosphate (ATP), creatine (Cr) and N-acetylaspartate (NAA) after HI. Mice that did not receive hypothermia exhibited ~80% reduction of CMRO2 at 24 h post-HI, coupled to a large cortical infarction. These results suggest that mitigation of post-HI uncoupling of OXPHOS is an early and/or pivotal effect of hypothermia. Further, optical measurement of CMRO2 may be a sensitive and non-invasive method to monitor brain damage in hypoxic-ischemic encephalopathy (HIE)10-15.

sentences: A phenomenological model of neuroimmune interactions in epileptogenesis Epilepsy can have many different causes and its development (epileptogenesis) involves a bewildering complexity of interacting processes. Here, we present a first-of-its-kind computational model to better understand the role of neuroimmune interactions in the development of acquired epilepsy. Our model describes the interactions between neuroinflammation, blood-brain barrier disruption, neuronal loss, circuit remodeling, and seizures. Formulated as a system of nonlinear differential equations, the model is validated using data from animal models that mimic human epileptogenesis caused by infection, status epilepticus, and blood-brain barrier disruption. The mathematical model successfully explains characteristic features of epileptogenesis such as its paradoxically long timescales (up to decades) despite short and transient injuries, or its dependence on the intensity of an injury. Furthermore, stochasticity in the model captures the variability of epileptogenesis outcomes in individuals exposed to identical injury. Notably, in line with the concept of degeneracy, our simulations reveal multiple routes towards epileptogenesis with neuronal loss as a sufficient but non-necessary component. We show that our framework allows for in silico predictions of therapeutic strategies, providing information on injury-specific therapeutic targets and optimal time windows for intervention.

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Single-cell mapping of tumor heterogeneity in pediatric rhabdomyosarcoma reveals developmental signatures with therapeutic relevance Rhabdomyosarcoma (RMS) is an aggressive human pediatric cancer. Despite robust expression of myogenic regulatory factors, RMS cells are blocked in a proliferative state and do not terminally differentiate. The extent to which the skeletal muscle lineage is represented in RMS tumors and the mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) and high-content imaging to resolve RMS heterogeneity. ScRNAseq and CyTOF analysis of a total of 17 patient-derived primary cultures and three cell lines uncovered plastic myogenic subpopulations that delineate a branched trajectory. The less aggressive embryonal RMS (eRMS) harbor primarily muscle stem cell (MuSC)-like cells and exhibit sparse commitment to differentiation. The more aggressive alveolar RMS (aRMS) comprise primarily actively cycling committed progenitors with a paucity of differentiated cells. The oncogenic fusion protein PAX3:FOXO1 sustains aRMS cells in the cycling trajectory loop, which we show can re-wired towards differentiation upon its downregulation or by dual pharmacological RAF and MEK inhibition. Our findings provide insights into the developmental states and trajectories underlying RMS progression and identify the RAS pathway as a promising target of differentiation therapy for human aRMS.

STATEMENT OF SIGNIFICANCEWe present the first comprehensive single-cell transcriptomic and proteomic atlas of pediatric rhabdomyosarcoma (RMS), in which we identify impaired myogenic trajectories with prognostic value. We demonstrate that RAS pathway inhibitors disrupt the oncogenic trajectory and induce terminal differentiation, revealing novel therapeutic targets for the aggressive alveolar RMS subtype.

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Functional selectivity for naturalistic social interaction perception in the human superior temporal sulcus Recognizing others social interactions is a crucial human ability. Using simple stimuli, previous studies have shown that social interactions are selectively processed in the superior temporal sulcus (STS), but prior work with movies has suggested that social interactions are processed in the medial prefrontal cortex (mPFC), part of the theory of mind network. It remains unknown to what extent social interaction selectivity is observed in real world stimuli when controlling for other covarying perceptual and social information, such as faces, voices, and theory of mind. The current study utilizes a functional magnetic resonance imaging (fMRI) movie paradigm and advanced machine learning methods to uncover the brain mechanisms uniquely underlying naturalistic social interaction perception. We analyzed two publicly available fMRI datasets, collected while both male and female human participants (n = 17 and 18) watched two different commercial movies in the MRI scanner. By performing voxel-wise encoding and variance partitioning analyses, we found that broad social-affective features predict neural responses in social brain regions, including the STS and mPFC. However, only the STS showed robust and unique selectivity specifically to social interactions, independent from other covarying features. This selectivity was observed across two separate fMRI datasets. These findings suggest that naturalistic social interaction perception recruits dedicated neural circuity in the STS, separate from the theory of mind network, and is a critical dimension of human social understanding.

Significance StatementSocial interaction perception guides our daily behavior, yet it is unknown how our brain processes real-world social interaction scenes. Here, we demonstrate that social brain areas, including the superior temporal sulcus (STS) and medial prefrontal cortex (mPFC), are sensitive to broad social-affective information in naturalistic movies, replicating prior results with controlled paradigms. We show for the first time however, that the STS, but not mPFC, selectively processes social interactions in natural movies, independent of other co-occurring perceptual and social features, including motion, presence of faces, speech, and mentalizing about others. Our results suggest that social interaction perception is a crucial dimension of social understanding represented in the human brain.

sentences: Oxidation-reduction and photophysical properties of isomeric forms of Safranin Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine Os utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.

sentences: Human Prefoldin Modulates Co-transcriptional Pre-mRNA Splicing Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.

sentences: Applications of the indole-alkaloid gramine shape the prokaryotic microbiota thriving at the barley root-soil interface Microbial communities proliferating at the root-soil interface, collectively referred to as the rhizosphere microbiota, represent an untapped beneficial resource for plant growth, development and health. Integral to a rational manipulation of the microbiota for sustainable agriculture is the identification of the molecular determinants of these communities. In plants, biosynthesis of allelochemicals is centre stage in defining inter-organismal relationships in the environment. Intriguingly, this process has been moulded by domestication and breeding selection. The indole-alkaloid gramine, whose occurrence in barley (Hordeum vulgare L.) is widespread among wild genotypes but has been counter selected in several modern varieties, is a paradigmatic example of this phenomenon. This prompted us to investigate how exogenous applications of gramine impacted on the rhizosphere microbiota of two, gramine-free, elite barley varieties grown in a reference agricultural soil. High throughput 16S rRNA gene amplicon sequencing revealed that applications of gramine interfere with the proliferation of a subset of soil microbes with a relatively broad phylogenetic assignment. Strikingly, growth of these bacteria appeared to be rescued by barley plants in a genotype- and dosage-independent manner. In parallel, we discovered that host recruitment cues can interfere with the impact of gramine application in a host genotype-dependent manner. Interestingly, this latter effect displayed a bias for members of the phyla Proteobacteria. These initial observations indicate that gramine can act as a determinant of the prokaryotic communities inhabiting the root-soil interface.

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

sentences: Modified defence peptides from horseshoe crab target and kill bacteria inside host cells Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two {beta}-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogues [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

sentences: Completion of neural crest cell production and emigration is regulated by retinoic acid-dependent inhibition of BMP signaling Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

sentences: No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination The impact of coronavirus disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated two of the most widely propagated claims to determine 1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities, and 2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from vaccinated murine pregnancies exhibit high circulating levels of anti-Spike and anti-RBD antibodies to SARS-CoV-2 consistent with maternal antibody status, indicating transplacental transfer. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

sentences: Phenotypic and Kinetic Changes of Myeloid Lineage Cells in Innate Response to Chikungunya Infection in Cynomolgus Macaques Chikungunya is an emerging threat, spreading across multiple continents. The immune response to infection can lead to protection and convalescence, or result in long term sequelae such as arthritis. Early innate immune events during acute infection have been characterized for some cell types, but more must be elucidated with respect to cellular responses of monocytes and other myeloid lineage cells. In addition to their roles in protection and inflammation resolution, monocytes and macrophages are sites for viral replication and reservoirs for the virus. They are also found in joints post-infection, possibly playing a role in long term CHIKV-induced pathology. We examined kinetic and phenotypic changes in myeloid lineage cells, including monocytes, in cynomolgus macaques early after infection with CHIKV. We found increased turnover of monocytes and mDCs early during infection, with an accompanying decreases in both cell types, as well as a simultaneous increase in pDC number. An increase in CD16 and CD14 was seen along with a decrease in monocyte HLA-DR expression within 3 days of infection, potentially indicating monocyte deactivation. A transient decrease in T cells, B cells and NK cells correlates with lymphocytopenia observed during human infections with CHIKV. CD4+ T cell turnover decreased in blood, indicating relocation of cells to effector sites. This data indicates CHIKV influences turnover rates and kinetics of myeloid lineage cells early during infection, and may prove useful in development of therapeutics and evaluation of infection-induced pathogenesis.

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Septins mediate a microtubule-actin crosstalk that enables actin growth on microtubules Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons (1, 2). Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin (1, 2). Septins are filamentous GTP-binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin and intermediate filaments (3, 4). Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Super-resolution imaging shows that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We show that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings provide the first evidence of septins directly mediating microtubule interactions with actin filaments, and reveal a new mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.

Significance StatementCellular morphogenesis and processes such as cell division and cell migration require the coordination of the actin and microtubule cytoskeletons. Despite its broader physiological significance, actin-microtubule crosstalk is molecularly and mechanistically poorly understood. Actin-microtubule crosstalk has been viewed primarily as the regulation or guidance of microtubule dynamics by actin filaments. Here, we have discovered a new mechanism by which actin filament growth is guided by microtubules. We report that septins, a poorly understood component of the cytoskeleton, can capture and link the polymerizing ends of actin filaments to microtubule polymers. We present evidence that this new septin-mediated mechanism is critical for the morphology of growth cones, which are structures that direct the growth of neuronal axons through actin-microtubule crosstalk.

sentences: Maturation of distinct neural components of the cognitive control network support early development of inhibitory control Crucial for goal-directed behavior is the capacity to suppress impulses and predominant behavioral responses, called inhibitory control (IC). This ability emerges in early childhood, and a distinction according to neutral ( cold) and emotional ( hot) contexts has been suggested. Here, we ask which maturational changes in the childs brain underlie the emergence of this critical ability. We relate behavioral changes in 3-and 4-year-olds  hot and  cold IC to brain maturation, using a multimodal approach that combines cortical and subcortical grey matter structure with white matter connectivity. Our results show that the maturation of distinct parts of the cognitive control brain network support early development of the different IC domains: Whereas  cold IC is related to frontoparietal regions and the left thalamus,  hot IC is associated with the left supramarginal gyrus and right thalamus. This dissociation of brain networks involved in  cold and  hot IC is confirmed by independent patterns of thalamocortical connectivity, supporting that IC in neutral and emotional settings relies on independent processes.

sentences: Role of mucin gene for growth in Anas platyrynchos - a novel report Mucin gene is expressed at the mucous membrane of the inner layer of the internal organs. Intestinal mucin 2 (MUC2), a major gel-forming mucin, represents a primary barrier component of mucus layers. This is the first report of role of mucin gene in growth traits in animals. In the current study, we had randomly studied Bengal ducks (Anas platyrynchos) reared from day old to 10 weeks of age under organized farm and studied the growth parameters as well as body weight and average daily body weight gain. We had characterized mucin gene for Bengal duck and observed glycosylation and EGF1 (EGF like domain signature) as important domain for growth traits in duck. We observed better expression profile for mucin gene in high growing ducks in comparison to that of lower growing ducks with real time PCR. Hence mucin gene may be employed as a marker for growth traits.

sentences: The BLMP-1 transcription factor promotes oscillatory gene expression to achieve timely molting Gene expression oscillators can coordinate developmental events in space and time. In C. elegans, a gene expression oscillator directs rhythmic accumulation of [~]25% of the transcriptome, and thus thousands of transcripts, presumably to control molting, a process of rhythmic skin regeneration. Recently, a reverse genetic screen identified several transcription factors important for molting. Here, we characterize one of these, BLMP-1, orthologous to the mammalian transcription repressor PRDM1. We find it to be important for timely molting, and oscillatory gene expression. We propose a dual function for BLMP-1 in shaping oscillatory gene expression and coupling it to a set of direct targets, which ensures cuticular integrity. With mammalian PRDM1/BLIMP1 promoting regular cycles of postnatal hair follicle regeneration, our findings point to the possible existence of a fundamentally conserved clock mechanism in control of rhythmic skin regeneration.

sentences: Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates survival of developing retinal neurons Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, large-scale transcriptomics reveals dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

sentences: A sinusoidal transform of the visual field in cortical area V2 The retinotopic maps of many visual cortical areas are thought to follow the fundamental principles that have been described for primary visual cortex (V1) where nearby points on the retina map to nearby points on the surface of V1, and orthogonal axes of the retinal surface are represented along orthogonal axes of the cortical surface. Here we demonstrate a striking departure from this conventional mapping in the secondary visual area (V2) of the tree shrew. Although local retinotopy is preserved, orthogonal axes of the retina are represented along the same axis of the cortical surface, an unexpected geometry explained by an orderly sinusoidal transform of the retinal surface. This sinusoidal topography is ideally suited for achieving uniform coverage in an elongated area like V2, is predicted by mathematical models designed to achieve wiring minimization, and provides a novel explanation for stripe-like patterns of intra-cortical connections and stimulus response properties in V2. Our findings suggest that cortical circuits flexibly implement solutions to sensory surface representation, with dramatic consequences for the large-scale layout of topographic maps.

sentences: Diversity of receptor expression in central and peripheral mouse neurons estimated from single cell RNA sequencing Because somatosensory PNS neurons, in particular nociceptors, are specially tuned to be able to detect a wide variety of both exogenous and endogenous signals, it is widely assumed that these neurons express a greater variety of receptor genes. Because cells detect such signals via cell surface receptors, we sought to formally test the hypothesis that PNS neurons might express a broader array of cell surface receptors than CNS neurons using existing single cell RNA sequencing resources from mouse. We focused our analysis on ion channels, G-protein coupled receptors (GPCRS), receptor tyrosine kinase and cytokine family receptors. In partial support of our hypothesis, we found that mouse PNS somatosensory, sympathetic and enteric neurons and CNS neurons have similar receptor expression diversity in families of receptors examined, with the exception of GPCRs and cytokine receptors which showed greater diversity in the PNS. Surprisingly, these differences were mostly driven by enteric and sympathetic neurons, not by somatosensory neurons or nociceptors. Secondary analysis revealed many receptors that are very specifically expressed in subsets of PNS neurons, including some that are unique among neurons for nociceptors. Finally, we sought to examine specific ligand-receptor interactions between T cells and PNS and CNS neurons. Again, we noted that most interactions between these cells are shared by CNS and PNS neurons despite the fact that T cells only enter the CNS under rare circumstances. Our findings demonstrate that both PNS and CNS neurons express an astonishing array of cell surface receptors and suggest that most neurons are tuned to receive signals from other cells types, in particular immune cells.

sentences: Cerebral hemodynamics: a mathematical model including autoregulation, baroreflex and extracranial peripheral circulation Cerebral autoregulation, the physiological capability to regulate cerebral blood flow, may be assisted by short-term mean arterial pressure control via baroreflex, which, among several effects, modulates total peripheral resistance. It is unclear, however, whether the resistance of the head and neck vasculatures is also affected by baroreflex and whether these extracranial vessels assist autoregulation. Since sensing technologies such as functional Near-Infrared Spectroscopy and noninvasive intracranial pressure monitoring by strain gauge may be influenced by superficial tissue, it is clinically relevant to understand the relations between intracranial and extracranial hemodynamics. Therefore, we created an autoregulation model consisting of arteries and arterioles regulated by the intralumial pressure and microcirculation regulated by local blood flow. As the first critical step to quantify the signal deterioration introduced by the extracranial circulation on superficial sensors, the extracranial peripheral circulation of the head and neck and baroreflex regulation of the peripheral vasculature and of heart rate were also included. During simulations of a bout of acute hypotension, the model predicts a rapid return of cerebral blood flow to baseline levels and a prolonged suppression of the blood flow to the external carotid vasculature, in accordance with experimental evidence. The inclusion of peripheral control via baroreflex at the external carotid vasculature did not assist cerebral autoregulation, thus we raise the hypothesis that baroreflex may act on the head and neck vasculatures but this action has negligible effects on regulation of cerebral blood flow. When autoregulation is impaired, results suggest that the blood flow of the brain and of the head and neck present similar dynamics, while they are weakly coupled when autoregulation is intact. The model also provides a mechanistic explanation of the protection brought by cerebral autoregulation to the microvasculature and to the brain parenchyma. Our model forms the foundation for predicting the interference introduced by the superficial tissue to nonivasive sensors.

sentences: Molecular transport and packing underlie increasing ribosome productivity in faster growing cells Faster growing cells must make proteins more quickly. Increasing ribosome abundance only partly accounts for increased protein production; the productivity of individual ribosomes must also increase, almost doubling via an unknown mechanism. To investigate, we model translation elongation in Escherichia coli by explicitly representing spatially-resolved transport and reactions among individual molecules each occupying finite volume. We predict that physical transport of individual ternary complexes accounts for ~80% of elongation latency. We find crowding increases at faster growth, which predictably decreases diffusion of ternary complexes, but that crowding also improves proximity between ternary complexes and ribosomes. Despite slowed diffusion, we predict that improved proximity wins out, offering a simple mechanism for how individual elongating ribosomes become more productive. We speculate how crowding imposes a physical limit on growth rate and likely undergirds cellular behavior more broadly. Our work reveals a fundamental mechanism arising from physical transport at the colloidal scale that cannot be obtained via kinetics-only modeling.

sentences: Striatal dopamine pulses follow a temporal discounting spectrum Dopamine input to striatum can encode reward prediction error, a critical signal for updating predictions of future rewards. However, it is unclear how this mechanism handles the need to make predictions, and provide feedback, over multiple time horizons: from seconds or less (if singing a song) to potentially hours or more (if hunting for food). Here we report that dopamine pulses in distinct striatal subregions convey reward prediction errors over distinct temporal scales. Dopamine dynamics systematically accelerated from ventral to dorsal-medial to dorsal-lateral striatum, in the tempo of their spontaneous fluctuations, their integration of prior rewards, and their discounting of future rewards. This spectrum of time scales for value computations can help achieve efficient learning and adaptive motivation for a wide range of behaviors.

sentences: Trial-by-trial fluctuations in post-stimulus attention during memory encoding predict subsequent associative context memory performance. Episodic memory formation rate varies over time partly due to fluctuations in attentional state during memory encoding. Emerging evidence suggests that fluctuations in pre- and/or post-stimulus attention during encoding impact subsequent memory performance. It remains unclear how these fluctuations may differentially impact the subsequent retrieval of items alone, compared to items + their contextual details (associative context memory). In this study, we explored this in 30 healthy younger adults (21-34 years old). We developed the Montreal Attention at Encoding (MAET) task where on each encoding trial, participants responded as quickly as possible to a central fixation cross that expanded in size after a random duration. They then had to encode a picture of an object and its spatial location. Memory for the object-location associations was tested during retrieval. Response time (RT) to the fixation cross presented prior to each object gauged pre-stimulus attention levels on a trial-by-trial basis, while RT to the fixation cross that ensued each object indexed post-stimulus attention levels. Within-subject logistic regressions were used to predict context and item memory performance from pre- and post-stimulus RTs. Results revealed that encoding pre-stimulus attentional levels did not differentially predict context vs. item memory. However, post-stimulus RTs did predict subsequent context retrieval such that, longer post-stimulus RT to the fixation was related to poorer subsequent context retrieval. This study introduces a novel paradigm for investigating the impact of attentional state at encoding on subsequent memory performance and indicate a link between post-stimulus delays in attention-related RT and associative encoding success.

sentences: A genome-wide mutational constraint map quantified from variation in 76,156 human genomes The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders, but attempts to assess constraint for non-protein-coding regions have proven more difficult. Here we aggregate, process, and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD), the largest public open-access human genome reference dataset, and use this dataset to build a mutational constraint map for the whole genome. We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation across the genome. As expected, proteincoding sequences overall are under stronger constraint than non-coding regions. Within the noncoding genome, constrained regions are enriched for regulatory elements and variants implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association, and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained genes, while non-coding constraint captures additional functional information underrecognized by gene constraint metrics. We demonstrate that this genome-wide constraint map provides an effective approach for characterizing the non-coding genome and improving the identification and interpretation of functional human genetic variation.

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Helicase-Like Functions in Phosphate Loop Containing Beta-Alpha Polypeptides The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple {beta} repeat proteins bind NTPs, but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one {beta}-(P-loop)- element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes comprise a plausible description of the sequence, structure and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Significance statementIt is widely assumed that todays large and complex proteins emerged from much shorter and simpler polypeptides. Yet the nature of these early precursors remains enigmatic. We describe polypeptides that contain one of the earliest protein motifs, a phosphate-binding loop, or P-loop, embedded in a single beta-alpha element. These P-loop prototypes show intriguing characteristics of a primordial world comprised of nucleic acids and peptides. They are  generalists capable of binding different phospho-ligands, including inorganic polyphosphates and single-stranded DNA. Nonetheless, in promoting double-stranded DNA unwinding and strand-exchange they resemble modern P-loop helicases and recombinases. Our study describes a missing link in the evolution of complex proteins - simple polypeptides that tangibly relate to contemporary P-loop enzymes in sequence, structure and function.

sentences: Chloride dynamics determine the optimal distribution of inhibitory synapses to minimise dendritic excitability AO_SCPLOWBSTRACTC_SCPLOWMany neurons in the mammalian central nervous system have complex dendritic arborisations and active dendritic conductances that enable these cells to perform sophisticated computations. How dendritically targeted inhibition affects local dendritic excitability is not fully understood. Here we use computational models of branched dendrites to investigate where GABAergic synapses should be placed to minimise dendritic excitability over time. To do so, we formulate a metric we term the "Inhibitory Level" (IL), which quantifies the effectiveness of synaptic inhibition for reducing the depolarising effect of nearby excitatory input. GABAergic synaptic inhibition is dependent on the reversal potential for GABAA receptors (EGABA), which is primarily set by the transmembrane chloride ion (Cl-) concentration gradient. We, therefore, investigated how variable EGABA and dynamic chloride affects dendritic inhibition. We found that the inhibitory effectiveness of dendritic GABAergic synapses accumulates at an encircled branch junction. The extent of inhibitory accumulation is dependent on the number of branches and location of synapses but is independent of EGABA. This accumulation occurs even for very distally placed inhibitory synapses when they are hyperpolarising - but not when they are shunting. When accounting for Cl- fluxes and dynamics in Cl- concentration, we observed that Cl- loading is detrimental to inhibitory effectiveness. This enabled us to determine the most inhibitory distribution of GABAergic synapses which is close to - but not at - a shared branch junction. This distribution balances a trade-off between a stronger combined inhibitory influence when synapses closely encircle a branch junction with the deleterious effects of increased Cl- loading that occurs when inhibitory synapses are co-located.

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Bacterial Sensitivity to Chlorhexidine and Povidone-Iodine Antiseptics Over Time: A Systematic Review and Meta-Analysis of Human-Derived Data BackgroundSurgical site infection (SSI) is the most common complication of surgery, increasing healthcare costs and hospital stay. Topical biocides such as chlorhexidine (CHX) and povidone-iodine (PVI) are used for skin antisepsis to minimise SSIs. There is an increasing concern of developing resistance to topical biocides, however the clinical implications of this remains unclear.

OutcomesThe objective of this review was to determine whether the Minimum Bactericidal Concentration (MBC) for topical preparations of CHX or PVI have changed over time, in microbes relevant to SSI.

MethodsWe searched for studies which reported the mean bactericidal concentration (MBC) of laboratory and clinical isolates of common SSI causing microbes to CHX and PVI. We excluded samples derived from non-humans and studies using antimicrobial solvents or mixtures of biocides with other active substances. MBC was pooled in random effects meta-analyses and change in MBC over time was explored using meta-regression.

Results79 studies were including, analysing 6218 microbes between 1976 and 2021. Most studies used CHX (93%) and there was insufficient data for meta-analysis of PVI. Enterobacteriales had the highest MBC for CHX (20 mg/L [95% CI 14, 25]; I2 95%) whilst MRSA had the lowest (3 mg/L [95% CI 1, 2]; I2 93%). There was no change in MBC of CHX to Staphylococci ({beta} 0.12 [-1.13, 1.37]; I2 99%) or Streptococci ({beta} 0.13 [-0.35, 0.62]; I2 97%).

ConclusionsThere is no evidence of change in susceptibility of common SSI-causing microbes to CHX over time. This study provides reassurance that the worldwide guidance that CHX should remain the first-choice agent for skin asepsis prior to surgery.

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Long-term patterns of an interconnected core marine microbiota BackgroundOcean microbes constitute [~]70% of the marine biomass, are responsible for [~]50% of the Earths primary production, and are crucial for global biogeochemical cycles. Marine microbiotas include core taxa that are usually key for ecosystem function. Despite their importance, core marine microbes are relatively unknown, which reflects the lack of consensus on how to identify them. So far, most core microbiotas have been defined based on species occurrence and abundance. Yet, species interactions are also important to identify core microbes, as communities include interacting species. Here, we investigate interconnected bacteria and small protists of the core pelagic microbiota populating a long-term marine-coastal observatory in the Mediterranean Sea over a decade.

ResultsCore microbes were defined as those present in >30% of the monthly samples over 10 years, with the strongest associations. The core microbiota included 259 Operational Taxonomic Units (OTUs) including 182 bacteria, 77 protists, and 1,411 strong and mostly positive ([~]95%) associations. Core bacteria tended to be associated with other bacteria, while core protists tended to be associated with bacteria. The richness and abundance of core OTUs varied annually, decreasing in stratified warmers waters and increasing in colder mixed waters. Most core OTUs had a preference for one season, mostly winter, which featured subnetworks with the highest connectivity. Groups of highly associated taxa tended to include protists and bacteria with predominance in the same season, particularly winter. A group of 13 highly-connected hub-OTUs, with potentially important ecological roles dominated in winter and spring. Similarly, 18 connector OTUs with a low degree but high centrality were mostly associated with summer or autumn and may represent transitions between seasonal communities.

ConclusionsWe found a relatively small and dynamic interconnected core microbiota in a model temperate marine-coastal site, with potential interactions being more deterministic in winter than in other seasons. These core microbes would be essential for the functioning of this ecosystem over the year. Other non-core taxa may also carry out important functions but would be redundant and non-essential. Our work contributes to the understanding of the dynamics and potential interactions of core microbes possibly sustaining ocean ecosystem function.

sentences: A large-sample crisis? Exaggerated false positives by popular differential expression methods We report a surprising phenomenon about identifying differentially expressed genes (DEGs) from population-level RNA-seq data: two popular bioinformatics methods, DESeq2 and edgeR, have unexpectedly high false discovery rates (FDRs). Via permutation analysis on an immunotherapy RNA-seq dataset, we observed that DESeq2 and edgeR identified even more DEGs after samples condition labels were randomly permuted. Motivated by this, we evaluated six DEG identification methods (DESeq2, edgeR, limma-voom, NOISeq, dearseq, and the Wilcoxon rank-sum test) on population-level RNA-seq datasets. We found that the FDR control was often failed by the three popular parametric methods--DESeq2, edgeR, and limma-voom-- and the new non-parametric method dearseq. In particular, the actual FDRs of DESeq2 and edgeR sometimes exceeded 20% when the target FDR threshold was only 5%. Although NOISeq, a non-parametric method used by GTEx, controlled the FDR better than the other four methods did, its power was much lower than that of the Wilcoxon rank-sum test, a classic nonparametric test that consistently controlled the FDR and achieved good power in our evaluation. Based on these results, for population-level RNA-seq studies, we recommend the Wilcoxon rank-sum test.

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Non-canonical Staphylococcus aureus pathogenicity island repression Mobile genetic elements (MGEs) control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of the Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation, never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.

SignificanceWhile most repressors controlling the transfer of mobile genetic elements are dimers, we demonstrate here that the Staphylococcal pathogenicity island 1 (SaPI1) is repressed by two tetramers, which have a novel structural fold in their body that has never been seen before in other proteins. Moreover, by solving the structure of the SaPI1 repressor in complex with its inducing protein Sri, we have demonstrated that Sri forces the SaPI1 repressor to adopt a conformation that is incompatible with DNA binding, explaining how SaPI1 is induced. Finally, our results demonstrate that this repression system is not exclusive of the SaPIs but widespread in nature. Our studies provide important insights understanding how SaPIs spread in nature.

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DENTIST - using long reads for closing assembly gaps at high accuracy BackgroundLong sequencing reads allow increasing contiguity and completeness of fragmented, short-read based genome assemblies by closing assembly gaps, ideally at high accuracy. While several gap closing methods have been developed, these methods often close an assembly gap with sequence that does not accurately represent the true sequence.

FindingsHere, we present DENTIST, a sensitive, highly accurate and automated pipeline method to close gaps in short-read assemblies with long error-prone reads. DENTIST comprehensively determines repetitive assembly regions to identify reliable and unambiguous alignments of long reads to the correct loci, integrates a consensus sequence computation step to obtain a high base accuracy for the inserted sequence, and validates the accuracy of closed gaps. Unlike previous benchmarks, we generated test assemblies that have gaps at the exact positions where real short-read assemblies have gaps. Generating such realistic benchmarks for Drosophila (134 Mb genome), Arabidopsis (119 Mb), hummingbird (1 Gb) and human (3 Gb) and using simulated or real PacBio continuous long reads, we show that DENTIST consistently achieves a substantially higher accuracy compared to previous methods, while having a similar sensitivity.

ConclusionDENTIST provides an accurate approach to improve the contiguity and completeness of fragmented assemblies with long reads. DENTISTs source code including a Snakemake workflow, conda package and Docker container is available at https://github.com/a-ludi/dentist. All test assemblies as a resource for future benchmarking are at https://bds.mpi-cbg.de/hillerlab/DENTIST/.

sentences: Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

sentences: Determination of nuclear position by the arrangement of actin filaments using deep generative networks The cell nucleus is a dynamic structure that changes locales during cellular processes such as proliferation, differentiation, or migration1, and its mispositioning is a hallmark of several disorders2,3. As with most mechanobiological activities of adherent cells, the repositioning and anchoring of the nucleus are presumed to be associated with the organization of the cytoskeleton, the network of protein filaments providing structural integrity to the cells4. However, this correlation between cytoskeleton organization and nuclear position has not, to date, been demonstrated, as it would require the parameterization of the extraordinarily intricate cytoskeletal fiber arrangements. Here, we show that this parameterization and demonstration can be achieved outside the limits of human conceptualization, using generative network and raw microscope images, relying on machine-driven interpretation and selection of parameterizable features. The developed transformer-based architecture was able to generate high-quality, completed images of more than 8,000 cells, using only information on actin filaments, predicting the presence of a nucleus and its exact localization in more than 70 per cent of instances. Our results demonstrate one of the most basic principles of mechanobiology with a remarkable level of significance. They also highlight the role of deep learning as a powerful tool in biology beyond data augmentation and analysis, capable of interpreting--unconstrained by the principles of human reasoning--complex biological systems from qualitative data.

sentences: Thermoregulatory morphodynamics of honeybee clusters During reproductive swarming, honeybees form clusters of more than 10,000 bees that hang from structures in the environment (e.g., tree branches) and are exposed to diurnal variations in ambient temperature for up to one week during the search for a new nesting site. Swarm clusters collectively modulate their morphology in response to these variations (i.e., expanding/contracting in response to heating/cooling) to maintain their internal temperature within a tolerable range and to avoid exhausting their honey stores prematurely. To understand the spatiotemporal aspects of thermoregulatory morphing, we measured the change in size and shape of swarm clusters over time and the internal temperature profiles in response to dynamic temperature ramp perturbations. We found that swarm clusters can achieve a twofold increase/decrease their volume/density when heated from 15{degrees}C to 30{degrees}C, but they do not reach an equilibrium size or shape when held at 30{degrees}C for 5 hours, long after the core temperature of the cluster has stabilized. Furthermore, the changes in cluster shape and size are hysteretic, contracting in response to cooling faster than expanding in response to heating. Although the contact diameter of the cluster increased continuously when the swarm is heated, the change in length of the swarm (base to tip) over time is non-monotonic. Consequently, the aspect ratio of the swarm fluctuated continuously even when held at a constant temperature. Taken together, our results quantify the hysteretic and anisotropic morphological responses of swarm clusters to ambient temperature variations while suggesting that both mechanical constraints and heat transfer govern the thermoregulatory morphing dynamics of swarm clusters.

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Prediction of RNA-protein interactions using a nucleotide language model MotivationThe accumulation of sequencing data has enabled researchers to predict the interactions between RNA sequences and RNA-binding proteins (RBPs) using novel machine learning techniques. However, existing models are often difficult to interpret and require additional information to sequences. Bidirectional encoder representations from Transformer (BERT) is a language-based deep learning model that is highly interpretable. Therefore, a model based on BERT architecture can potentially overcome such limitations.

ResultsHere, we propose BERT-RBP as a model to predict RNA-RBP interactions by adapting the BERT architecture pre-trained on a human reference genome. Our model outperformed state-of-the-art prediction models using the eCLIP-seq data of 154 RBPs. The detailed analysis further revealed that BERT-RBP could recognize both the transcript region type and RNA secondary structure only from sequence information. Overall, the results provide insights into the fine-tuning mechanism of BERT in biological contexts and provide evidence of the applicability of the model to other RNA-related problems.

AvailabilityPython source codes are freely available at https://github.com/kkyamada/bert-rbp.

Contactmhamada@waseda.jp

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Patterns of Saliency and Semantic Features Distinguish Gaze of Expert and Novice Viewers of Surveillance Footage When viewing the actions of others, we not only see patterns of body movements, but we also "see" the intentions and social relations of people, enabling us to understand the surrounding social environment. Previous research has shown that experienced forensic examiners--Closed Circuit Television (CCTV) operators--convey superior performance in identifying and predicting hostile intentions from surveillance footages than novices. However, it remains largely unknown what visual content CCTV operators actively attend to when viewing surveillance footage, and whether CCTV operators develop different strategies for active information seeking from what novices do. In this study, we conducted computational analysis for the gaze-centered stimuli captured by experienced CCTV operators and novices eye movements when they viewed the same surveillance footage. These analyses examined how low-level visual features and object-level semantic features contribute to attentive gaze patterns associated with the two groups of participants. Low-level image features were extracted by a visual saliency model, whereas object-level semantic features were extracted by a deep convolutional neural network (DCNN), AlexNet, from gaze-centered regions. We found that visual regions attended by CCTV operators versus by novices can be reliably classified by patterns of saliency features and DCNN features. Additionally, CCTV operators showed greater inter-subject correlation in attending to saliency features and DCNN features than did novices. These results suggest that the looking behavior of CCTV operators differs from novices by actively attending to different patterns of saliency and semantic features in both low-level and high-level visual processing. Expertise in selectively attending to informative features at different levels of visual hierarchy may play an important role in facilitating the efficient detection of social relationships between agents and the prediction of harmful intentions.

Author SummaryImagine seeing a person walking toward another person menacingly on the street, we may instantly feel that some physical confrontation will happen in the next second. However, it remains unclear how we efficiently infer social intentions and outcomes from the observed dynamic visual input. To answer this question, CCTV experts, who have years of experience on observing social scenes and making online predictions of the action outcomes, provide a unique perspective. Here, we collected experts and novices eye movements when observing different action sequences and compared the attended visual information between groups. A saliency model was used to compare low-level visual features such as luminance and color, and a deep convolutional neural network was used to extract object-level semantic visual features. Our findings showed that experts obtained different patterns of low-level and semantic-level features in visual processing compared to novices. Thus, the expertise in selectively attending to informative features at different levels of visual hierarchy may play an important role in facilitating the efficient detection of social relationships between agents and the prediction of harmful intentions.

sentences: Shed the light on virus: virucidal effects of 405 nm visible light on SARS-CoV-2 and influenza A virus. Germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit its use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) as well as the influenza A virus to 405nm, visible light in the absence of exogenous photosensitizers indicating a potential porphyrin-independent alternative mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are generally safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.

sentences: Molecular Insights into the Differential Dynamics of SARS-CoV-2 Variants of Concern (VOCs) Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected the lives and livelihood of millions of individuals around the world. It has mutated several times after its first inception, with an estimated two mutations occurring every month. Although we have been successful in developing vaccines against the virus, emergence of variants has enabled it to escape therapy. Few of the generated variants are also reported to be more infectious than the wild-type (WT). In this study, we analyze the attributes of all RBD/ACE2 complexes for the reported VOCs, namely, Alpha, Beta, Gamma, and Delta through computer simulations. Results indicate differences in orientation and binding energies of the VOCs from the WT. Overall, it was observed that electrostatic interactions play a major role in the binding of the complexes. Detailed residue level energetics revealed that the most prominent changes in interaction energies were seen particularly at the mutated residues which were present at RBD/ACE2 interface. We found that the Delta variant is one of the most tightly bound variants of SARS-CoV-2 with dynamics similar to WT. High binding affinity of RBD towards ACE2 is indicative of an increase in the viral transmission and infectivity. The details presented in our study would prove extremely useful for the design and development of effective therapeutic strategies for the emerging variants of the virus.

sentences: Non-specific effects of a CINNAMATE-4-HYDROXYLASE inhibitor on auxin homeostasis Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although being a powerful tool, off-target effects of these inhibitors are easily overlooked in a complex biological setting. Here we illustrate the implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is often used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the auxin catabolite indole-3-acetic acid (IAA)-Asp. By competing for the same enzyme, PA interferes with auxin conjugation, resulting in an increase in cellular auxin concentrations. These increased auxin levels likely further contribute to an increase in adventitious rooting previously observed upon PA-treatment. Despite the focus on GH3.6 in this report, PA is conjugated by an array of enzymes and their subsequent reduced activity on native substrates could potentially affect a whole set of physiological processes in the plant. We conclude that surrogate occupation of the endogenous conjugation machinery in the plant by exogenous compounds is likely a more general phenomenon that is rarely considered in pharmacological studies. Our results hereby provide an important basis for future reference in studies using chemical inhibitors.

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Genome mosaicism in field strains of Mycoplasma bovis as footprints of in-host horizontal chromosomal transfer Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinitive variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016-2018) we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during co-infections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs) and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control.

IMPORTANCEMycoplasma bovis is a major pathogen of cattle with significant detrimental economic and animal welfare on cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Despite these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.

sentences: Performance Scaling for Structural MRI Surface Parcellations: A Machine Learning Analysis in the ABCD Study The use of pre-defined parcellations on surface-based representations of the brain as a method for data reduction is common across neuroimaging studies. In particular, prediction-based studies typically employ parcellation-driven summaries of brain measures as input to predictive algorithms, but the choice of parcellation and its influence on performance is often ignored. Here we employed pre-processed structural magnetic resonance imaging data (sMRI) from the ABCD Study(R) to examine the relationship between 220 parcellations and out-of-sample predictive performance across 45 phenotypic measures in a large sample of 9-10-year-old children (N=9,432). Choice of Machine Learning (ML) pipeline and use of alternative multiple parcellation-based strategies were also assessed. Relative parcellation performance was dependent on the spatial resolution of the parcellation, with larger numbers of parcels (up to [~]4000) outperforming coarser parcellations, according to a power-law scaling of between 1/4 and 1/3. Performance was further influenced by the type of parcellation, ML pipeline, and general strategy, with existing literature-based parcellations, a support vector based pipeline, and ensembling across multiple parcellations, respectively, as the highest performing. These findings highlight the choice of parcellation as an important influence on downstream predictive performance, showing in some cases that switching to a higher resolution parcellation can yield a relatively large boost to performance.

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Spatial and molecular anatomy of germ layers in the gastrulating primate embryo During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors and germ layer morphogenesis. While the mouse model has been instrumental for our understanding of mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a morphological and molecular survey of spatiotemporal dynamics of cell types populating the non-human primate embryos during gastrulation. We performed serial sections of Cynomolgus monkeys (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21, and reconstructed three-dimensional digital models based on high-resolution anatomical atlas that revealed the dynamic changes in the geography of the mesoderm and primitive streaks. Spatial transcriptomics identified unique gene profiles that correspond to distinct germ layers and cross-species spatiotemporal transcriptome analysis revealed a developmental coordinate of germ layer segregation between mouse and primate. Furthermore, we identified species-specific transcription programs during gastrulation. These results offer important insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

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C_LIO_LICreated a three-dimensional digital template from serial sections of five developmental stages
C_LIO_LIA two-dimensional spatiotemporal transcriptome of the germ layers of gastrulating embryos
C_LIO_LICross-species comparison infers conservation of functional attributes of regulome and signaling activity in germ layer formation
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sentences: Allosteric modulators enhance agonist efficacy by increasing the residence time of a GPCR in the active state Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity, and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.

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Height, but not binding epitope, affects the potency of synthetic TCR agonists Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCR{beta} Fab antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

STATEMENT OF SIGNIFICANCEWe report geometric requirements for potent T cell activation by synthetic TCR ligands that mimic biophysical properties of the native pMHC ligand, but have the additional ability to activate polyclonal T cell populations. We find that increasing the space between apposed membranes at TCR binding events decreases ligand potency, but that changing the ligands binding epitope on the TCR has essentially no effect. The observed decrease in potency with increased ligand height is attributed to the longer ligands attenuated ability to trigger TCR at binding events.

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Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled inline (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid-{beta} (A{beta}) plaques.

HighlightsO_LIX-ray phase-contrast tomography (XPCT) enables myelin mapping of the whole brain
C_LIO_LIXPCT detects and quantifies white-matter injuries in a range of diseases
C_LIO_LIFiber directions and anisotropy metrics can be retrieved from XPCT data
C_LIO_LIXPCT is compatible with subsequent conventional histology of brain samples
C_LIO_LIXPCT is a powerful virtual histology tool that requires minimal sample preparation
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Graphical Abstract

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sentences: Clonal diversification and histogenesis of malignant germ cell tumours Germ cell tumours (GCTs) are a collection of benign and malignant neoplasms derived from primordial germ cells. They are uniquely able to recapitulate embryonic and extraembryonic tissues, which carries prognostic and therapeutic significance. The developmental pathways underpinning GCT initiation and histogenesis are incompletely understood. Here, we studied the relationship of histogenesis and clonal diversification in GCTs by analysing the genome and transcriptome of 547 microdissected histological units. We found that the extensive diversification of tissues and genetic subclones were not correlated. However, we identified unifying features including the retention of fetal developmental transcripts across tissues, expression changes on chromosome 12p, and a conserved somatic evolutionary sequence of whole genome duplication followed by clonal diversification. Whilst this pattern was preserved across all GCTs, the developmental timing of the duplication varied between prepubertal and postpubertal cases. In addition, tumours of younger children exhibited distinct substitution signatures, including a novel one, which may lend themselves as potential biomarkers for risk stratification. Our findings portray the extensive diversification of GCT tissues and genetic subclones as randomly distributed, whilst identifying overarching tissue and tumour transcriptional and genomic features.

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The ChiS-family DNA-binding domain contains a cryptic helix-turn-helix variant Sequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 [A]. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight "turn" of the HTH is replaced with an insertion containing a {beta}-sheet, a variant which we term the "helix-sheet-helix". Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.

ImportanceRegulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

sentences: FGF8-mediated signaling regulates tooth developmental pace during odontogenesis The developing human and mouse teeth constitute an ideal model system to study the regulatory mechanism underlying organ growth control due to the fact that their teeth share highly conserved and well-characterized developmental processes and their developmental tempo vary notably. In the current study, we manipulated heterogenous recombination between human and mouse dental tissues and demonstrate that the dental mesenchyme dominates the tooth developmental tempo and FGF8 could be a critical player during this developmental process. Forced activation of FGF8 signaling in the dental mesenchyme of mice promoted cell proliferation, prevented cell apoptosis via p38 and perhaps PI3K-Akt intracellular signaling, and impelled the transition of the cell cycle from G1- to S-phase in the tooth germ, resulting in the slowdown of the tooth developmental pace. Our results provide compelling evidence that extrinsic signals can profoundly affect tooth developmental tempo and the dental mesenchymal FGF8 could be a pivotal factor in controlling developmental pace in a non-cell-autonomous manner during mammalian odontogenesis.

sentences: Limited selection towards lighter seeds in Danish grasslands over an eight-year period Time-series analysis of cover data from 236 Danish grassland sites demonstrated community selection on seed mass. Across four grassland habitat types and during an eight-year period there was significant community selection towards lighter seeds. In the analysis, the continuous seed size variable was used to group plant species into functional types. This method was chosen in order to account for the sampling process of the abundance data, and is in contrast to most other analyses of trait selection, where the community weighted mean of the traits is used as the dependent variable. Generally, little is known on the underlying causes of possible plant trait community selection in semi-natural grasslands that are not subjected to successional processes.

sentences: Mycobacterial Serine/Threonine phosphatase PstP is phospho-regulated and localized to mediate control of cell wall metabolism The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven Serine/Threonine protein kinases (STPKs) phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated but the role of its phosphorylation in regulating its activity has been unclear. In this study we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two peptidoglycan regulators of Mtb, FhaA and Wag31. We also show that a phospho-mimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulatory substrates FhaA, Wag31, CwlM, PknB and PknA, and that the corresponding mutation in Mycobacterium smegmatis (Msmeg) causes mis-regulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.

sentences: Effects of Ancestry and Hypopigmentation Alleles on Skin Color in a Caribbean Native American Population Interest in the genetic basis of variation in skin pigmentation in Native American populations led us to seek indigenous populations of the Western Hemisphere with African and minimal European admixture to study the effect of Native American ancestry on skin color. Admixture analysis from DNA collected from 458 individuals in the Kalinago territory of the Commonwealth of Dominica showed shared ancestry with East Asians at K=3 and 55% Native American, 32% African, and 11% European ancestry at K=6, the highest Native American ancestry of Caribbean populations. Skin pigmentation was 20 to 80 melanin units, averaging 46. Three albino individuals were homozygous for multi-nucleotide polymorphism OCA2NW273KV of African origin, whose population allele frequency was 0.03 and single allele effect size was -8 melanin units. Hypopigmenting allele frequencies for SLC24A5A111T and SLC45A2L374F were 0.14 and 0.05, whose single allele effect sizes were -6 and -3, respectively. Skin color plots of individuals lacking known hypopigmenting alleles suggests that Native American Ancestry reduced pigmentation by more than 20 melanin units (low and high estimates 21.8 and 28.5). Shared ancestry with East Asians at K=3 suggests potential sharing of one or more pigmentation alleles.

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Effect sizes and test-retest reliability of the fMRI-based Neurologic Pain Signature Identifying biomarkers that predict mental states with large effect sizes and high test-retest reliability is a growing priority for fMRI research. We examined a well-established multivariate brain measure that tracks pain induced by nociceptive input, the Neurologic Pain Signature (NPS). In N = 295 participants across eight studies, NPS responses showed a very large effect size in predicting within-person single-trial pain reports (d = 1.45) and medium effect size in predicting individual differences in pain reports (d = 0.49). The NPS showed excellent shortterm (within-day) test-retest reliability (ICC = 0.84, with average 69.5 trials/person). Reliability scaled with the number of trials within-person, with [≥]60 trials required for excellent test-retest reliability. Reliability was tested in two additional studies across 5-day (N = 29, ICC = 0.74, 30 trials/person) and 1-month (N = 40, ICC = 0.46, 5 trials/person) test-retest intervals. The combination of strong within-person correlations and only modest between-person correlations between the NPS and pain reports indicate that the two measures have different sources of between-person variance. The NPS is not a surrogate for individual differences in pain reports but can serve as a reliable measure of pain-related physiology and mechanistic target for interventions.

Significance statementCurrent efforts towards translating brain biomarkers require identifying brain measures that can strongly and reliably predict outcomes of interest. We systematically examined the performance of a well-established brain activity pattern, the Neurological Pain Signature (NPS), in a large and diverse sample of participants. The NPS showed excellent reliability, and the reliability scaled with the number of trials within-person. The NPS responses showed strong correlations with pain reports at the within-person level but only modest correlations at the between-person level. The findings suggest that the NPS is not a surrogate for individual differences in pain reports but can serve as a reliable measure of a pain-related physiological target.

Author NoteThis project was supported by grants R01MH076136 (T.D.W.), R01DA046064, R01EB026549, and R01DA035484. Elizabeth A. Reynolds Losin was supported by a Mentored Research Scientist Development award from National Institute On Drug Abuse of the National Institutes of Health (K01DA045735). Lauren Y. Atlas was supported in part by funding from the Intramural Research Program of the National Center for Complementary and Integrative Health. Yoni K. Ashar was supported by NCATS Grant # TL1-TR-002386. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Code for all analyses and figures is available at https://github.com/XiaochunHan/NPS_measurement_properties. Data for all analyses and figures is available at https://osf.io/v9px7/.

sentences: Comparative population genetic structure of two ixodid tick species (Ixodes ovatus and Haemaphysalis flava) in Niigata Prefecture, Japan Ixodid tick species such as Ixodes ovatus and Haemaphysalis flava are essential vectors of tick-borne diseases in Japan. In this study, we investigated the population genetic structures and gene flow of I. ovatus and H. flava as affected by the tick host mobility. We hypothesized that I. ovatus and H. flava may have differences in their genetic structure due to the low mobility of small rodent hosts of I. ovatus at the immature stage in contrast to the mediated dispersal of avian hosts for immature H. flava. We collected 307 adult I. ovatus and 220 adult H. flava from 29 and 17 locations across Niigata Prefecture, Japan. We investigated the genetic structure at two mitochondrial loci (cox1, 16S rRNA gene). For I. ovatus, pairwise FST and analysis of molecular variance (AMOVA) analyses of cox1 sequences indicated significant genetic variation among populations. Both cox1 and 16S rRNA markers showed non-significant genetic variation among locations for H. flava. The Bayesian tree and haplotype network of cox1 marker for I. ovatus samples in Niigata Prefecture found 3 genetic groups wherein most haplotypes in group 2 were distributed in low altitudinal areas. When we added cox1 sequences of I. ovatus from China to the phylogenetic analysis, three genetic groups (China 1, China 2, and Niigata and Hokkaido, Japan) were formed in the tree suggesting the potential for cryptic species in the genetic group in Japan. Our results support our hypothesis and suggest that the host preference of ticks at the immature stage may influence the genetic structure and gene flow of the ticks. This information is vital in understanding the tick-host interactions in the field to better understand the tick-borne disease transmission and in designing an effective tick control program.

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Assessing Reproducibility of High-throughput Experiments in the Case of Missing Data. High-throughput experiments are an essential part of modern biological and biomedical research. The outcomes of high-throughput biological experiments often have a lot of missing observations due to signals below detection levels. For example, most single-cell RNA-seq (scRNA-seq) protocols experience high levels of dropout due to the small amount of starting material, leading to a majority of reported expression levels being zero. Though missing data contain information about reproducibility, they are often excluded in the reproducibility assessment, potentially generating misleading assessments.

In this paper, we develop a regression model to assess how the reproducibility of high-throughput experiments is affected by the choices of operational factors (e.g., platform or sequencing depth) when a large number of measurements are missing. Using a latent variable approach, we extend correspondence curve regression (CCR), a recently proposed method for assessing the effects of operational factors to reproducibility, to incorporate missing values. Using simulations, we show that our method is more accurate in detecting differences in reproducibility than existing measures of reproducibility. We illustrate the usefulness of our method using a single-cell RNA-seq dataset collected on HCT116 cells. We compare the reproducibility of different library preparation platforms and study the effect of sequencing depth on reproducibility, thereby determining the cost-effective sequencing depth that is required to achieve sufficient reproducibility.

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Geminiviral βC1 orchestrates organellar genomic instability to augment viral infection by hijacking host RecA Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defence against pathogens including viruses. Geminiviruses disrupt chloroplast homeostasis as a basic strategy for their successful infection inducing vein-clearing, mosaics and chlorosis in infected plants. Here we show that a geminiviral pathogenicity determinant protein {beta}C1 directly interferes with plastid homeostasis. {beta}C1 was capable of inducing organelle-specific nuclease to degrade plastid genome as well as diverted functions of RecA1 protein, a major player in plastid genome maintenance. {beta}C1 interacted with RecA1 in plants and its homolog in bacteria to reduce the ability of host cells to maintain genomic integrity under stresses. Further, reduction in the coding capacity of plastids severely affected retrograde signalling necessary for viral perception and activation of defense. Induction of chloroplast-specific nuclease by {beta}C1 is similar to phosphate starvation-response in which nucleotides are recycled to augment synthesis of new, potentially viral, DNA. These results indicate the presence of a novel strategy in which a viral protein alters host defence by targeting regulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

One line summary{beta}C1 alters plastid genome metabolism.

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Meet me in the middle: median temperatures impact cyanobacteria and photoautotrophy in eruptive Yellowstone hot springs Geographic isolation can be a main driver of microbial evolution in hot springs while temperature plays a role on local scales. For example, cyanobacteria, particularly high temperature Synechococcus spp., have undergone ecological diversification along temperature gradients in hot spring outflow channels. While water flow, and thus temperature, is largely stable in many hot springs, flow can vary in geysing/eruptive hot springs resulting in large temperature fluctuations (sometimes more than 40{degrees}C). However, the role of large temperature fluctuations in driving diversification of cyanobacteria in eruptive hot springs has not been explored. Here, we examined phototroph community composition and potential photoautotrophic activity in two alkaline eruptive hot springs with similar geochemistry in the Lower Geyser Basin in Yellowstone National Park, WY. We observed distinct cyanobacterial amplicon sequencing variants (ASVs) consistent with allopatry and levels of light-dependent inorganic carbon uptake rates similar to other hot springs, despite large temperature fluctuations. Our data suggests median temperatures may drive phototroph fitness in eruptive hot springs while future studies are necessary to determine the evolutionary consequences of thriving under continuously fluctuating temperatures. We propose that large temperature swings in eruptive hot springs offer unique environments to examine the role of allopatry vs. physical and chemical characteristics of ecosystems in driving cyanobacteria evolution and add to the debate regarding the ecology of thermal adaptation and the potential for narrowing niche breadth with increasing temperature.

ImportanceHot spring cyanobacteria have long been model systems for examining ecological diversification as well as characterizing microbial adaptation and evolution to extreme environments. These studies have reported cyanobacterial diversification in hot spring outflow channels that can be defined by distinct temperature ranges. Our study builds on these previous studies by examining cyanobacteria in geysing hot springs. Geysing hot springs result in outflow channel that experience regular and large temperature fluctuations. While community composition is similar between geysing and nongeysing hot spring outflow channels, our data suggests median, rather than high temperature, drive the fitness of cyanobacteria in geysing hot springs. We propose that large temperature swings may result in patterns of ecological diversification that are distinct from more stable outflows.

sentences: Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale The axonal cytoskeleton is organized in a highly periodic structure, the membrane-associated periodic skeleton (MPS), which is essential to maintain the structure and function of the axon. Here, we use stimulated emission depletion microscopy (STED) of primary rat cortical neurons in microfluidic chambers to analyze the temporal and spatial sequence of MPS formation at the distal end of growing axons and during regeneration after axotomy. We demonstrate that the MPS does not extend continuously into the growing axon but develops from patches of periodic {beta}-spectrin II arrangements that grow and coalesce into a continuous scaffold. We estimate that the underlying sequence of nucleation, elongation, and subsequent coalescence of periodic {beta}-spectrin II patches takes around 15 hours. Strikingly, we find that development of the MPS occurs faster in regenerating axons after axotomy and note marked differences in the morphology of the growth cone and adjacent axonal regions between regenerating and unlesioned axons. Moreover, we find that inhibition of the spectrin-cleaving enzyme calpain accelerates MPS formation in regenerating axons and increases the number of regenerating axons after axotomy. Taken together, we provide here a detailed nanoscale analysis of MPS development in growing axons.

sentences: Otolith-based discrimination of cod ecotypes and the effect of growth rate. Otolith shape has previously been used to identify ecotypes within the Icelandic cod (Gadus morhua) stock, using DST profiles to validate the results. Fish otolith shape variation has repeatedly been found to be largely determined by growth rate. To examine the effect of growth rate on the relationship between otolith shape and cod ecotypes (using the Pan I genotype as a proxy for ecotype), 826 archived sagittal otoliths collected over a 52 year sampling period were retrieved, the individual growth rate calculated, and otolith shape described using both Normalized Elliptic Fourier transform and Discrete Wavelet transform. Discriminant functions of otolith shape yielded high ecotype classification success, whether using Fourier or Wavelet descriptors, but only when excluding a heterozygous genotype from the analysis. The otolith shape variability of this genotype lowered the classification success, while otolith shape, in turn, was significantly affected by growth rate and cohort. Growth rate differences previously reported for the ecotypes were present, but were less marked than expected and indeed, growth rate variance attributable to ecotype identity was dwarfed by cohort- and location-related variance in growth. Such a strong effect of growth rate suggests that cod ecotype discrimination based on otolith shape is sensitive to both temporal and spatial variations in growth, which can mask the effect of ecotype-related growth rate differences on otolith shape.

sentences: Host phenology can select for multiple stable parasite virulence strategies Host phenology is an important driver of parasite transmission dynamics and evolution. Prior research has demonstrated that host phenology can drive monocyclic, obligate-killer parasites to evolve an intermediate virulence strategy where all parasites kill their host just before the season ends to limit the death of parasite progeny in the environment. The impact of host seasonality on parasites that are not constrained to a monocyclic life-cycle, however, cannot be inferred from these results. Here we present a mathematical model that demonstrates that many, but not all, seasonal host activity patterns support multiple evolutionarily stable parasite strategies (ESS), although these strategies cannot coexist in the same system. The specific monocyclic and polycyclic parasite evolutionarily stable strategies in each phenological pattern are interspersed with less-fit mono- and polycyclic strategies (evolutionary repellors). The ESS that dominates each system at equilibrium is a function of the strategy of the parasite introduced into the system. The results demonstrate that host phenology can, in theory, maintain diverse parasite strategies among isolated geographic locations.

sentences: Enhanced loss of retinoic acid network genes in Xenopus laevis achieves a tighter signal regulation Retinoic acid (RA) is a major regulatory signal during embryogenesis produced from vitamin A (retinol) by an extensive, autoregulating metabolic and signaling network to prevent fluctuations that result in developmental malformations. Xenopus laevis is an allotetraploid hybrid frog species whose genome includes L (long) and S (short) chromosomes from the originating species. Evolutionarily, the X. laevis subgenomes have been losing either L or S homoeologs in about 43% of genes to generate singletons. In the RA network, out of the 47 genes, about 46% have lost one of the homoeologs, like the genome average. In contrast, RA metabolism genes from storage (retinyl esters) to retinaldehyde production exhibit enhanced gene loss with 75% singletons out of 28 genes. The effect of this gene loss on RA signaling autoregulation was studied. Employing transient RA manipulations, homoeolog gene pairs were identified in which one homeolog exhibits enhanced responses or looser regulation than the other, while in other pairs both homoeologs exhibit similar RA responses. CRISPR /Cas9 targeting of individual homoeologs to reduce their activity supports the hypothesis where the RA metabolic network gene loss results in tighter network regulation and more efficient RA robustness responses to overcome complex regulation conditions.

sentences: IKZF3 deficiency potentiates chimeric antigen receptor T cells targeting solid tumors Chimeric antigen receptor (CAR) T cell therapy has been successful in treating hematological malignancy, but solid tumors remain refractory. Here, we demonstrated that knocking out transcription factor IKZF3 in HER2-specific CAR T cells targeting breast cancer cells did not affect proliferation or differentiation of the CAR T cells in the absence of tumors, but markedly enhanced killing of the cancer cells in vitro and in a xenograft model. Furthermore, IKZF3 KO had similar effects on the CD133-specific CAR T cells targeting glioblastoma cells. AlphaLISA and RNA-seq analyses indicate that IKZF3 KO increased the expression of genes involved in cytokine signaling, chemotaxis and cytotoxicity. Our results suggest a general strategy for enhancing CAR T efficacy on solid tumors.

sentences: LAP1 regulates nuclear plasticity to enable constrained migration Metastasis involves dissemination of cancer cells away from a primary tumour and colonisation at distal sites. During this process, cancer cells must negotiate multiple physical constraints imposed by the microenvironment and tissue structure. The biophysical properties of the nucleus must be tuned since they pose a challenge to constrained migration. By analysing nuclear genes upregulated during the acquisition of metastatic potential, we discovered increased expression of the inner nuclear membrane protein LAP1 in metastatic melanoma cells and at the invasive fronts of human primary tumours and in metastases. Human cells express two LAP1 isoforms (LAP1B and LAP1C), which differ in their amino terminus. We found that whereas the longer isoform, LAP1B, binds more strongly to nuclear lamins and has restricted motility within the nuclear envelope, the shorter isoform, LAP1C, favours nuclear envelope blebbing and allows migration through constraints. We propose that LAP1 renders the nucleus plastic and enhances melanoma aggressiveness.

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Computational analysis of memory consolidation following inhibitory avoidance (IA) training in adult and infant rats: critical roles of CaMKIIα and MeCP2 Key features of long-term memory (LTM), such as its stability and persistence, are acquired during processes collectively referred to as consolidation. The dynamics of biological changes during consolidation are complex. In adult rodents, consolidation exhibits distinct periods during which the engram is more or less resistant to disruption. Moreover, the ability to consolidate memories differs during developmental periods. Although the molecular mechanisms underlying consolidation are poorly understood, the initial stages rely on interacting signaling pathways that regulate gene expression, including brain-derived neurotrophic factor (BDNF) and Ca2+/calmodulin-dependent protein kinase II  (CaMKII) dependent feedback loops. We investigated the ways in which these pathways may contribute to developmental and dynamical features of consolidation. A computational model of molecular processes underlying consolidation following inhibitory avoidance (IA) training in rats was developed. Differential equations described the actions of CaMKII, multiple feedback loops regulating BDNF expression, and several transcription factors including methyl-CpG binding protein 2 (MeCP2), histone deacetylase 2 (HDAC2), and SIN3 transcription regulator family member A (Sin3a). This model provides novel explanations for the (apparent) rapid forgetting of infantile memory and the temporal progression of memory consolidation in adults. Simulations predict that dual effects of MeCP2 on the expression of bdnf, and interaction between MeCP2 and CaMKII, play critical roles in the rapid forgetting of infantile memory and the progress of memory resistance to disruptions. These insights suggest new potential targets of therapy for memory impairment.

Author SummaryLong-term memories (LTMs) are enduring and resistant to disruption These features are acquired via processes collectively referred to as consolidation. In adults, the initial stages of consolidation follow complex dynamics that are believed to emerge from interacting biochemical signaling pathways [1], including BDNF and CaMKII dependent feedback loops. Similarly, the acquisition of ability to consolidate memory in infantile animals is believed to emerge from the functional maturation of these molecular pathways [2]. Here, the ways in which these pathways contribute to consolidation were investigated using a computational model. This model provides novel explanations for the apparent rapid forgetting of infantile memory and for development of resistance to disruption during memory consolidation.

sentences: The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA-RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.

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Standing genetic variation of the AvrPm17 avirulence gene in powdery mildew limits the effectiveness of an introgressed rye resistance gene in wheat Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable - a phenomenon that remains poorly understood as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used QTL mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of re-occurring gene conversion events and are members of a mildew sub-lineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sub-lineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangle complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations becomes an integral part of introgression breeding to ensure effective and durable resistance in wheat.

Significance StatementDomesticated and wild wheat relatives provide an important source of new immune receptors for wheat resistance breeding against fungal pathogens. The durability of these resistance genes is variable and difficult to predict, yet it is crucial for effective resistance breeding. We identified a fungal effector protein recognised by an immune receptor introgressed from rye to wheat. We found that variants of the effector allowing the fungus to overcome the resistance are ancient. They were already present in the wheat powdery mildew gene pool before the introgression of the immune receptor and are therefore responsible for the rapid resistance breakdown. Our study demonstrates that the effort to identify new resistance genes should be accompanied by studies of avirulence genes on the pathogen side.

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Evidence for Deleterious Antigenic Imprinting in SARS-CoV-2 Immune Response A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity.

ImportanceInfections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.

sentences: A curated data resource of 214K metagenomes for characterization of the global resistome The growing threat of antimicrobial resistance (AMR) calls for new epidemiological surveillance methods, as well as a deeper understanding of how antimicrobial resistance genes (ARGs) have transmitted around the world. The large pool of sequencing data available in public repositories provides an excellent resource for monitoring the temporal and spatial dissemination of AMR in different ecological settings. However, only a limited number of research groups globally have the computational resources allowing analyses of such data. We retrieved 442 Tbp of sequencing reads from 214,095 metagenomic samples from the European Nucleotide Archive (ENA) and aligned them using a uniform approach against ARGs and 16S/18S rRNA genes. Here, we present the results of this extensive computational analysis and share the counts of reads aligned. Over 6.76 {middle dot} 108 read fragments were assigned to ARGs and 3.21 {middle dot} 109 to rRNA genes, where we observed distinct differences in both the abundance of ARGs and the link between microbiome and resistome compositions across various sampling types. This collection is another step towards establishing a global surveillance of AMR and can serve as a resource for further research into the environmental spread and dynamic changes of ARGs.

sentences: Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37{o}C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

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The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

SUMMARY STATEMENTCellular invasion through the basement membrane by the C. elegans anchor cell requires both BAF and PBAF SWI/SNF assemblies to arrest the cell cycle and promote the expression of pro-invasive genes.

sentences: Therapeutic targeting of SLC6A8 creatine transporter inhibits KRAS mutant and wildtype colon cancer and modulates human creatine levels Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 creatine transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels and induces tumor cell apoptosis in CRC. RGX-202 suppressed tumor growth across KRAS wild-type and KRAS mutant xenograft, syngeneic and patient-derived xenograft colorectal cancers. Anti-tumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5- fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in metastatic CRC patients enrolled in a Phase-1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of pre-clinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, revealing a critical target for CRC.

sentences: Met-HER3 crosstalk supports proliferation via MPZL3 in MET-amplified cancer cells Receptor tyrosine kinases (RTKs) are recognized as targets of precision medicine in human cancer upon their gene amplification or constitutive activation, resulting in increased downstream signal complexity including heterotypic crosstalk with other RTKs. The Met RTK exhibits such reciprocal crosstalk with several members of the human EGFR (HER) family of RTKs when amplified in cancer cells. We show that Met signaling converges on HER3 tyrosine phosphorylation across a panel of seven MET-amplified cancer cell lines and that HER3 is required for cancer cell expansion and oncogenic capacity in-vitro and in-vivo. Gene expression analysis of HER3-depleted cells identified MPZL3, encoding a single-pass transmembrane protein, as a HER3-dependent effector in multiple MET-amplified cancer cell lines. MPZL3 interacts with HER3 and MPZL3 loss phenocopies HER3 loss in MET-amplified cells, while MPZL3 overexpression rescues proliferation upon HER3 depletion. Together, these data support an oncogenic role for a HER3-MPZL3 axis in MET-amplified cancers.

sentences: Cooperation of two opposite flagella allows high-speed swimming and active turning in zoospore Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, characteristics of individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its pattern from sinusoidal waves to power and recovery strokes, similar to Chlamydomonass breaststroke, to reorient its body to a new direction. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated "run-and-tumble" motility class exhibited by peritrichous bacteria.

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A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 delta variant Although efficacious vaccines have significantly reduced the morbidity and mortality due to COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of two highly potently neutralizing mAbs (THSC20.HVTR04 and THSC20.HVTR26) from an Indian convalescent donor, that neutralize SARS-CoV-2 VOCs at picomolar concentrations including the delta variant (B.1.617.2). These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein thereby preventing the virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

HighlightsO_LIIdentification of an Indian convalescent donor prior to emergence of SARS-CoV-2 Delta variant whose plasma demonstrated neutralization breadth across SARS-CoV-2 variants of concern (VOCs).
C_LIO_LITwo (THSC20.HVTR04 and THSC20.HVTR26) monoclonal antibodies isolated from peripheral memory B cells potently neutralize SARS-CoV-2 VOCs: Alpha, Beta, Gamma, Delta and VOIs: Kappa and Delta Plus.
C_LIO_LITHSC20.HVTR04 and THSC20.HVTR26 target non-competing epitopes on the receptor binding domain (RBD) and represent distinct germline lineages.
C_LIO_LIPassive transfer of THSC20.HVTR04 and THSC20.HVTR26 mAbs demonstrated protection against Delta virus challenge in K18-hACE2 mice at low antibody doses.
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Graphical Abstract

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pop-1/TCF, ref-2/ZIC and T-box factors regulate the development of anterior cells in the C. elegans embryo Patterning of the anterior-posterior axis is fundamental to animal development. The Wnt pathway plays a major role in this process by activating the expression of posterior genes in animals from worms to humans. This observation raises the question of whether the Wnt pathway or other regulators control the expression of the many anterior-expressed genes. We found that the expression of five anterior-specific genes in Caenorhabditis elegans embryos depends on the Wnt pathway effectors pop-1/TCF and sys-1/{beta}-catenin. We focused further on one of these anterior genes, ref-2/ZIC, a conserved transcription factor expressed in multiple anterior lineages. Live imaging of ref-2 mutant embryos identified defects in cell division timing and position in anterior lineages. Cis-regulatory dissection identified three ref-2 transcriptional enhancers, one of which is necessary and sufficient for anterior-specific expression. This enhancer is activated by the T-box transcription factors TBX-37 and TBX-38, and surprisingly, concatemerized TBX-37/38 binding sites are sufficient to drive anterior-biased expression alone, despite the broad expression of TBX-37 and TBX-38. Taken together, our results highlight the diverse mechanisms used to regulate anterior expression patterns in the embryo.

Graphical Abstract

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sentences: In situ architecture of human kinetochore-microtubule interface visualized by cryo-electron tomography Faithful segregation of chromosomes during mitosis relies on a carefully coordinated and intricate interplay between the centromere, kinetochore, and spindle microtubules. Despite its importance, the architecture of this interface remains elusive. Here we used in situ cryo-electron tomography to visualize the native architecture of the kinetochore-microtubule interface in human U2OS cells at different stages of mitosis. We find that the centromere forms a pocket-like structure around kinetochore microtubules. Two morphologically distinct fibrillar densities form end-on and side-on connections to the plus-ends of microtubules within this centromeric pocket. Our data suggest a dynamic kinetochore-microtubule interface with multiple interactions between outer kinetochore components and spindle microtubules.

sentences: Identification of differentially distributed gene expression and distinct sets of cancer-related genes identified by changes in mean and variability There is increasing evidence that changes in the variability or overall distribution of gene expression are important both in normal biology and in diseases, particularly cancer. Genes whose expression differs in variability or distribution without a difference in mean are ignored by traditional differential expression-based analyses. Using a Bayesian hierarchical model that provides tests for both differential variability and differential distribution for bulk RNA-seq data, we report here an investigation into differential variability and distribution in cancer. Analysis of eight paired tumour-normal datasets from The Cancer Genome Atlas confirms that differential variability and distribution are able to identify cancer-related genes. We further demonstrate that differential variability identifies cancer-related genes that are missed by differential expression analysis, and that differential expression and differential variability identify functionally distinct sets of genes. These results suggest that differential variability analysis may provide insights into genetic aspects of cancer that would not be revealed by differential expression, and that differential distribution analysis may allow for more comprehensive identification of cancer-related genes than analyses based on changes in mean or variability alone.

sentences: Taking fright: the decline of Australian Pied Oystercatchers Haematopus longirostris at South Ballina Beach, New South Wales This study reviewed data from the Richmond River Area Pied Oystercatcher Protection Program 1997-2013 and Richmond River Area Shorebird Protection Program 2014-2018, on the far north coast of the State of New South Wales (NSW), Australia. The Australian Pied Oystercatcher Haematopus longirostris breeding population size for South Ballina Beach has decreased from 15-16 pairs in 1994-1996 to 7-9 pairs in 2016-2018. This is despite control of the European Red Fox Vulpes vulpes successfully reducing depredation of eggs and chicks and > 207 oystercatchers fledging from beaches in the Richmond River area in 1997-2018. The negative trend for South Ballina is contrasted with the positive trend for Bombing Range Beach, where the population size has increased from 4-5 pairs in 2002-2004 to 8-9 pairs in 2016-2018. Vehicle-based recreation has increased at South Ballina in the past two decades versus Bombing Range has remained closed to the public. It is proposed that human recreation disturbance is preventing recruitment into the South Ballina oystercatcher breeding population. Without strong protection of habitat from such disturbance, the NSW oystercatcher breeding population size is predicted to continue to decrease.

sentences: Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimers disease is processively cut by {gamma}-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis--composed of a helical peptide inhibitor linked to a transition-state analog--that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both exosite and active site as demonstrated by inhibitor cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with {gamma}-secretase, ultimately aiding rational drug design.

sentences: The first transchromosomic rat model with human chromosome 21 shows robust Down syndrome features Progress in earlier detection and symptom management has increased life expectancy and quality of life in people with Down syndrome (DS). However, no drug has been approved to help individuals with DS live independently and fully. Although rat models could support more robust physiological, behavioral, and toxicology analysis than mouse models during preclinical validation, no DS rat model is available due to technical challenges. We developed the first transchromosomic rat model of DS, TcHSA21rat, which contains a freely segregating, EGFP-inserted, human chromosome 21 (HSA21) with >93% of its protein coding genes. RNA-Seq of neonatal forebrains demonstrates that TcHSA21rat not only expresses HSA21 genes but also has an imbalance in global gene expression. Using EGFP as a marker for trisomic cells, flow cytometry analyses of peripheral blood cells from 361 adult TcHSA21rat animals show that 81% of animals retain HSA21 in >80% of cells, the criterion for a "Down syndrome karyotype" in people. TcHSA21rat exhibits learning and memory deficits and shows increased anxiety and hyperactivity. TcHSA21rat recapitulates well-characterized DS brain morphology, including smaller brain volume and reduced cerebellar size. In addition, the rat model shows reduced cerebellar foliation, a prominent feature of DS that is not observed in DS mouse models. Moreover, TcHSA21rat exhibits anomalies in craniofacial morphology, heart development, husbandry, and stature. TcHSA21rat is a robust DS animal model that can facilitate DS basic research and provide a unique tool for preclinical validation to accelerate DS drug development.

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Interpretable and tractable models of transcriptional noise for the rational design of single-molecule quantification experiments The question of how cell-to-cell differences in transcription rate affect RNA count distributions is fundamental for understanding biological processes underlying transcription. We argue that answering this question requires quantitative models that are both interpretable (describing concrete biophysical phenomena) and tractable (amenable to mathematical analysis). This enables the identification of experiments which best discriminate between competing hypotheses. As a proof of principle, we introduce a simple but flexible class of models involving a stochastic transcription rate coupled to a discrete stochastic RNA transcription and splicing process, and compare and contrast two biologically plausible hypotheses about transcription rate variation. One assumes variation is due to DNA experiencing mechanical strain, while the other assumes it is due to regulator number fluctuations. Although biophysically distinct, these models are mathematically similar, and we show they are hard to distinguish without comparing whole predicted probability distributions. Our work illustrates the importance of theory-guided data collection, and introduces a general framework for constructing and solving mathematically nontrivial continuous-discrete stochastic models.

Significance StatementThe interpretation of transcriptomic observations requires detailed models of biophysical noise that can be compared and fit to experimental data. Models of intrinsic noise, describing stochasticity in molecular reactions, and extrinsic noise, describing cell-to-cell variation, are particularly common. However, integrating and solving them is challenging, and previous results are largely limited to summary statistics. We examine two mechanistically grounded stochastic models of transcriptional variation and demonstrate that (1) well-known regimes naturally emerge in limiting cases, and (2) the choice of noise model significantly affects the RNA distributions, but not the lower moments, offering a route to model identification and inference. This approach provides a simple and biophysically interpretable means to construct and unify models of transcriptional variation.

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Distinct evolutionary trajectories of SARS-CoV-2 interacting proteins in bats and primates identify important host determinants of COVID-19 The COVID-19 pandemic is caused by SARS-CoV-2, a novel coronavirus that spilled from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional-genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. Here, we performed high-throughput evolutionary analyses of 334 SARS- CoV-2 interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms-race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo important interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified functional adaptations involved in SARS- CoV-2 infection in bats and primates, critically enlightening modern genetic determinants of virus susceptibility and severity.

Key findingsO_LIEvolutionary history of 334 SARS-CoV-2 interacting proteins (VIPs) in bats and primates identifying how the past has shaped modern viral reservoirs and humans - results publicly-available in an online resource.
C_LIO_LIIdentification of 81 primate and 38 bat VIPs with signatures of adaptive evolution. The common ones among species delineate a core adaptive interactome, while the ones displaying distinct evolutionary trajectories enlighten host lineage-specific determinants.
C_LIO_LIEvidence of primate specific adaptation of the entry factor TMPRSS2 pointing to its host- specific in vivo importance and predicting molecular interfaces.
C_LIO_LIFYCO1 sites associated with severe COVID-19 in human (GWAS) display hallmarks of ancient adaptive evolution in primates, highlighting its importance in SARS-CoV-2 replication or pathogenesis and differences with the bat reservoir.
C_LIO_LIIdentification of adaptive evolution in the bats multifunctional RIPK1 at residues that may differentially regulate inflammation.
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Functional coupling of TRPM2 and NMDARs exacerbates excitotoxicity in ischemic brain injury Excitotoxicity caused by NMDA receptors (NMDARs) is a major cause of neuronal death in ischemic stroke. However, past efforts of directly targeting NMDARs have unfortunately failed in clinical ischemic stroke trials. Here we reveal an unexpected mechanism underlying NMDARs-mediated neurotoxicity, which leads to identification of a novel target and development of an effective therapeutic peptide for ischemic stroke. We show that NMDARs excitotoxicity upon ischemic insults is mediated by physical and functional coupling to TRPM2. The physical interaction of TRPM2 with NMDARs results in markedly increase in the surface expression of NMDARs, leading to enhanced NMDAR function and increased neuronal death. We identified a specific NMDAR-interacting domain on TRPM2, and developed a cell-permeable peptide to uncouple TRPM2-NMDARs. The disrupting-peptide protects neurons against ischemic injury in vitro and protects mice against ischemic stroke in vivo. These findings provide an unconventional strategy to eliminate excitotoxic neuronal death without directly targeting NMDARs.

HIGHLIGHTSO_LITRPM2 physically and functionally interacts with NMDARs
C_LIO_LIInteraction of TRPM2 with NMDARs exacerbates NMDARs extrasynaptic excitotoxicity by increasing NMDARs surface expression during ischemic injury
C_LIO_LITRPM2 recruits PKC{gamma} to the interacting complexes to increase NMDARs surface expression
C_LIO_LIUncoupling the interaction between TRPM2 and NMDARs with a disrupting peptide (TAT-EE3) protects neurons against ischemic stroke in vitro and in vivo
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GRAPHIC ABSTRACT

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Protistan grazing impacts microbial communities and carbon cycling at deep-sea hydrothermal vents Microbial eukaryotes (or protists) in marine ecosystems are a link between microbial primary producers and all higher trophic levels. The rate at which heterotrophic protistan grazers consume microbial prey and recycle organic matter is an important factor that influences marine microbial food webs and carbon cycling. At deep-sea hydrothermal vents, chemosynthetic bacteria and archaea form the base of a food web that functions in the absence of sunlight, but the role of protistan grazers in these highly productive ecosystems is largely unexplored. Here, we pair grazing experiments with a molecular survey to quantify protistan grazing and to characterize the composition of vent-associated protists in low-temperature venting fluids from Gorda Ridge in the North East (NE) Pacific Ocean. Results reveal protists exert higher predation pressure at vents compared to the surrounding deep seawater environment and may account for consuming 28-62% of the daily stock of prokaryotic biomass within the hydrothermal vent food web. The vent-associated protistan community was more species rich relative to the background deep sea, and patterns in the distribution and co-occurrence of vent microbes provide additional insights into potential predator-prey interactions. Ciliates, followed by dinoflagellates, Syndiniales, rhizaria, and stramenopiles dominated the vent protist community and included bacterivorous species, species known to host symbionts, and parasites. Our findings provide an estimate of protistan grazing pressure within hydrothermal vent food webs, highlighting the role that diverse deep-sea protistan communities have in carbon cycling.

SignificanceHeterotrophic protists are ubiquitous in all aquatic ecosystems and represent an important ecological link because they transfer organic carbon from primary producers to higher trophic levels. Here, we quantify the predator-prey trophic interaction among protistan grazers and microbial prey at multiple sites of hydrothermal venting near the Gorda Ridge spreading center in the NE Pacific Ocean. Grazing pressure was higher at the site of active diffuse flow and was carried out by a highly diverse assemblage of protistan species; elevated grazing rates are attributed to higher concentrations of chemosynthetic microorganisms and biological diversity localized to hydrothermal vent environments.

sentences: MSX2 safeguards syncytiotrophoblast fate of human trophoblast stem cells The majority of placental pathologies are associated with failures in trophoblast differentiation, yet the underlying transcriptional regulation is poorly understood. Here, we use human trophoblast stem cells to elucidate the function of the transcription factor MSX2 in trophoblast specification. We show that depletion of MSX2 de-represses the syncytiotrophoblast program, while forced expression of MSX2 blocks it. We demonstrate that a large proportion of the affected genes are directly bound and regulated by MSX2 and identify components of the SWI/SNF complex as its strong interactors. Our findings uncover the pivotal role of MSX2 in cell fate decisions that govern human placental development and function.

sentences: Systematically quantifying morphological features reveals constraints on organoid phenotypes Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoids morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoids morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

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The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides.

SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.

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GeneTonic: an R/Bioconductor package for streamlining the interpretation of RNA-seq data BackgroundThe interpretation of results from transcriptome profiling experiments via RNA sequencing (RNA-seq) can be a complex task, where the essential information is distributed among different tabular and list formats - normalized expression values, results from differential expression analysis, and results from functional enrichment analyses. A number of tools and databases are widely used for the purpose of identification of relevant functional patterns, yet often their contextualization within the data and results at hand is not straightforward, especially if these analytic components are not combined together efficiently.

ResultsWe developed the GeneTonic software package, which serves as a comprehensive toolkit for streamlining the interpretation of functional enrichment analyses, by fully leveraging the information of expression values in a differential expression context. GeneTonic is implemented in R and Shiny, leveraging packages that enable HTML-based interactive visualizations for executing drilldown tasks seamlessly, viewing the data at a level of increased detail. GeneTonic is integrated with the core classes of existing Bioconductor workflows, and can accept the output of many widely used tools for pathway analysis, making this approach applicable to a wide range of use cases. Users can effectively navigate interlinked components (otherwise available as flat text or spreadsheet tables), bookmark features of interest during the exploration sessions, and obtain at the end a tailored HTML report, thus combining the benefits of both interactivity and reproducibility.

ConclusionGeneTonic is distributed as an R package in the Bioconductor project (https://bioconductor.org/packages/GeneTonic/) under the MIT license. Offering both birds-eye views of the components of transcriptome data analysis and the detailed inspection of single genes, individual signatures, and their relationships, GeneTonic aims at simplifying the process of interpretation of complex and compelling RNA-seq datasets for many researchers with different expertise profiles.

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High survival following bleaching highlights the resilience of a highly disturbed region of the Great Barrier Reef Natural bleaching events provide an opportunity to examine how local scale environmental variation influences bleaching severity and recovery. During the 2020 marine heatwave, we documented widespread and severe coral bleaching (75 - 98% of coral cover) throughout the Keppel Islands in the Southern inshore Great Barrier Reef. Acropora, Pocillopora and Porites were the most severely affected genera, while Montipora was comparatively less susceptible. Site-specific heat-exposure metrics were not correlated with Acropora bleaching severity, but recovery was faster at sites that experienced lower heat exposure. Despite severe bleaching and exposure to accumulated heat that often results in coral mortality (degree heating weeks [~] 4 - 8), cover remained stable. Approximately 94% of fate-tracked Acropora millepora colonies survived, perhaps owing to reduced irradiance stress from high turbidity, heterotrophic feeding, and large tidal flows that can increase mass transfer. Severe bleaching followed by rapid recovery, and the continuing dominance of Acropora populations in the Keppel Islands is indicative of high resilience. These coral communities have survived an 0.8 {degrees}C increase in average temperatures over the last 150 years. However, recovery following the 2020 bleaching was driven by the easing of thermal stress, which may challenge their recovery potential under further warming.

Open Research StatementData are not yet provided but are being compiled. Upon acceptance data will be archived on GitHub.

sentences: Reproducibility of protein X-ray diffuse scattering and potential utility for modeling atomic displacement parameters Protein structure and dynamics can be probed using X-ray crystallography. Whereas the Bragg peaks are only sensitive to the average unit-cell electron density, the signal between the Bragg peaks -- diffuse scattering -- is sensitive to spatial correlations in electron-density variations. Although diffuse scattering contains valuable information about protein dynamics, the diffuse signal is more difficult to isolate from the background compared to the Bragg signal, and the reproducibility of diffuse signal is not yet well understood. We present a systematic study of the reproducibility of diffuse scattering from isocyanide hydratase (ICH) in three different protein forms. Both replicate diffuse datasets and datasets obtained from different mutants were similar in pairwise comparisons (Pearson correlation coefficient (CC) [≥]0.8). The data were processed in a manner inspired by previously published methods using custom software with modular design, enabling us to perform an analysis of various data processing choices to determine how to obtain the highest quality data as assessed using unbiased measures of symmetry and reproducibility. The diffuse data then were used to characterize atomic mobility using a liquid-like motions (LLM) model. This characterization was able to discriminate between distinct anisotropic atomic displacement parameter (ADP) models arising from different anisotropic scaling choices that agreed comparably with the Bragg data. Our results emphasize the importance of data reproducibility as a model-free measure of diffuse data quality, illustrate the ability of LLM analysis of diffuse scattering to select among alternative ADP models, and offer insights into the design of successful diffuse scattering experiments.

sentences: Patient-Derived Organoids Recapitulate Intrinsic Immune Landscapes and Progenitor Populations of Glioblastoma. Glioblastoma stem cells (GSCs) are highly self-renewing, resistant to therapy, and are able to form lethal tumors1, 2. Tumor organoids have been developed to study tumor evolution1-4, and while GSCs can form organoids for glioblastoma multiforme, our understanding of their intrinsic immune, metabolic, genetic, and molecular programs is limited. To address this, we deeply characterized GSC-derived GBM organoids using a modified protocol (GBMOsm) from several patient-derived GSCs and found they develop into complex 3D tissues with unique self-organization, cancerous metabolic states, and burdensome genetic landscapes. We discovered that GBMOsc recapitulate the presence of two important cell populations thought to drive GBM progression, SATB2+ and HOPX+ progenitors. Despite being devoid of immune cells, transcriptomic analysis across GBMOsc revealed an immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We determined that SATB2+ and HOPX+ populations contribute to this immune and interferon landscape in GBM in vivo and GBMOsm. Our work deepens our understanding of the intrinsic molecular and cellular architecture of GSC-derived GBMO and defines a novel GBMOsc intrinsic immune-like program.

sentences: eSCAN: Scan Regulatory Regions for Aggregate Association Testing using Whole Genome Sequencing Data Multiple statistical methods for aggregate association testing have been developed for whole genome sequencing (WGS) data. Many aggregate variants in a given genomic window and ignore existing knowledge to define test regions, resulting in many identified regions not clearly linked to genes and thus limiting biological understanding. Functional information from new technologies (such as Hi-C and its derivatives), which can help link enhancers to their effector genes, can be leveraged to predefine variant sets for aggregate testing in WGS data. Here we propose the eSCAN (Scan the Enhancers) method for genome-wide assessment of enhancer regions in sequencing studies, combining the advantages of dynamic window selection in SCANG, a previously developed method, with the advantages of incorporating putative regulatory regions from annotation. eSCAN, by searching in putative enhancer, increases statistical power and aids mechanistic interpretation, as demonstrated by extensive simulation studies. We also apply eSCAN for blood cell traits using TOPMed WGS data. Results from real data analysis show that eSCAN is able to capture more significant signals, and these signals are of shorter length (indicating higher resolution fine-mapping capability) and drive association of larger regions detected by other methods.

sentences: Quaternary structure independent folding of voltage-gated ion channel pore domain subunits Every voltage-gated ion channel (VGIC) superfamily member has an ion conducting pore consisting of four pore domain (PD) subunits that are each built from a common plan comprising an antiparallel transmembrane helix pair, a short, obliquely positioned helix (the pore helix), and selectivity filter. The extent to which this structure, the VGIC-PD fold, relies on the extensive quaternary interactions observed in PD assemblies is unclear. Here, we present crystal structures of three bacterial voltage-gated sodium channel (BacNaO_SCPLOWVC_SCPLOW) pores that adopt a surprising set of non-canonical quaternary structures and yet maintain the native tertiary structure of the PD monomer. This context-independent structural robustness demonstrates that the VGIC-PD fold, the fundamental VGIC structural building block, can adopt its native-like tertiary fold independent of native quaternary interactions. In line with this observation, we find that the VGIC-PD fold is not only present throughout the VGIC superfamily and other channel classes but has homologs in diverse transmembrane and soluble proteins. Characterization of the structures of two synthetic Fabs (sFabs) that recognize the VGIC-PD fold shows that such sFabs can bind purified full-length channels and indicates that non-canonical quaternary PD assemblies can occur in the context of complete VGICs. Together, our data demonstrate that the VGIC-PD structure can fold independently of higher-order assembly interactions and suggest that full-length VGIC PDs can access previously unknown non-canonical quaternary states. These PD properties have deep implications for understanding how the complex quaternary architectures of VGIC superfamily members are achieved and point to possible evolutionary origins of this fundamental VGIC structural element.

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Testing microbiome associations with censored survival outcomes at both the community and individual taxon levels BackgroundFinding microbiome associations with possibly censored survival times is an important problem, especially as specific taxa could serve as biomarkers for disease prognosis or as targets for therapeutic interventions. The two existing methods for survival outcomes, MiRKAT-S and OMiSA, are restricted to testing associations at the community level and do not provide results at the individual taxon level. An ad hoc approach testing each taxon with a survival outcome using the Cox proportional hazard model may not perform well in the microbiome setting with sparse count data and small sample sizes.

MethodsWe have previously developed the linear decomposition model (LDM) that unifies community-level and taxon-level tests into one framework. Here we extend the LDM to test survival outcomes. We propose to use the Martingale residuals or the deviance residuals obtained from the Cox model as continuous covariates in the LDM. We further construct tests that combine the results of analyzing each set of residuals separately. Finally, we extend PERMANOVA, the most commonly used distance-based method for testing community-level hypotheses, to handle survival outcomes in a similar manner.

ResultsUsing simulated data, we showed that the LDM-based tests preserved the false discovery rate for testing individual taxa and had good sensitivity. The LDM-based community-level tests and PERMANOVA-based tests had comparable or better power than MiRKAT-S and OMiSA. An analysis of data on the association of the gut microbiome and the time to acute graft-versus-host disease revealed several dozen associated taxa that would not have been achievable by any community-level test, as well as improved community-level tests by the LDM and PERMANOVA over those obtained using MiRKAT-S and OMiSA.

Availability and ImplementationThe new methods described here have been added to our R package LDM, which is available on GitHub at https://github.com/yijuanhu/LDM.

sentences: Genetic Analysis Suggests a Surface of PAT-4 (ILK) that Interacts with UNC-112 (kindlin) The transmembrane protein integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a four protein complex (Kindlin, Integrin linked kinase (ILK), PINCH, and parvin). C. elegans muscle provides an excellent model to study integrin adhesion complexes. In C. elegans, UNC-112 (Kindlin) binds to the cytoplasmic tail of PAT-3 ({beta}-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 (ILK) and UNC-112 (Kindlin), we took a genetic approach. First, we isolated mutant PAT-4 proteins that cannot bind to UNC-112. Then, we isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, the mutant PAT-4 protein could localize to attachment structures. Third, overexpression of a mutant PAT-4 protein results in disorganization of the adhesion plaques at muscle cell boundaries, and co-expression of the UNC-112 supressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.

sentences: 3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells C/EBP{beta} is a potent regulator of RAS-induced senescence (RIS) and the SASP. C/EBP{beta} is post- translationally activated in RIS cells by the effector kinases ERK1/2 and CK2, but in tumor cells activation is suppressed by the CEBPB 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 colocalize with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site act to suppress C/EBP{beta}-mediated senescence, while a separate 3UTR region inhibits SASP induction. Accordingly, KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas. Our findings identify kinase-proximal mRNA decay as a novel mechanism to inhibit C/EBP{beta} activating modifications in tumor cells to facilitate senescence bypass.

sentences: Using dilution grazing assays to measure size fractionated phytoplankton mortality rates across the freshwater-to-marine continuum There is a need for a unified grazing method that can be used across the freshwater-to-marine continuum. To accomplish this, this research utilized dilution grazing assays across the freshwater-to-marine continuum and across the oligotrophic-to-hypereutrophic gradient by measuring size fractions of dilution-based mortality. This was investigated by using 0.7 {micro}m and 0.2 {micro}m prefiltered water and major ion solutions (MIS) as diluent media for use in the Landry-Hassett grazing bioassays run in lake, river, estuarine (riverine and lagoonal), and oceanic shelf systems. Because MIS does not include vitamins that would be in prefiltered natural water, vitamin effects on grazing rate determination were also investigated for the MIS bioassays. Results show that the dilution grazing method can be broadly applied across the freshwater-to-marine continuum and across trophic gradients.

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Probing the association between resting state brain network dynamics and psychological resilience This study aimed at replicating a previously reported negative correlation between node flexibility and psychological resilience, i.e., the ability to retain mental health in the face of stress and adversity. To this end, we used multiband resting-state BOLD fMRI (TR = .675 sec) from 52 participants who had filled out three psychological questionnaires assessing resilience. Time-resolved functional connectivity was calculated by performing a sliding window approach on averaged time series parcellated according to different established atlases. Multilayer modularity detection was performed to track network reconfigurations over time and node flexibility was calculated as the number of times a node changes community assignment. In addition, node promiscuity (the fraction of communities a node participates in) and node degree (as proxy for time-varying connectivity) were calculated to extend previous work. We found no substantial correlations between resilience and node flexibility. We observed a small number of correlations between the two other brain measures and resilience scores, that were however very inconsistently distributed across brain measures, differences in temporal sampling, and parcellation schemes. This heterogeneity calls into question the existence of previously postulated associations between resilience and brain network flexibility and highlights how results may be influenced by specific analysis choices.

Author SummaryWe tested the replicability and generalizability of a previously proposed negative association between dynamic brain network reconfigurations derived from multilayer modularity detection (node flexibility) and psychological resilience. Using multiband resting-state BOLD fMRI data and exploring several parcellation schemes, sliding window approaches, and temporal resolutions of the data, we could not replicate previously reported findings regarding the association between node flexibility and resilience. By extending this work to other measures of brain dynamics (node promiscuity, degree) we observe a rather inconsistent pattern of correlations with resilience, that strongly varies across analysis choices. We conclude that further research is needed to understand the network neuroscience basis of mental health and discuss several reasons that may account for the variability in results.

sentences: Improved exercise anaerobic power and less central fatigue in CD73 knockout mice Ecto-5-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill graded test to evaluate running power, oxygen uptake [Formula], and respiratory exchange ratio (RER) - the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and [Formula] in the treadmill were similar between CD73-KO and WT mice; [Formula] also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7{+/-}4.2% larger critical power (large effect size, P<0.05) and 3.8{+/-}0.4% increase of maximum RER (small effect size, P<0.05). Thus, KO of CD73 was ergogenic, i.e., it increased physical performance.

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Patterns of parent and offspring gene expression reflect canalization, not plasticity, in response to environmental stress Parenting buffers offspring from hostile environments, but it is not clear how or if the genes that underlie parenting change their expression under environmental stress. We recently demonstrated that for the subsocial carrion beetle, Nicrophorus orbicollis, temperature during parenting does not affect parenting phenotypes. Here, we ask if transcriptional changes associated with parenting are likewise robust to environmental stress. The absence of a transcriptional response for parenting under stress would suggest that the genetic  programs for parenting and being parented are canalized. Conversely, a robust transcriptional response would suggest that plasticity of underlying gene expression is critical for maintaining behavioral stability, and that these mechanisms provide a potential target for selection in the face of environmental change. We test these alternatives by characterizing gene expression of parents and offspring with and without parent-offspring interactions under a benign and a stressful temperature. We found that parent-offspring interactions elicit distinct transcriptional responses of parents and larvae irrespective of temperature. We further detected robust changes of gene expression in beetles breeding at 24{degrees} C compared to 20{degrees} C irrespective of family interaction. However, no strong interaction between parent-offspring interaction and temperature was detected for either parents or larvae. We therefore conclude that canalization, not plasticity of gene expression, most likely explains the absence of behavioral plasticity under thermal stress. This result suggests that species may not have the genetic variation needed to respond to all environmental change, especially for complex phenotypes.

IMPACT STATEMENTParenting is thought to evolve to buffer offspring from harsh or inhospitable conditions. However, once evolved, does parenting continue to buffer against new environmental challenges such as might occur under global warming? One way organisms achieve different phenotypes without evolving different genes for each trait is by changing timing or extent of gene expression. Changes in gene expression influencing physiological traits such as hypoxia have been shown to facilitate adaptation to changing environments. It is less clear if changes in gene expression are also important for behavioral plasticity in complex social traits, such as parental care. In this study, we ask whether families of the subsocial burying beetle, Nicrophorus orbicollis, show altered expression of parent or offspring genes that may help compensate for reduced offspring success under global warming. To test the extent that gene expression underlies behavioral plasticity in parenting and offspring responses to parenting, we characterize the gene expression profiles of whole families (males, females, and larvae) with and without parent-offspring interactions under two thermal conditions: 20{degrees} C and 24{degrees} C. We found that heat stress predominantly induced downregulation of genes, consistent with a constrained acclimation response. However, when temperature was considered in conjunction with parent-offspring interactions, genetic plasticity was extremely limited. That is, the genes underlying parent-offspring interactions did not show strong signatures of differential expression under more stressful thermal conditions. Given that we previously found no effects of temperature on parental behavior, we suggest that constrained, not plastic gene expression, underpins stability of parent-offspring interactions and limits the buffering capacity for family interactions under environmental stress. Insights from this study deepen our understanding of the interplay between genetic and phenotypic plasticity in complex behavioral traits and how traits such as parenting may limit responses to novel environmental challenges.

sentences: The EEG spectral fingerprints of meditation and mind wandering differ between experienced meditators and novices Previous literature suggests that meditation training is associated with changes in participants experience during meditation practice. In this study, we assess whether putative differences in the experience of meditation between meditators and non-meditators are reflected in EEG spectral modulations. For this purpose, we recorded electroencephalography (EEG) during rest and two breath focus meditations (with and without experience sampling) in a group of 29 adult participants with more than 3 years of meditation experience and a control group of 29 participants without any meditation experience. Experience sampling in one of the meditation conditions allowed us to disentangle periods of breath focus from mind wandering (i.e. moments of distraction driven by task-irrelevant thoughts) during meditation practice. Overall, meditators reported a greater level of focus and reduced mind wandering during meditation practice than controls. In line with these reports, EEG spectral modulations associated to meditation and mind wandering also differed significantly between meditators and controls. While meditators (but not controls) showed a significant decrease in individual alpha frequency and amplitude and a steeper 1/f slope during meditation relative to rest, controls (but not meditators) showed a relative increase in individual alpha amplitude during mind wandering relative to breath focus periods. Together, our results show that the experience of meditation changes with training and that this is reflected in oscillatory and non-oscillatory components of brain activity.

sentences: Induced neural differentiation of human mesenchymal stem cells affects lipid metabolism pathways Neuronal membranes contain exceptionally high concentrations of long-chain polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), which are essential for neuronal development and function. Adult bone-marrow-derived mesenchymal stem cells (MSC) can be induced to possess some neuronal characteristics. Here we examined the effects of neuronal induction on the PUFA metabolism specific pathways. Differentiated cells contained ~30% less ARA than MSC. The expression of specific ARA metabolizing enzymes was upregulated, notably that of prostaglandin E2 synthase which increased more than 15-fold, concomitantly with a 3-fold increase in the concentration of PGE2 in the medium. Moreover, induced differentiation was associated with enhanced incorporation of exogenous DHA, upregulation of acyl-CoA synthases, fatty acid binding proteins, choline kinase (CK) and phosphatidylserine synthases as well as increased total cellular phospholipids (PL). These findings suggest that active ARA metabolites may be important in the differentiation process and that neuronal induction prepares the resulting cells for increased DHA incorporation through the action of specific enzymes.

sentences: Wnt-dependent activation of ERK mediates repression of chondrocyte fate during calvarial development. Wnt signaling regulates cell fate decisions in diverse contexts during development, and loss of Wnt signaling in the cranial mesenchyme results in a robust and binary cell fate switch from cranial bone to ectopic cartilage. The Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and Wnt signaling pathways are activated during calvarial osteoblast cell fate selection. Here, we test the hypothesis that ERK signaling is a mediator of Wnt-dependent cell fate decisions in the cranial mesenchyme. First, we show that loss of Erk1/2 in the cranial mesenchyme results in a diminished domain of osteoblast marker expression and increased expression of cartilage fate markers and ectopic cartilage formation in the frontal bone primordia. Second, we show that mesenchyme Wnt/{beta}-catenin signaling and Wntless are required for ERK activation in calvarial osteoblasts. Third, we demonstrate that Wnt and ERK signaling pathways function together to repress Sox9 expression in mouse cranial mesenchyme. Our results demonstrate a link between the Wnt and ERK signaling pathways in regulating lineage selection in a subset of calvarial cells and provide new insights into Wnt-dependent cell fate decisions.

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overlappingCGM: Automatic detection and analysis of overlapping co-expressed gene modules BackgroundWhen it comes to the co-expressed gene module detection, its typical challenges consist of overlap between identified modules and local co-expression in a subset of biological samples. The nature of module detection is the use of unsupervised clustering approaches and algorithms. Those methods are advanced undoubtedly, but the selection of a certain clustering method for sample- and gene-clustering tasks is separate, in which the latter task is often more complicated.

ResultsThis study presented an R-package, Overlapping CoExpressed gene Module (oCEM), armed with the decomposition methods to solve the challenges above. We also developed a novel auxiliary statistical approach to select the optimal number of principal components using a permutation procedure. We showed that oCEM outperformed state-of-the-art techniques in the ability to detect biologically relevant modules additionally.

ConclusionsoCEM helped non-technical users easily perform complicated statistical analyses and then gain robust results. oCEM and its applications, along with example data, were freely provided at https://github.com/huynguyen250896/oCEM.

sentences: V1 receptive field structure contributes to neuronal response latency The timing of neuronal responses is considered to be important for information transferring and communication across individual neurons. However, the sources of variabilities in the timing of neuronal responses are not well understood and sometimes over-interpreted. A systematic variability in the response latencies of the primary visual cortex has been reported in presence of drifting grating stimulus. Whereas the response latencies are systematically dependent on stimulus orientation. To understand the underlying mechanism of these systematic latencies, we recorded the neuronal response of the cat visual cortex, area 17, and simulated the response latency of V1 neurons, with two geometric models. We showed that outputs of these two models significantly predict the response latencies of the electrophysiology recording during orientation tasks. The periodic patterns created in the raster plots were dependent on the relative position of the stimulus rotation center and the receptive-field sub-regions. We argue the position of stimulus is contributing to systematic response latencies, dependent on drifting orientation. Therefore, we provide a toolbox based on our geometrical model for determining the exact location of RF sub-regions. Our result indicates that a major source of neuronal variability is the lack of fine-tuning in the task parameters. Considering the simplicity of the orientation selectivity task, we argue fine-tuning of stimulus properties is crucial for deduction of neural variability in higher-order cortical areas and understanding their neural dynamics.

sentences: Specific sequence of arrival promotes coexistence via spatial niche preemption by the weak competitor Historical contingency, such as the order of species arrival, can modify competitive outcomes via niche modification or preemption. However, how these mechanisms ultimately modify stabilising niche and average fitness differences remains largely unknown. By experimentally assembling two congeneric spider mite species feeding on tomato plants during two generations, we show that order of arrival affects species competitive ability and changes the outcome of competition. Contrary to expectations, order of arrival did not cause positive frequency dependent priority effects. Instead, coexistence was predicted when the inferior competitor (Tetranychus urticae) arrived first. In that case, T. urticae colonised the preferred feeding stratum (leaves) of T. evansi leading to spatial niche preemption, which equalised fitness and reduced niche differences, driving community assembly to a close-to-neutrality scenario. Our study demonstrates how the order of species arrival and the spatial context of competitive interactions can jointly determine whether species can coexist.

sentences: Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected Patients RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

sentences: Cell-free prototyping enables implementation of optimized reverse beta-oxidation pathways in heterotrophic and autotrophic bacteria Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse {beta}-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimized and implemented r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screened 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generated designer strains for the selective production of butanoic acid (4.9 {+/-}0.1 gL-1), hexanoic acid (3.06 {+/-} 0.03 gL-1) and 1-hexanol (1.0 {+/-} 0.1 gL-1) at the best performance reported to date in this bacterium. We also generated Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5-L continuous fermentation. Our strategy enables optimization of rBOX derived products for biomanufacturing and industrial biotechnology.

sentences: Dengue virus clinical isolates sustain viability of infected hepatic cells by counteracting apoptosis-mediated DNA breakage NS1, a virotoxin, abundantly present in Dengue patients blood, is a major player behind disease patho-biogenesis including plasma leakage and damage to the liver. Despite the presence of NS1 in blood, Dengue is asymptomatic and self-limiting in [≥]80% Dengue virus (DV) infected people. We investigated this observation and found that plasmid-mediated NS1 expression and secretion in liver cells (Huh7) are sufficient to cause programmed cell death (apoptosis) and associated cellular DNA breakage. However, liver or kidney cell lines infected with DV and secreting equivalent amounts of NS1 didnt exhibit apoptotic DNA breakage. In fact, DV-infected cells showed better survival than cells in which only NS1 was transiently expressed by transfection. We also found that DV can even prevent chemical-induced apoptotic DNA damage in infected host cells. So, DV thwarts host antiviral defence i.e. apoptosis, by counteracting cellular DNA breakages and keeps the infected cells metabolically active to prolong virus replication.

sentences: Pairwise Relative Distance (PRED) is an intuitive and robust metric for assessing vector similarity and class separability Scientific studies often require assessment of similarity between ordered sets of values. Each set, containing one value for every dimension or class of data, can be conveniently represented as a vector. The commonly used metrics for vector similarity include angle-based metrics, such as cosine similarity or Pearson correlation, which compare the relative patterns of values, and distance-based metrics, such as the Euclidean distance, which compare the magnitudes of values. Here we evaluate a newly proposed metric, pairwise relative distance (PRED), which considers both relative patterns and magnitudes to provide a single measure of vector similarity. PRED essentially reveals whether the vectors are so similar that their values across the classes are separable. By comparing PRED to other common metrics in a variety of applications, we show that PRED provides a stable chance level irrespective of the number of classes, is invariant to global translation and scaling operations on data, has high dynamic range and low variability in handling noisy data, and can handle multi-dimensional data, as in the case of vectors containing temporal or population responses for each class. We also found that PRED can be adapted to function as a reliable metric of class separability even for datasets that lack the vector structure and simply contain multiple values for each class.

sentences: Overground Gait Kinematics and Muscle Activation Patterns in the Yucatan Mini Pig A growing number of spinal cord injury, neuromodulation, and cell therapy studies on porcine models, especially the Yucatan minipigs (YMPs), have been recently reported. This is due to the large similarities between human and porcine neuroanatomy and biomechanics. To assess treatment modalities and locomotor recovery in this model, there is an obvious need for detailed characterization of normative overground gait in neurologically intact YMPs. The objective of this study was to assess gait biomechanics and the effect of overground walking speed on gait parameters, kinematics, and electromyographic (EMG) activity in the hindlimb muscles of YMPs. Nine neurologically-intact adult YMPs were trained to walk overground in a straight line. Whole-body kinematics and EMG activity of hindlimb muscles were recorded and analyzed at 6 different speed ranges (0.4-0.59, 0.6-0.79, 0.8-0.99, 1.0-1.19, 1.2-1.39, and 1.4-1.6 m/s). A MATLAB program was developed to detect strides and gait events automatically from motion-captured data. Significant decreases in stride duration, stance and swing times and an increase in stride length were observed with increasing speed. A transition in gait pattern occurred at the 1.0m/s walking speed. Significant increases in the range of motion of the knee and ankle joints were observed at higher speeds. Also, the points of minimum and maximum knee and ankle joint angles occurred earlier in the gait cycle at higher speeds. The onset of EMG activity in the biceps femoris muscle occurred significantly earlier in the gait cycle with increasing speed. A comprehensive characterization of overground walking in neurologically-intact YMPs is provided. These normative measures set the basis against which the effects of future interventions on locomotor capacity in YMPs can be compared.

sentences: Organ- and function-specific anatomical organization and bioelectronic modulation of the vagus nerve Afferent and efferent fibers in the vagus nerve travel inside fascicles and form branches to innervate organs and regulate organ functions. The spatial organization of fibers and fascicles, with respect to the functions they mediate and the organs they innervate, is unknown. Accordingly, it is unknown whether such organization can be leveraged by bioelectronic devices for function- and organ-specific neurostimulation. To characterize the functional microscopic anatomy of the vagus, we developed a pipeline consisting of micro-computed tomography imaging of fascicles and quantitative immunohistochemistry of single fibers. We found that, in swine, the vagus contains clusters of fascicles, organized along 2 overlapping functional axes: one axis specific to sensory vs. motor somatic and visceral functions, with clusters increasingly separating in the cephalad direction, and a second axis specific to innervated organs, including larynx, lungs and heart, with increasing separation in the caudad direction. Large myelinated afferents and efferents, and small unmyelinated efferents occupy large areas of specific clusters of fascicles; small unmyelinated afferents are numerous and occupy small areas across every fascicle. To test whether fiber populations can be selectively modulated, we developed a multi-contact cuff electrode accommodating the nerve fascicular structure. Stimulation targeting separate nerve sections evokes compound action potentials from fibers of distinct functional types and elicits differential organ responses, including laryngeal muscle contraction, cough reflex, and changes in breathing and heart rate. Our results indicate that vagus fibers are anatomically organized according to functions they mediate and organs they innervate and can be differentially modulated by spatially selective nerve stimulation.

sentences: Nanoengineered DNA origami with repurposed TOP1 inhibitors targeting myeloid cells for the mitigation of neuroinflammation Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Here, we screened a library of compounds and identified the topoisomerase 1 (TOP1) inhibitor camptothecin (CPT) as a promising drug candidate for microglial modulation. CPT and its FDA-approved analog topotecan (TPT) inhibited inflammatory responses in microglia and macrophages, and ameliorated neuroinflammation in mice. Transcriptomic analysis of sorted microglia revealed an altered transcriptional phenotype following TPT treatment, with Ikzf1 identified as a potential target. Importantly, TOP1 expression was found elevated in several neuroinflammatory conditions, including human MS brains. To achieve targeted delivery to myeloid cells we designed a nanosystem using DNA origami and loaded TPT into it (TopoGami). TopoGami also significantly suppressed the inflammatory response in microglia and mitigated disease progression in MS-like mice. Our findings suggest that TOP1 inhibition represents a therapeutic strategy for neuroinflammatory diseases, and the proposed nanosystem may foster future research and drug development with a demand to target myeloid cells.

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A multi-omics study to investigate tacrolimus nephrotoxicity mechanisms BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics.

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Caregiving quality modulates neuroendocrine and immunological markers in young children in foster care who have experienced early adversity BackgroundEarly adversity is believed to alter the bodys stress response systems, putting children at increased risk for somatic and mental health problems. However, it remains unclear whether such alterations normalize under improved caregiving experiences. Thus, the goal of the present study was to investigate i) whether children in foster care show endocrine and immunological alterations relative to children living with their biological families, ii) whether these alterations change over time spent with the foster family and iii) whether the alterations are modulated by current caregiving experiences.

MethodsA total of 94 children in foster care and 157 biological children, aged two to seven years, took part in a longitudinal study with three assessments conducted over a 12-month study period. At the initial assessment, children lived for an average of 18 months with their current foster families. Childrens cortisol, dehydroepiandrosterone (DHEA) and progesterone concentrations and cortisol/DHEA ratios were measured in scalp hair and childrens secretory immunoglobulin A (sIgA) levels in saliva. Caregiving quality was assessed based on caregiver-reports and observational measures of caregiver-child interactions.

ResultsChildren in foster care had lower cortisol/DHEA ratios and higher progesterone concentrations than biological children, while no group differences were found for cortisol, DHEA or sIgA. Time spent with the current foster family did not significantly influence the childs endocrine or immunological markers. Importantly, caregiving quality modulated cortisol/DHEA ratios and sIgA concentrations: children in foster care of lower caregiving quality had lower cortisol/DHEA ratios than children in foster care of higher caregiving quality and showed decreasing, rather than increasing, sIgA concentrations across the study period.

ConclusionsOur results indicate that caregiving quality in the foster family may have an important modulating effect on selected indicators of the childs stress response and could thereby mitigate the possible consequences of early childhood adversity.

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The Arabidopsis Framework Model version 2 predicts the organism-level effects of circadian clock gene mis-regulation Predicting a multicellular organisms phenotype quantitatively from its genotype is challenging, as genetic effects must propagate across scales. Circadian clocks are intracellular regulators that control temporal gene expression patterns and hence metabolism, physiology and behaviour. Here we explain and predict canonical phenotypes of circadian timing in a multicellular, model organism. We used diverse metabolic and physiological data to combine and extend mathematical models of rhythmic gene expression, photoperiod-dependent flowering, elongation growth and starch metabolism within a Framework Model for the vegetative growth of Arabidopsis thaliana, sharing the model and data files in a structured, public resource. The calibrated model predicted the effect of altered circadian timing upon each particular phenotype in clock-mutant plants under standard laboratory conditions. Altered night-time metabolism of stored starch accounted for most of the decrease in whole-plant biomass, as previously proposed. Mobilisation of a secondary store of malate and fumarate was also mis-regulated, accounting for any remaining biomass defect. We test three candidate mechanisms for the accumulation of these organic acids. Our results link genotype through specific processes to higher-level phenotypes, formalising our understanding of a subtle, pleiotropic syndrome at the whole-organism level, and validating the systems approach to understand complex traits starting from intracellular circuits.

This work updates the first biorXiv version, February 2017, https://doi.org/10.1101/105437, with an expanded description and additional analysis of the same core data sets and the same FMv2 model, summary tables and supporting, follow-on data from three further studies with further collaborators. This biorXiv revision constitutes the second version of this report.

sentences: Coat color mismatch improves survival of a keystone boreal herbivore: energetic advantages exceed lost camouflage Climate warming is causing asynchronies between animal phenology and environments. Mismatched traits, like coat color change mismatched with snow, can decrease survival. However, coat change does not serve a singular adaptive benefit of camouflage, and alternate coat change functions may confer advantages that supersede mismatch costs. We found that mismatch reduced rather than increased, autumn mortality risk of snowshoe hares in Yukon by 86.5 %. We suggest that the increased coat insulation and lower metabolic rates of winter acclimatized hares confer energetic advantages to white mismatched hares that reduce their mortality risk. We found that white mismatched hares forage 17-77 minutes less per day than matched brown hares between 0 and -10 {degrees}C, thus lowering their predation risk and increasing survival. We found no effect of mismatch on spring mortality risk, where mismatch occurred at warmer temperatures, suggesting a potential temperature limit where the costs of conspicuousness outweigh energetic benefits.

sentences: MyD88 Mediates Colitis- and RANKL-induced Microfold Cell Differentiation Intestinal microfold (M) cells are critical for sampling antigen in the gut and initiating the intestinal mucosal immune response. In this study, we found that the differentiation efficiency of M cells was closely related to the colitis severity. The expression levels of M cells differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines expression originated from dextran sulfate sodium (DSS) induction and Salmonella infection. Compared with wild-type (WT) mice, MyD88-/- mice exhibited significantly lower expression levels of M cells differentiation-related genes. However, DSS could induce colitis in MyD88-/- mice but failed to promote M cells differentiation. Furthermore, the receptor activator of the Nuclear Factor-{kappa}B ligand (RANKL) induced M cells differentiation in murine intestinal organoids prepared from both WT and MyD88-/- mice. However, less M cells differentiation were found in MyD88-/- mice as compared with WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related M cells differentiation.

sentences: A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs.

sentences: Discordant population structure inferred from male- and female-type mtDNAs from Limecola balthica, a bivalve species characterized by doubly uniparental inheritance of mitochondria Doubly Uniparental Inheritance (DUI) of mitochondria is a remarkable exception to the Strictly Maternal Inheritance (SMI) in metazoans. In species characterized by DUI --almost exclusively gonochoric bivalve mollusks--, females (F) transmit mitochondria to offspring of both sexes, while males (M) pass on their mitochondria exclusively to their sons. Under DUI, males are heteroplasmic, somatic tissues containing F-transmitted mtDNA and gametic cells containing M-transmitted mtDNAs. The aforementioned transmission routes make M- and F-transmitted mtDNA interesting as sex-specific markers which can differ in their effective population sizes, mutation rates, and selective constraints. For these reasons, looking at both markers can provide significant insight into the genetic structure of populations and investigate its determinants. In this study, we document differences in genetic diversity, divergence, inter-populational genetic differentiation and biogeographic structure between M- and F-type cox1 mt genes in the Baltic tellin (Limecola balthica) to test whether cox1m and cox1f genes bear the marks of similar phylogeographic histories. Both markers were sequenced for 313 male individuals sampled from the Baltic Sea to the Gironde Estuary (Southern France). Haplotype diversity and net divergence were over twice higher in cox1m compared to cox1f. A strong southward decrease in nucleotide diversity was observed only at cox1m. Genetic differentiation between northern and southern populations was nearly 3 times higher at cox1m compared to cox1f (global {Phi}ST = 0.447 and 0.126 respectively) and the geographic localization of the strongest genetic break significantly differed between the markers (Finistere Peninsula at cox1f; Cotentin Peninsula at cox1m). A higher mutation rate, relaxed negative selective pressure and differences in effective population sizes (depending on locations) at cox1m could explain differences in population genetic structure. As both F- and M-type mtDNAs interact with nuclear genes for oxidative phosphorylation and ATP production, geographical discordances in genetic clines could be linked to mito-nuclear genetic incompatibilities in this system.

sentences: nf-core/mag: a best-practice pipeline for metagenome hybrid assembly and binning The analysis of shotgun metagenomic data provides valuable insights into microbial communities, while allowing resolution at individual genome level. In absence of complete reference genomes, this requires the reconstruction of metagenome assembled genomes (MAGs) from sequencing reads. We present the nf-core/mag pipeline for metagenome assembly, binning and taxonomic classification. It can optionally combine short and long reads to increase assembly continuity and utilize sample-wise group-information for co-assembly and genome binning. The pipeline is easy to install - all dependencies are provided within containers -, portable and reproducible. It is written in Nextflow and developed as part of the nf-core initiative for best-practice pipeline development. All code is hosted on GitHub under the nf-core organization https://github.com/nf-core/mag and released under the MIT license.

sentences: Evaluation of the stereochemical quality of predicted RNA 3D models in the RNA-Puzzles submissions In silico prediction is a well-established approach to derive a general shape of an RNA molecule based on its sequence or secondary structure. This paper reports an analysis of the stereochemical quality of the RNA three-dimensional models predicted using dedicated computer programs. The stereochemistry of 1,052 RNA 3D structures, including 1,030 models predicted by fully automated and human-guided approaches within 22 RNA-Puzzles challenges and reference structures, is analysed. The evaluation is based on standards of RNA stereochemistry that the Protein Data Bank requires from deposited experimental structures. Deviations from standard bond lengths and angles, planarity or chirality are quantified. A reduction in the number of such deviations should help in the improvement of RNA 3D structure modelling approaches.

sentences: Representational integration and differentiation in the human hippocampus following goal-directed navigation As we learn, dynamic memory processes build structured knowledge across our experiences. Such knowledge enables the formation of internal models of the world that we use to plan, make decisions, and act. Recent theorizing posits that mnemonic mechanisms of differentiation and integration - which at one level may seem to be at odds - both contribute to the emergence of structured knowledge. We tested this possibility using fMRI as human participants learned to navigate within local and global virtual environments over the course of three days. Pattern similarity analyses on entorhinal cortex, hippocampus, and ventromedial prefrontal cortex patterns revealed evidence that differentiation and integration work concurrently to build local and global environmental representations, and that variability in integration relates to differences in navigation efficiency. These results offer new insights into the neural machinery and the underlying mechanisms that translate experiences into structured knowledge that allows us to navigate to achieve goals.

sentences: NucleoMap: a computational tool for identifying nucleosomes in ultra-high resolution contact maps 1Although poorly positioned nucleosomes are ubiquitous in the prokaryote genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling profiling of nucleosomes in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome binding preferences, read density, and pairing information, NucleoMap precisely locates nucleosomes in both eukaryotic and prokaryotic genomes and outperforms existing nucleosome identification methods in precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also predict two tetra-nucleosome folding structures in human embryonic stem cells using machine learning methods and analysis their distribution at different structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the original datas contact distance profile.

sentences: Molecular basis of uracil/H+ symport mechanism operated by the FurE/NCS1 transporter Transporters mediate the uptake or efflux of solutes, metabolites and drugs across the cell membrane. The FurE symporter of uracil-allantoin-uric acid/H+ of Aspergillus nidulans has been used as a model eukaryotic transporter to address structure-function relationships and the mechanism of transport in the NCS1/APC family of transporters. Extensive genetic, functional and cellular studies, as well as homology modeling based on a prokaryotic transporter with a similar fold (Mhp1) have provided important information on specific structural elements of FurE. However, the exact mechanism by which substrates and proton or other cations are translocated by FurE or transporters with a similar fold remains elusive. In this study, we reveal the binding mode, translocation and release pathway of uracil/H+ from the extracellular space to the cytoplasm, using novel metadynamics calculations and rationally designed mutational analysis. In particular, Metadynamics Free Energy Surface maps provide the relative order of internalization of uracil and proton, permitting also selection of intermediate conformational states related to transport cycle. Funnel Metadynamics allow the sampling of specific interactions of both uracil and proton with residues during their internalization, generating a holistic model of the transport events in conjunction with experimental mutation studies. Our work not only complements and extends the existing knowledge on FurE same-fold transporters, but also challenges the so-called rocking bundle mechanism associated with biomedically interesting members of the APC superfamily of transporters.

sentences: A Comparison of Methods to Harmonize Cortical Thickness Measurements Across Scanners and Sites Results of neuroimaging datasets aggregated from multiple sites may be biased by site- specific profiles in participants demographic and clinical characteristics, as well as MRI acquisition protocols and scanning platforms. We compared the impact of four different harmonization methods on results obtained from analyses of cortical thickness data: (1) linear mixed-effects model (LME) that models site-specific random intercepts (LMEINT), (2) LME that models both site-specific random intercepts and age-related random slopes (LMEINT+SLP), (3) ComBat, and (4) ComBat with a generalized additive model (ComBat-GAM). Our test case for comparing harmonization methods was cortical thickness data aggregated from 29 sites, which included 1,343 cases with posttraumatic stress disorder (PTSD) (6.2-81.8 years old) and 2,067 trauma-exposed controls without PTSD (6.3-85.2 years old). We found that, compared to the other data harmonization methods, data processed with ComBat-GAM were more sensitive to the detection of significant case-control differences in regional cortical thickness (X2(3) = 34.339, p < 0.001), and case-control differences in age-related cortical thinning (X2(3) = 15.128, p = 0.002). Specifically, ComBat-GAM led to larger effect size estimates of cortical thickness reductions (corrected p-values < 0.001), smaller age-appropriate declines (corrected p-values < 0.001), and lower female to male contrast (corrected p-values < 0.001) in cases compared to controls relative to other harmonization methods. Harmonization with ComBat-GAM also led to greater estimates of age-related declines in cortical thickness (corrected p-values < 0.001) in both cases and controls compared to other harmonization methods. Our results support the use of ComBat-GAM for harmonizing cortical thickness data aggregated from multiple sites and scanners to minimize confounds and increase statistical power.

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Fast adaptation of cooperative channels engendersHopf bifurcations in auditory hair cells Since the pioneering work of Thomas Gold published in 1948, it has been known that we owe our sensitive sense of hearing to a process in the inner ear that can amplify incident sounds on a cycle-by-cycle basis. Termed the active process, it uses energy to counteract the viscous dissipation associated with sound-evoked vibrations of the ears mechanotransduction apparatus. Despite its importance, the mechanism of the active process and the proximate source of energy that powers it have remained elusive--especially at the high frequencies characteristic of mammalian hearing. This is partly due to our insufficient understanding of the mechanotransduction process in hair cells, the sensory receptors and amplifiers of the inner ear. It has previously been proposed that a cyclical binding of Ca2+ ions to individual mechanotransduction channels could power the active process. That model, however, relied on tailored reaction rates that structurally forced the direction of the cycle. Here, we ground our study on our previous model of hair-cell mechanotransduction, which relied on the cooperative gating of pairs of channels, and incorporate into it the cyclical binding of Ca2+ ions. With a single binding site per channel and reaction rates drawn from thermodynamic principles, our model shows that hair cells behave as nonlinear oscillators that exhibit Hopf bifurcations, dynamical instabilities long understood to be signatures of the active process. Using realistic parameter values, we find bifurcations at frequencies in the kilohertz range with physiological Ca2+ concentrations. In contrast to the myosin-based mechanism, responsible for low-frequency relaxation oscillations in the vestibular hair cells of amphibians, the current model relies on the electrochemical gradient of Ca2+ as the only energy source for the active process and on the relative motion of cooperative channels within the stereociliary membrane as the single mechanical driver. Equipped with these two mechanisms, a hair bundle proves capable of operating at frequencies in the kilohertz range, characteristic of mammalian hearing.

SIGNIFICANCEHow the inner ear amplifies incident sounds at audible frequencies of several kilohertz is a key question that has remained unanswered despite decades of research into several candidate mechanisms. Here, we model the behavior of hair cells, the sensory receptors of the inner ear, and show that they can undergo oscillatory instabilities called Hopf bifurcations due to the effect of Ca2+ on the cooperative opening and closing of mechanotransduction ion channels. Close to the bifurcation point, a hair cell behaves as a nonlinear oscillator that can amplify its input on a cycle-by-cycle basis. We find that our proposed mechanism can operate in the kilohertz range.

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A tomato prolyl-4-hydroxylase causes relocation of abscission zone and alters abscission kinetics The detachment of organs is controlled by highly regulated molecular mechanisms. The position of the tomato abscission zone (AZ) is defined by the ratio of the proximal to distal part of the pedicel. In this study, the ratio was altered due to a shift in the position of the AZ which was attributed to shorter and longer pedicels of SlP4H3 RNAi and OEX lines due to changes on cell division and expansion in AZ and distal part. This might be associated with LM2- and JIM8-AGPs which increased in OEX and decreased in RNAi lines throughout the pedicel. The JIM13 AGPs were downregulated in the flower AZ of OEX lines, pointing to a role on abscission regulation. In addition, Co-IP in flower AZ with SlP4H3-GFP fusion proteins showed interaction with LM2-, JIM13- and JIM8-epitopes suggesting proline hydroxylation by SlP4H3. The lower content of methyl-esterified HGs and higher of demethyl-esterified HGs in the AZs of RNAi lines might be responsible for increased rigidity of the AZ cell walls, accounting for the higher force required for AZ tissue detachment to occur. Moreover, ethylene-induced flower abscission was accelerated in the RNAi lines and delayed in OEX lines, while exactly the opposite response was observed in the red ripe fruit AZs. This was partly attributed to alterations in the expression of cell wall hydrolases. Overall, these results indicate that P4Hs might regulate molecular and structural features of cell walls in the AZ as well as abscission progression by regulating the structure and function of AGPs.

Sentence SummaryAlterations on the expression of tomato prolyl 4 hydroxylase 3 shifts the position of the pedicel abscission zone and alters ethylene induced abscission progression according to the developmental context of the pedicel.

sentences: The extinction of threatened species by anthropogenic threats endangers the functional diversity of turtles and crocodiles The Anthropocene is tightly associated with a drastic loss of species worldwide, and consequently, with the disappearance of key ecosystem functions. The on-going reduction in species functionality is driven by global and local threats. The orders Testudines (turtles and tortoises) and Crocodilia (crocodilians and alligators) contain numerous threatened, long-lived species for which their functional diversity and potential erosion by anthropogenic impacts remains unknown. Here, we examine 259 of the existing 375 species of Testudines and Crocodilia quantifying their life history strategies (i.e., schedules of survival, development, and reproduction) from open-access data on their demography, ancestry, and threats. We find that the loss of functional diversity in simulated extinction scenarios of threatened species is greater than expected by chance. Moreover, the effects of unsustainable local consumption, diseases, and pollution are associated to specific functional strategies. Meanwhile climate change, habitat disturbance, and global trade affect all species independent of their life history strategy. Importantly, the loss of functional diversity for threatened species by habitat disturbance is twice greater than for all other threats. Our findings highlight the key importance of conservation programmes focused on preserving the functional diversity of life history strategies jointly with the phylogenetic representativity of these highly threatened groups.

sentences: Structure of the human FERRY Rab5 effector complex Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction is linked to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and the early endosome in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, in which two arm-like appendages, each consisting of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. We demonstrate that the coiled-coil domains of Fy-2 are flexible and project into opposite directions from the FERRY complex core. While the C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils together with Fy-5 bind mRNA. Thus, Fy-2 serves as binding hub that connects not only all five complex subunits, but also mediates the binding to mRNA and to the early endosome via Rab5. The FERRY structure provides novel mechanistic insight into long-distance mRNA transport.

sentences: Cooperative escape in ants and robots The solution of complex problems by the collective action of simple agents in both biologically evolved and synthetically engineered systems involves cooperative action. Understanding the resulting emergent solutions requires integrating across the organismal behaviors of many individuals. Here we investigate an ecologically relevant collective task in black carpenter ants Camponotus pennsylvanicus: escape from a soft, erodible confining corral. Individual ants show a transition from individual exploratory excavation at random locations to spatially localized collective exploitative excavation and escape from the corral. A minimal continuum theory that coarse-grains over individual actions and considers their integrated influence on the environment leads to the emergence of an effective phase space of behaviors in terms of excavation strength and cooperation intensity. To test the theory over the range of predicted behaviors, we used custom-built robots (RAnts) that respond to stimuli and show the emergence (and failure) of cooperative excavation and escape. Overall, our approach shows how the cooperative completion of tasks can arise from relatively simple rules that involve the interaction of simple agents with a dynamically changing environment that serves as an enabler and modulator of behavior.

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Novel genotyping algorithms for rare variants significantly improve the accuracy of Applied Biosystems™ Axiom™ array genotyping calls ObjectiveTo significantly improve the positive predictive value (PPV) and sensitivity of Applied Biosystems Axiom array variant calling, by means of novel improvement to genotyping algorithms and careful quality control of array probesets. The improvement makes array genotyping more suitable for very rare variants.

DesignRetrospective evaluation of UK Biobank array data re-genotyped with improved algorithms for rare variants.

Participants488,359 people recruited to the UK Biobank with Axiom array genotyping data including 200,630 with exome sequencing data.

Main Outcome MeasuresA comparison of genotyping calls from array data to genotyping calls on a subset of variants with exome sequencing data.

ResultsAxiom genotyping [18] performed well, based on comparison to sequencing data, for over 100,000 common variants directly genotyped on the Axiom UK Biobank array and also exome sequenced by the UK Biobank Exome Sequencing Consortium. However, in a comparison to the initial exome sequencing results of the first 50K individuals, Weedon et al. [1] observed that when grouping these variants by the minor allele frequency (MAF) observed in UK Biobank, the concordance with sequencing and resulting positive predictive value (PPV) decreased with the number of heterozygous (Het) array calls per variant. An improved genotyping algorithm, Rare Heterozygous Adjustment (RHA) [16], released mid-2020 for genotyping on Axiom arrays, significantly improves PPV in all MAF ranges for the 50K data as well as when compared to the exome sequencing of 200K individuals, released after Weedon et al. [1] performed their comparison. The RHA algorithm improved PPVs in the 200K data in the lowest three frequency groups [0, 0.001%), [0.001%, 0.005%) and [0.005%, 0.01%) to 83%, 82% and 88%; respectively. PPV was above 95% for higher MAF ranges without algorithm improvement. PPVs are somewhat higher in the 200K dataset, due to a different "truth set" from exome sequencing and because monomorphic exome loci are not included in the joint genotyping calls for the 200K data set, as explained in the methods section.

Sensitivity was higher in the 200K data set than in the original 50K data as well, especially for low MAF ranges. This increase is in part due to the larger data set over which sensitivity could be computed and in part due to the different WES algorithms used for the 200K data [7]. Filtering of a relatively small number of non-performing probesets (determined without reference to the exome sequencing data) significantly improved sensitivities for all MAF ranges, resulting in 70%, 88% and 94% respectively in the three lowest MAF ranges and greater than 98% and 99.9% for the two higher MAF ranges ([0.01%, 1%), [1%, 50%]).

ConclusionsImproved algorithms for genotyping along with enhanced quality control of array probesets, significantly improve the positive predictive value and the sensitivity of array data, making it suitable for the detection of very rare variants. The probeset filtering methods developed have resulted in better probe designs for arrays and the new genotyping algorithm is part of the standard algorithm for all Axiom arrays since early 2020.

sentences: Recruitment of CTCF to an Fto enhancer is responsible for transgenerational inheritance of obesity Transgenerational transmission of epiphenotypes is poorly understood. Here we show that exposure of pregnant mouse F0 females to BPA results in obesity in the F2 progeny due to increased food intake and leptin resistance. This epiphenotype can be transmitted up to the F6 generation and disappears in F7. Analyses of chromatin accessibility in sperm of the F1-F6 generations reveals alterations in the binding of CTCF at two enhancers of the Fto gene in obese but not control animals that correlates with transmission of obesity. Deletion of the CTCF site in Fto results in mice that fail to become obese when exposed to BPA. These Fto enhancers show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite controlling AgRP/NPY neurons. Single-nucleus and immunofluorescence analyses in the arcuate nucleus of the hypothalamus suggest that exposure to BPA results in expansion of the number of orexigenic AgRP neurons. This expansion correlates with increased accessibility of the Fto proximal enhancer in radial glia-like neural stem cells (RG-NSCs), which give rise to AgRP/NPY neurons, and in mature oligodendrocytes. The results provide a molecular mechanism for transgenerational inheritance in mammals and suggest that both genetic and epigenetic alterations of Fto can lead to the same phenotypic outcomes.

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Heat stress prevented the biomass and yield stimulation caused by elevated CO2 in two well-watered wheat cultivars To investigate the interactive effects of elevated CO2 and heat stress (HS), we grew two contrasting wheat cultivars, early-maturing Scout and high-tillering Yitpi, under non-limiting water and nutrients at ambient (aCO2, 450 ppm) or elevated (eCO2, 650 ppm) CO2 and 22{degrees}C in the glasshouse. Plants were exposed to two 3-day HS cycles at the vegetative (38.1{degrees}C) and/or flowering (33.5{degrees}C) stage.

At aCO2, both wheat cultivars showed similar responses of photosynthesis and mesophyll conductance to temperature and produced similar grain yield. Relative to aCO2, eCO2 enhanced photosynthesis rate and reduced stomatal conductance and maximal carboxylation rate (Vcmax). During HS, high temperature stimulated photosynthesis at eCO2 in both cultivars, while eCO2 stimulated photosynthesis in Scout. Electron transport rate (Jmax) was unaffected by any treatment. eCO2 equally enhanced biomass and grain yield of both cultivars in control, but not HS, plants. HS reduced biomass and yield of Scout at eCO2. Yitpi, the cultivar with higher grain nitrogen, underwent a trade-off between grain yield and nitrogen. In conclusion, eCO2 improved photosynthesis of control and HS wheat, and improved biomass and grain yield of control plants only. Under well-watered conditions, HS was not detrimental to photosynthesis or growth but precluded a yield response to eCO2.

Key messageHigh temperatures increased photosynthetic rates only at eCO2 and photosynthesis was upregulated after recovery from heat stress at eCO2 in Scout suggesting that eCO2 increased optimum temperature of photosynthesis.

sentences: Fine human genetic map based on UK10K data set Recombination is a major force that shapes genetic diversity. Determination of recombination rate is important and can theoretically be improved by increasing the sample size. However, it is challenging to estimate recombination rates when the sample size is extraordinarily large because of computational burden. In this study, we used a refined artificial intelligence approach to estimate the recombination rate of the human genome using the UK10K human genomic dataset with 7,562 genomic sequences and its three subsets with 200, 400 and 2,000 genomic sequences under the Out-of-Africa demography model. We not only obtained an accurate human genetic map, but also found that the fluctuation of estimated recombination rate is reduced along the human genome when the sample size is increased. UK10K recombination activity is less concentrated than its subsets. Our results demonstrate how the sample size affects the estimated recombination rate, and analyses of a larger number of genomes result in a more precise estimation of recombination rate.

sentences: Evolutionary origins of sexual dimorphism : Lessons from female-limited mimicry in butterflies. The striking female-limited mimicry observed in some butterfly species is a text-book example of sexually-dimorphic trait submitted to intense natural selection. Two main evolutionary hypotheses, based on natural and sexual selection respectively, have been proposed. Predation pressure favouring mimicry toward defended species could be higher in females because of their slower flight, and thus overcome developmental constraints favouring the ancestral trait that limits the evolution of mimicry in males but not in females. Alternatively, the evolution of mimicry in males could be limited by females preference for non-mimetic males. However, the evolutionary origin of female preference for non-mimetic males remains unclear. Here, we hypothesise that costly sexual interactions between individuals from distinct sympatric species might intensify because of mimicry, therefore promoting female preference for non-mimetic trait. Using a mathematical model, we compare the evolution of female-limited mimicry when assuming either alternative selective hypotheses. We show that the patterns of divergence of male and female trait from the ancestral traits can differ between these selection regimes. We specifically highlight that divergence in females trait is not a signature of the effect of natural selection. Our results also evidence why female-limited mimicry is more frequently observed in Batesian mimics.

sentences: Chemogenetic activation of nigrostriatal dopamine neurons in freely moving common marmosets To interrogate particular neuronal pathways in non-human primates under natural and stress-free conditions, we applied designer receptors exclusively activated by designer drugs (DREADDs) technology to common marmosets. We injected adeno-associated virus vectors expressing the excitatory DREADD hM3Dq into the unilateral substantia nigra in three marmosets. Using multi-tracer positron emission tomography imaging, we detected DREADD expression in vivo, which was confirmed in nigrostriatal dopamine neurons by immunohisto-chemistry, and assessed activation of the substantia nigra and dopamine release following agonist administration. The marmosets rotated in a contralateral direction relative to the activated side 30-90 min after consuming food containing the highly potent DREADD agonist deschloroclozapine (DCZ), but not on the following days without DCZ. These results indicate that non-invasive and reversible DREADD manipulation will extend the utility of marmoset as a primate model for linking neuronal activity and natural behavior in various contexts.

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Recognition of copy-back defective interfering rabies virus genomes by RIG-I triggers the antiviral response against vaccine strains Rabies virus (RABV) is a lethal neurotropic virus that causes 60,000 human deaths every year around the world. A typical feature of RABV infection is the suppression of type I and III interferon (IFN)-mediated antiviral response. However, molecular mechanisms leading to RABV sensing by RIG-I-like receptors (RLR) to initiate IFN signaling remain elusive. Here, we showed that RABV RNAs are recognized by RIG-I (retinoic acid-inducible gene I) sensor resulting in an IFN response of the infected cells but that this global feature was differently modulated according to the type of RABV used. RNAs from pathogenic RABV strain, THA, were poorly detected in the cytosol by RIG-I and therefore mediated a weak antiviral response. On the opposite, we revealed a strong interferon activity triggered by the RNAs of the attenuated RABV vaccine SAD strain mediated by RIG-I. Using next-generation sequencing (NGS) combined with bioinformatics tools, we characterized two major 5copy-back defective interfering (5cb DI) genomes generated during SAD replication. Furthermore, we identified a specific interaction of 5cb DI genomes and RIG-I that correlated with a high stimulation of the type I IFN signaling. This study indicates that RNAs from a wild-type RABV poorly activate the RIG-I pathway, while the presence of 5cb DIs in vaccine SAD strain serves as an intrinsic adjuvant that strengthens its efficiency by enhancing RIG-I detection and therefore strongly stimulates the IFN response.

HighlightsO_LIRABV pathogenic strain replication in vitro is characterized by the absence of defective interfering genomes thus induces a weak RLR-mediated innate immunity antiviral response.
C_LIO_LIRABV vaccine attenuated strain shows a high release of 5 copy-back defective interfering genomes during replication in vitro and therefore enhances a strong antiviral response upon infection.
C_LIO_LIRIG-I is the main sensor for RABV RNA detection within cells.
C_LI

sentences: EARLY AND MID-GESTATION ZIKA VIRUS (ZIKV) INFECTION IN THE OLIVE BABOON (PAPIO ANUBIS) LEADS TO FETAL CNS PATHOLOGY BY TERM GESTATION Zika virus (ZIKV) infection in pregnancy can produce catastrophic teratogenic damage to the developing fetus including microcephaly and congenital Zika syndrome (CZS). We previously described fetal CNS pathology occurring by three weeks post-ZIKV infection in Olive baboons at mid-gestation, including neuroinflammation, loss of radial glia (RG), RG fibers, neuroprogenitor cells (NPCs) resulting in disrupted NPC migration. In the present study, we explored fetal brain pathologies at term gestation resulting from ZIKV infection during either first or second trimester in the Olive baboon. In all dams, viremia resolved after 7 days post infection (dpi). One first trimester infected dam aborted at 5 dpi. All dams developed IgM and IgG response to ZIKV with ZIKV IgG detected in fetal serum. Substantial placental pathology and inflammation was observed including disruption of syncytiotrophoblast layers, delayed villous maturation, partially or fully thrombosed vessels, calcium mineralization and fibrin deposits. In the uterus, ZIKV was detected in [3/4] first trimester but not in second trimester infected dams. While ZIKV was not detected in any fetal tissue at term, all fetuses exhibited varying degrees of neuropathology. Fetal brains from ZIKV infected dams exhibited a range of gross brain pathologies including irregularities of the major gyri and sulci of the cerebral cortex and cerebellar pathology. Frontal cortices of ZIKV fetuses showed a general disorganization of the six-layered cortex with degree of disorganization varying among the fetuses from the two groups. Frontal cortices from first but not second trimester infections exhibited increased microglia and astrocyte numbers (white matter) in both trimester infections. In the cerebellum, increased microglia were observed in both first and second trimester infected fetuses and decreased oligodendrocyte precursor cell populations in the cerebellar white matter in first trimester infections. In general, our observations are in accordance with those described in human ZIKV infected fetuses.

sentences: Whole-genome sequencing of Chinese native goat offers biological insights into cashmere fiber formation Cashmere evolved naturally in the goat, and almost all breeds of goat can produce more or less cashmere fibers. However, the genetic alterations underlying cashmere trait selection are still unclear.We sequenced 120 Chinese native goat including two cashmere goat breeds (Ujumain, Chaidamu) and six ordinary goat breeds (Jining Gray, Matou, Guizhou Black, Jintang Black, Yunnan Black Bone, Chengdu Brown). The genome-wide selective sweep of cashmere goat and ordinary goat revealed a novel set of candidate genes as well as pathways, such as Nuclear factor kappa-B and Wnt Signaling pathways. Of them, the LHX2 gene regulating hair follicle development, was evident from the strongest selection signal when comparing the Uhumqin cashmere goat and ordinary goat. Interestingly, we identified a 582bp deletion at 367 kb upstream of LHX2 with higher frequency in cashmere goats and their ancient relatives. This mutation probably rises along the breeding procedures, and is putatively responsible for cashmere production and diameter, as revealed by association studies. Luciferase assay shows that the deletion, which acts as an insulator, restrains the expression of LHX2 by interfering its upstream enhancers.Our study discovers a novel insulator of the LHX2 involved in regulating cashmere production and diameter, which would be beneficial to understanding hair follicle development and regeneration. Our findings also provide new insights into the genetic formation of cashmere, and facilitate subsequent molecular breeding for cashmere goat improvement.

sentences: Scalable, methanol-free manufacturing of the SARS-CoV-2 receptor binding domain in engineered Komagataella phaffii Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply on-going demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol-induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5x by alleviating protein folding stress. Removal of methanol from the production process enabled scale up to a 1,200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.

sentences: A second view on the evolution of flight in stick and leaf insects (Phasmatodea) The re-evolution of complex characters is generally considered impossible, yet, studies of recent years have provided several examples of phenotypic reversals shown to violate Dollos law. Along these lines, the regain of wings in stick and leaf insects (Phasmatodea) was hypothesised to have occurred several times independently after an ancestral loss, a scenario controversially discussed among evolutionary biologists. Here, we revisit the recovery of wings by reconstructing a phylogeny based on a comprehensive taxon sample of over 500 representative phasmatodean species to infer the evolutionary history of wings. We additionally explored the presence of ocelli, the photoreceptive organs used for flight stabilisation in winged insects, which might provide further information for interpreting flight evolution. Our findings support an ancestral loss of wings and that the ancestors of most major lineages were wingless. While the evolution of ocelli was estimated to be dependent on the presence of (fully-developed) wings, ocelli are nevertheless absent in the majority of all examined winged species and only appear in the members of few subordinate clades, albeit winged and volant taxa are found in every lineage. The disjunct distribution of ocelli substantiates the hypothesis on trait reacquisition and that wings were regained in Phasmatodea.

sentences: Phase separation of tunable biomolecular condensates predicted by an interacting particle model Phase separation is emerging as key principle in the spatiotemporal organization of living cells. Given its relevance in the regulation of numerous biological functions, including gene transcription and chromatin architecture, modeling biomolecular condensation is gaining interest. Yet, most models developed so far rely on specific descriptions and/or experimentally inaccessible properties. Here we propose a theoretical model, where phase separation is explained by means of interaction probabilities between particles. With minimum model requirements, particle condensates emerge above a critical interaction probability. We tested the model predictions with single molecule experiments of tunable transcription factor condensates in the nucleus of living cells. Phase separation, condensate sizes, diffusion behavior, and mobility parameters, quantified by data analysis and machine learning, are fully recapitulated by our model. Our combined theoretical and experimental approach provides a general framework to investigate the biophysical parameters controlling phase separation in living cells and in other soft matter-based interacting systems.

sentences: Angiotensin II regulates the neural expression of subjective fear in humans There has been growing interest in using pharmacological fMRI to identify novel treatments for fear, yet these models are limited with respect to determining process-specific effects on the actual subjective experience of fear which represents the key symptom why patients seek treatment. Combining a selective angiotensin II type 1 receptor (ATR1) antagonist with a fear vigilance fMRI paradigm and neurofunctional decoding in n = 87 individuals we demonstrate that AT1R blockade selectively reduces the neurofunctional reactivity to fear-inducing visual oddballs in terms of attenuating dorsolateral prefrontal activity and amygdala-ventral anterior cingulate (vACC) communication, and further show that the observed effect is due to reduced subjective fear, but not threat or non-specific negative affect, using a decoding analysis. Our findings demonstrate a highly specific role of AT1R in subjective fear experience and demonstrate the feasibility of a precision approach to the affective characterization of novel receptor targets for fear in humans.

sentences: Systematic multi-level analysis of an organelle proteome reveals new peroxisomal functions Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover the peroxi-ome of the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by [~]40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

sentences: Detection of F1 hybrids from single-genome data reveals frequent hybridization in Hymenoptera and particularly ants Hybridization occupies a central role in many fundamental evolutionary processes, such as speciation or adaptation. Yet, despite its pivotal importance in evolution, little is known about the actual prevalence and distribution of hybridization across the tree of life. Here we develop and implement a new statistical method enabling the detection of F1 hybrids from single-individual genome sequencing data. Using simulations and sequencing data from known hybrid systems, we first demonstrate the specificity of the method, and identify its statistical limits. Next, we showcase the method by applying it to available sequencing data from more than 1500 species of Arthropods, including Hymenoptera, Hemiptera, Coleoptera, Diptera and Archnida. Among these taxa, we find Hymenoptera, and especially ants, to display the highest number of candidate F1 hybrids, suggesting higher rates of recent hybridization in these groups. The prevalence of F1 hybrids was heterogeneously distributed across ants, with taxa including many candidates tending to harbor specific ecological and life history traits. This work shows how large-scale genomic comparative studies of recent hybridization can be implemented, uncovering the determinants of hybridization frequency across whole taxa.

sentences: Pro-resolving lipid mediators within brain esterified lipid pools are reduced in female rats chronically exposed to traffic-related air pollution or genetically susceptible to Alzheimer's Disease phenotype Traffic-related air pollution (TRAP) is a risk factor for Alzheimers disease (AD) where neuroinflammation underlies disease progression and pathogenesis. Unresolved inflammation in AD is known to be exacerbated by brain deficits in unesterified pro-resolving lipid mediators enzymatically synthesized from polyunsaturated fatty acids. Recently, we reported that in the brain, unesterified pro-resolving lipid mediators which are bioactive, can also be supplied from less bioactive esterified lipid pools such as neutral lipids (NLs) and phospholipids (PLs). It is not known whether esterified pro-resolving lipid mediators are affected by AD pathology and exacerbated by TRAP exposure. In the present study we addressed this data gap using TgF344-AD male and female rats that express human AD risk genes and their wildtype littermates exposed to filtered air (FA) or TRAP from 1 to 15 months of age. Esterified lipid mediators within NLs and PLs were quantified by mass-spectrometry. We observed a significant reduction in pro-resolving lipid mediators in both NLs and PLs of female TgF344-AD rats compared to wildtype controls. TRAP exposure also reduced pro-resolving lipids in the female brain, mainly in PL pools, but did not exacerbate changes observed in TgF344-AD rats. Minimal changes were observed in males. Our findings indicate that AD genotype and chronic TRAP exposure result in sex-specific deficits in brain esterified pro-resolving lipid mediators, the pool that supplies free and bioactive lipid mediators. These data provide new information on lipid-mediated mechanisms regulating impaired inflammation resolution in AD, and show for the first time that chronic TRAP exposure targets the same lipid network implicated in AD.

sentences: A Phylogenomic Framework for Charting the Diversity and Evolution of Giant Viruses Large DNA viruses of the phylum Nucleocytoviricota have recently emerged as important members of ecosystems around the globe that challenge traditional views of viral complexity. Numerous members of this phylum that cannot be classified within established families have recently been reported, and there is presently a strong need for a robust phylogenomic and taxonomic framework for these viruses. Here we report a comprehensive phylogenomic analysis of the Nucleocytoviricota, present a set of giant virus orthologous groups (GVOGs) together with a benchmarked reference phylogeny, and delineate a hierarchical taxonomy within this phylum. We show that the majority of Nucleocytoviricota diversity can be partitioned into 6 orders, 32 families, and 344 genera, substantially expanding the number of currently recognized taxonomic ranks for these viruses. We integrate our results within a megataxonomy that has been adopted for all viruses to establish a unifying framework for the study of Nucleocytoviricota diversity, evolution, and environmental distribution.

sentences: Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, mechanisms of autophagosome maturation, i.e., delivery and fusion with the vacuole, remain largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1s function is evolutionarily conserved in plants as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by connecting autophagosomes with the ESCRT-I component VPS23, leading to the formation of amphisomes. Disrupting the VPS23-CFS1 interaction affects autophagic flux and renders plants sensitive to starvation stress. Altogether, our results reveal a deeply conserved mechanism of vacuolar delivery in plants that is mediated by amphisomes.

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

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High regional and intra-generic variation in susceptibility to mass bleaching in Indo-Pacific coral species AimMass bleaching is a major threat to reef-building corals and the ecosystems they underpin. Here, we identified regional variation in the nature of this threat in terms of the bleaching-susceptibility of individual coral species on some Indian Ocean and Pacific Ocean reefs.

Location22 sites in the central Great Barrier Reef, Australia (GBR) and 30 sites in the central Maldives Archipelago (MA).

Time period2002 for the GBR and 2016 for the MA.

Major taxa studiedCorals (Order Scleractinia).

MethodsFollowing marine heat-wave conditions, timed in-situ surveys were used to record bleaching responses (tissue colour) of large samples of individual coral colonies. Responses of 106 shared species were analysed for sites with similar levels of temperature stress, depth of occurrence and mortality. In each region, phylogenetic mixed models were used to partition the effects on responses of species of deep-time phylogeny, contemporary history and local-scale, among-site variability.

ResultsRelative susceptibility to bleaching varied widely between regions: only 27 of the 106 shared species were in the same quartile for relative susceptibility in both regions. Few species were highly susceptible in both regions. Closely related species varied widely in their individual susceptibilities. Phylogenetic effects were moderate in both regions, but contemporary phenotypic effects indicative of recent evolution and acclimatization were greater in the MA, consistent with a stronger history of recent bleaching.

Main conclusionsThe high regional and intra-generic variation in coral bleaching-susceptibility described here suggests there may be important differences in the extent to which these Indian and Pacific Ocean coral populations are exhibiting responses to deep-time evolutionary changes on the one hand, versus recent adaptation, on the other. There is a concerning scarcity of this type of data, by which coral species most at risk from bleaching in particular regions may be more accurately identified.

sentences: A novel auxin-inducible degron system for rapid, cell cycle-specific targeted proteolysis The OsTIR1/auxin-inducible degron (AID) system allows "on demand" selective and reversible protein degradation upon exposure to the phytohormone auxin. In the current format, this technology does not allow to study the effect of acute protein depletion selectively in one phase of the cell cycle, as auxin similarly affects all the treated cells irrespectively of their proliferation status. Therefore, the AID system requires coupling with cell synchronization techniques, which can alter the basal biological status of the studied cell population. Here, we introduce a new AID system to Regulate OsTIR1 Levels based on the Cell Cycle Status (ROLECCS system), which induces proteolysis of both exogenously transfected and endogenous gene-edited targets in specific phases of the cell cycle. This new tool paves the way to studying the differential roles that target proteins may have in specific phases of the cell cycle.

sentences: Polygenic score accuracy in ancient samples: quantifying the effects of allelic turnover Polygenic scores link the genotypes of ancient individuals to their phenotypes, which are often unobservable, offering a tantalizing opportunity to reconstruct complex trait evolution. In practice, however, interpretation of ancient polygenic scores is subject to numerous assumptions. For one, the genome-wide association (GWA) studies from which polygenic scores are derived, can only estimate effect sizes for loci segregating in contemporary populations. Therefore, a GWA study may not correctly identify all loci relevant to trait variation in the ancient population. In addition, the frequencies of trait-associated loci may have changed in the intervening years. Here, we devise a theoretical framework to quantify the effect of this allelic turnover on the statistical properties of polygenic scores as functions of population genetic dynamics, trait architecture, power to detect significant loci, and the age of the ancient sample. We model the allele frequencies of loci underlying trait variation using the Wright-Fisher diffusion, and employ the spectral representation of its transition density to find analytical expressions for several error metrics, including the correlation between an ancient individuals polygenic score and true phenotype, referred to as polygenic score accuracy. Our theory also applies to a two-population scenario and demonstrates that allelic turnover alone may explain a substantial percentage of the reduced accuracy observed in cross-population predictions, akin to those performed in human genetics. Finally, we use simulations to explore the effects of recent directional selection, a bias-inducing process, on the statistics of interest. We find that even in the presence of bias, weak selection induces minimal deviations from our neutral expectations for the decay of polygenic score accuracy. By quantifying the limitations of polygenic scores in an explicit evolutionary context, our work lays the foundation for the development of more sophisticated statistical procedures to analyze both temporally and geographically resolved polygenic scores.

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Effects of background food on alternative grain uptake and zinc phosphide efficacy in wild house mice BACKGROUNDHouse mice (Mus musculus) cause significant, ongoing losses to grain crops in Australia, particularly during mouse plagues. Zinc phosphide (ZnP) coated grain is used for control, but with variable success. In a laboratory setting, we tested if mice would (1) switch from consumption of one grain type to another when presented with an alternative, and (2) consume ZnP-treated grains when presented as a choice with a different grain.

RESULTSMice readily switched from their background grain to an alternative grain, preferring cereals (wheat or barley) over lentils. Mice readily consumed ZnP-coated barley grains. Their mortality rate was significantly higher (86%, n=30) in the presence of a less-favoured grain (lentils) compared to their mortality rate (47%, n=29; and 53%, n=30) in the presence of a more-favoured grain (wheat and barley, respectively). Mice died between 4-112 h (median = 18 h) after consuming one or more toxic grains. Independent analysis of ZnP-coated grains showed variable toxin loading indicating that consumption of a single grain would not guarantee intake of a lethal dose. There was also a strong and rapid behavioural aversion if mice did not consume a lethal dose on the first night.

CONCLUSIONSThe registered dose rate of 25 g ZnP/kg wheat; [~] 1 mg ZnP/grain in Australia needs to be re-evaluated to determine what factors may be contributing to variation in efficacy. Further field research is also required to understand the complex association between ZnP dose, and quantity and quality of background food on efficacy of ZnP baits.

sentences: A rapid F0 CRISPR screen in zebrafish to identify regulators of neuronal development in the enteric nervous system The enteric nervous system (ENS) provides the intrinsic innervation of the gastrointestinal (GI) tract with millions of neurons and diverse neuronal subtypes and glial cells. The ENS regulates essential gut functions such as motility, nutrient uptake, and immune response, but basic information about the genes that control ENS neuronal specification and differentiation remains largely unknown. Deficits in ENS neuron numbers and composition cause gut dysfunction with debilitating GI symptoms, and are associated with e.g. Hirschsprung disease, inflammatory gut diseases, autism spectrum disorder, and neurodegenerative diseases such as Parkinsons disease. The genetic basis of most of these ENS disorders remains unknown. Recent transcriptomic analyses have identified many candidate genes for regulating ENS neurogenesis. However, functional evaluation of these candidate genes significantly lags because experimental testing of their role in ENS neurogenesis is time-consuming and expensive. Here, we have developed a rapid, scalable F0 CRISPR genome editing screen in zebrafish to functionally determine which candidate genes control neuronal development in the ENS. Proof-of-concept experiments targeting the known ENS regulators sox10 and ret phenocopy stable mutants with high efficiency and precision showing that our approach is reliable to identify regulators ENS neurogenesis using F0 guide RNA-injected larvae (F0 crispants). We then evaluate the role of 10 transcription factor genes for regulating ENS neurogenesis and function. Pools of guide RNAs targeting 2-3 candidate genes are co-injected with Cas9 protein into one-cell stage phox2bb:GFP transgenic zebrafish embryos to directly assess qualitative change in ENS neuron numbers compared to controls in 6-day old F0 crispants. Target genes from crispant pools exhibiting reduced ENS neuronal numbers were then tested individually to identify the responsible gene(s). We identify five transcription factors that show a reduction in ENS neurons indicating an influence on enteric progenitor cell differentiation into ENS neurons. Adding a simple and efficient test to further assess crispant gut motility, we find that loss-of-function of two of the transcription factor genes reduced intestinal transit of fluorescently labeled food through the gut. In summary, our novel, multistep, yet straight-forward CRISPR screening approach in zebrafish enables testing the genetic basis of ENS developmental and disease gene functions that will facilitate high-throughput evaluation of the manifold candidate genes emerging from transcriptomic, genome-wide association or other ENS-omics studies. Such in vivo ENS crispant screens will contribute to a better understanding of ENS neuronal development regulation in vertebrates and what goes awry in ENS disorders.

sentences: Phylogeny of the Formicoxenus genus-group (Hymenoptera: Formicidae) reveals isolated lineages of Leptothorax acervorum in the Iberian Peninsula predating the Last Glacial Maximum The Formicoxenus genus-group comprises six genera within the tribe Crematogastrini. The group is well known for repeated evolution of social parasitism among closely related taxa and cold-adapted species with large distribution ranges in the Nearctic and Palearctic regions. Previous analyses based on nuclear markers (ultraconserved elements, UCEs) and mitochondrial genes suggest close relationship between Formicoxenus Mayr, 1855, Leptothorax Mayr, 1855 and Harpagoxenus Forel, 1893. However, scant sampling has limited phylogenetic assessment of these genera. Also, previous phylogeographic analyses of L. acervorum (Fabricius, 1793) have been limited to its West-Palearctic range of distribution, which has provided a narrow view on recolonization, population structure and existing refugia of the species. Here, we inferred the phylogenenetic history of genera within the Formicoxenus genus-group and reconstructed the phylogeography of L. acervorum with more extensive sampling. We employed four datasets consisting of whole genomes and sequences of the COI. The topologies of previous nuclear and our inferences based on mitochondrial genomes were overall congruent. Further, Formicoxenus may not be monophyletic. We found several monophyletic lineages that do not correspond to the current species described within Leptothorax, especially in the Nearctic region. We identified a monophyletic L. acervorum lineage that comprises both Nearctic and Palearctic locations. The most recent expansion within L. acervorum probably occurred within the last 0.5 Ma with isolated populations predating the Last Glacial Maximum (LGM), which are localized in at least two refugial areas (Pyrenean and Northern plateau) in the Iberian Peninsula. The patterns recovered suggest a shared glacial refugium in the Iberian Peninsula with cold-adapted trees that currently share high-altitude environments in this region.

sentences: Qualifying a human Liver-Chip for predictive toxicology: Performance assessment and economic implications Human organ-on-a-chip (Organ-Chip) technology has the potential to disrupt preclinical drug discovery and improve success in drug development by recapitulating organ-level pathophysiology and clinical responses. The Innovation and Quality (IQ) consortium formed by multiple pharmaceutical and biotechnology companies to confront this challenge has published guidelines that define criteria for qualifying preclinical models, however, systematic and quantitative evaluation of the predictive value of Organ-Chips has not yet been reported. Here, 780 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury caused by small molecules identified as benchmarks by the IQ consortium. The Liver-Chip met the qualification guidelines across a blinded set of 27 known hepatotoxic and non-toxic drugs with a sensitivity of 80%, increasing to 87% when protein binding is corrected, and a specificity of 100%. A computational economic value analysis suggests that with this performance the Liver-Chip could generate $3 billion annually for the pharmaceutical industry due to increased R&D productivity.

sentences: Inference of B cell clonal families using heavy/light chain pairing information AO_SCPLOWBSTRACTC_SCPLOWNext generation sequencing of B cell receptor (BCR) repertoires has become a ubiquitous tool for understanding the antibody-mediated immune response: it is now common to have large volumes of sequence data coding for both the heavy and light chain subunits of the BCR. However, until the recent development of high throughput methods of preserving heavy/light chain pairing information, these samples contained no explicit information on which heavy chain sequence pairs with which light chain sequence. One of the first steps in analyzing such BCR repertoire samples is grouping sequences into clonally related families, where each stems from a single rearrangement event. Many methods of accomplishing this have been developed, however, none so far has taken full advantage of the newly-available pairing information. This information can dramatically improve clustering performance, especially for the case of the light chain; the light chain has traditionally been challenging for clonal family inference because of its low diversity and consequent abundance of non-clonal families with indistinguishable naive rearrangements. Here we present a method of incorporating this pairing information into the clustering process in order to arrive at a more accurate partition of the data into clonally related families. We also demonstrate two methods of fixing imperfect pairing information, which may allow for simplified sample preparation and increased sequencing depth. Finally, we describe several other improvements to the partis software package (https://github.com/psathyrella/partis).

sentences: Contraction-dependent intracortical inhibition supports gravity-efficient motor control Efficient control of voluntary movements along the gravity axis requires adapted shifts in muscular contraction modes. In daily life, rising the arm up involves shortening (i.e., concentric) contractions of arm flexors, while the reverse movement can rely on lengthening (i.e., eccentric) contractions of the same muscles with the help of gravity force. Although this muscular-control mode is universal, the neuromuscular mechanisms that subserve the control of such gravity-oriented movements remain unknown. In this study, we designed two neurophysiological experiments that allowed tracking modulations of cortical, spinal, and muscular outputs of arm flexors while healthy humans carried out vertical pointing movements. In conditions where upward and downward movements revealed kinematic features reminiscent of optimal motor commands (i.e., directional asymmetries), we report fine contraction-dependent modulations of the corticospinal output. The overall corticospinal excitability dropped during lengthening contractions (downward movements) compared with shortening contractions (upward movements). Specifically, we did not observe any change in spinal motoneuron responsiveness from cervicomedullary stimulations but a specific increase in intracortical inhibition during lengthening vs. shortening contractions. We discuss these fine contraction-dependent modulations of the supraspinal motor output in the light of feedforward mechanisms that may support gravity-tuned motor control. Generally, these results shed a new perspective on the neural policy that optimize movement control along the gravity axis.

sentences:
FGFR2 fusion protein-driven mouse models of intrahepatic cholangiocarcinoma unveil a necessary role for Erk signaling Background and aimsAbout 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration sufficient to ignite oncogenic FF activation. In line, FGFR-specific tyrosine kinase inhibitors (F-TKI) were shown to provide clinical benefit in FF+ iCCA, although responses were partial and/or limited by resistance mechanisms, including the FF V565F gatekeeper mutation. Herein we present an FF-driven murine iCCA model and exploit its potential for pre-clinical studies on FF therapeutic targeting.

MethodsFour iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by intrahepatic/subcutaneous transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies.

ResultsTumors diagnosed as CCA were obtained upon transplantation of FF-expressing liver organoids. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or presence of V565F mutation. Double blockade of FF-Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo.

ConclusionsFF-driven iCCA pathogenesis was successfully modelled in murine Tp53-/- background. This model revealed biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for improving precision-based approaches against human FF+ iCCA.

Abbreviations used in this paper: ANOVA, analysis of variance; Bap1, BRCA1-Associated-Protein 1; Cdkn2a, cyclin-dependent kinase inhibitor 2A; Cftr, cystic fibrosis transmembrane conductance regulator; Ck19, cytokeratin 19; Cyp3A, cytochrome P450, family 3, subfamily A; EGF, Epidermal growth factor; EGFR, Epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; Erk, extracellular signal-regulated kinase; FGFR2, fibroblast growth factor receptor 2; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor-bound 2; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; H&E, hematoxylin and eosin; HepPar1, hepatocyte Paraffin 1; Hnf4-7, hepatocyte nuclear factor 4 alpha; Hprt, hypoxanthine-guanine phosphoribosyl transferase; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NTRK, neurotrophic Tyrosine Kinase; Parp, Poly (ADP-ribose) polymerase; RECIST, response evaluation criteria in solid tumors; SHP2, Src homology phosphatase 2; Ttr, transthyretin.

sentences: Blood-based untargeted metabolomics in Relapsing-Remitting Multiple Sclerosis revealed the testable therapeutic target Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 relapsing-remitting patients and 14 healthy age-matched controls. Out of 632 known metabolites detected, 60 were significantly altered in relapsing-remitting MS (RRMS). Bioinformatics analysis identified an altered "metabotype" in RRMS patients, represented by 4 changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these 4 pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient derived peripheral blood mononuclear cells, showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor, 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the anti-inflammatory phenotype of monocytes/macrophage in the central nervous system. Our study suggests that targeting glycolysis offers a potential target for MS.

sentences: Multiple knockout mutants reveal a high redundancy of phytotoxic compounds that determine necrotrophic pathogenesis of Botrytis cinerea Botrytis cinerea is a major pathogen of more than 1400 plant species. During infection, the kills host cells during infection and spreads through necrotic tissue, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas protocol was established which allowed serial marker-free mutagenesis to generate Botrytis mutants lacking up to 12 different CDIPs. Infection analysis revealed a decrease in virulence with increasing numbers of knockouts, and differences in the effects of knockouts on different host plants. The on planta secretomes obtained from these mutants revealed substantial remaining necrotic activity after infiltration into leaves. Our study has addressed for the first time the functional redundancy of virulence factors of a fungal pathogen, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins and metabolites to achieve necrotrophic infection of a wide variety of host plants.

sentences: Strategies of Mycoplasma ovipneumoniae to evade immune clearance by alveolar macrophages Chronic nonprogressive pneumonia is a prevalent disease that infects many young sheep. Mycoplasma ovipneumoniae was isolated from the lungs of sheep with chronic nonprogressive pneumonia. Evidences showed that it might associate with the development and duration of chronic pneumonia, moreover, sheep infected with M. ovipneumoniae are easily infected by other organisms, suggesting that M. ovipneumoniae may play an immunosuppressive role during infection. However, the mechanism is still poorly understood. The infection occurs in the airway, where resident alveolar macrophages first encounter M. ovipneumoniae. Therefore, primary alveolar macrophages (AMs) were collected from the lungs of healthy adult sheep, and the (iTRAQ) protein assay was used to investigate the immunosuppressive effects of M. ovipneumoniae on sheep AMs. The RAW264.7 cells were used to confirm the findings. The results showed that M. ovipneumoniae promoted higher expression of anti-apoptotic proteins and lower expression of apoptosis-related proteins in the infected AMs. Moreover, the number of infected AMs increased. However, M. ovipneumoniae reduced ATP levels in AMs and impaired late endosome maturation and phagolysosome fusion. Furthermore, M. ovipneumoniae inhibited the autophagy pathway via the Akt-mTOR axis in AMs. These findings indicated that M. ovipneumoniae had distinctive strategies to evade elimination caused by the AMs. The findings might explain the chronic infection and co-infection in sheep infected by M. ovipneumoniae.

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Repurposing of the tamoxifen metabolites to treat methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecalis infections Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM) and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyse the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp.

MRSE (n=17) and Enterococcus spp. (E. faecalis n=8, and E. faecium n=10) strains were used. MIC of the mixture of DTAM, HTAM and ENDX, and vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays.

MIC50 and MIC90 of tamoxifen metabolites were 1 mg/L for MRSE strains and 2 mg/L for Enterococcus spp. strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 2x and 4xMIC for MRSE (SE385 and SE742) and E. faecalis (EVR1 and EVR2) strains. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of SE385 and EVR2 strains.

Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drugs treatment against these bacterial pathogens.

sentences: Sulfur-mediated chalcogen versus hydrogen bonds in proteins: a seesaw effect in the conformational space Divalent sulfur (S) form chalcogen bond (Ch-bond) via its {sigma}-holes and hydrogen bond (H-bond) via its lone-pairs. Relevance of these interactions and their interplay for protein structure and function is unclear. Based on the analyses of the crystal structures of small organic/organometallic molecules and proteins, and their Molecular Electrostatic Surface Potential, we show that the reciprocity of the substituent-dependent strength of the {sigma}-holes and lone-pairs correlate with the formation of either Ch-bond or H-bond. In proteins, disulfide-bonded cystine preferentially forms Ch-bond, metal-chelated cysteine forms H-bond, while methionine forms either of them with comparable frequencies. This has implications to the positioning of these residues and their role in protein structure and function. Computational analyses reveal that the S-mediated interactions stabilize protein secondary structures by mechanisms such as helix capping, protecting free {beta}-sheet edges by negative-design, and augmenting the stability of {beta}-turns. We find that Ch-bond can be as strong as H-bond. The study highlights the importance of S-mediated Ch-bond and H-bond for understanding protein folding and function, development of improved strategies for protein/peptide structure prediction and design, and structure-based drug discovery.

sentences: Velodrome: Out-of-Distribution Generalization from Labeled and Unlabeled Gene Expression Data for Drug Response Prediction Data discrepancy between preclinical and clinical datasets poses a major challenge for accurate drug response prediction based on gene expression data. Different methods of transfer learning have been proposed to address this data discrepancy. These methods generally use cell lines as source domains and patients, patient-derived xenografts, or other cell lines as target domains. However, they assume that they have access to the target domain during training or fine-tuning and they can only take labeled source domains as input. The former is a strong assumption that is not satisfied during deployment of these models in the clinic. The latter means these methods rely on labeled source domains which are of limited size. To avoid these assumptions, we formulate drug response prediction as an out-of-distribution generalization problem which does not assume that the target domain is accessible during training. Moreover, to exploit unlabeled source domain data, which tends to be much more plentiful than labeled data, we adopt a semi-supervised approach. We propose Velodrome, a semi-supervised method of out-of-distribution generalization that takes labeled and unlabeled data from different resources as input and makes generalizable predictions. Velodrome achieves this goal by introducing an objective function that combines a supervised loss for accurate prediction, an alignment loss for generalization, and a consistency loss to incorporate unlabeled samples. Our experimental results demonstrate that Velodrome outperforms state-of-the-art pharmacogenomics and transfer learning baselines on cell lines, patient-derived xenografts, and patients. Finally, we showed that Velodrome models generalize to different tissue types that were well-represented, under-represented, or completely absent in the training data. Overall, our results suggest that Velodrome may guide precision oncology more accurately.

sentences: A UVB-responsive common variant at chr7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor gene (AHR) Genome-wide association studies have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP, and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). As ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWAS identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor gene (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. As AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus, and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression, and altering melanocyte growth phenotypes upon exposure.

sentences: Impact of hedonic value of stimuli on sampling dynamics during a preference test The decision of whether to continue with a current action or to stop and consider alternatives is ever present in the life of an animal. Such continuous-time decision making lies at the heart of food preference tests whose outcomes are typically quantified by a single variable, the total amount consumed. However, the dynamics that give rise to such a quantity in terms of durations of bouts of sampling at a stimulus before pauses, and the impact of alternative stimuli on those bout durations and subsequent actions following a pause, can contain a richness of behavior that is not captured in a single palatability measure. Here we carry out multiple analyses of these dynamics, with a particular focus on assessing how the hedonic value of one taste stimulus impacts the behavior of a rat sampling a second taste stimulus during a preference test. We find evidence for an explicit competitive interaction between bout durations, such that the more palatable a stimulus the longer the bout durations when the rat samples the stimulus and the shorter the bout durations at the alternative. Such competition is reproduced in a model of a neural circuit that could underlie the continuous decision of when to end a sampling bout. We find that the competitive impact on bout durations is relatively short-lived whereas a competitive impact on the choice of which stimulus to approach following a pause persists. Such a discrepancy in the timescales for the decay of the impact of the alternative stimulus suggests different neural processes are involved in the choice of which stimulus to approach versus the choice of how long to sample from it. Since these two choices together combine to determine net consumption and therefore the inferred palatability or preference of a gustatory stimulus, our results suggest that palatability is not a unitary quantity but the result of at least two distinct, context-dependent neural processes.

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Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2 The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.

IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

sentences: LORE homomerization is required for 3-OH-C10:0 induced immune signaling Perception and processing of various internal and external signals is essential for all living organisms. Plants have an expanded and diversified repertoire of cell surface-localized receptor-like kinases (RLKs) that transduce signals across the plasma membrane. RLKs often assemble into higher-order receptor complexes with co-receptors, regulators and scaffolds to convert extracellular stimuli into cellular responses. To date, the only S-domain-RLK from Arabidopsis thaliana with a known ligand and function is AtLORE, a pattern recognition receptor that senses bacterial 3-hydroxy fatty acids of medium chain length, such as 3-hydroxy decanoic acid (3-OH-C10:0), to activate pattern-triggered immunity. Here we show that AtLORE forms receptor homomers, which is essential for 3-OH-C10:0-induced immune signaling. AtLORE homomerization is mediated by the transmembrane and extracellular domain. We show natural variation in the perception of 3-OH-C10:0 within the Brassicaceae family. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3-OH-C10:0, although they possess a putative LORE orthologue. We found that LORE orthologues of these 3-OH-C10:0 nonresponsive species have defective extracellular domains that can bind the 3-OH-C10:0 ligand but lack the ability to homomerize. Our findings shed light on the activation mechanisms of AtLORE and explain natural variation of 3-OH-C10:0 perception within the Brassicaceae family.

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Genomic characteristics of recently recognized Vibrio cholerae El Tor lineages associated with cholera in Bangladesh, 1991-2017 Comparative genomic analysis of Vibrio cholerae El Tor associated with endemic cholera in Asia revealed two distinct lineages, one dominant in Bangladesh and the other in India. An in depth whole genome study of V. cholerae El Tor clinical strains isolated during endemic cholera in Bangladesh (1991 - 2017) included reference genome sequence data obtained online. Core genome phylogeny established using single nucleotide polymorphisms (SNPs) showed V. cholerae El Tor strains comprised two lineages, BD-1 and BD-2, which, according to Bayesian phylodynamic analysis, originated from paraphyletic group BD-0 around 1981. BD-1 and BD-2 lineages overlapped temporally but were negatively associated as causative agents of cholera 2004-2017. Genome wide association study (GWAS) revealed 140 SNPs and 31 indels, resulting in gene alleles unique to BD-1 and BD-2. Regression analysis of root to tip distance and year of isolation indicated early BD-0 strains at the base, whereas BD-1 and BD-2 subsequently emerged and progressed by accumulating SNPs. Pangenome analysis provided evidence of gene acquisition by both BD-1 and BD-2, of which six crucial proteins of known function were predominant in BD-2. BD-1 and BD-2 diverged and have distinctively different genomic traits, namely heterogeneity in VSP-2, VPI-1, mobile elements, toxin encoding elements, and total gene abundance. In addition, the observed phage-inducible chromosomal island-like element (PLE1), and SXT ICE elements (ICETET) in BD-2 presumably provided a fitness advantage for the lineage to outcompete BD-1 as the etiological agent of the endemic cholera in Bangladesh, with implications for global cholera epidemiology.

ImportanceCholera is a global disease with specific reference to the Bay of Bengal Ganges Delta where Vibrio cholerae O1 El Tor, causative agent of the disease showed two circulating lineages, one dominant in Bangladesh and the other in India. Results of in-depth genomic study of V. cholerae associated with endemic cholera during the past 27 years (1991 - 2017) indicate emergence and succession of the two lineages, BD-1 and BD-2, arising from a common ancestral paraphylatic group, BD-0, comprising the early strains and short-term evolution of the bacterium in Bangladesh. Among the two V. cholerae lineages, BD-2 supersedes BD-1 and is predominant in the most recent endemic cholera in Bangladesh. The BD-2 lineage contained significantly more SNPs and indels, and showed richness in gene abundance, including antimicrobial resistance genes, gene cassettes, and PLE to fight against bacteriophage infection, acquired over time. These findings have important epidemic implications at a global scale.

sentences: Effects of domestication on neophobia: A comparison between the domesticated Bengalese finch (Lonchura striata var. domestica) and its wild ancestor, the white-rumped munia (Lonchura striata) Bengalese finches (Lonchura striata var. domestica) have more complex song traits than their wild ancestors, white-rumped munias (Lonchura striata). Domesticated finches are likely able to allocate more resources to song development rather than allocating resources to mechanisms aimed at coping with predation, which are no longer needed under domesticated conditions. Here, we aimed to examine the effects of changes in selection pressure due to domestication on the behaviour of Bengalese finches and to contemplate the possible evolutionary mechanisms underlying these changes. To do so, we compared neophobic responses to novel-object conditions as an assessment of reactions to potential predators. We studied groups of Bengalese finches and white-rumped munias and found that Bengalese finches were more likely to eat the food provided to them under novel-object conditions. Bengalese finches had a shorter latency time to eat, and this latency time was less affected by the novel object in the case of Bengalese finches compared to white-rumped munias. Therefore, Bengalese finches have reduced neophobic responses due to domestication. The behavioural strategies of white-rumped munias appear to be more suitable for natural environments, which include unpredictable risks, whereas Bengalese finches have likely adapted their behaviour to the conditions of artificial selection.

sentences: Sector search strategies for odor trail tracking Terrestrial animals such as ants, mice and dogs often use surface-bound scent trails to establish navigation routes or to find food and mates, yet their tracking strategies are poorly understood. Tracking behavior features zig-zagging paths with animals often staying in close contact with the trail. Upon sustained loss of contact, animals execute a characteristic sequence of sweeping "casts" - wide oscillations with increasing amplitude. Here, we provide a unified description of trail-tracking behavior by introducing an optimization framework where animals search in the angular sector defined by their estimate of the trails heading and its uncertainty. In silico experiments using reinforcement learning based on this hypothesis recapitulate experimentally observed tracking patterns. We show that search geometry imposes limits on the tracking speed, and quantify its dependence on trail statistics and memory of past contacts. By formulating trail-tracking as a Bellman-type sequential optimization problem, we quantify the basic geometric elements of optimal sector search strategy, effectively explaining why and when casting is necessary. We propose a set of experiments to infer how tracking animals acquire, integrate and respond to past information on the tracked trail. More generally, we define navigational strategies relevant for animals and bio-mimetic robots, and formulate trail-tracking as a novel behavioral paradigm for learning, memory and planning.

sentences: Intranasal oxytocin modulates very early visual processing of emotional faces Functional imaging and behavioural studies have shown that the neuropeptide oxytocin influences processing of emotional faces. However, it is not clear whether these effects reflect modulation at an early or late stage of affective processing. We investigated the effects of oxytocin administration on early and late visual evoked potentials (VEP) in response to faces with neutral, fearful and happy expressions. In addition, we measured multifocal VEP and its associated nonlinearities to ascertain whether any changes observed in electrophysiology were indicative of a generalised effect or of one tied strictly to emotional processing. In a randomized, double-blind, cross-over design, 27 healthy male participants self-administered a nasal spray of either oxytocin (24 IU) or placebo. At very early latencies (40-60ms), oxytocin reduced right-temporal responses to fearful faces (d = .51), and central responses to both fearful (d = .48) and neutral faces (d = .54). For left occipito-temporal electrode sites, oxytocin decreased P100 reactivity to fearful expressions (d = 0.72). Oxytocin also decreased the amplitudes of the vertex positive potential (140-180ms) and late positive potential (400-600ms), regardless of whether the faces had fearful, happy or neutral expressions. The mfVEP showed no signs of selective magno-or parvo-cellular peak modulation comparing OXT with placebo with either low or high contrast stimulation. These results suggest that at early stages of visual processing, nasal oxytocin modulates responses to facial emotions, whereas at later stages of visual processing, it appears to influence more general face processing mechanisms. In addition, the measurable effects of OXT appear to be not a result of generalized brain change, but systematically related to emotional processing.

sentences: Principal component analysis reveals multiple consistent responses to naturalistic stimuli in children and adults Functional MRI (fMRI) study of naturalistic conditions, e.g. movie watching, usually focuses on shared responses across subjects. However, individual differences have been attracting increasing attention in search of group differences or associations with behavioral outcomes. Individual differences are typically studied by directly modeling the pair-wise intersubject correlation matrix or projecting the relations onto a single dimension. We contend that it is critical to examine whether there are one or more consistent responses underlying the whole sample, because multiple components, if exist, may undermine the intersubject relations using the previous methods. We propose to use principal component analysis (PCA) to examine the heterogeneity of brain responses across subjects and project the individual variability into higher dimensions. By analyzing an fMRI dataset of children and adults watching a cartoon movie, we showed evidence of two consistent responses in the supramarginal gyrus and other regions. While the first components in many regions represented a response pattern mostly in older children and adults, the second components mainly represented the younger children. The second components in the supramarginal network resembled a delayed version of the first PCs for 4 seconds (2 TR), indicating slower responses in the younger children than the older children and adults. The analyses highlight the importance of identifying multiple consistent responses in responses to naturalistic stimuli. This PCA-based approach could be complementary to the commonly used intersubject correlation to analyze movie watching data.

sentences: Quantifying Numerical and Spatial Reliability of Amygdala and Hippocampal Subdivisions in FreeSurfer On-going, large-scale neuroimaging initiatives can aid in uncovering neurobiological causes and correlates of poor mental health, disease pathology, and many other important conditions. As projects grow in scale with hundreds, even thousands, of individual participants and scans collected, quantification of brain structures by automated algorithms is becoming the only truly tractable approach. Here, we assessed the spatial and numerical reliability for newly deployed automated segmentation of hippocampal subfields and amygdala nuclei in FreeSurfer 7. In a sample of participants with repeated structural imaging scans (N=923), we found numerical reliability (as assessed by intraclass correlations, ICCs) was reasonable: [~]95% of hippocampal subfields have "excellent" numerical reliability (ICCs[≥]0.90), however, only 67% of amygdala subnuclei met this same threshold. Spatial reliability was similarly reasonable, with 58% of hippocampal subfields and 44% of amygdala subnuclei having Dice coefficients[≥]0.70. Notably, multiple regions had poor numerical and/or spatial reliability. We also examined correlations between spatial reliability and person-level factors (e.g., participant age; T1 image quality). Both sex and image scan quality were related to variations in spatial reliability metrics. Examined collectively, our work suggests caution should be exercised for a few hippocampal subfields and amygdala nuclei with more variable reliability.

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CellProfiler 4: Improvements in Speed, Utility and Usability BackgroundImaging data contains a substantial amount of information which can be difficult to evaluate by eye. With the expansion of high throughput microscopy methodologies producing increasingly large datasets, automated and objective analysis of the resulting images is essential to effectively extract biological information from this data. CellProfiler is a free, open source image analysis program which enables researchers to generate modular pipelines with which to process microscopy images into interpretable measurements.

ResultsHerein we describe CellProfiler 4, a new version of this software with expanded functionality. Based on user feedback, we have made several user interface refinements to improve the usability of the software. We introduced new modules to expand the capabilities of the software. We also evaluated performance and made targeted optimizations to reduce the time and cost associated with running common large-scale analysis pipelines.

ConclusionsCellProfiler 4 provides significantly improved performance in complex workflows compared to previous versions. This release will ensure that researchers will have continued access to CellProfilers powerful computational tools in the coming years.

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C. elegans DSB-3 Reveals Conservation and Divergence among Protein Complexes Promoting Meiotic Double-Strand Breaks Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure for their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form meiotic crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2 and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have co-evolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

Significance StatementFaithful inheritance of chromosomes during meiosis depends on the formation and repair of double-strand DNA breaks (DSBs), which are generated through the activity of a topoisomerase-like protein known as Spo11. Spo11 exhibits strong conservation throughout eukaryotes, presumably reflecting constraints imposed by its biochemical activity, but auxiliary proteins that collaborate with Spo11 to promote and regulate DSB formation are less well conserved. Here we investigate a cohort of proteins comprising a complex required for meiotic DSB formation in Caenorhabditis elegans, providing evidence for both conservation with and divergence from homologous complexes in other organisms. This work highlights the evolutionary malleability of protein complexes that serve essential, yet auxiliary, roles in fundamental biological processes that are central to reproduction.

sentences: Structured Modulations of Ongoing Variability by Task and Development In carefully designed experiments, cognitive scientists interpret the mean event-related potentials (ERP) in terms of cognitive operations. However, the huge signal variability from one trial to the next, questions the representability of such mean events. We explored here whether this variability is an unwanted noise, or an informative part of the neural response. We took advantage of the rapid changes in the visual system during human infancy and analyzed the variability of visual responses to central and lateralized faces in 2-to 6-month-old infants and adults using high-density electroencephalography (EEG). We observed that neural trajectories of individual trials always remain very far from ERP components, only moderately bending their direction with a substantial temporal jitter across trials. However, single trial trajectories displayed characteristic patterns of acceleration and deceleration when approaching ERP components, as if they were under the active influence of steering forces causing transient attraction and stabilization. These dynamic events could only partly be accounted for by induced microstate transitions or phase reset phenomena. Furthermore, these structured modulations of response variability, both between and within trials, had a rich sequential organization, which, in infants, was modulated by the task difficulty. Our approaches to characterize Event Related Variability (ERV) expand and reinterpret classic ERP analyses, making them compliant with pervasive neural variability and providing a more faithful description of neural events following stimulus presentation.

sentences: An estimate of the deepest branches of the tree of life from ancient vertically-evolving genes Core gene phylogenies provide a window into early evolution, but different gene sets and analytical methods have yielded substantially different views of the tree of life. Trees inferred from a small set of universal core genes have typically supported a long branch separating the archaeal and bacterial domains. By contrast, recent analyses of a broader set of non-ribosomal genes have suggested that Archaea may be less divergent from Bacteria, and that estimates of inter-domain distance are inflated due to accelerated evolution of ribosomal proteins along the inter-domain branch. Resolving this debate is key to determining the diversity of the archaeal and bacterial domains, the shape of the tree of life, and our understanding of the early course of cellular evolution. Here, we investigate the evolutionary history of the marker genes key to the debate. We show that estimates of a reduced Archaea-Bacteria (AB) branch length result from inter-domain gene transfers and hidden paralogy in the expanded marker gene set. By contrast, analysis of a broad range of manually curated marker gene datasets from an evenly sampled set of 700 Archaea and Bacteria reveal that current methods likely underestimate the AB branch length due to substitutional saturation and poor model fit; that the best-performing phylogenetic markers tend to support longer inter-domain branch lengths; and that the AB branch lengths of ribosomal and non-ribosomal marker genes are statistically indistinguishable. Furthermore, our phylogeny inferred from the 27 highest-ranked marker genes recovers a clade of DPANN at the base of the Archaea, and places CPR within Bacteria as the sister group to the Chloroflexota.

sentences: An environmental control box for serial crystallography enables multi-dimensional experiments We present a new environmental enclosure for fixed-target, serial crystallography enabling full control of both the temperature and humidity. While maintaining the relative humidity to within a percent, this enclosure provides access to X-ray diffraction experiments in a wide temperature range from below 10 {degrees}C to above 80 {degrees}C. Coupled with the LAMA method, time-resolved serial crystallography experiments can now be carried out at truly physiological temperatures, providing fundamentally new insight into protein function. Using the hyperthermophile enzyme xylose isomerase, we demonstrate changes in the electron density as a function of increasing temperature and time. This method provides the necessary tools to successfully carry out multi-dimensional serial crystallography.

sentences: SBP-SITA: A sequence-based prediction tools for S-itaconation As a recently-reported post-translational modification, S-itaconation plays an important role in inflammation suppression. In order to understand its regulatory mechanism in many life activities, the essential step is the recognition of S-itaconation. However, it is difficult to identify S-itaconation in the proteome for the high cost, which limits further investigation. In this study, we constructed an ensemble algorithm based on Soft Voting Classifier. The area under the ROC curve (AUC) value 0.73 for ensemble model. Accordingly, we constructed the on-line prediction tool dubbed SBP-SITA for easily identifying Cystine sites. SBP-SITA is available at http://www.bioinfogo.org/sbp-sita.

sentences: Ageing impairs the airway epithelium defence response to SARS-CoV-2 Age-dependent differences in the clinical response to SARS-CoV-2 infection is well-documented1-3 however the underlying molecular mechanisms involved are poorly understood. We infected fully differentiated human nasal epithelium cultures derived from healthy children (1-12 years old), young adults (26-34 years old) and older adults (56-62 years old) with SARS-COV-2 to identify age-related cell-intrinsic differences that may influence viral entry, replication and host defence response. We integrated imaging, transcriptomics, proteomics and biochemical assays revealing age-related changes in transcriptional regulation that impact viral replication, effectiveness of host responses and therapeutic drug targets. Viral load was lowest in infected older adult cultures despite the highest expression of SARS-CoV-2 entry and detection factors. We showed this was likely due to lower expression of hijacked host machinery essential for viral replication. Unlike the nasal epithelium of young adults and children, global host response and induction of the interferon signalling was profoundly impaired in older adults, which preferentially expressed proinflammatory cytokines mirroring the "cytokine storm" seen in severe COVID-194,5. In silico screening of our virus-host-drug network identified drug classes with higher efficacy in older adults. Collectively, our data suggests that cellular alterations that occur during ageing impact the ability for the host nasal epithelium to respond to SARS-CoV-2 infection which could guide future therapeutic strategies.

sentences: MHC class IIa haplotypes derived by high throughput SNP screening in an isolated sheep population Investigating the current evolutionary processes acting on a highly polymorphic gene region, such as the major histocompatibility complex (MHC), requires extensive population data for both genotypes and phenotypes. The MHC consists of several tightly linked loci with both allelic and gene content variation, making it challenging to genotype. Eight class IIa haplotypes have previously been identified in the Soay sheep (Ovis aries) of St. Kilda using Sanger sequencing and cloning, but no single locus is representative of all haplotypes. Here, we exploit the closed nature of the island population of Soay sheep and its limited haplotypic variation to identify a panel of SNPs that enable imputation of MHC haplotypes. We compared MHC class IIa haplotypes determined by Sanger sequence-based genotyping of 135 individuals to their SNP profiles generated using the Ovine Infinium HD BeadChip. A panel of 11 SNPs could reliably determine MHC diplotypes, and two additional SNPs within the DQA1 gene enabled detection of a recombinant haplotype affecting only the SNPs downstream of the expressed genes. The panel of 13 SNPs was genotyped in 5951 Soay sheep, of which 5349 passed quality control. Using the Soay sheep pedigree, we were able to trace the origin and inheritance of the recombinant SNP haplotype. This SNP-based method has enabled the rapid generation of locus-specific MHC genotypes for large numbers of Soay sheep. This volume of high-quality genotypes in a well-characterized population of free-living sheep will be valuable for investigating the mechanisms maintaining diversity at the MHC.

sentences: Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with loss of proteostasis Chromatin instability and loss of protein homeostasis (proteostasis) are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of S.cerevisiae, proteostasis decline occurred specifically in the fraction of cells with decreased stability at the ribosomal DNA (rDNA) region. A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)- binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability. We further found that loss of rDNA stability contributes to age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates negatively impact nucleolar integrity and global proteostasis and hence limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis decline and highlight the importance of cell-to-cell variability in aging processes.

sentences: Acitretin mitigates uroporphyrin-induced bone defects in congenital erythropoietic porphyria models Congenital erythropoietic porphyria (CEP) is a rare genetic disorder leading to accumulation of uro/coproporphyrin-I in tissues due to inhibition of uroporphyrinogen-III synthase. Clinical manifestations of CEP include bone fragility, severe photosensitivity and photomutilation. Currently there is no specific treatment for CEP, except bone marrow transplantation, and there is an unmet need for treating this orphan disease. Fluorescent porphyrins cause protein aggregation, which led us to hypothesize that uroporphyrin-I accumulation leads to protein aggregation and CEP-related bone phenotype. We developed a zebrafish model that phenocopies features of CEP. As in human patients, uroporphyrin-I accumulated in the bones of zebrafish, leading to impaired bone development. Furthermore, in an osteoblast-like cell line, uroporphyrin-I decreased mineralization, aggregated bone matrix proteins, activated endoplasmic reticulum stress and disrupted autophagy. Using high-throughput drug screening, we identified acitretin, a second-generation retinoid, and showed that it reduced uroporphyrin-I accumulation and its deleterious effects on bones. Our findings provide a new CEP experimental model and a potential repurposed therapeutic.

sentences: Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality The precise molecular mechanisms behind life-threatening lung abnormalities during severe SARS-CoV-2 infections are still unclear. To address this challenge, we performed whole transcriptome sequencing of lung autopsies from 31 patients suffering from severe COVID-19 related complications and 10 uninfected controls. Using a metatranscriptome analysis of lung tissue samples we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant "classical" signature (n=23) showed upregulation of unfolded protein response, steroid biosynthesis and complement activation supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) potentially representing "Cytokine Release Syndrome" (CRS) showed upregulation of cytokines such IL1 and CCL19 but absence of complement activation and muted inflammation. Further, dissecting expression of individual genes within enriched pathways for patient signature suggests heterogeneity in host response to the primary infection. We found that the majority of patients cleared the SARS-CoV-2 infection, but all suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Staphylococcus cohnii in "classical" patients and Pasteurella multocida in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients that can be identified through the status of the complement activation, presence of specific cytokines and characteristic microbiome. This information can be used to design personalized therapy to treat COVID-19 related complications corresponding to patient signature such as using the identified drug molecules or mitigating specific secondary infections.

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Identification of Fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no FDA-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 years of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo, and that it inhibits activation of the NLRP3-ASC inflammasome in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other anti-depressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies triangulate to link fluoxetine as a potential drug repurposing candidate for a major unmet medical need that causes blindness in millions of people in the United States and across the world.

Significance StatementDry age-related macular degeneration (AMD) affects the vision of millions of people worldwide. There is currently no FDA-approved treatment for dry AMD. The inflammasome components NLRP3 and ASC have been implicated in the pathogenesis of dry AMD. We report that fluoxetine, which is approved for the treatment of clinical depression, directly binds the NLRP3 protein and prevents the assembly and activation of the NLRP3-ASC inflammasome. As a result, it also blocks the degeneration of retinal pigmented epithelium (RPE) cells in an animal model of dry AMD. Furthermore, we demonstrate through an analysis of health insurance databases that use of this FDA-approved anti-depressant drug is associated with reduced incidence of dry AMD. These studies identify that fluoxetine is a potential repurposing candidate for AMD, a prevalent cause of blindness.

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UFold: Fast and Accurate RNA Secondary Structure Prediction with Deep Learning MotivationFor many RNA molecules, the secondary structure is essential for the correct function of the RNA. Predicting RNA secondary structure from nucleotide sequences is a long-standing problem in genomics, but the prediction performance has reached a plateau over time. Traditional RNA secondary structure prediction algorithms are primarily based on thermodynamic models through free energy minimization, which imposes strong prior assumptions and is slow to run.

ResultsHere we propose a deep learning-based method, called UFold, for RNA secondary structure prediction, trained directly on annotated data without any thermodynamic assumptions. UFold improves substantially upon previous models, with approximately 10~30% improvement over traditional thermodynamic models and 14% improvement over other learning-based methods. It achieves an F1 score of 0.91 on base pair prediction accuracy on an RNA structure prediction benchmark dataset. UFold is also fast with an inference time about 160ms per sequence up to 1600bp length. We provide an online web server that implements UFold for RNA structure prediction and is made freely available.

AvailabilityAn online web server running UFold is available at https://ufold.ics.uci.edu. Code is available at https://github.com/uci-cbcl/UFold.

Contactxhx@uci.edu

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Transcriptome Mining Reveals a Spectrum of RNA Viruses in Primitive Plants Current knowledge of plant viruses stems largely from those affecting economically important plants. Yet, plant species in cultivation represent a small and bias subset of the plant kingdom. Here, we describe virus diversity and abundance from a survey of 1079 transcriptomes from species across the breadth of the plant kingdom (Archaeplastida) by analysing open-source data from the One Thousand Plant Transcriptomes Initiative (1KP). We identified 104 potentially novel viruses, of which 40% comprised single-stranded positive-sense RNA viruses across eight orders, including members of the Hepelivirales, Tymovirales, Cryppavirales, Martellivirales and Picornavirales. One-third of the newly described viruses comprised double-stranded RNA viruses from the orders Durnavirales and Ghabrivirales. The remaining were negative-sense RNA viruses from the Rhabdoviridae, Aspiviridae, Yueviridae, Phenuiviridae and the newly proposed Viridisbunyaviridae. Our analysis considerably expands the known host range of 13 virus families to include lower plants (e.g., Benyviridae and Secoviridae) and four virus families to include algae hosts (e.g., Tymoviridae and Chrysoviridae). The discovery of the first 30 kDa movement protein in a non-vascular plant, suggests that the acquisition of plant virus movement proteins occurred prior to the emergence of the plant vascular system. More broadly, however, a co-phylogeny analysis revealed that the evolutionary history of these families is largely driven by cross-species transmission events. Together, these data highlight that numerous RNA virus families are associated with older evolutionary plant lineages than previously thought and that the scarcity of RNA viruses found in lower plants to date likely reflects a lack of investigation rather than their absence.

ImportanceOur knowledge of plant viruses is mainly limited to those infecting economically important host species. In particular, we know little about those viruses infecting primitive plant lineages such as the ferns, lycophytes, bryophytes and charophytes. To expand this understanding, we conducted a broad-scale viral survey of species across the breadth of the plant kingdom. We find that primitive plants harbour a wide diversity of RNA viruses including some that are sufficiently divergent to comprise a new virus family. The primitive plant virome we reveal offers key insights into the evolutionary history of core plant virus gene modules and genome segments. More broadly, this work emphasises that the scarcity of viruses found in these species to date likely reflects the absence of research in this area.

sentences: Isolation and characterization of fucosylated extracellular vesicles based on a novel GlyExo-Capture technique Extracellular vesicles (EVs) derived from tumor cells have recently garnered great interest due to their diagnostic and therapeutic potential. The presence of different glycosylation at the EVs surface potentiates the need for efficient glycosylated-EVs isolation. Here, we developed a GlyExo-Capture technique for capturing fucosylated-EVs from serum and cell supernatants robustly. Lens culinaris lectin (LCA)-immobilized beads were found to capture approximately 60% of the total EVs from HepG2 cells. The capture efficiency was reduced to lower than 40% in non-tumorigenic MIHA cells. Notably, the cellular uptake pattern of highly fucosylated-EVs was markedly different from EVs with low fucosylation. The disclosure of enriched fucosylated-EVs miRNA cargos by next-generation deep sequencing revealed 75 differentially expressed miRNAs in hepatocellular carcinoma (HCC). Among them, a 4-miRNA panel was chosen and yielded an area under the ROC curve (AUC) of 0.86 and 0.84 for the detection of HCC from non-HCC controls in testing sample and an independent validation sample, respectively. The 4-miRNA signature was independent of AFP and a combined model with AFP yielded an increased AUC of 0.92. In conclusion, we developed a high-throughput method for capturing fucosylated-EVs efficiently and shed light on using fucosylated-EVs as potential sources of miRNA for cancer biomarker detection.

sentences: Dynamic causal communication channels between neocortical areas Dynamic pathways of information flow between distributed brain regions underlie the diversity of behaviour. However, it remains unclear how neuronal activity in one area causally influences ongoing population activity in another, and how such interactions change over time. Here we introduce a causal approach to quantify cortical interactions by pairing simultaneous electrophysiological recordings with neural perturbations. We found that the influence visual cortical areas had on each other was surprisingly variable over time. Both feedforward and feedback pathways reliably affected different subpopulations of target neurons at different moments during processing of a visual stimulus, resulting in dynamically rotating communication dimensions between the two cortical areas. The influence of feedback on primary visual cortex (V1) became even more dynamic when visual stimuli were associated with a reward, impacting different subsets of V1 neurons within tens of milliseconds. This, in turn, controlled the geometry of V1 population activity in a behaviourally relevant manner. Thus, distributed neural populations interact through dynamically reorganizing and context-dependent communication channels to evaluate sensory information.

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Machine learning to extract muscle fascicle length changes from dynamic ultrasound images in real-time Background and objectiveDynamic muscle fascicle length measurements through B-mode ultrasound have become popular for the non-invasive physiological insights they provide regarding musculoskeletal structure-function. However, current practices typically require time consuming post-processing to track muscle length changes from B-mode images. A real-time measurement tool would not only save processing time but would also help pave the way toward closed-loop applications based on feedback signals driven by in vivo muscle length change patterns. In this paper, we benchmark an approach that combines traditional machine learning (ML) models with B-mode ultrasound recordings to obtain muscle fascicle length changes in real-time. To gauge the utility of this framework for  in-the-loop applications, we evaluate accuracy of the extracted muscle length change signals against time-series derived from a standard, post-hoc automated tracking algorithm.

MethodsWe collected B-mode ultrasound data from the soleus muscle of six participants performing five defined ankle motion tasks: (a) seated, constrained ankle plantarflexion, (b) seated, free ankle dorsi/plantarflexion, (c) weight-bearing, calf raises (d) walking, and then a (e) mix. We trained machine learning (ML) models by pairing muscle fascicle lengths obtained from standardized automated tracking software (UltraTrack) with the respective B-mode ultrasound image input to the tracker, frame-by-frame. Then we conducted hyperparameter optimizations for five different ML models using a grid search to find the best performing parameters for a combination of high correlation and low RMSE between ML and UltraTrack processed muscle fascicle length trajectories. Finally, using the global best model/hyperparameter settings, we comprehensively evaluated training-testing outcomes within subject (i.e., train and test on same subject), cross subject (i.e., train on one subject, test on another) and within/direct cross task (i.e., train and test on same subject, but different task).

ResultsSupport vector machine (SVM) was the best performing model with an average r = 0.70 {+/-}0.34 and average RMSE = 2.86 {+/-}2.55 mm across all direct training conditions and average r = 0.65 {+/-}0.35 and average RMSE = 3.28 {+/-}2.64 mm when optimized for all cross-participant conditions. Comparisons between ML vs. UltraTrack (i.e., ground truth) tracked muscle fascicle length versus time data indicated that ML tracked images reliably capture the salient qualitative features in ground truth length change data, even when correlation values are on the lower end. Furthermore, in the direct training, calf raises condition, which is most comparable to previous studies validating automated tracking performance during isolated contractions on a dynamometer, our ML approach yielded 0.90 average correlation, in line with other accepted tracking methods in the field.

ConclusionsBy combining B-mode ultrasound and classical ML models, we demonstrate it is possible to achieve real-time tracking of human soleus muscle fascicles across a number of functionally relevant contractile conditions. This novel sensing modality paves the way for muscle physiology in-the-loop applications that could be used to modify gait via biofeedback or unlock novel wearable device control techniques that could enable restored or augmented locomotion performance.

sentences: An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

sentences: Ventral Hippocampal Infusions of C20:0 Ceramide Stimulate Microglia and Induce Anhedonia-like Behavior in Female and Male Rats Increased long-chain C20:0 ceramides have been found in the serum of patients with depression. Moreover, ceramides are linked with increased microglia reactivity and inflammatory cytokine production, which are associated with depression. Since ceramides can readily cross the blood brain barrier, peripheral C20:0 ceramides could enter the brain, activate microglia, and induce depressive-like behavior. In this study, we determined whether localized infusion of C20:0 ceramides into the ventral hippocampus (VH) of rats is sufficient to activate microglia and induce depressive-like behaviors. Adult male and female rats received infusions of C20:0 ceramides or vehicle solution every other day for 2 weeks. After the third infusion, C20:0-infused animals showed reduced sucrose preference suggesting anhedonia-like behavior. However, 4-6 days after the last infusion, animals infused with C20:0 ceramides did not show anhedonia-like or despair-like behavior suggesting the behavioral effect was reversible. After the final behavioral test, C20:0-infused animals had more Iba-1+ microglia in the VH. Despite the increased number of VH microglia, the expression of inflammatory genes was not increased. In conclusion, our data suggest that localized increases in C20:0 ceramides in the VH are sufficient to induce microglia activation and reversible anhedonia-like behavior.

sentences: Parameter estimation for correlated Ornstein-Uhlenbeck time-series In this article, we develop a Maximum likelihood (ML) approach to estimate parameters from correlated time traces that originate from coupled Ornstein-Uhlenbeck processes. The most common technique to characterize the correlation between time-series is to calculate the Pearson correlation coefficient. Here we show that for time series with memory (or a characteristic relaxation time), our method gives more reliable results, but also results in coupling coefficients and their uncertainties given the data. We investigate how these uncertainties depend on the number of samples, the relaxation times and sampling time. To validate our analytic results, we performed simulations over a wide range of correlation coefficients both using our maximum likelihood solutions and Markov-Chain Monte-Carlo (MCMC) simulations. We found that both ML and MCMC result in the same parameter estimations. We also found that when analyzing the same data, the ML and MCMC uncertainties are strongly correlated, while ML underestimates the uncertainties by a factor of 1.5 to 3 over a large range of parameters. For large datasets, we can therfore use the less computationally expensive maximum likelihood method to run over the whole dataset, and then we can use MCMC on a few samples to determine the factor by which the ML method underestimates the uncertainties. To illustrate the application of our method, we apply it to time series of brain activation using fMRI measurements of the human default mode network. We show that our method significantly improves the interpretation of multi-subject measurements of correlations between brain regions by providing parameter confidence intervals for individual measurements, which allows for distinguishing between the variance from differences between subjects from variance due to measurement error.

sentences: Gradients in cellular metabolism regulate development of tonotopy along the chick cochlea. In vertebrates with elongated auditory organs, mechanosensory hair cells (HCs) are organised such that complex sounds are broken down into their component frequencies along the basal-to-apical long (tonotopic) axis. To generate the frequency-specific characteristics required at the appropriate positions, nascent HCs must determine their tonotopic properties during development. This relies on complex signalling between the developing HC and its local niche along the axis within the auditory organ. Here we apply NAD(P)H fluorescence lifetime imaging (FLIM) and live imaging of mitochondria to reveal metabolic gradients along the tonotopic axis of the developing cochlea. We further show that re-shaping these gradients during development, by inhibiting cytosolic glucose metabolism, alters normal Bmp7 and Chordin like-1 signalling leading to flattening of tonotopic patterning along the axis. Our work supports a causal link between morphogen signalling and metabolic reprogramming in specifying tonotopic morphologies in auditory HCs.

sentences: scDeepHash: An automatic cell type annotation and cell retrieval method for large-scale scRNA-seq datasets using neural network-based hashing Single-cell RNA-sequencing technologies measure transcriptomic expressions, which quantifies cell-to-cell heterogeneity at an unprecedented resolution. As these technologies become more readily available, the number of scRNA-seq datasets increases drastically. Prior works have demonstrated that bias-free, holistic single-cell profiling infrastructures are essential to the emerging automatic cell-type annotation methods. We propose scDeepHash, a scalable scRNA-seq analytic tool that employs content-based deep hashing to index single-cell gene expressions. scDeepHash allows for fast and accurate automated cell-type annotation and similar-cell retrieval. We also demonstrate the performance of scDeepHash by benchmarking it against current state-of-the-art methods across multiple public scRNA-seq datasets.

sentences: Plakoglobin regulates adipocyte differentiation independently of the Wnt/β-catenin signalling pathway The scaffold protein 14-3-3{zeta} is an established regulator of adipogenesis and postnatal adiposity. We and others have demonstrated that the 14-3-3{zeta} interactome to be diverse and dynamic, and it can be examined to identify novel regulators of physiological processes, including adipogenesis. In the present study, we sought to determine if factors that influence adipogenesis could be identified in the 14-3-3{zeta} interactome found in white adipose tissue of lean or obese TAP-tagged-14-3-3{zeta} overexpressing mice. Using mass spectrometry, changes in the abundance of novel, as well as established, adipogenic factors within the 14-3-3{zeta} interactome could detected. One novel candidate is plakoglobin, the homolog of the known adipogenic inhibitor {beta}-catenin, and herein, we report that plakoglobin is involved in adipocyte differentiation. Plakoglobin is expressed in murine 3T3-L1 cells and is primarily localized to the nucleus, where its abundance decreases during adipogenesis. Ectopic overexpression and siRNA-mediated depletion of plakoglobin had dual effects on inhibiting adipogenesis and reducing PPAR{gamma}2 expression. Plakoglobin depletion in human adipose-derived stem cells also impaired adipogenesis and reduced lipid accumulation post-differentiation. Transcriptional assays indicated that plakoglobin does not participate in Wnt/{beta}-catenin signaling, as its depletion did not affect Wnt3a-mediated SUPERTOPFlash activity. Taken together, our results establish plakoglobin as a novel regulator of adipogenesis in vitro and highlights the ability of using the 14-3-3{zeta} interactome to discover undiscovered pro-obesogenic factors.

sentences: Acetyltransferases GCN5 and PCAF are required for B cell maturation B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacks both GCN5 and PCAF in B cells. We found that double-deficient mice possess low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We conclude that both GCN5 and PCAF are required for B cell development in vivo.

sentences: Enhancing top-down proteomics of brain tissue with FAIMS Proteomic investigations of Alzheimers and Parkinsons disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a proteins "intact" state. Top-down proteomics (TDP) overcomes this limitation, however it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, offline fractionation techniques are commonly used to reduce sample complexity, limiting throughput. A higher throughput alternative is online fractionation, such as gas phase high-field asymmetric waveform ion mobility spectrometry (FAIMS). Utilizing a high complexity sample derived from Alzheimers disease brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS at -50 compensation voltage (CV) more than doubled the mean number of non-redundant proteoforms observed (1,833 {+/-} 17, n = 3), compared to without (754 {+/-} 35 proteoforms). We also found FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (A{beta}) proteoforms, including the aggregation-prone A{beta}1-42 variant which is strongly linked to Alzheimers disease.

sentences: Applying biostimulants boosts forage productivity without affecting soil biotic and abiotic parameters on a Central Coast California rangeland There is increasing interest in using biostimulant products, such as microbial inoculants and humic substances, to help manage rangelands regeneratively. Understanding how plant and soil communities on rangelands respond to these products is therefore important. In this study, we examined the combined effects of a commercial inoculant and humic product that are currently on the market, and asked whether they influenced rangeland forage productivity and quality, soil microbial biomass and community composition, and abiotic soil parameters in Central Coastal California. We found that forage productivity and some metrics of forage quality responded positively to the foliar application of a commercial microbial inoculant and humic product, but that these benefits were not mirrored by changes belowground in the microbial community or abiotic parameters. Depending on the goals of using the products, this could be seen as a winning scenario and suggests microbial inoculants and humic products could warrant attention as a potential tool for regenerative stewardship of rangelands. While our study derives from one ranch and therefore requires confirmation of its ubiquity prior to broadscale adoption, our results provide new insights into the usefulness of this approach for managing rangeland productivity in Californias Central Coast.

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Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes. Influenza A virus (IAV) genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins (vRNPs). Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes (TNTs) as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion.

Author SummaryInfluenza A viruses infect epithelial cells of the upper and lower respiratory tract in humans. Infection is propagated by the generation of viral particles from infected cells, which disseminate within the tissue. Disseminating particles can encounter obstacles in the extracellular environment, including mucus, ciliary movement, antibody neutralization and uptake by phagocytic immune cells. An alternative mode of spread, which avoids these hazards, involves direct transport of viral components between cells. This cell-cell spread of infection is not a well understood process. In this study we demonstrate that the host factor Rab11a mediates the transport of viral genomes in the cell-cell spread of infection. Rab11a is already known to play a pro-viral role in the transport of viral genomes to the plasma membrane for assembly into virus particles. Here, we see that this same transport mechanism is co-opted for direct cell-cell spread through cellular connections called tunneling nanotubes. We show that complexes of Rab11a and viral components can be trafficked across tunneling nanotubes, transmitting infection without the formation of virus particles. Importantly, this route of spread often seeds viral genomes from multiple donor cells into recipient cells, which in turn increases viral genetic diversity.

sentences: Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.

sentences: Parenteral glucose supply and pharmacological glycolysis inhibition determine the clinical fate of infected preterm newborns Preterm infants are susceptible to bloodstream infection that can lead to sepsis. High parenteral glucose supplement is commonly used to support their growth and energy expenditure, but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal sepsis induced by Staphylococcus epidermidis infection, we demonstrate the delicate interplay between immunity and energy metabolism to regulate the host infection response. Circulating glucose levels, glycolysis and inflammatory response to infection are closely connected across the states of tolerance, resistance and immunoparalysis. Further, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis and inflammation, leading to lactate acidosis and sepsis, whereas glucose restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, pharmacological glycolysis inhibition during normoglycemia enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose controls immune cell metabolism and function, in turn determining the clinical fate of infected preterm neonates. This also questions the current practice of parenteral glucose supply for infected preterm infants.

sentences: Vangl2, a core component of the WNT/PCP pathway, regulates adult hippocampal neurogenesis and age-related decline in cognitive flexibility Decline in episodic memory is one of the hallmarks of aging and represents one of the most important health problems facing western societies. A key structure in episodic memory is the hippocampal formation and the dentate gyrus in particular, as the continuous production of new dentate granule neurons in this brain region was found to play a crucial role in memory and in age-related decline in memory. As such, understanding the molecular processes that regulate the relationship between adult neurogenesis and aging of memory function holds great therapeutic potential. Recently, we found that Vang-gogh like 2 (Vangl2), a core component of the planar cell polarity signaling pathway, is enriched in the dentate gyrus of adult mice. In this context, we sought to evaluate the involvement of this effector of the Wnt/PCP pathway in both adult neurogenesis and memory abilities in adult and middle-aged mice. Using a heterozygous mouse model carrying a dominant negative mutation in Vangl2 gene, we show that alteration in Vangl2 expression decreases the survival of adult-born granule cells and advances the onset of decrease in cognitive flexibility. Inability of mutant mice to erase old irrelevant information to the benefit of new relevant ones highlights a key role of Vangl2 in interference-based forgetting. Taken together, our findings show for the first that Vangl2 activity may constitute an interesting target to prevent age-related decline in hippocampal plasticity and memory.

sentences: Extracellular cues accelerate neurogenesis of induced pluripotent stem cell derived neurons Neurogenesis is a complex process encompassing neuronal progenitor cell expansion/proliferation and differentiation, followed by neuron maturation. In vivo models are most commonly used to study neurogenesis; however, human induced pluripotent stem cell-derived (iPSC) neurons are increasingly used to establish cellular models of human neurological processes. Unfortunately, the differentiation and maturation of iPSC-derived neurons varies in methodology, is asynchronous, and has restricted experimental utility because of extended differentiation/maturation times. To accelerate and standardize iPS neuronal maturation, we differentiated and matured feeder layer-free iPSC-derived neuronal cultures under physiological oxygen levels (5%), and modified the underlying extracellular matrix and medium composition. Our results demonstrate that calretinin gene expression occurred earlier under our optimized iPS conditions and the corresponding "neurogenesis burst" associated with proliferative expansion occurred more synchronously, reliably emerging two and three weeks after differentiation. As expected, the expression of mature neuronal markers (i.e., NeuN+/Calbindin+) started at 4-weeks post-differentiation. qPCR microarray, western blot and single cell analyses using high content imaging indicated that 4-week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Our data demonstrate that extracellular cues influence the kinetics of neurogenesis models and that feeder layer-free iPSC-derived neurogenesis can be reproducibly miniaturized.

sentences: Brain-inspired spiking neural network controller for a neurorobotic whisker system It is common for animals to use self-generated movements to actively sense the surrounding environment. For instance, rodents rhythmically move their whiskers to explore the space close to their body. The mouse whisker system has become a standard model to study active sensing and sensorimotor integration through feedback loops. In this work, we developed a bioinspired spiking neural network model of the sensorimotor peripheral whisker system, modelling trigeminal ganglion, trigeminal nuclei, facial nuclei, and central pattern generator neuronal populations. This network was embedded in a virtual mouse robot, exploiting the Neurorobotics Platform, a simulation platform offering a virtual environment to develop and test robots driven by brain-inspired controllers. Eventually, the peripheral whisker system was properly connected to an adaptive cerebellar network controller. The whole system was able to drive active whisking with learning capability, matching neural correlates of behaviour experimentally recorded in mice.

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Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase.

ImportanceBiofuel production from non-food crops is an important strategy for reducing carbon emissions from the transportation industry but it has not yet become commercially viable. An important avenue to improve biofuel production is to enhance the characteristics of fermentation organisms by genetic engineering. To make genetic modifications successful, we must gain sufficient information about the genome and metabolism of the organism to enable rational design and engineering. Here, we improved understanding of Zymomonas mobilis, a promising biofuel producing bacterium, by identifying a metabolic pathway and associated gene that lead to byproduct formation. This information may be used in the future for genetic engineering to reduce byproduct formation during biofuel production.

sentences: Dopamine enhances model-free credit assignment through boosting of retrospective model-based inference Dopamine is implicated in signalling model-free (MF) reward prediction errors and various aspects of model-based (MB) credit assignment and choice. Recently, we showed that cooperative interactions between MB and MF systems include guidance of MF credit assignment by MB inference. Here, we used a double-blind, placebo-controlled, within-subjects design to test the hypothesis that enhancing dopamine levels, using levodopa, boosts the guidance of MF credit assignment by MB inference. We found that levodopa enhanced retrospective guidance of MF credit assignment by MB inference, without impacting on MF and MB influences per se. This drug effect positively correlated with working memory, but only in a context where reward needed to be recalled for MF credit assignment. The dopaminergic enhancement in MB-MF interactions correlated negatively with a dopamine-dependent change in MB credit assignment, possibly reflecting a potential trade-off between these two components of behavioural control. Thus, our findings demonstrate that dopamine boosts MB inference during guidance of MF learning, supported in part by working memory, but trading-off with a dopaminergic enhancement of MB credit assignment. The findings highlight a novel role for a DA influence on MB-MF interactions.

sentences: CCR1 regulatory variants linked to pulmonary macrophage recruitment in severe COVID-19 Genome-wide association studies have identified 3p21.31 as the main risk locus for severe disease in COVID-19 patients, although underlying biological mechanisms remain elusive. We performed a comprehensive epigenomic dissection of the 3p21.31 locus, identifying a CTCF-dependent tissue-specific 3D regulatory chromatin hub that controls the activity of several tissue-homing chemokine receptor (CCR) genes in monocytes and macrophages. Risk SNPs colocalized with regulatory elements and were linked to increased expression of CCR1, CCR2 and CCR5 in monocytes and macrophages. As excessive organ infiltration of inflammatory monocytes and macrophages is a hallmark of severe COVID-19, our findings provide a rationale for the genetic association of 3p21.31 variants with elevated risk of hospitalization upon SARS-CoV-2 infection.

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Development of an in-situ printing system with human platelet lysate based bioink to treat corneal perforation PurposeCorneal perforation is a clinical emergency. Tissue glue to seal the perforation, and supplementary topical medication represents existing standard treatment. Previously, our group developed a transparent human platelet lysate (hPL)-based biomaterial that showed good cell compatibility and accelerated corneal epithelial cells healing in-vitro. This study aims to develop a novel treatment method for corneal perforation using this biomaterial.

MethodsRheometry was used to measure the hPL-based biomaterial behaviour at room and corneal surface temperatures. Its adhesiveness to porcine skin and burst pressure limit were also measured. Based on rheological behaviour, a hand-held biopen was developed to extrude it onto the cornea. An animal trial (5 New Zealand white rabbits) to compare impact of the biomaterial and cyanoacrylate glue (control group) on a 2mm perforation was conducted to evaluate safety and efficacy.

ResultsThe hPL-based biomaterial showed higher adhesiveness compared to commercial fibrin glue and withstood burst pressure approximately 6.4x higher than routine intraocular pressure. Treatment rabbits had lower pain scores and faster recovery, despite generating similar scar-forming structure compared to controls. No secondary corneal ulcer was generated in rabbits treated with the bio-adhesive.

ConclusionsThis study reports a novel in-situ printing system capable of delivering a hPL-based, transparent bio-adhesive and successfully treating small corneal perforations. Bio-adhesive-treated rabbits recovered faster and required no additional analgesia. Both groups showed scarred corneal tissue after healing, however no infection and inflammation was observed by 3 weeks. The delivery system was easy to use and may represent an alternative treatment for corneal perforation.

HighlightsO_LIThis study presents a novel in situ printing system to treat corneal perforation
C_LIO_LIThe system is comprised of a human platelet lysate-based bio-adhesive and a pen-like hand held delivery system
C_LIO_LIMechanical tests showed our transparent bio-adhesive has a higher adhesiveness compared to existing treatments and burst pressure threshold approximately 6.4 times higher than normal intraocular pressure.
C_LIO_LIIn vivo rabbit trial showed that compared to cyanoacrylate glue, the bio-adhesive was safer, faster healing and led to less pain in rabbits.
C_LI

sentences: BionetBF: A Novel Bloom Filter for Faster Membership Identification of Paired Biological Network Data Biological network represents the interaction or relationship between the biological entities such as proteins and genes of a biological process. A biological network with thousands of millions of vertices makes its processing complex and challenging. In this article, we propose a novel Bloom Filter called Biological network Bloom Filter (BionetBF) fast membership identification of the biological network edges or paired biological data. BionetBF is capable of executing millions of operations within a second while occupying a tiny amount of main memory. We have conducted rigorous experiments to prove the performance of BionetBF with large datasets. The experiment is conducted using 12 generated datasets and three biological network datasets. It takes less than 8 sec for insertion and query of 40 million paired data. It demonstrates higher performance while maintaining a 0.001 false positive probability. BionetBF is also compared with other filters: Cuckoo Filter and Libbloom, where BionetBF proves its supremacy by exhibiting higher performance with a smaller sized memory compared with large sized filters of Cuckoo Filter and Libbloom.

sentences: Perivascular macrophages collaborate to facilitate chemotherapy resistance in cancer A subset of tumor associated macrophages (TAMs) identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) reside proximal to blood vasculature and contribute to disease progression. Using a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we show that Lyve-1+ TAMs, which co-express heme oxygenase-1, form coordinated multi-cellular  nest structures in the perivascular niche. We show that TAM nest formation is dependent on IL-6 and a communication axis involving CCR5 and its cognate ligands CCL3/4. We demonstrate that Lyve-1+ TAM nests are associated with CD8+ T-cell exclusion from the tumor and the resistance to immune-stimulating chemotherapeutics. This study highlights an unappreciated collaboration between TAMs and uncovers a spatially driven therapeutic resistance mechanism of these cells in cancer which can be therapeutically targeted.

sentences: Dynamic neural reconstructions of attended object location and features using EEG We live in a dynamic world and our environment contains far more stimuli than our brain can process at one time. Visual attention allows us to select relevant information and ignore irrelevant information. What happens when attention is shifted from one item to another? To answer this question, it is critical to have tools that can accurately recover neural representations of both feature and location information, and do so across shifts of attention with high temporal resolution. In the current study, we used human electroencephalography (EEG) and machine learning methods to explore how neural representations of object features and locations update across dynamic shifts of attention. We demonstrate that EEG can be used to create simultaneous timecourses of the neural representations of attended features (timepoint-by-timepoint inverted encoding model reconstructions) and attended location (timepoint-by-timepoint decoding) during both stable periods and across dynamic spatial shifts of attention. On each trial two oriented gratings were presented that flickered at the same frequency but had different orientations; participants were cued to attend to one of them, and on half of the trials received a shift cue in the middle of the trial. We trained the models on a stable period from Hold attention trials, and then reconstructed/decoded the attended orientation/location information at each timepoint on Shift attention trials. Our results showed that both feature reconstructions and location decoding dynamically track the shift of attention, and that there may be timepoints during the shifting of attention when (1) feature and location representations become uncoupled, and (2) both the previously-attended and currently-attended orientations are represented with roughly equal strength. The results offer insight into our understanding of attentional shifts, and the noninvasive techniques developed in the current study lend themselves well to a wide variety of future applications.

sentences: Collagen remodeling dictates pancreatic cancer bioenergetics and outcome through DDR1 activation or degradation Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses by liver metastasis1. Cancer-associated fibroblasts (CAF), extracellular matrix (ECM), and type I collagen (Col I) support2-5 or restrain PDAC progression and may impede blood supply and nutrient availability6-8. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers escape nutrient limitation remain poorly understood. Here we show that matrix metalloprotease (MMP)-cleaved or intact Col I (cCol I and iCol I, respectively) exert opposing effects on PDAC bioenergetics, macropinocytosis (MP), tumor growth and liver metastasis. While cCol I activates DDR1 (discoidin domain receptor-1)-NF-{kappa}B-p62-NRF2 signaling to promote PDAC growth, iCol I triggers DDR1 degradation and restrains PDAC growth. Patients whose tumors are enriched in iCol I and low in DDR1 and NRF2 have improved median survival compared to those enriched in cCol I, DDR1 and NRF2. Inhibition of DDR1-stimulated NF-{kappa}B or mitochondrial biogenesis blocked tumorigenesis in wildtype mice but not in mice expressing MMP-resistant Col I. In summary, the diverse effects of tumor stroma on PDAC growth, metastasis, and patient survival are mediated through the Col I-DDR1-NF-{kappa}B-NRF2-mitochondrial biogenesis pathway, presenting multiple new opportunities for PDAC therapy.

sentences: Noise-augmented directional clustering of genetic association data identifies distinct mechanisms underlying obesity Clustering genetic variants based on their associations with different traits can provide insight into their underlying biological mechanisms. Existing clustering approaches typically group variants based on the similarity of their association estimates for various traits. We present a new procedure for clustering variants based on their proportional associations with different traits, which is more reflective of the underlying mechanisms to which they relate. The method is based on a mixture model approach for directional clustering and includes a noise cluster that provides robustness to outliers. The procedure performs well across a range of simulation scenarios. In an applied setting, clustering genetic variants associated with body mass index generates groups reflective of distinct biological pathways. Mendelian randomization analyses support that the clusters vary in their effect on coronary heart disease, including one cluster that represents elevated body mass index with a favourable metabolic profile and reduced coronary heart disease risk. Analysis of the biological pathways underlying this cluster identifies inflammation as playing a key role in mediating the effects of increased body mass index on coronary heart disease.

sentences: Human myelin protein P2: From crystallography to time-lapse membrane imaging and neuropathy-associated variants Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patient families. To shed light on the structure and function of the corresponding P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations, and lipid binding assays. The crystal and solution structures of the I50del, M114T, and V115A variants of P2 showed only minor differences to the wild-type protein, whereas the thermal stability of the disease variants was reduced. Lipid vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed slightly altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated membrane blebbing induced by P2, which could be related to its function in stacking of two curved membrane surfaces in myelin in vivo. All variants caused blebbing of membranes on similar timescales. In order to better understand the links between structure, dynamics, and function, the crystal structure of perdeuterated P2 was refined from room temperature data collected using both neutrons and X-rays, and the results were compared to molecular dynamics simulations and cryocooled crystal structures. Taken together, our data indicate similar properties of all known CMT variants of human P2; while crystal structures are nearly identical, stability and function of the disease variants are impaired compared to the wild-type protein. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

sentences: Real-time single-molecule observation of chaperone-assisted protein folding The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an individual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

sentences: New fossils of Jurassic ophiurid brittle stars (Ophiuroidea; Ophiurida) provide evidence for early clade evolution in the deep sea 1.Understanding of the evolutionary history of the ophiuroids, or brittle stars, is hampered by a patchy knowledge of the fossil record. Especially the stem members of the living clades are poorly known, resulting in blurry concepts of the early clade evolution and imprecise estimates of divergence ages. Here, we describe new ophiuroid fossil from the Lower Jurassic of France, Luxembourg, and Austria and introduce the new taxa Ophiogojira labadiei gen. et sp. nov. from lower Pliensbachian shallow sublittoral deposits, Ophiogojira andreui gen. et sp. nov. from lower Toarcian shallow sublittoral deposits, and Ophioduplantiera noctiluca gen. et sp. nov. from late Sinemurian to lower Pliensbachian bathyal deposits. A Bayesian morphological phylogenetic analysis shows that Ophiogojira holds a basal position within the order Ophiurida, whereas Ophioduplantiera has a more crownward position within the ophiurid family Ophiuridae. The position of Ophioduplantiera in the evolutionary tree suggests that family-level divergences within the Ophiurida must have occurred before the late Sinemurian, and that ancient slope environments played an important role in fostering early clade evolution.

sentences: Tensor-decomposition--based unsupervised feature extraction in single-cell multiomics data analysis Analysis of single-cell multiomics datasets is a novel topic and is considerably challenging because such datasets contain a large number of features with numerous missing values. In this study, we implemented a recently proposed tensor-decomposition (TD)-based unsupervised feature extraction (FE) technique to address this difficult problem. The technique can successfully integrate single-cell multiomics data composed of gene expression, DNA methylation, and accessibility. Although the last two have large dimensions, as many as ten million, containing only a few percentages of non-zero values, TD-based unsupervised FE can integrate three omics datasets without filling missing values. Together with UMAP, which is used frequently when embedding single-cell measurements into two-dimensional space, TD-based unsupervised FE can produce two-dimensional embedding coincident with classification when integrating single-cell omics datasets. Genes selected based on TD-based unsupervised FE were also significantly related to reasonable biological roles.

sentences: Diversification dynamics and (non-)parallel evolution along an ecological gradient in African cichlid fishes Understanding the drivers and dynamics of diversification is a central topic in evolutionary biology. Here, we investigated the dynamics of diversification in the cichlid fish Astatotilapia burtoni that diverged along a lake-stream environmental gradient. Whole-genome and morphometric analyses revealed that divergent selection was essential at the early stages of diversification, but that periods in allopatry were likely involved towards the completion of speciation. While morphological differentiation was continuous, genomic differentiation was not, as shown by two clearly separated categories of genomic differentiation. Reproductive isolation increased along a continuum of genomic divergence, with a "grey zone" of speciation at [~]0.1% net nucleotide divergence. The quantification of the extent of (non-)parallelism in nine lake-stream population pairs from four cichlid species by means of multivariate analyses revealed one parallel axis of genomic and morphological differentiation among seven lake-stream systems. Finally, we found that parallelism was higher when ancestral lake populations were more similar.

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Fine mapping without phenotyping: Identification of selection targets in secondary Evolve and Resequence experiments Evolve and Resequence (E&R) studies investigate the genomic selection response of populations in an Experimental Evolution setup. Despite the popularity of E&R, empirical studies in sexually reproducing organisms typically suffer from an excess of candidate loci due to linkage disequilibrium, and single gene or SNP resolution is the exception rather than the rule. Recently, so-called "secondary E&R" has been suggested as promising experimental follow-up procedure to confirm putatively selected regions from a primary E&R study. Secondary E&R provides also the opportunity to increase mapping resolution by allowing for additional recombination events, which separate the selection target from neutral hitchhikers. Here, we use computer simulations to assess the effect of different crossing schemes, population size, experimental duration, and number of replicates on the power and resolution of secondary E&R. We find that the crossing scheme and population size are crucial factors determining power and resolution of secondary E&R: a simple crossing scheme with few founder lines consistently outcompetes crossing schemes where evolved populations from a primary E&R experiment are mixed with a complex ancestral founder population. Regardless of the experimental design tested, a population size of at least 4,800 individuals, which is roughly 5 times larger than population sizes in typical E&R studies, is required to achieve a power of at least 75%. Our study provides an important step towards improved experimental designs aiming to characterize causative SNPs in Experimental Evolution studies.

SignificanceDespite the popularity of Evolve and Resequence (E&R) to investigate genomic selection responses, most studies that use sexually reproducing organisms have broad selection signatures and an excess of candidate loci due to linkage disequilibrium. In this study, we use computer simulations and statistical modelling to evaluate the effects of different experimental and population genetic parameters on the success of potential follow-up experiments (=secondary E&R) aiming to validate and fine-map selection signatures of primary studies. We found that a large population size in combination with a simple crossing scheme is key to the success of secondary E&R in Drosophila.

sentences: The population frequency of human mitochondrial DNA variants is highly dependent upon mutational bias Next-generation sequencing can quickly reveal genetic variation potentially linked to heritable disease. As databases encompassing human variation continue to expand, rare variants have been of high interest, since the frequency of a variant is expected to be low if the genetic change leads to a loss of fitness or fecundity. However, the use of variant frequency when seeking genomic changes linked to disease remains very challenging. Here, we explore the role of selection in controlling human variant frequency using the HelixMT database, which encompasses hundreds of thousands of mitochondrial DNA (mtDNA) samples. We find that a substantial number of synonymous substitutions, which have no effect on protein sequence, were never encountered in this large study, while many other synonymous changes are found at very low frequencies. Further analyses of human and mammalian mtDNA datasets indicate that the population frequency of synonymous variants is predominantly determined by mutational biases rather than by strong selection acting upon nucleotide choice. Our work has important implications that extend to the interpretation of variant frequency for non-synonymous substitutions.

sentences: Host-pathogen Immune Feedbacks Can Explain Widely Divergent Outcomes from Similar Infections A longstanding question in infection biology is why two very similar individuals, with very similar pathogen exposures, may have very different outcomes. Recent experiments have found that even isogenic Drosophila melanogaster hosts, given identical inoculations of some bacterial pathogens at suitable doses, can experience very similar initial bacteria proliferation but then diverge to either a lethal infection or a sustained chronic infection with much lower pathogen load. We hypothesized that divergent infection outcomes are a natural result of mutual negative feedbacks between pathogens and the host immune response. Here we test this hypothesis in silico by constructing process-based dynamic models for bacterial population growth, host immune induction, and the feedbacks between them, based on common mechanisms of immune system response. Mathematical analysis of a minimal conceptual model confirms our qualitative hypothesis that mutual negative feedbacks can magnify small differences among hosts into life-or-death differences in outcome. However, explaining observed features of chronic infections requires an extension of the model to include induced pathogen modifications that shield themselves from host immune responses at the cost of reduced proliferation rate. Our analysis thus generates new, testable predictions about the mechanisms underlying bimodal infection outcomes.

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Nanopore sequencing methods detect cell-free DNA associated with minimal residual disease and central nervous system infiltration in pediatric Acute Lymphoblastic Leukemia Acute Lymphoblastic Leukemia (ALL) patients that are positive for minimal residual disease (MRD) after therapy or have leukemic infiltration into the central nervous system (CNS) are considered high risk and receive intensive chemotherapy regimens. Current methods to diagnose MRD and CNS infiltration rely on detecting leukemic cells in patient samples using pathology, flow cytometry, or next-generation sequencing. However, leukemic blasts may persist in the patient but not be physically present in bone marrow biopsy or biofluid sample, leading to inaccurate or delayed patient diagnosis. We have developed a nanopore sequencing workflow to detect B-ALL-associated cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) samples. Quantitation of B-cell specific VDJ recombination events in cfDNA samples defined B-ALL clonal heterogeneity. This workflow allowed us to track the response of individual B-ALL clones throughout treatment. Detection of cfDNA also predicted the clinical diagnosis of MRD and CNS disease. Importantly, we identified patients diagnosed as CNS negative who had low B-cell derived cell-free DNA levels in their CSF sample that correlated with B-cell clones present in the bone marrow. These data suggest that cfDNA assays may be useful in detecting the presence of ALL in the patient even when blasts are not in the biofluid sample. Nanopore analysis of cell-free DNA is a simple, rapid, and inexpensive assay that can serve as a valuable complement to traditional clinical diagnostic approaches for ALL.

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Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via interplay among all chromatin layers including DNA, histones post-translational modifications (hPTMs), and chromatin-associated proteins. Here we employ multi-layered proteomics to characterize chromatin-mediated interactions of repair proteins, signatures of hPTMs, and the DNA-bound proteome during DNA double-strand break repair at high temporal resolution. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining or homologous recombination (HR) revealing histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and PARP inhibitor sensitivity. Furthermore, we dynamically profile numerous hPTMs at {gamma}H2AX-mononucleosomes during the DDR. Integration of these complementary data implicated G9A-mediated monomethylation of H3K56 in HR. Collectively, we provide a dynamic chromatin-centered view of DDR, while representing a valuable resource for the use of PARP inhibitors in cancer.

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RgCop-A regularized copula based method for gene selection in single cell rna-seq data Gene selection in unannotated large single cell RNA sequencing (scRNA-seq) data is important and crucial step in the preliminary step of downstream analysis. The existing approaches are primarily based on high variation (highly variable genes) or significant high expression (highly expressed genes) failed to provide stable and predictive feature set due to technical noise present in the data. Here, we propose RgCop, a novel regularized copula based method for gene selection from large single cell RNA-seq data. RgCop utilizes copula correlation (Ccor), a robust equitable dependence measure that captures multivariate dependency among a set of genes in single cell expression data. We raise an objective function by adding a l1 regularization term with Ccor to penalizes the redundant co-efficient of features/genes, resulting non-redundant effective features/genes set. Results show a significant improvement in the clustering/classification performance of real life scRNA-seq data over the other state-of-the-art. RgCop performs extremely well in capturing dependence among the features of noisy data due to the scale invariant property of copula, thereby improving the stability of the method. Moreover, the differentially expressed (DE) genes identified from the clusters of scRNA-seq data are found to provide an accurate annotation of cells. Finally, the features/genes obtained from RgCop can able to annotate the unknown cells with high accuracy.

AvailabilityCorresponding software is available in: https://github.com/Snehalikalall/RgCop

Contactsumanta.ray@cwi.nl

sentences: Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters The loss of smell related to SARS-CoV-2 infection is one of the most prevalent symptoms of COVID-19. It is now clear that this symptom is related to the massive infection by SARS-CoV-2 of the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamster, we show here that while apoptosis remains at a low level in damaged infected epithelium, the latter is invaded by innate immunity cells. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we reduced the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease of SARS-CoV-2 infection levels in the nasal cavity. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus.

sentences: STOmicsDB: a database of Spatial Transcriptomic data Recent technological development in spatial transcriptomics allows researchers to measure gene expression of cells and their spatial locations at the almost single-cell level, which generates detailed biological insight into biological processes. However, specialized spatial transcriptomics databases are rare. Here, we present the Spatial TranscriptOmics DataBase (STOmicsDB), a user-friendly database with multifunctions including search of relevant publications and tools, public dataset visualization, customized specialized databases, new data archive, and online analysis. The current version of STOmicsDB consists of 141 curated spatial transcript datasets covering 12 species, and includes 5,618 spatial multi-omics publications and 674 tools. STOmicsDB is freely accessible at https://db.cngb.org/stomics/.

sentences: Single-cell multimodal modeling with deep parametric inference The paired measurement of multiple modalities, known as the multimodal analysis, is an exciting frontier for connecting single-cell genomics with epitopes and functions. Mapping of transcriptomes in single-cells and the integration with cell phenotypes enable a better understanding of cellular states. However, assembling these paired omics into a unified representation of the cellular state remains challenging with the unique technical characteristics of each measurement. In this study, we built a deep parameter inference model (DPI) based on the properties of single-cell multimodal data. DPI is a complete single-cell multimodal omics analysis framework, which has built in multimodal data preprocessing, multimodal data integration, multimodal data reconstruction, reference and query, disturbance prediction and other analysis functions.

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Novel Paju Apodemus Paramyxovirus 1 and 2, Harbored by Apodemus agrarius in The Republic of Korea Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a potential threat to public health. Currently, 78 species and 17 genera of paramyxoviruses are classified and harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Jeilongvirus has been proposed as a novel paramyxovirus genus containing J-, Beilong, and Tailam viruses, found in wild rodents. Using RT-PCR, 824 Apodemus agrarius individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was detected in 108 (13.1%) rodents captured at 14 trapping sites in Korea. We first present two genetically distinct novel paramyxoviruses (genus Jeilongvirus), Paju Apodemus paramyxoviruses 1 (PAPV-1) and 2 (PAPV-2), from A. agrarius. Six PAPV strains were completely sequenced using next-generation and Sanger sequencing. PAPV-1 genome comprised 19,716 nucleotides, with eight genes (3'-N-P/V/C-M-F-SH-TM-G-L-5'), whereas PAPV-2 genome contained 17,475 nucleotides, with seven genes (3'-N-P/V/C-M-F-TM-G-L-5'). The disparity between PAPV-1 and -2 revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogenies of PAPV-1 and -2 belong to distinct genetic lineages of Jeilongvirus despite being from the same natural host. PAPV-1 clustered with Beilong and Tailam viruses, while PAPV-2 formed a genetic lineage with Mount Mabu Lophuromys virus-1. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. Therefore, this study provides profound insights into the molecular epidemiology, virus-host interactions, and zoonotic potential of novel rodent-borne paramyxoviruses.

ImportanceParamyxoviruses are a critical public health and socio-economic burden to humans. Rodents play a crucial role in transmitting pathogens to humans. In the last decade, novel paramyxoviruses have been discovered in different rodents. Here, we found that Apodemus agrarius harbored two distinct genotypes of the novel paramyxoviruses, Paju Apodemus paramyxovirues 1 (PAPV-1) and 2 (PAPV-2), possessing unique genome structures that are responsible for encoding TM and G proteins of different sizes. In addition, PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III IFNs, ISGs, and proinflammatory cytokines. Thus, this study provides significant insights into molecular prevalence, virus-host interactions of paramyxoviruses. These observations raise the awareness of physicians and scientists about the emergence of new rodent-borne paramyxoviruses.

sentences: A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 {micro}m. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

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Mean field approximation of network of biophysical neurons driven by conductance-based ion exchange dynamics Numerous network and whole brain modeling approaches make use of mean-field models. Their relative simplicity allows studying network dynamics at a large scale. They correspond to lumped descriptions of neuronal assemblies connected via synapses. mean-field models do not consider the ionic composition of the extracellular space, which can change in physiological and pathological conditions, with strong effects on neuron activity. Here we derive a mean-field model of a population of Hodgkin-Huxley type neurons, which links the neuronal intra- and extra-cellular ion concentrations to the mean membrane potential and the mean synaptic input in terms of the synaptic conductance. The model can generate various physiological brain activities including multi-stability at resting states, as well as pathological spiking and bursting behaviors, and depolarization block. The results from the analytical solution of the mean-field model agree with the mean behavior of numerical simulations of large-scale networks of neurons. The mean-field model is analytically exact for non-autonomous ion concentration variables and provides a mean-field approximation in the thermodynamic limit, for locally homogeneous mesoscopic networks of biophysical neurons driven by an ion-exchange mechanism. These results may provide the missing link between high-level neural mass approaches which are used in the brain network modeling and physiological parameters that drive the neuronal dynamics.

Significance StatementIn this study, we applied mathematical formalism to estimate the mean-field behaviors of a large neuronal ensemble taking into account the ion-exchange between the intracellular and extracellular space. The model demonstrates different brain activities including resting state, spiking behavior, and seizure, as a function of the extracellular ion concentration. The relevant parameter regime for different brain activities is extracted mainly in terms of ion concentration and heterogeneity of individual neurons in the network. This neural mass model enables studying the influence of changes in extracellular ionic conditions on whole brain dynamics in health and disease. The effect of external stimulus current and conductance-based coupling of neural masses are also analyzed.

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Hot and cold spots of pest-induced US urban tree death, 2020-2050 O_LIUrban trees are important nature-based solutions for future wellbeing and livability but are at high risk of mortality from insect pests. In the United States (US), 82% of the population live in urban settings and this number is growing, making urban tree mortality a matter of concern for most of its population. Until now, the magnitudes and spatial distributions of risks were unknown.
C_LIO_LIHere, we combine new models of street tree populations in [~]30,000 US communities, species-specific spread predictions for 57 invasive insect species, and estimates of tree death due to insect exposure for 48 host tree genera.
C_LIO_LIWe estimate that 1.4 million street trees will be killed by invasive insects from 2020 through 2050, costing an annualized average of US$ 30M. However, these estimates hide substantial variation: 23% of urban centers will experience 95% of all insect-induced mortality. Further, 90% of all mortality will be due to emerald ash borer (Agrilus planipennis, EAB), which is expected to kill virtually all ash trees (Fraxinus spp.) in >6000 communities.
C_LIO_LIWe define an EAB high-impact zone spanning 902,500km2, largely within the southern and central US, within which we predict the death of 98.8% of all ash trees. "Mortality hotspot cities" include Milwaukee, WI; Chicago, IL; and New York, NY.
C_LIO_LIWe identify Asian wood borers of maple and oak trees as the highest risk future invaders, where a new establishment could cost US$ 4.9B over 30 years.
C_LIO_LIPolicy implications: To plan effective mitigation, managers must know which tree species in which communities will be at the greatest risk, as well as the highest-risk insects. We provide the first country-wide, spatial forecast of urban tree mortality due to invasive insect pests. This framework identifies dominant pest insects and spatial impact hotspots, which can provide the basis for spatial prioritization of spread control efforts such as quarantines and biological control release sites. Our results highlight the need for EAB early-detection efforts as far from current infestations as Seattle, WA. Further, these findings produce a list of biotic and spatiotemporal risk factors for future high-impact US urban forest insect pests.
C_LI

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Identification and differentiation of Pseudomonas species in field samples using an rpoD amplicon sequencing methodology Species of the genus Pseudomonas are used for several biotechnological purposes, including plant biocontrol and bioremediation. To exploit the Pseudomonas genus in environmental, agricultural or industrial settings, the organisms must be profiled at species level as their bioactivity potential differs markedly between species. Standard 16S rRNA gene amplicon profiling does not allow for accurate species differentiation. Thus, the purpose of this study was to develop an amplicon-based high-resolution method targeting a 760 nt region of the rpoD gene enabling taxonomic differentiation of Pseudomonas species in soil samples. The method was benchmarked on a sixteen membered Pseudomonas species mock community. All 16 species were correctly and semi-quantitatively identified using rpoD gene amplicons, whereas 16S rRNA V3V4 amplicon sequencing only correctly identified one species. We analysed the Pseudomonas profile in thirteen soil samples in northern Zealand, Denmark, where samples were collected from grassland (3 samples) and agriculture soil (10 samples). Pseudomonas species represented up to 0.7% of the microbial community, of which each sampling site contained a unique Pseudomonas composition. Thirty culturable Pseudomonas strains were isolated from each grassland site and ten from each agriculture site and identified by Sanger sequencing of the rpoD gene. In all cases, the rpoD-amplicon approach identified more species than found by cultivation, including hard-to-culture non-fluorescent pseudomonads, as well as more than found by 16S rRNA V3V4 amplicon sequencing. Thus, rpoD profiling can be used for species profiling of Pseudomonas, and large scale prospecting of bioactive Pseudomonas may be guided by initial screening using this method.

ImportanceA high throughput sequence-based method for profiling of Pseudomonas species in soil microbiomes was developed and identified more species than 16S rRNA gene sequencing or cultivation. Pseudomonas species are used as biocontrol organisms and plant-growth promoting agents, and the method will allow tracing of specific species of Pseudomonas as well as enable screening of environmental samples for further isolation and exploitation.

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Phylogeny of the genus Dolichopus Latreille, 1796 and testing for phylogenetic signal in morphological characters and morphometric data Four groups of characters were used to evaluate phylogenetic relationships among 50 species of the genus Dolichopus: 810 nucleotide characters (for cytochrome oxidase I), 18 continuous characters generated from a geometric morphometric analysis of wing shape, 12 relative characters of the leg morphometry and 42 traditional morphological characters. Subsequently, the common database was used to construct a phylogenetic genus tree and to study the presence of a phylogenetic signal in each group. In this study it was shown that the following characters have a significant phylogenetic signal: the thickening of the costal vein at the insertion point with R1, the color of the fore coxa, the presence of male secondary sexual characters on the forelegs, the color of the femora, the presence of long cilia on the hind femora, and the color of the lower postocular cilia. Traits of male wing shape, such us the relative position of the apex of M1+2, the location of the posterior cossvein, the origin of the radial veins, and the position of the anal vein base, also showed a high phylogenetic signal. In addition, in males, the relative lengths of the first segment of the fore and mid tarsi showed a clear correlation with molecular data, which were interconnected with the presence of males leg modifications. In females, morphometric traits exhibited a less significant phylogenetic signal than in males, although, in most cases, the same traits evinced a high phylogenetic signal.

StatementsWe (corresponding authors) certify that we have participated sufficiently in the conception and design of this work and the analysis of the data (wherever applicable), as well as the writing of the manuscript, to take public responsibility for it. We believe the manuscript represents valid work. We have reviewed the final version of the manuscript and approve it for publication. Neither has the manuscript nor one with substantially similar content under my authorship been published nor is being considered for publication elsewhere, except as described in an attachment. Furthermore, we attest that we will produce the data upon which the manuscript is based for examination by the editors or their assignees, if requested.

The present paper has not been submitted to another journal. All co-authors are aware of the present submission. All co-authors agree to the publication. The work was funded by RFBR and NSFC according to the research project No 20-54-53005.

sentences: Impact of Type II LRRK2 inhibitors on signalling and mitophagy Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinsons disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

sentences: Microstructural variation in the human striatum using non-negative matrix factorization The striatum is a major subcortical connection hub that has been heavily implicated in a wide array of motor and cognitive functions. Here, we developed a normative multimodal, data-driven microstructural parcellation of the striatum using multiple magnetic resonance imaging-based metrics (mean diffusivity, fractional anisotropy, and the ratio between T1- and T2-weighted structural) from the Human Connectome Project Young Adult dataset (n=329 unrelated participants, age range: 22-35, F/M: 185/144). We further explored the biological and functional relationships of this parcellation by relating our findings to motor and cognitive performance in tasks known to involve the striatum as well as demographics. We identified 5 spatially distinct striatal components, for each hemisphere. We also show the gain in component stability when using multimodal versus unimodal metrics. Our findings suggest distinct microstructural patterns in the human striatum that are largely symmetric and that relate mostly to age and sex. Our work also highlights the putative functional relevance of these striatal components to different designations based on a Neurosynth meta-analysis.

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Fertilizer reduction with bio-organic fertilizer to regulate the root soil microbial community structure to improve Baby Chinese cabbage yield Using high-throughput sequencing, this study aimed to explore the response of soil microbial community and Baby Chinese cabbage yield to the reduction of chemical fertilizers combined with bio-organic fertilizer in the Gansu plateau, China. Our experiments consisted of conventional fertilizer (CK), 30% chemical fertilizer reduction + 6,000 kg bio-organic fertilizer (T1), 30% chemical fertilizer reduction + 9,000 kg bio-organic fertilizer (T2), 40% chemical fertilizer reduction + 6,000 kg bio-organic fertilizer (T3), and 40% chemical fertilizer reduction + 9,000 kg bio-organic fertilizer (T4). Compared with CK, soil microbial diversity and richness were higher for all treatments with added bio-organic fertilizer. PCoA showed that the bacterial and fungal communities in T2 and T4 were similar to each other. Redundancy and Spearmans correlation analyses of microbial communities and soil physicochemical properties revealed that reductions in chemical fertilizer rate combined with bio-organic fertilizer had a stronger impact on the fungal than the bacterial community. They also increased the relative abundance of the dominant bacterial and fungal phyla. Baby Chinese cabbage yield was relatively higher under the combined bio-organic fertilizer plus reduced chemical fertilizer rate with T2 showing the highest yield. Therefore, this approach is feasible for sustainable agricultural, cost-effective and profitable crop production.

ImportanceO_LIBio-organic + moderately reduced chemical fertilizer raised Chinese cabbage yield
C_LIO_LIBio-organic + chemical fertilizer was more efficacious than either one alone
C_LIO_LIPresence of bio-organic fertilizer enhanced overall rhizosphere physicochemistry
C_LIO_LIBio-organic fertilizer improved beneficial bacterial & fungal abundance & diversity
C_LIO_LIFertilizer combination sustainably & cost-effectively improves crop & soil quality
C_LI

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Three models of vaccination strategies against cryptococcosis in immunocompromised hosts using heat-killed Cryptococcus neoformans Δsgl1 Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans {Delta}sgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4+ T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans {Delta}sgl1-vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans {Delta}sgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans {Delta}sgl1 subsequent to WT challenge significantly improve the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans {Delta}sgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK {Delta}sgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans {Delta}gcs1. Remarkably, we show that administration of HK {Delta}sgl1 prevents mice from reactivating {Delta}gcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4+ T cells. Our results suggest that HK {Delta}sgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency.

ImportanceCryptococcosis results in [~]180,000 global deaths per year in immunocompromised individuals. Current antifungal treatment options are potentially toxic, lacking in areas of need, and exhibit limited efficacy. In addition to these lackluster therapeutic options, no fungal vaccines are currently available for clinical use. Due to the increasing rate of immunocompromised individuals, there is a dire need for the development of improved antifungal therapeutics. Presently, we have demonstrated the high efficacy of a clinically relevant heat-killed mutant strain of Cryptococcus neoformans in inducing advantageous host protection in three models of vaccination against cryptococcosis during immunodeficiencies most associated with this disease.

sentences: Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

sentences: Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution Live imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLEs nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

sentences: Search behavior of individual foragers involves neurotransmitter systems characteristic for social scouting In honey bees search behavior occurs as social and solitary behavior. In the context of foraging, searching for food sources is performed by behavioral specialized foragers, the scouts. When the scouts have found a new food source, they recruit other foragers (recruits). These recruits never search for a new food source on their own. However, when the food source is experimentally removed, they start searching for that food source. Our study provides a detailed description of this solitary search behavior and the variation of this behavior among individual foragers. Furthermore, mass spectrometric measurement showed that the initiation and performance of this solitary search behavior is associated with changes in glutamate, GABA, histamine, aspartate and the catecholaminergic system in the optic lobes and central brain area. These findings strikingly correspond with the results of an earlier study that showed that scouts and recruits differ in the expression of glutamate and GABA receptors. Together, the results of both studies provide first clear support for the hypothesis that behavioral specialization in honey bees is based on adjusting modulatory systems involved in solitary behavior to increase the probability or frequency of that behavior.

sentences: A population-level invasion by transposable elements triggers genome expansion in a fungal pathogen AO_SCPLOWBSTRACTC_SCPLOWGenome evolution is driven by the activity of transposable elements (TEs). The spread of TEs can have deleterious effects including the destabilization of genome integrity and expansions. However, the precise triggers of genome expansions remain poorly understood because genome size evolution is typically investigated only among deeply divergent lineages. Here, we use a large population genomics dataset of 284 individuals from populations across the globe of Zymoseptoria tritici, a major fungal wheat pathogen. We built a robust map of genome-wide TE insertions and deletions to track a total of 2,456 polymorphic loci within the species. We show that purifying selection substantially depressed TE frequencies in most populations but some rare TEs have recently risen in frequency and likely confer benefits. We found that specific TE families have undergone a substantial genome-wide expansion from the pathogens center of origin to more recently founded populations. The most dramatic increase in TE insertions occurred between a pair of North American populations collected in the same field at an interval of 25 years. We find that both genome-wide counts of TE insertions and genome size have increased with colonization bottlenecks. Hence, the demographic history likely played a major role in shaping genome evolution within the species. We show that both the activation of specific TEs and relaxed purifying selection underpin this incipient expansion of the genome. Our study establishes a model to recapitulate TE-driven genome evolution over deeper evolutionary timescales.

sentences: High-Speed Imaging Reveals the Bimodal Nature of Dense Core Vesicle Exocytosis and Jet Flow through the Fusion Pore During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Osmotic forces contribute to exocytosis but release through the pore is thought to occur by diffusion. Heterogeneity of fusion pore behavior has also suggested stochastic variation in a common exocytic mechanism, implying a lack of biological control. Imaging at millisecond resolution to observe the first events in exocytosis, we find that fusion pore duration is bimodal rather than stochastic. Loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. In addition, dual imaging shows a delay in the entry of external dye relative to release that indicates discharge at high velocity rather than strictly by diffusion.

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PpRPK2 modulates auxin homeostasis and transport to specify stem cell identity and plant shape in the moss Physcomitrella Plant shape is determined by the activity of stem cells in the growing tips, and evolutionary changes in shape are linked to changes in stem cell function. The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, acting via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL-like (WOX) proteins in bryophytes, raising questions about the evolution of stem cell function and the role of the CLAVATA pathway.

Here we show that the moss (Physcomitrella) CLAVATA pathway affects stem cell activity and overall plant shape by modulating hormone homeostasis. CLAVATA pathway components are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching patterns and plant spread. The PpRPK2 receptor-like kinase plays the major role and is expressed more strongly than other receptor-encoding genes. Pprpk2 mutants have abnormal responses to cytokinin, and auxin transport inhibition and show reduced PIN auxin transporter expression.

We propose a model whereby PpRPK2 modulates PIN activity to determine stem cell identity and overall plant form in Physcomitrella. Our data indicate that CLAVATA-mediated auxin homeostasis is a fundamental property of plant stem cell function likely exhibited by the last shared common ancestor of land plants.

sentences: Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for anti-malarial intervention deployment. Insecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes, including some commonly implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around Lake Victoria, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of the Cyp6aa1 gene are common in An. gambiae across Africa and, given the enzymes metabolic activity, are likely to be a useful diagnostic for high levels of pyrethroid resistance.

sentences: Comparative infection and pathogenesis of SARS-CoV-2 Omicron and Delta variants in aged and young Syrian hamsters Coronavirus disease 2019 continues to batter the world with the unceasing introduction of new variants of the causative virus, SARS-CoV-2. In order to understand differences in disease caused by variants of concern and to develop variant-specific vaccines, suitable small animal models are required that mimic disease progression in humans at various stages of life. In this study, we compared the dynamics of infection with two SARS-CoV-2 variants of concern (Delta and Omicron) in aged (>1 year 3 months old) and young (<5 weeks old) Syrian hamsters (Mesocricetus auratus). We show that no weight loss occurred in Omicron infected groups regardless of age, while infection with the Delta variant caused weight loss of up to 10% by day 7 post-infection with slower and incomplete recovery in the aged group. Omicron replicated to similar levels as Delta in the lungs, trachea and nasal turbinates, with no significant differences in the tissue viral loads of aged versus young animals for either variant. In contrast to rare necrosis observed in Omicron-infected animals regardless of age, severe necrosis was observed in the olfactory epithelium in Delta-infected animals. Omicron infection also resulted in mild pulmonary disease in both young and aged animals compared to the moderate acute necrotizing bronchointerstitial pneumonia seen in Delta-infected animals. These results suggest that Omicron infection results in an attenuated clinical disease outlook in Syrian hamsters compared to infection with the Delta variant irrespective of age.

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The non-specific Lipid Transfer Protein (nsLTP) is involved at early and late stages of symbiosis between Alnus glutinosa and Frankia alni. Alnus glutinosa response to Frankia alni is driven by several sequential physiological modifications that include calcium spiking, root hair deformation, penetration, induction of primordium, formation and growth of nodule. Here, we have conducted a transcriptomic study to analyse plant responses to Frankia alni at early stages of symbiosis establishment.

Forty-two genes were significantly activated by either with a Frankia culture supernatant or with living cells separated from the roots by a dialysis membrane permitted to identify plant genes which expression changes upon early contact with Frankia. Most of these genes encode biological processes, including oxidative stress and response to stimuli. The most upregulated gene is the non-specific lipid transfer protein (nsLTP) encoding gene with a fold change of 141. Physiological experiments showed that nsLTP increases Frankia nitrogen fixation at sub-lethal concentration. Immunohistochemistry experiments conducted at an early infection stage indicated that nsLTP protein is localized at the deformed root hair region after Frankia inoculation and later in nodules, precisely around bacterial vesicles. Taken together, these results suggest that nsLTP acts at early and late stages of symbiosis, probably by increasing nitrogen uptake by Frankia.

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SOPHIE: viral outbreak investigation and transmission history reconstruction in a joint phylogenetic and network theory framework Genomic epidemiology is now widely used for viral outbreak investigations. Still, this methodology faces many challenges. First, few methods account for intra-host viral diversity. Second, maximum parsimony principle continues to be employed, even though maximum likelihood or Bayesian models are usually more consistent. Third, many methods utilize case-specific data, such as sampling times or infection exposure intervals. This impedes study of persistent infections in vulnerable groups, where such information has a limited use. Finally, most methods implicitly assume that transmission events are independent, while common source outbreaks violate this assumption.

We propose a maximum likelihood framework SOPHIE (SOcial and PHilogenetic Investigation of Epidemics) based on integration of phylogenetic and random graph models. It infers transmission networks from viral phylogenies and expected properties of inter-host social networks modelled as random graphs with given expected degree distributions. SOPHIE is scalable, accounts for intra-host diversity and accurately infers transmissions without case-specific epidemiological data. SOPHIE code is freely available at https://github.com/compbel/SOPHIE/

sentences: Uncertainty in cell fate decision making: Lessons from potential landscapes of bifurcation systems Cell fate decision making is known to be a complex process and is still far from being understood. The intrinsic complexity, but also features such as molecular noise represent challenges for modelling these systems. Waddingtons epigenetic landscape has become the overriding metaphor for developmental processes: it both serves as pictorial representation, and can be related to mathematical models. In this work we investigate how the landscape is affected by noise in the underlying system. Specifically, we focus on those systems where minor changes in the parameters cause major changes in the stability properties of the system, especially bifurcations. We analyse and quantify the changes in the landscapes shape as the effects of noise increase. We find ample evidence for intricate interplay between noise and dynamics which can lead to qualitative change in a systems dynamics and hence the corresponding landscape. In particular, we find that the effects can be most pronounced in the vicinity of the bifurcation point of the underlying deterministic dynamical systems, which would correspond to the cell fate decision event in cellular differentiation processes.

sentences: Adaptive introgression and de novo mutations increase access to novel fitness peaks on the fitness landscape during a vertebrate adaptive radiation Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation.

sentences: Cross-modal perceptual enhancement of unisensory targets is uni-directional and does not affect temporal expectations Previous studies demonstrated that redundant target stimuli can enhance performance due to multisensory interplay and interactively facilitate performance enhancements due to temporal expectations (TE; faster and accurate reactions to temporally expected targets). Here we tested whether other types of multisensory interactions - i.e. interactions evoked by temporally flanking irrelevant stimuli - can result in similar performance patterns and boost not only unisensory target perception (multi-vs. unisensory sequences) but also unisensory temporal expectations (expected vs. unexpected). To test our hypothesis, we presented sequences of 12 stimuli (10 Hz) which either consisted of auditory (A), visual (V) or alternating auditory-visual stimuli (e.g. A-V-A-V-...) with either auditory (AV(A)) or visual (AV(V)) targets. Participants had to discriminate target frequency which was unpredictable by temporal regularities (expected vs. unexpected target positions) and by stimulation sequence (A, V, AV(A), AV(V)). Moreover, we ran two experiments in which we presented redundant multisensory targets and manipulated the speed of the stimulation sequence (10 vs. 15 Hz stimulus trains) to control whether the results of Experiment 1 depended on sequence speed. Performance for unisensory targets was affected by temporally flanking distractors, with multisensory interactions selectively improving unisensory visual target perception. Yet, only redundant multisensory targets reliably affected TEs. Together, these results indicate that cross-modal facilitation of unisensory target perception in fast stimulus streams is uni-directional, but also differs from multisensory interactions evoked by redundant targets; more specifically, it appears to be context-dependent (task, design etc.) whether unisensory stimulation (unlike redundant target stimulation) allows for the generation of temporal expectations.

sentences: Tactile suppression stems from sensation-specific sensorimotor predictions The ability to sample sensory information with our hands is crucial for smooth and efficient interactions with the world. Despite this important role of touch, tactile sensations on a moving hand are perceived weaker than when presented on the same but stationary hand.1-3 This phenomenon of tactile suppression has been explained by predictive mechanisms, such as forward models, that estimate future sensory states of the body on the basis of the motor command and suppress the associated predicted sensory feedback.4 The origins of tactile suppression have sparked a lot of debate, with contemporary accounts claiming that suppression is independent of predictive mechanisms and is instead akin to unspecific gating.5 Here, we target this debate and provide evidence for sensation-specific tactile suppression due to sensorimotor predictions. Participants stroked with their finger over textured surfaces that caused predictable vibrotactile feedback signals on that finger. Shortly before touching the texture, we applied external vibrotactile probes on the moving finger that either matched or mismatched the frequency generated by the stroking movement. We found stronger suppression of the probes that matched the predicted sensory feedback. These results show that tactile suppression is not limited to unspecific gating but is specifically tuned to the predicted sensory states of a movement.

sentences: An information theoretic method to resolve millisecond-scale spike timing precision in a comprehensive motor program Sensory inputs in nervous systems are often encoded at the millisecond scale in a temporally precise code. There is now a growing appreciation for the prevalence of precise timing encoding in motor systems. Animals from moths to birds control motor outputs using precise spike timing, but we largely do not know at what scale timing matters in these circuits due to the difficulty of recording a complete set of spike-resolved motor signals and relatively few methods for assessing spike timing precision. We introduce a method to estimate spike timing precision in motor circuits using continuous MI estimation at increasing levels of added uniform noise. This method can assess spike timing precision at fine scales for encoding rich motor output variation. We demonstrate the advantages of this approach compared to a previously established discrete information theoretic method of assessing spike timing precision. We use this method to analyze a data set of simultaneous turning (yaw) torque output and EMG recordings from the 10 primary muscles of Manduca sexta as tethered moths visually tracked a robotic flower moving with a 1 Hz sinusoidal trajectory. We know that all 10 muscles in this motor program encode the majority of information about yaw torque in spike timings, but we do not know whether individual muscles receive information encoded at different levels of precision. Using the continuous MI method, we demonstrate that the scale of temporal precision in all motor units in this insect flight circuit is at the sub-millisecond or millisecond-scale, with variation in precision scale present between muscle types. This method can be applied broadly to estimate spike timing precision in sensory and motor circuits in both invertebrates and vertebrates.

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Staphylococcus aureus overcomes anaerobe-derived short-chain fatty acid stress via FadX and the CodY regulon Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coA-transferase, was significantly increased, leading to our hypothesis that short chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was frequently observed. Whole genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its co-colonizing microbiota and metabolites they exchange, and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth.

ImportanceS. aureus is an important CRS pathogen, yet is found in the upper airways of 30-50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, F. nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress, and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions, and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

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Degradation of Photoreceptor Outer Segments by the Retinal Pigment Epithelium Requires Pigment Epithelium-derived Factor Receptor (PEDF-R) PurposeTo examine the contribution of PEDF-R to the phagocytosis process. Previously, we identified PEDF-R, the protein encoded by the PNPLA2 gene, as a phospholipase A2 in the retinal pigment epithelium (RPE). During phagocytosis, RPE cells ingest abundant phospholipids and protein in the form of photoreceptor outer segment (POS) tips, which are then hydrolyzed. The role of PEDF-R in RPE phagocytosis is not known.

MethodsMice in which PNPLA2 was conditionally knocked out in the RPE were generated (cKO). Mouse RPE/choroid explants were cultured. Human ARPE-19 cells were transfected with siPNPLA2 silencing duplexes. POS were isolated from bovine retinas. The phospholipase A2 inhibitor bromoenol lactone was used. Transmission electron microscopy, immunofluorescence, lipid labeling, pulse-chase experiments, western blots, and free fatty acid and {beta}-hydroxybutyrate assays were performed.

ResultsThe RPE of the cKO mice accumulated lipids as well as more abundant and larger rhodopsin particles compared to littermate controls. Upon POS exposure, RPE explants from cKO mice released less {beta}-hydroxybutyrate compared to controls. After POS ingestion during phagocytosis, rhodopsin degradation was stalled both in cells treated with bromoenol lactone and in PNPLA2-knocked-down cells relative to their corresponding controls. Phospholipase A2 inhibition lowered {beta}-hydroxybutyrate release from phagocytic RPE cells. PNPLA2 knock down also resulted in a decline in fatty acids and {beta}-hydroxybutyrate release from phagocytic RPE cells.

ConclusionsPEDF-R downregulation delayed POS digestion during phagocytosis. The findings imply that efficiency of RPE phagocytosis depends on PEDF-R, thus identifying a novel contribution of this protein to POS degradation in the RPE.

sentences: Improved sensitivity and resolution of ATAC-seq differential DNA accessibility analysis Eukaryotic genomes are packaged into chromatin, and the extent of its compaction must be modulated to allow several biological processes such as gene transcription. The regulatory elements of expressed genes are typically in relatively accessible chromatin, and several studies have revealed a reliable correlation between the abundance of mRNA transcripts and the degree of DNA accessibility at the regulatory elements of their coding genes. In consequence, the genome-wide profiling of DNA accessibility by methods such as ATAC-seq can help in the study of gene regulatory networks by serving as a proxy for gene expression and by helping identify important gene cis-regulatory elements and the trans-acting factors that bind them. The predominant approach used to identify differentially accessible genomic loci from ATAC-seq data obtained in two conditions of interest is comparable to that employed in RNA-seq gene expression profiling studies: accessible regions are identified through peak calling and treated like "genes", then sequenced DNA fragments (originating from two neighboring transposase insertion events) that overlap them are counted and subjected to abundance modeling, which then allows to identify those that have a significant difference between the two conditions. We reasoned that this approach could be improved in terms of sensitivity and resolution by introducing two changes: bypassing peak calling, using instead a genome-wide sliding window quantification approach, and counting transposase insertion sites, instead of fragments originating from two neighboring insertion sites. We present the development of this approach, which we term "widaR", for Window- and Insertion-based Differential Accessibility in R, using a murine skeletal myoblast differentiation dataset. Reproducible R code is provided.

sentences: Contribution of quasi-fibrillar superstructures in peroxide quenching by collagen peptides derived from fish processing by-products and their application as natural food additives This study attempts to identify the significant role played by the secondary structure of collagen-derived peptides that are involved in lipid peroxide quenching in food products. Collagen was extracted from the skin of Perch and swim bladder of Rohu at 45-78% efficiency. It was identified as type-I based on a high molecular weight (110kDa) and its ion-exchange elution profile. The collagen samples were enzymatically hydrolyzed and collagen hydrolysate (CH) was extracted with an efficiency of 0.67-0.74g/g of collagen. The CH samples displayed a molecular weight in the range of 8.2-9.7kDa and exhibited a higher abundance of charges resulting in higher solubility. The structural studies revealed that the CH peptides existed in polyproline-II helix and formed a mimic-triple helix in a wide range of pH. In neutral and alkaline pH, the mimic helices joined to form a hierarchical quasi-fibrillar network that was smaller than collagen fibrils but also more dynamic. The CH exhibited >95% degradation in 15h through simulated digestion. The CH were able to decrease peroxide formation by 84.5-98.9% in commercially available cod liver and almond oil and increased the shelf life of soya bean oil by a factor of 5 after 6 months of storage. The addition of CH to cultured cells quenched peroxide ions generated in situ and decreased stressor activity by a factor of 12. The reason behind the high efficacy of CH was deciphered to be the proximal charge stabilization by the quasi-fibrillar network, which allowed efficient peroxide quenching and long-term stability.

sentences: Intralocus sexual conflict can maintain alternative reproductive tactics Alternative reproductive tactics (ARTs) are ubiquitous throughout the animal kingdom and widely regarded as an outcome of high variance in reproductive success. Proximate mechanisms underlying ARTs include genetically based polymorphisms, environmentally induced polymorphisms, and those mediated by a combination of genetic and environmental factors. However, few ultimate mechanisms have been proposed to explain the maintenance of ARTs over time, the most important of which have been disruptive and negative frequency-dependent selection. Here we explore the role that intralocus sexual conflict may play in the maintenance of sex-specific ARTs. We use a genetically explicit individual-based model in which body size influences both female fecundity and male tactic through a shared genetic architecture. By modeling ART maintenance under varying selection regimes and levels of sex-specific gene expression, we explore the conditions under which intralocus sexual conflict can maintain a hypothetical ART defined by larger (alpha) and smaller (beta) tactics. Our models consistently revealed that sexual conflict can result in the persistence of a sex-specific polymorphism over hundreds of generations, even in the absence of negative frequency-dependent selection. ARTs were maintained through correlated selection when one male ART has lower fitness but produces daughters with higher fitness. These results highlight the importance of understanding selection on both sexes when attempting to explain the maintenance of ARTs. Our results are consistent with a growing literature documenting genetic correlations between male ARTs and female fitness, suggesting that the maintenance of sex-specific ARTs through intralocus sexual conflict may be common and widespread in nature.

sentences: Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, they accumulate and contribute to persistent inflammation after severe injury. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making them vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.

sentences: Rapid and parallel adaptive mutations in spike S1 drive clade success in SARS-CoV-2 Given the importance of variant SARS-CoV-2 viruses with altered receptor-binding or antigenic phenotypes, we sought to quantify the degree to which adaptive evolution is driving accumulation of mutations in the SARS-CoV-2 genome. Here we assessed adaptive evolution across genes in the SARS-CoV-2 genome by correlating clade growth with mutation accumulation as well as by comparing rates of nonsynonymous to synonymous divergence, clustering of mutations across the SARS-CoV-2 phylogeny and degree of convergent evolution of individual mutations. We find that spike S1 is the focus of adaptive evolution, but also identify positively-selected mutations in other genes that are sculpting the evolutionary trajectory of SARS-CoV-2. Adaptive changes in S1 accumulated rapidly, resulting in a remarkably high ratio of nonsynonymous to synonymous divergence that is 2.5X greater than that observed in HA1 at the beginning of the 2009 H1N1 pandemic.

sentences: The 14-3-3 Proteins Bmh1 and Bmh2 Are Key Regulators of Meiotic Commitment Induction of meiosis requires exogenous signals that activate internal gene regulatory networks. Meiotic commitment ensures the irreversible continuation of meiosis, even upon withdrawal of the meiosis inducing signals. Budding yeast cells have a unique property in that cells enter meiosis when starved, but if given nutrient-rich medium prior to the commitment point, they exit meiosis and enter mitosis. After the meiotic commitment point in prometaphase I, cells remain in meiosis even with addition of nutrients. Despite the importance of meiotic commitment in ensuring the production of gametes, only a few genes involved in the commitment process are known. We performed a genome-scale screen in budding yeast and discovered new regulators of meiotic commitment including Bcy1, which is involved in nutrient sensing, the meiosis-specific kinase Ime2, Polo kinase Cdc5, and the 14-3-3 proteins Bmh1 and Bmh2. Importantly, we found that Bmh1 and Bmh2 are involved in multiple processes throughout meiosis including the maintenance of the middle meiosis transcription factor Ndt80, activation of Cdc5, and interaction with an RNA-binding protein Pes4, which is important for regulating the timing of translation of several mRNAs in meiosis II. This study identifies a meiotic commitment regulatory network with the 14-3-3 proteins functioning as central regulators.

sentences: Metaviromes reveal the dynamics of Pseudomonas host-specific phages cultured and uncultured by plaque assay Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or the ones with highest ability to plaque on solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in envi-ronmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage communitys shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media corresponds to the metaviromic data. In this study, we observed a significant shift (p = 0.015) of the 47 identified putative Pseudomonas phages with a minimum 2-fold change above 0 in read abundance when adding a Pseudomonas syringae DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the Pseudomonas strain, was highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlights how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.

sentences: Structural Basis of Substrate Recognition by the Mitochondrial ADP/ATP Transporter Specific import of ADP and export of ATP by ADP/ATP carrier (AAC) across the inner mitochondrial membrane are crucial for sustainable energy supply in all eukaryotes. However, mechanism for highly specific substrate recognition in the dynamic transport process remains largely elusive. Here, unguided MD simulations of 22 microseconds in total reveal that AAC in ground c-state uses the second basic patch (K91K95R187), tyrosine ladder (Y186Y190Y194), F191 and N115 in the upper region of the cavity to specifically recognize ADP and confer selectivity for ADP over ATP. Mutations of these residues in yeast AAC2 reduce ADP transport across the L. lactis membrane and induce defects in OXPHOS and ATP production in yeast. Sequence analyses also suggest that AAC and other adenine nucleotide transporters use the upper region of the cavity, rather than the central binding site to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC, and together with central binding site support early biochemical findings about existence of two substrate binding sites. Our results imply that using different sites for substrate recognition and conformational transition could be a smart strategy for transporters to cope with substrate recognition problem in the highly dynamic transport process.

sentences: Two-photon microscopy in vivo reveals brain vessel type-specific loss of glycocalyx caused by apoM/S1P signaling impairment. Increases in adsorptive mediated transcytosis (AMT) at the blood-brain barrier (BBB) are linked to many brain disorders. In a healthy brain, AMT is suppressed by sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) signaling. Low levels of S1P lead to a rise in AMT, but the mechanisms are incompletely understood. Here, we explored whether the rises in AMT are caused by the loss of the endothelial glycocalyx (gcx). We used two-photon microscopy in mice with low S1P plasma levels (Apom-/-) and developed a novel photobleaching approach to measure gcx in vivo at distinct classes of cerebral microvessels, i.e., arterioles, capillaries and venules. We show that S1P signaling impairment reduced gcx in arterioles but not in other vessel segments. The location of gcx loss corresponded to the vascular topology of AMT increases. The S1PR1 agonist SEW2871 restores low levels of AMT in Apom-/- mice but did not restore the gcx within the same time window. We propose that while the gcx loss may contribute to AMT increase, restoring gcx is not necessary for AMT to return to normal. These data establish a new imaging method to study gcx in the living mouse brain, demonstrate zonation of gcx in cerebral microvessels, and suggest differences in vascular susceptibility to gcx loss in disease states.

sentences: Psychedelics and schizophrenia: Distinct alterations to Bayesian inference Schizophrenia and states induced by certain psychotomimetic drugs may share some physiological and phenomenological properties, but they differ in fundamental ways: one is a crippling chronic mental disease, while the others are temporary, pharmacologically-induced states presently being explored as treatments for mental illnesses. Building towards a deeper understanding of these different alterations of normal consciousness, here we compare the changes in neural dynamics induced by LSD and ketamine (in healthy volunteers) against those associated with schizophrenia, as observed in resting-state M/EEG recordings. While both conditions exhibit increased neural signal diversity, our findings reveal that this is accompanied by an increased transfer entropy from the front to the back of the brain in schizophrenia, versus an overall reduction under the two drugs. Furthermore, we show that these effects can be reproduced via different alterations of standard Bayesian inference applied on a computational model based on the predictive processing framework. In particular, the effects observed under the drugs are modelled as a reduction of the precision of the priors, while the effects of schizophrenia correspond to an increased precision of sensory information. These findings shed new light on the similarities and differences between schizophrenia and two psychotomimetic drug states, and have potential implications for the study of consciousness and future mental health treatments.

sentences: Molecular Dynamics Study on the Effects of Charged Amino Acid Distribution Under low pH Condition to the Unfolding of Hen Egg White Lysozyme and Formation of Beta Strands. Aggregation of unfolded or misfolded proteins into amyloid fibrils can cause various diseases in humans. However, the fibrils synthesized in vitro can be developed toward useful biomaterials under some physicochemical conditions. In this study, atomistic molecular dynamics simulations were performed to address the mechanism of beta-sheet formation of the unfolded hen egg white lysozyme (HEWL) under a high temperature and low pH. Simulations of the protonated HEWL at pH 2 and the non-protonated HEWL at pH 7 were performed at the highly elevated temperature of 450 K to accelerate the unfolding, followed by the 333 K temperature in some previous in vitro studies. The simulations showed that HEWL unfolded faster and refolded into structures with higher beta-strand contents at pH 2. The mechanism of beta-strand formation at the earlier stage of amyloidosis was addressed in terms of the radial distribution of amino acids, affected by the high protonation level at a low pH.

sentences: SIEVE: joint inference of single-nucleotide variants and cell phylogeny from single-cell DNA sequencing data Single-cell DNA sequencing (scDNA-seq) has enabled the identification of single nucleotide somatic variants and the reconstruction of cell phylogenies. However, statistical phylogenetic models for cell phylogeny reconstruction from raw sequencing data are still in their infancy. Here we present SIEVE (SIngle-cell EVolution Explorer), a statistical method for the joint inference of somatic variants and cell phylogeny under the finite-sites assumption from scDNA-seq reads. SIEVE leverages raw read counts for all nucleotides at candidate variant sites, and corrects the acquisition bias of branch lengths. In our simulations, SIEVE outperforms other methods both in phylogenetic accuracy and variant calling accuracy. We apply SIEVE to three scDNA-seq datasets, for colorectal (CRC) and triple-negative breast cancer (TNBC), one of them generated by us. On simulated data, SIEVE reliably infers homo-and heterozygous somatic variants. The analysis of real data uncovers that double mutant genotypes are rare in CRC but unexpectedly frequent in TNBC samples.

sentences: A conditional survival distribution-based method for censored data imputation: overcoming the hurdle in machine learning-based survival analysis Data analyses by machine learning (ML) algorithms are gaining popularity in biomedical research. When time-to-event data are of interest, censoring is common and needs to be properly addressed. Most ML methods cannot conveniently and appropriately take the censoring information into consideration, potentially leading to inaccurate or biased results. We aim to develop a general-purpose method for imputing censored survival data, facilitating downstream ML analysis. In this study, we propose a novel method of imputing the survival times for censored observations. The proposal is based on their conditional survival distributions (CondiS) derived from Kaplan-Meier estimators. CondiS can replace censored observations with their best approximations from the statistical model, allowing for direct application of ML methods. When covariates are available, we extend CondiS by incorporating the covariate information through ML modeling (CondiS-X), which further improves the accuracy of the imputed survival time. Compared with existing methods with similar purposes, the proposed methods achieved smaller prediction errors and higher concordance with the underlying true survival times in extensive simulation studies. We also demonstrated the usage and advantages of the proposed methods through two real-world cancer datasets. The major advantage of CondiS is that it allows for the direct application of standard ML techniques for analysis once the censored survival times are imputed. We present a user-friendly R package to implement our method, which is a useful tool for ML-based biomedical research in this era of big data.

sentences: STAG2 promotes the myelination transcriptional program in oligodendrocytes Cohesin folds chromosomes via DNA loop extrusion. Cohesin-mediated chromosome loops regulate transcription by shaping long-range enhancer-promoter interactions, among other mechanisms. Mutations of cohesin subunits and regulators cause human developmental diseases termed cohesinopathy. Vertebrate cohesin consists of SMC1, SMC3, RAD21, and either STAG1 or STAG2. To probe the physiological functions of cohesin, we created conditional knockout (cKO) mice with Stag2 deleted in the nervous system. Stag2 cKO mice exhibit growth retardation, neurological defects, and premature death, in part due to insufficient myelination of nerve fibers. Stag2 cKO oligodendrocytes exhibit delayed maturation and downregulation of myelination-related genes. Stag2 loss reduces promoter-anchored loops at downregulated genes in oligodendrocytes. Thus, STAG2-cohesin generates promoter-anchored loops at myelination-promoting genes to facilitate their transcription. Our study implicates defective myelination as a contributing factor to cohesinopathy and establishes oligodendrocytes as a relevant cell type to explore the mechanisms by which cohesin regulates transcription.

sentences: An intranasal vaccine durably protects against SARS-CoV-2 variants in mice SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy and the end of the COVID-19 pandemic. We recently reported the protective activity of a single-dose intranasally-administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose-response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351 and B.1.1.28 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.

sentences: Model-Based Meta-Analysis of Relapsing Mouse Model Studies from the Critical Path to Tuberculosis Drug Regimens Initiative Database Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease-related cause of death worldwide, necessitating the development of new and improved treatment regimens. Non-clinical evaluation of candidate drug combinations via the relapsing mouse model (RMM) is an important step in regimen development, through which candidate regimens that provide the greatest decrease in probability of relapse following treatment in mice may be identified for further development. Although RMM studies are a critical tool to evaluate regimen efficacy, making comprehensive "apples to apples" comparisons of regimen performance in the RMM has been a challenge, in large part due to the need to evaluate and adjust for variability across studies arising from differences in design and execution. To address this knowledge gap, we performed a model-based meta-analysis on data for 17 unique regimens obtained from a total of 1592 mice across 28 RMM studies. Specifically, a mixed-effects logistic regression model was developed that described the treatment duration-dependent probability of relapse for each regimen and identified relevant covariates contributing to inter-study variability. Using the model, covariate-normalized metrics of interest, namely treatment duration required to reach 50% and 10% relapse probability, were derived and used to compare relative regimen performance. Overall, the model-based meta-analysis approach presented herein enables cross-study comparison of efficacy in the RMM, and provides a framework whereby data from emerging studies may be analyzed in the context of historical data to aid in selecting candidate drug combinations for clinical evaluation as TB drug regimens.

sentences: Vestibular Agnosia is linked to abnormal functional brain networks Vestibular Agnosia - where peripheral vestibular activation triggers the usual reflex nystagmus response but with attenuated or no self-motion perception - is found in brain disease with disrupted cortical network functioning, e.g. traumatic brain injury (TBI) or neurodegeneration (Parkinsons Disease). Patients with acute focal hemispheric lesions (e.g. stroke) do not manifest vestibular agnosia. Thus brain network mapping techniques, e.g. resting state functional MRI (rsfMRI), are needed to interrogate functional brain networks mediating vestibular agnosia. Whole-brain rsfMRI was acquired from 39 prospectively recruited acute TBI patients with preserved peripheral vestibular function, along with self-motion perceptual thresholds during passive yaw rotations in the dark. Following quality-control checks, 25 patient scans were analyzed. TBI patients were classified as having vestibular agnosia (n = 11) or not (n = 14) via laboratory testing of self-motion perception. Using independent component analysis, we found altered functional connectivity in the right superior longitudinal fasciculus and left rostral prefrontal cortex in vestibular agnosia. Moreover, regions of interest analyses showed both inter-hemispheric and intra-hemispheric network disruption in vestibular agnosia. In conclusion, our results show that vestibular agnosia is mediated by bilateral anterior and posterior network dysfunction and reveal the distributed brain mechanisms mediating vestibular self-motion perception.

sentences: A waveform-independent measure of recurrent neural activity Rhythmic neural activity, so called oscillations, play a key role for neural information transmission, processing and storage. Neural oscillations in distinct frequency bands are central to physiological brain function and alterations thereof have been associated with several neurological and psychiatric disorders. The most common methods to analyse neural oscillations, e.g. short-term Fourier transform or wavelet analysis, assume that measured neural activity is composed of a series of symmetric prototypical waveforms, e.g. sinusoids. However, usually the models generating the signal, including waveform shapes of experimentally measured neural activity are unknown. Decomposing asymmetric waveforms of nonlinear origin using these classic methods may result in spurious harmonics visible in the estimated frequency spectra. Here, we introduce a new method for capturing rhythmic brain activity based on recurrences of similar states in phase-space. This method allows for a time-resolved estimation of amplitude fluctuations of recurrent activity irrespective of or specific to waveform-shapes. The algorithm is derived from the well-established field of recurrence analysis, which has rarely been adopted in neuroscience. In this paper, we show its advantages and limitations in comparison to short-time Fourier transform and wavelet convolution using periodic signals of different waveform shapes. Further, we demonstrate its application using experimental data, i.e. intracranial electrophysiological recordings from the human motor cortex of one epilepsy patient.

sentences: The extracellular loops of OmpA control the slow rate of in vitro folding Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test the effect of three different loop types on outer membrane protein A (OmpA) folding. We designed twelve 5-residue loops and experimentally tested the effect of replacing the long loops of outer membrane protein OmpA with the designed loops. Our studies succeeded in creating the smallest known outer membrane barrel. We find that significant changes in OmpA loops do not have a strong overall effect on OmpA folding. However, when decomposing folding into a fast rate and a slow rate we find that changes in loops strongly affect the slow rate of OmpA folding. Extracellular loop types with higher levels of hydrogen bonds had more instances of increasing the slow folding rate and extracellular loop types with low levels of hydrogen bonds had more instances of decreasing the slow folding rate. Having the slow rate affected by loop composition is consistent with the slow rate being associated with the insertion step of outer membrane protein folding.

sentences: RNASeq analysis of a Pax3-expressing myoblast clone in-vitro and effect of culture surface stiffness on differentiation. Skeletal muscle satellite cells cultured on soft surfaces (12kPa) show improved differentiation than cells cultured on stiff surfaces (approximately 100kPa). To better understand the reasons for this, we performed an RNA-Seq analysis for a single satellite cell clone (C1F) derived from the H2kb-tsA58 immortomouse, which differentiates into myotubes under tightly regulated conditions (withdrawal of {gamma}-interferon, 37{degrees}C). As expected, the largest change in overall gene expression occurred at day 1, as cells switch from proliferation to differentiation. Surprisingly, further analysis showed that proliferating C1F cells express Pax3 and not Pax7, confirmed by immunostaining, yet their subsequent differentiation into myotubes is normal, and enhanced on softer surfaces, as evidenced by significantly higher expression levels of myogenic regulatory factors, sarcomeric genes, enhanced fusion and improved myofibrillogenesis. Levels of RNA encoding extracellular matrix structural constituents and related genes were consistently upregulated on hard surfaces, suggesting that a consequence of differentiating satellite cells on hard surfaces is that they attempt to manipulate their niche prior to differentiating. This comprehensive RNASeq dataset will be a useful resource for understanding Pax3 expressing cells.

sentences: Light-dependent modulation of protein localization and function in living bacteria cells Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a proteins subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.

sentences: Improving localization precision via restricting biomolecule confined stochastic motion in SMLM Single-molecule localization microscopy (SMLM) boosts its applications when combined with the studies of cells, in which nanometer-sized biomolecules are irresolvable due to diffraction limit unless being subjected to SMLM. Although being well immobilized, given the nanometer sizes of biological molecules, they are still capable of movement stochastically around their immobilized sites. The influence of such motion on image quality and possible improvements have not yet been systematically investigated. Here, we accessed the biomolecule stochastic motion in SMLM by calculating the displacements between different localizations from the same molecule in single-molecule samples of Alexa Fluor-647-conjugated oligonucleotides. We found that, for most molecules, localization displacements at random frame intervals are remarkably larger than those between temporally neighbouring frames despite of drift correction, showing that biomolecule stochastic motion is involved in SMLM. Furthermore, the localization displacements were observed to increase with frame intervals and then saturate, suggesting biomolecule stochastic motion is confined within a finite area. Moreover, we showed that the localization precision is deteriorated by enlarging molecule sizes and improved by sample post-fixation. This study reveals confined stochastic motion of biomolecules increase localization uncertainty in SMLM, and improved localization precision can be achieved via restricting biomolecule stochastic motion.

sentences: The Size of the Stag Determines the Level of Cooperation In the last 12,000 years, human societies have scaled up from small bands to large states of millions and even billions. Many modern societies and even groups of societies cooperate on large-scale projects with relatively low levels of conflict, but the scale and intensity of cooperation varies dramatically between societies. Here we attempt to formalize dynamics that may be driving this rapid increase in cooperation and the differences we see between societies. Our model extends an N-person stag hunt to include population growth dynamics, "stags" with different sized payoffs, and competition for these stags. An increasing number of cooperators is required to access larger stags. The payoff from these stags in turn increases carrying capacity, which increases competition for the stag. As population size increases, new cooperative thresholds are attainable, and as population size shrinks, previously attainable thresholds fall out of reach. Among other predictions, we show that when a new threshold is accessible to a population, the level of cooperation will increase to reach this threshold. However, when the next threshold is out of reach, cooperation decreases as individuals refrain from costly cooperation, preferring a smaller stag. This model offers a framework for understanding the rapid increase in the scale of human cooperation and decline of violence, differences between societies, and challenges to future cooperation.

sentences: Accurate de novo identification of biosynthetic gene clusters with GECCO Biosynthetic gene clusters (BGCs) are enticing targets for (meta)genomic mining efforts, as they may encode novel, specialized metabolites with potential uses in medicine and biotechnology. Here, we describe GECCO (GEne Cluster prediction with COnditional random fields; https://gecco.embl.de), a high-precision, scalable method for identifying novel BGCs in (meta)genomic data using conditional random fields (CRFs). Based on an extensive evaluation of de novo BGC prediction, we found GECCO to be more accurate and over 3x faster than a state-of-the-art deep learning approach. When applied to over 12,000 genomes, GECCO identified nearly twice as many BGCs compared to a rule-based approach, while achieving higher accuracy than other machine learning approaches. Introspection of the GECCO CRF revealed that its predictions rely on protein domains with both known and novel associations to secondary metabolism. The method developed here represents a scalable, interpretable machine learning approach, which can identify BGCs de novo with high precision.

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Topography of corticopontine projections is controlled by postmitotic expression of the area-mapping gene Nr2f1 Axonal projections from layer V neurons of distinct neocortical areas are topographically organized into discrete clusters within the pontine nuclei during the establishment of voluntary movements. However, the molecular determinants controlling corticopontine connectivity are insufficiently understood. Here, we show that an intrinsic cortical genetic program driven by Nr2f1 graded expression is directly implicated in the organization of corticopontine topographic mapping. Transgenic mice lacking cortical expression of Nr2f1 and exhibiting areal organization defects were used as model systems to investigate the arrangement of corticopontine projections. Combining three-dimensional digital brain atlas tools, Cre-dependent mouse lines, and axonal tracing, we show that Nr2f1 expression in postmitotic neurons spatially and temporally controls somatosensory topographic projections, whereas expression in progenitor cells influences the ratio between corticopontine and corticospinal fibres passing the pontine nuclei. We conclude that cortical gradients of area patterning genes are directly implicated in the establishment of a topographic somatotopic mapping from the cortex onto pontine nuclei.

Summary statementCortical gradient expression of the area patterning gene Nr2f1 spatially and temporally controls corticopontine topographic connectivity in layer V projection neurons.

sentences:
Phylogeny, evolution, and classification of the ant genus Lasius, the tribe Lasiini, and the subfamily Formicinae (Hymenoptera: Formicidae) Within the Formicidae, the higher classification of nearly all subfamilies has been recently revised due to the findings of molecular phylogenetics. Here, we integrate morphology and molecular data to holistically address the evolution and classification of the ant genus Lasius, its tribe Lasiini, and their subfamily Formicinae. We accomplish this through a critical re-examination of morphology of extant and fossil taxa, molecular phylogenetic analyses, total-evidence dating under fossilized birth-death process, phylogeography, and ancestral state estimation. We use these results to provide revised taxonomic definitions for the Lasiini and select genera, and we provide a key to the genera of the Lasiini with emphasis on the Lasius genus group. We find that the crown Lasiini originated around the end of the Cretaceous on the Eurasian continent and is divisible into four morphologically distinct clades: Cladomyrma, the Lasius genus group, the Prenolepis genus group, and a previously undetected lineage we name XXX gen. n. The crown of the Lasius genus group is considerably younger than that of the Prenolepis genus group, indicating that extinction has played a major role in the evolution of the former clade. Lasius itself is divided into two well-supported monophyletic groups which are approximately equally speciose. We present evidence that temporary social parasitism and fungiculture arose in Lasius two times independently. Additionally, we recover the paraphyly of three Lasius subgenera and propose replacing all subgenera with an informal species group classification: Lasius = Acanthomyops syn. rev., = Austrolasius syn. n., = Cautolasius syn. n., = Chthonolasius syn. n., = Dendrolasius syn. n. Total-evidence analysis reveals that the Baltic-region amber fossil species {dagger}Lasius pumilus and {dagger}Pseudolasius boreus are misplaced to genus; we therefore designate {dagger}XXX gen. n. for the former and {dagger}XXX gen. n. for the latter. Further, we transfer {dagger}XXX and {dagger}Glaphyromyrmex out of the tribe, considering the former to be incertae sedis in the subfamily, and the latter a member of the Formicini (tribal transfer). Two final taxonomic actions are deemed necessary: synonymy of Lasius escamole Reza, 1925 with Liometopum apiculatum Mayr, 1870 syn. n. (subfamilial transfer), and transfer of Paratrechina kohli to Anoplolepis (tribal transfer, forming A. kohli (Forel, 1916) n. comb.).

Summary of taxonomic actionsO_LISubgenera of Lasius synonymized: Lasius = Acanthomyops syn. rev. = Austrolasius syn. n. = Cautolasius syn. n. = Chthonolasius syn. n. = Dendrolasius syn. n.
C_LIO_LILasius myrmidon transferred to XXX gen. n. (Lasiini, XXX genus group).
C_LIO_LI{dagger}Lasius pumilus transferred to {dagger}XXX gen. n. (Lasiini, XXX genus group).
C_LIO_LI{dagger}Pseudolasius boreus transferred to {dagger}XXX gen. n. (incertae sedis in Formicinae) (tribal transfer).
C_LIO_LI{dagger}Glaphyromyrmex transferred to the Formicini from the Lasiini (tribal transfer).
C_LIO_LILasius escamole Reza, 1925 synonymized with Liometopum apiculatum Mayr, 1870, syn. n. (subfamilial transfer).
C_LIO_LIParatrechina kohli (Forel, 1916) transferred to Anoplolepis (Plagiolepidini) (genus and tribal transfer).
C_LI

sentences:
A Large-Scale Brain Network Mechanism for Increased Seizure Propensity in Alzheimer's Disease People with Alzheimers disease (AD) are 6-10 times more likely to develop seizures than the healthy aging population. Leading hypotheses largely consider increased excitability of local cortical tissue as primarily responsible for increased seizure prevalence in AD. However, both local dynamics and large-scale brain network structure are believed to be crucial for determining seizure likelihood and phenotype. In this study, we combine computational modelling with electrophysiological data to demonstrate a potential large-scale brain network mechanism for increased seizure propensity in people with AD. EEG was recorded from 21 people with probable AD and 26 healthy controls. At the time of EEG acquisition, all participants were free from seizures. Whole brain functional connectivity derived from source-reconstructed EEG recordings was used to build subject-specific brain network models of seizure transitions using an approach previously validated on participants with epilepsy vs controls. As cortical tissue excitability was increased in the simulations, network models of AD simulations were more likely to transition into seizures than simulations from healthy controls. Our results suggest an increased group-level probability of developing seizures at a future time for AD participants. We subsequently used the model to assess seizure propensity of different regions across the cortex. We found the most important regions for seizure generation were those typically burdened by amyloid-beta at the early stages of AD, as previously reported by in-vivo and post-mortem staging of amyloid plaques. These included cingulate, medial temporal, and orbital regions. Analysis of these spatial distributions also give potential insight into mechanisms of increased susceptibility to generalized (as opposed to focal) seizures in AD vs controls. This research suggests avenues for future studies testing patients with seizures, e.g. co-morbid AD/epilepsy patients, and comparisons with PET and MRI scans to relate regional seizure propensity with amyloid/tau pathology and cortical atrophy.

Author summaryPeople with Alzheimers disease (AD) are more likely to develop seizures than cognitively healthy people. In this study, we aimed to understand whether whole-brain network structure is related to this increased seizure likelihood. We used electroencephalography (EEG) to estimate brain networks from people with AD and healthy controls. We subsequently inserted these networks into a model brain and simulated disease progression by increasing the excitability of brain tissue. We found the simulated AD brains were more likely to develop seizures than the simulated control brains. No participants had seizures when we collected data, so our results suggest an increased probability of developing seizures at a future time for AD participants. Therefore functional brain network structure may play a role in increased seizure likelihood in AD. We also used the model to examine which brain regions were most important for generating seizures, and found that the seizure-generating regions corresponded to those typically affected in early AD. Our results also provide a potential explanation for why people with AD are more likely to have generalized seizures (i.e. seizures involving the whole brain, as opposed to  focal seizures which only involve certain areas) than the general population with epilepsy.

sentences: Cannabinoid receptor 1 is required for neurodevelopment of striosome-dendron bouquets Cannabinoid receptor 1 (CB1R) has strong effects on neurogenesis and axon pathfinding in the prenatal brain. Endocannabinoids that activate CB1R are abundant in the early postnatal brain and in mothers milk, but few studies have investigated their function in newborns. We examined postnatal CB1R expression in the major striatonigral circuit from striosomes of the striatum to the dopamine-containing neurons of the substantia nigra. CB1R enrichment was first detectable between postnatal days 5 and 7, and this timing coincided with the formation of  striosome-dendron bouquets, the elaborate anatomical structures by which striosomal neurons control dopaminergic cell activity through inhibitory synapses. In Cnr1-/- knockouts lacking CB1R expression, striosome-dendron bouquets were markedly disorganized by postnatal day 11 and at adulthood, suggesting a postnatal pathfinding connectivity function for CB1R in connecting striosomal axons and dopaminergic neurons analogous to CB1Rs prenatal function in other brain regions. Our finding that CB1R plays a major role in postnatal wiring of the striatonigral dopamine system, with lasting consequences at least in mice, points to a crucial need to determine whether lactating mothers use of CB1R agonists (e.g., in marijuana) or antagonists (e.g., type 2 diabetes therapies) can disrupt brain development in nursing offspring.

sentences: Low and high frequency intracranial neural signals match in the human associative cortex In vivo intracranial recordings of neural activity offer a unique opportunity to understand human brain function. Intracranial electrophysiological (iEEG) activity related to sensory, cognitive or motor events manifests mostly in two types of signals: event-related local field potentials in lower frequency bands (<30 Hz, LF) and broadband activity in the higher end of the frequency spectrum (>30 Hz, High frequency, HF). While most current studies rely exclusively on HF, thought to be more focal and closely related to spiking activity, the relationship between HF and LF signals is unclear, especially in human associative cortex. Here we provide a large-scale in-depth investigation of the spatial and functional relationship between these 2 signals based on intracranial recordings from 121 individual brains (8000 recording sites). We measure selective responses to complex ecologically salient visual stimuli - human faces - across a wide cortical territory in the ventral occipito-temporal cortex (VOTC), with a frequency-tagging method providing high signal-to-noise ratio (SNR) and the same objective quantification of signal and noise for the two frequency ranges. While LF face-selective activity has higher SNR across the VOTC, leading to a larger number of significant electrode contacts especially in the anterior temporal lobe, LF and HF display highly similar spatial, functional, and timing properties. Specifically, and contrary to a widespread assumption, our results point to nearly identical spatial distribution and local spatial extent of LF and HF activity at equal SNR. These observations go a long way towards clarifying the relationship between the two main iEEG signals and reestablish the informative value of LF iEEG to understand human brain function.

sentences: Mild CF Lung Disease is Associated with Bacterial Community Stability Microbial communities in the airways of persons with CF (pwCF) are variable, may include genera that are not typically associated with CF, and their composition can be difficult to correlate with long-term disease outcomes. Leveraging two large datasets characterizing sputum communities of 167 pwCF and associated metadata, we identify five bacterial community types. These communities explain 24% of the variability in lung function in this cohort, far more than single factors like Simpson diversity, which explains only 4%. Subjects with Pseudomonas-dominated communities tended to be older and have reduced percent predicted FEV1 (ppFEV1) than subjects with Streptococcus-dominated communities, consistent with previous findings. To assess the predictive power of these five communities in a longitudinal setting, we used random forests to classify 346 additional samples from 24 subjects observed 8 years on average in a range of clinical states. Subjects with mild disease were more likely to be observed at baseline, that is, not in the context of a pulmonary exacerbation, and community structure in these subjects was more self-similar over time, as measured by Bray-Curtis distance. Interestingly, we found that subjects with mild disease were more likely to remain in a mixed Pseudomonas community, providing some support for the climax-attack model of the CF airway. In contrast, patients with worse outcomes were more likely to show shifts among community types. Our results suggest that bacterial community instability may be a risk factor for lung function decline and indicates the need to better understand factors that drive shifts in community composition.

sentences: Arousal elevation drives the development of oscillatory vocal output Adult behaviors, such as vocal production, often exhibit temporal regularity. In contrast, their immature forms are more irregular. We ask whether the coupling of motor behaviors with arousal changes give rise to temporal regularity. Do they drive the transition from variable to regular motor output over the course of development? We used marmoset monkey vocal production to explore this putative influence of arousal on the nonlinear changes in their developing vocal output patterns. Based on a detailed analysis of vocal and arousal dynamics in marmosets, we put forth a general model incorporating arousal and auditory-feedback loops for spontaneous vocal production. Using this model, we show that a stable oscillation can emerge as the baseline arousal increases, predicting the transition from stochastic to periodic oscillations observed during marmoset vocal development. We further provide a solution for how this model can explain vocal development as the joint consequence of energetic growth and social feedback. Together, we put forth a plausible mechanism for the development of arousal-mediated adaptive behavior.

sentences: GM1 asymmetry in the membrane stabilizes pores Cell membranes are highly asymmetric and their stability against poration is crucial for survival. We investigated the influence of membrane asymmetry on electroporation of giant vesicles with membranes doped with GM1, a ganglioside asymmetri-cally enriched in neuronal cell membranes. Compared to symmetric membranes, the lifetimes of micronsized pores are about an order of magnitude longer suggesting that pores are stabilized by GM1. Internal membrane nanotubes caused by the GM1 asymmetry, obstruct and additionally slow down pore closure, effectively reducing pore edge tension and leading to leaky membranes. Our results point to the drastic effects this ganglioside can have on pore resealing in biotechnology applications based on poration as well as on membrane repair processes.

sentences: High Expression of Glycolytic Genes in Clinical Glioblastoma Patients Correlates with Lower Survival Glioblastoma (GBM), the most aggressive brain tumor, is associated with a median survival at diagnosis of 16-20 months and limited treatment options. The key hallmark of GBM is altered tumor metabolism and marked increase in the rate of glycolysis. Aerobic glycolysis along with elevated glucose consumption and lactate production supports rapid cell proliferation and GBM growth. In this study, we examined the gene expression profile of metabolic targets in GBM samples from patients with low grade glioma (LGG) and GBM. We found that gene expression of glycolytic enzymes is up-regulated in GBM samples and significantly associated with an elevated risk for developing GBM. Our findings of clinical outcomes showed that GBM patients with high expression of HK2 and PKM2 in the glycolysis related genes and low expression of genes involved in mitochondrial metabolism-SDHB and COX5A related to tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), respectively, was associated with poor patient overall survival. Surprisingly, expression levels of genes involved in mitochondrial oxidative metabolism are markedly increased in GBM compared to LGG but was lower compared to normal brain. The fact that in GBM the expression levels of TCA cycle and OXPHOS-related genes are higher than those in LGG patients suggests the metabolic shift in GBM cells when progressing from LGG to GBM. These results are an important step forward in our understanding of the role of metabolic reprogramming in glioma as drivers of the tumor and could be potential prognostic targets in GBM therapies.

sentences: Profiling of histone H1 variants and changes on genome architecture and gene expression upon histone H1 depletion in breast cancer cells Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local DNA GC content and that their distribution is robust with respect to multiple H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of Topologically Associating Domains or TADs. Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants within a cell line and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells.

sentences: Reconstitution of Kinesin-1 Activation Autoinhibition is an important regulatory mechanism for cytoskeletal motor proteins. Kinesin-1 (kinesin hereafter), the ubiquitous plus-end directed microtubule motor, is thought to be controlled by a complicated autoinihibition mechanism, but the molecular details remain unclear. Conformational changes mediated by intramolecular interactions between the C-terminal tail and N-terminal motor domains of the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The dimeric KHC also binds two copies of the kinesin light chains (KLCs), which have been implicated in both autoinhibition and cargo-dependent activation of the tetrameric motor complex, although the precise mechanisms remain opaque. Using in vitro reconstitution, we show that the KLC strongly inhibits the kinesin-microtubule interaction via an independent mechanism from the tail-motor interaction within KHC. Kinesin cargo-adaptor proteins that bind KLC activated processive movement of the kinesin tetramer but the landing rate of these activated complexes remained low. The addition of MAP7, which specifically binds to the KHC, strongly enhanced activated motor motility by dramatically increasing the landing rate and processivity of the activated kinesin motors. Our results support a model whereby the activity of the kinesin tetramer is regulated by independent tail- and KLC-based inhibition mechanisms, and that cargo-adaptor binding to the KLC directly releases both of these inhibitions. However, we find that a third component, a non-motor MAP is required for robust activity of the activated motor. Thus, human kinesin activity is regulated by a two-factor mechanism comprised of intramolecular allosteric regulation, as well as intermolecular kinesin-adaptor and kinesin-MAP interactions.

sentences: ISM-FLUX: single-step MINFLUX with an array detector Single-molecule localization based on the concept of MINFLUX allows for molecular resolution imaging and tracking. However, MINFLUX has a limited field-of-view and therefore requires a precise and photon-costly pre-localization step. We propose ISM-FLUX, a localization technique that combines structured illumination with structured detection. We show through simulations that replacing the point-detector with a small (e.g. 5 x 5 pixels) single-photon camera and sequentially exciting the sample with four spatially separated doughnut-shaped beams, a localization precision between 1 and 15 nm can be obtained over a field-of-view (FOV) of more than 800 nm with 100 photons. The large FOV, induced by the camera, makes the pre-localization step obsolete. We calculate the effect of different parameters, such as the position of the doughnut beams and the number of pixels, on the localization precision. We predict that the combination of a good localization precision and the experimental simplicity of ISM-FLUX will help the wide adoption of MINFLUX based microscopy.

sentences: Pancreatic α and β cells are globally phase-locked The Ca2+ modulated pulsatile secretion of glucagon and insulin by pancreatic  and {beta} cells plays a key role in glucose homeostasis. However, how  and {beta} cells coordinate via paracrine interaction to produce various Ca2+ oscillation patterns is still elusive. Using a microfluidic device and transgenic mice in which  and {beta} cells were labeled with different colors, we were able to record islet Ca2+ signals at single cell level for long times. Upon glucose stimulation, we observed heterogeneous Ca2+ oscillation patterns intrinsic to each islet. After a transient period, the oscillations of  and {beta} cells were globally phase-locked, i.e., the two types of cells in an islet each oscillate synchronously but with a phase shift between the two. While the activation of  cells displayed a fixed time delay of ~20 s to that of {beta} cells, {beta} cells activated with a tunable delay after the  cells. As a result, the tunable phase shift between  and {beta} cells set the islet oscillation period and pattern. Furthermore, we demonstrated that the phase shift can be modulated by glucagon. A mathematical model of islet Ca2+ oscillation taking into consideration of the paracrine interaction was constructed, which quantitatively agreed with the experimental data. Our study highlights the importance of cell-cell interaction to generate stable but tunable islet oscillation patterns.

sentences: Mechanically Sensitive HSF1 is a Key Regulator of Left-Right Symmetry Breaking in Zebrafish Embryos The left-right symmetry breaking of vertebrate embryos requires fluid flow (called nodal flow in zebrafish). However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. In this paper, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kuppfers vesicle at the early stage of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 could be immediately activated by fluid shear stress. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and the Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress in vitro and in vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation triggered by mechanical clues during embryonic development and other physiological and pathological transformations.

sentences: Population and Individual Firing Behaviors in Sparsely Synchronized Rhythms in The Hippocampal Dentate Gyrus We investigate population and individual firing behaviors in sparsely synchronized rhythms (SSRs) in a spiking neural network of the hippocampal dentate gyrus (DG). The main encoding granule cells (GCs) are grouped into lamellar clusters. In each GC cluster, there is one inhibitory (I) basket cell (BC) along with excitatory (E) GCs, and they form the E-I loop. Winner-take-all competition, leading to sparse activation of the GCs, occurs in each GC cluster. Such sparsity has been thought to enhance pattern separation performed in the DG. During the winner-take-all competition, SSRs are found to appear in each population of the GCs and the BCs through interaction of excitation of the GCs with inhibition of the BCs. Sparsely synchronized spiking stripes appear successively with the population frequency fp (= 13 Hz) in the raster plots of spikes. We also note that excitatory hilar mossy cells (MCs) control the firing activity of the GC-BC loop by providing excitation to both the GCs and the BCs. SSR also appears in the population of MCs via interaction with the GCs (i.e., GC-MC loop). Population behaviors in the SSRs are quantitatively characterized in terms of the synchronization measures. In addition, we investigate individual firing activity of GCs, BCs, and MCs in the SSRs. Individual GCs exhibit random spike skipping, leading to a multi-peaked inter-spike-interval histogram, which is well characterized in terms of the random phase-locking degree. In this case, population-averaged mean-firing-rate [Formula] is less than the population frequency fp. On the other hand, both BCs and MCs show "intrastripe" burstings within stripes, together with "interstripe" random spike skipping. Thus, the population-averaged [Formula] (X = MC and BC) is larger than fp, in contrast to the case of the GCs. MC loss may occur during epileptogenesis. With decreasing the fraction of the MCs, changes in the population and individual firings in the SSRs are also studied. Finally, quantitative association between the population/individual firing behaviors in the SSRs and the winner-take-all competition is discussed.

sentences: Time-Dependent Image Restoration of Low-SNR Live Cell Ca2+ Fluorescence Microscopy Data Live cell Ca2+ fluorescence microscopy is a cornerstone of cellular signaling analysis and imaging. The demand for high spatial and temporal imaging resolution is, however, intrinsically linked to a low signal-to-noise ratio (SNR) of the acquired spatio-temporal image data, which impedes subsequent image analysis. Advanced deconvolution and image restoration algorithms can partly mitigate the corresponding problems, but are usually defined only for static images. Frame-by-frame application to spatio-temporal image data neglects inter-frame contextual relationships and temporal consistency of the imaged biological processes. Here, we propose a variational approach to time-dependent image restoration built on entropy-based regularization specifically suited to process low- and lowest-SNR fluorescence microscopy data. The advantage of the presented approach is demonstrated by means of four data sets: synthetic data for in-depth evaluation of the algorithm behavior; two data sets acquired for analysis of initial Ca2+ microdomains in T cells; and, to illustrate transferability of the methodical concept to different applications, one dataset depicting spontaneous Ca2+ signaling in jGCaMP7b-expressing astrocytes. To foster re-use and reproducibility, the source code is made publicly available.

sentences: Ensemble cryo-electron microscopy reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex, or RTC) associated with two copies of nsp13 (nsp132-RTC). One copy of nsp13 interacts with the template RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backwards on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here, we use cryo-electron microscopy and molecular dynamics simulations to analyze the nsp132-RTC, revealing four distinct conformational states of the helicases. The results suggest a mechanism for the nsp132-RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

sentences: BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae Human bromodomain-containing BET family members are promising targets for therapy of cancer and immunoinflammatory diseases, but their mechanisms of action and functional redundancies are poorly understood. Bdf1/2, yeast homologues of the human BET factors, were previously proposed to target transcription factor TFIID to acetylated histone H4, analogous to bromodomains that are present within the largest subunit of metazoan TFIID. We investigated the genome-wide roles of Bdf1/2 and found that their important contributions to transcription extend beyond TFIID function, as transcription of many genes is more sensitive to Bdf1/2 than to TFIID depletion. Bdf1/2 co-occupy the majority of yeast promoters and affect preinitiation complex formation through recruitment of TFIID, Mediator and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and processive elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results establish Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes.

sentences: RSC primes the quiescent genome for hypertranscription upon cell cycle re-entry Quiescence is a reversible G0 state essential for differentiation, regeneration, stem cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.

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

sentences: Thalamic state controls timing and synchronization of primary somatosensory cortical representations in the awake mouse The thalamus controls transmission of sensory signals from periphery to cortex, ultimately shaping perception. Despite this significant role, dynamic thalamic gating and the consequences for downstream cortical sensory representations have not been well studied in the awake brain. We optogenetically modulated the ventro-posterior medial thalamus in the vibrissa pathway of the awake mouse, and measured spiking activity in the thalamus, and activity in primary somatosensory cortex (S1) using extracellular electrophysiology and genetically encoded voltage imaging. Thalamic hyperpolarization significantly enhanced thalamic sensory-evoked bursting, yet surprisingly the S1 cortical response was not amplified, but instead timing precision was significantly increased, spatial activation more focused, and there was an increased synchronization of cortical inhibitory neurons. A thalamocortical network model implicates the modulation of precise timing of feedforward thalamic population spiking, presenting a highly sensitive, timing-based gating of sensory signaling to cortex.

sentences: Perceptual consequences of interocular imbalances in the duration of temporal integration Temporal differences in visual information processing between the eyes can cause dramatic misperceptions of motion and depth. Processing delays between the eyes cause the Pulfrich effect: oscillating targets in the frontal plane are misperceived as moving along near-elliptical motion trajectories in depth (Pulfrich, 1922). Here, we explain a previously reported but poorly understood variant: the anomalous Pulfrich effect. When this variant is perceived, the illusory motion trajectory appears oriented left- or right-side back in depth, rather than aligned with the true direction of motion. Our data indicate that this perceived misalignment is due to interocular differences in neural temporal integration periods, as opposed to interocular differences in delay. For oscillating motion, differences in the duration of temporal integration dampen the effective motion amplitude in one eye relative to the other. In a dynamic analog of the Geometric effect in stereo-surface-orientation perception (Ogle, 1950), the different motion amplitudes cause the perceived misorientation of the motion trajectories. Forced-choice psychophysical experiments, conducted with either different spatial frequencies and/or different onscreen motion damping in the two eyes, show that the perceived misorientation in depth is associated with the eye having greater motion damping. A target-tracking experiment provided more direct evidence that the anomalous Pulfrich effect is caused by interocular differences in temporal integration and delay. These findings highlight the computational hurdles posed to the visual system by temporal differences in sensory processing. Future work will explore how the visual system overcomes these challenges to achieve accurate perception.

sentences: Tatajuba -- Exploring the distribution of homopolymer tracts Length variation of homopolymeric tracts, which induces phase variation, is known to regulate gene expression leading to phenotypic variation in a wide range of bacterial species. There is no specialised bioinformatics software which can, at scale, exhaustively explore and describe these features from sequencing data. Identifying these is non-trivial as sequencing and bioinformatics methods are prone to introducing artefacts when presented with homopolymeric tracts due to the decreased base diversity. We present tatajuba, which can automatically identify potential homopolymeric tracts and their putative phenotypic impact, allowing for rapid investigation. We use it to detect all tracts in two separate datasets, one of Campylobacter jejuni and one of three Bordetella species, and to highlight those tracts that are polymorphic across samples. With this we confirm homopolymer tract variation with phenotypic impact found in previous studies and additionally find many more with potential variability. The software is written in C and is available under the open source license GNU GPL version 3 from https://github.com/quadram-institute-bioscience/tatajuba.

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Multimodal image fusion via deep generative models Recently, it has become progressively more evident that classic diagnostic labels are unable to accurately and reliably describe the complexity and variability of several clinical phenotypes. This is particularly true for a broad range of neuropsychiatric illnesses such as depression and anxiety disorders or behavioural phenotypes such as aggression and antisocial personality. Patient heterogeneity can be better described and conceptualized by grouping individuals into novel categories, which are based on empirically-derived sections of intersecting continua that span both across and beyond traditional categorical borders. In this context, neuroimaging data carry a wealth of spatiotemporally resolved information about each patients brain. However, they are usually heavily collapsed a priori through procedures which are not learned as part of model training, and consequently not optimized for the downstream prediction task. This is due to the fact that every individual participant usually comes with multiple whole-brain 3D imaging modalities often accompanied by a deep genotypic and phenotypic characterization, hence posing formidable computational challenges.

In this paper we design and validate a deep learning architecture based on generative models rooted in a modular approach and separable convolutional blocks (which result in a 20-fold decrease in parameter utilization) in order to a) fuse multiple 3D neuroimaging modalities on a voxel-wise level, b) efficiently convert them into informative latent embeddings through heavy dimensionality reduction, c) maintain excellent generalizability and minimal information loss. As proof of concept, we test our architecture on the well characterized Human Connectome Project database (n=974 healthy subjects), demonstrating that our latent embeddings can be clustered into easily separable subject strata which, in turn, map to extremely different phenotypical information (including organic, neuropsychological, personality variables) which was not included in the embedding creation process.

The ability to extract meaningful and separable phenotypic information from brain images alone can aid in creating multi-dimensional biomarkers able to chart spatio-temporal trajectories which may correspond to different pathophysiological mechanisms unidentifiable to traditional data analysis approaches. In turn, this may be of aid in predicting disease evolution as well as drug response, hence supporting mechanistic disease understanding and also empowering clinical trials.

sentences: Verbally mediated cooperation is consistently associated with inter-brain synchrony in frontal and temporoparietal areas: A mini-review and meta-analysis Single-brain neuroimaging studies have shown that human cooperation is associated with neural activity in frontal and temporoparietal regions. However, it remains unclear whether single-brain studies are informative about cooperation in real life, where people interact dynamically. Such dynamic interactions have become the focus of inter-brain studies. An advantageous technique in this regard is functional near-infrared spectroscopy (fNIRS) because it is less susceptible to movement artifacts than more conventional techniques like EEG or fMRI. We conducted a systematic review and the first quantitative meta-analysis of fNIRS hyperscanning of cooperation, based on thirteen studies with 890 participants. Overall, the meta-analysis revealed evidence of statistically significant inter-brain synchrony while people were cooperating, with large overall effect sizes in both frontal and temporoparietal areas. All thirteen studies observed significant inter-brain synchrony in the prefrontal cortex (PFC), suggesting that this region is particularly relevant for cooperative behavior. The consistency in these findings is unlikely to be due to task-related activations, given that the relevant studies used diverse cooperation tasks. Together, the present findings support the importance of inter-brain synchronization of frontal and temporoparietal regions in interpersonal cooperation. Moreover, the present article highlights the usefulness of meta-analyses as a tool for discerning patterns in inter-brain dynamics.

sentences: Transcription factor competition facilitates self-sustained oscillations in single gene genetic circuits Genetic feedback loops can be used by cells as a means to regulate internal processes or keep track of time. It is often thought that, for a genetic circuit to display self-sustained oscillations, a degree of cooperativity is needed in the binding and unbinding of actor species. This cooperativity is usually modeled using a Hill function, regardless of the actual promoter architecture. Moreover, genetic circuits do not operate in isolation and often transcription factors are shared between different promoters. In this work we show how mathematical modelling of genetic feedback loops can be facilitated with a mechanistic fold-change function that takes into account the titration effect caused by competing binding sites for transcription factors. The model shows how the titration effect aids self-sustained oscillations in a minimal genetic feedback loop: a gene that produces its own repressor directly -- without cooperative transcription factor binding. The use of delay-differential equations leads to a stability contour that predicts whether a genetic feedback loop will show self-sustained oscillations, even when taking the bursty nature of transcription into account.

sentences: Temporal control of the integrated stress response by a stochastic molecular switch Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

sentences: Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor Synapses maintain both action potential-evoked and spontaneous neurotransmitter release, however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release. In non-neuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of non-photobleached probes within 10-seconds. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

sentences: Interpretation of southern hemisphere humpback whale diet  via stable isotopes; implications of tissue-specific analysis Blubber and skin are commonly used tissues in stable isotope analysis for the purpose of investigating cetacean diet. Critical comparison of tissue-specific isotopic signals is, however, lacking resulting in uncertainty surrounding the representativeness and therefore utility of different tissues for accurate determination of recent foraging. This study used remotely biopsied blubber and skin tissues from southern hemisphere humpback whales for strategic comparison of {delta}13C and {delta}15N values. Samples were collected 2008-2018 as part of long-term monitoring under the Humpback Whale Sentinel Program. Blubber tissues were lipid-extracted prior to analysis, whilst mathematical lipid-correction was performed on skin samples. Isotopic values from paired blubber and skin samples from the same individuals were compared to assess whether tissues could be used interchangeably for isotope analysis and dietary interpretation. Significant differences were observed for both {delta}13C and {delta}15N, flagging previously undocumented methodological considerations, and the need for method validation and standardisation in application of these approaches. This study therefore advances methodological aspects of cetacean dietary analysis. This is of elevated importance in the context of rapidly changing ocean ecosystems.

sentences: KIBRA-PKCγ signaling pathway modulates memory performance in mice and humans Human memory is a polygenic cognitive trait that is fundamental to individual competence. Genome-wide association studies (GWAS) have identified KIBRA as a novel gene associated with human memory performance. KIBRA interacts with AMPA receptors (AMPARs) and proteins essential for synaptic plasticity. The deletion of Kibra in mice impairs synaptic plasticity and learning and memory. However, the molecular basis through which KIBRA regulates dynamic AMPAR trafficking underlying synaptic plasticity is still unknown. Here we report that KIBRA interacts with the neuronal specific kinase PKC{gamma} to modulate AMPAR trafficking upon learning, and KIBRA-PKC{gamma} signaling pathway also associates with human memory performance. We find PKC{gamma} is an essential kinase that phosphorylates AMPARs upon learning, and the loss of KIBRA in mouse brain impedes PKC{gamma}-AMPAR interaction. Activation of PKC{gamma} enables KIBRA to recruit phosphorylated AMPARs to the synapse to promote LTP and learning. We further performed transcriptomic and genetic analyses in human postmortem brain samples, and behavioral and fMRI evaluations in living human subjects, to demonstrate the genetic interactions between KIBRA and PRKCG on memory performance and memory associated physiological engagement of the hippocampal memory system. Overall, our results support that the KIBRA-PKC{gamma} signaling pathway is crucial for modulating memory performance in mice and humans.

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A hippocampal-anterior thalamic circuit associated with learning rate in ageing Memory normally declines with ageing and these age-related cognitive changes are associated with changes in brain structure. Episodic memory retrieval has been widely studied during ageing, whereas learning has received less attention. Here we examined the neural correlates of learning rate in ageing. Our study sample consisted of 982 cognitively healthy female and male older participants from the Vallecas Project cohort, without a clinical diagnosis of mild cognitive impairment or dementia. The learning rate across three consecutive verbal memory (Free and Cued Selective Reminding Test) recall trials was used as a predictor of grey matter (GM) and white matter (WM) volume using voxel-based morphometry, and WM microstructure using tract-based spatial statistics on fractional anisotropy (FA) and mean diffusivity (MD) measures. Immediate recall improved by 1.4 items per trial on average, and this learning rate was faster in women and negatively associated with age. Structurally, hippocampal and anterior thalamic GM volume correlated positively with learning rate. Learning also correlated with the integrity of WM microstructure (high FA and low MD) in an extensive network of tracts including bilateral anterior thalamic radiation, fornix, and long-range tracts. These results suggest that structural GM and WM characteristics, centred on a hippocampal-anterior thalamic circuit, support learning capacity in ageing. Specifically, reduced volume and microstructure may explain some of the age-related memory deficits that result from impaired learning.

Significance statementA detailed understanding of age-related memory changes is crucial in an increasingly ageing population, in which memory decline is prevalent. Whilst memory performance is usually quantified by the ability to correctly retrieve information, memory impairment could be caused by a reduced ability to learn or encode information. In a cohort of cognitively healthy ageing participants, we found an association between verbal learning rate and grey matter volume and white matter microstructure in the limbic system. These findings establish the neural underpinnings of the often-overlooked learning phase of the memory process and have important implications for our understanding of neurobiological changes in healthy ageing and their cognitive phenotypes.

sentences: Bottom-up and top-down effects of temperature on body growth, population size spectra and yield in a size-structured food web Resolving the combined effect of climate warming and exploitation in a food web context is key for predicting future biomass production, size-structure, and potential yields of marine fishes. Previous studies based on mechanistic size-based food web models have found that bottom-up processes are important drivers of size-structure and fisheries yield in changing climates. However, we know less about the joint effects of  bottom-up and  top-down effects of temperature: how do temperature effects propagate from individual-level physiology through food webs and alter the size-structure of exploited species in a community? Here we assess how a species-resolved size-based food web is affected by warming through both these pathways, and by exploitation. We parameterize a dynamic size spectrum food web model inspired by the offshore Baltic Sea food web, and investigate how individual growth rates, size-structure, relative abundances of species and yields are affected by warming. The magnitude of warming is based on projections by the regional coupled model system RCA4-NEMO and the RCP 8.5 emission scenario, and we evaluate different scenarios of temperature dependence on fish physiology and resource productivity. When accounting for temperature-effects on physiology in addition to on basal productivity, projected size-at-age in 2050 increases on average for all fish species, mainly for young fish, compared to scenarios without warming. In contrast, size-at-age decreases when temperature affects resource dynamics only, and the decline is largest for young fish. Faster growth rates due to warming, however, do not always translate to larger yields, as lower resource carrying capacities with increasing temperature tend to result in declines in the abundance of larger fish and hence spawning stock biomass - the part of the population exposed to fishing. These results show that to understand how global warming impacts the size structure of fish communities, both direct metabolic effects and indirect effects of temperature via basal resources must be accounted for.

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AI-Generated Virtual Libraries Could Help Uncover RNA-Specific Regions of Chemical Space RNAs can recognize small-molecule ligands. However, the extent to which the molecules that they recognize differ from those recognized by proteins remains an open question. Cheminformatics analysis of experimentally validated RNA binders strongly suggests that RNA binders occupy a specific region of chemical space. However, less than 100 validated small molecule ligands are currently known. Here, we demonstrate how structure-based approaches could be used to navigate vast regions of the chemical space specific to ligand binding sites in five highly-structured RNAs. Our method involves using generative-AI to design target- and site-specific virtual libraries and then analyzing them using similar cheminformatics approaches as those used to assess experimentally validated RNA binders. Despite employing a completely orthogonal strategy, our results essentially reproduce the trends observed by analyzing the experimentally validated RNA binders. Large-scale generation of target and site-specific libraries may therefore prove to be helpful in simultaneously mapping the regions of chemical space unique to RNA and generating libraries that could be mined to identify novel RNA binders.

TOC IMAGE O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

sentences: Predicting proprioceptive cortical anatomy and neural coding with topographic autoencoders Proprioception is one of the least understood senses yet fundamental for the control of movement. Even basic questions of how limb pose is represented in the somatosensory cortex are unclear. We developed a variational autoencoder with topographic lateral connectivity (topo-VAE) to compute a putative cortical map from a large set of natural movement data. Although not fitted to neural data, our model reproduces two sets of observations from monkey centre-out reaching: 1. The shape and velocity dependence of proprioceptive receptive fields in hand-centered coordinates despite the model having no knowledge of arm kinematics or hand coordinate systems. 2. The distribution of neuronal preferred directions (PDs) recorded from multi-electrode arrays. The model makes several testable predictions: 1. Encoding across the cortex has a blob-and-pinwheel-type geometry PDs. 2. Few neurons will encode just a single joint. Topo-VAE provides a principled basis for understanding of sensorimotor representations, and the theoretical basis of neural manifolds, with applications to the restoration of sensory feedback in brain-computer interfaces and the control of humanoid robots.

sentences: Activity in the dorsomedial striatum decreases with improvement in motor coordination It has been suggested that the dorsomedial striatum (DMS) facilitates the early stages of motor learning for goal-directed actions, whereas at later stages, control is transferred to the dorsolateral striatum (DLS), which enables the motor actions to become a skill or habit. It is unknown whether these striatal regions are simultaneously active while expertise is acquired during skill learning. To address this question, we developed the "treadmill training task" to track changes in mouse locomotor coordination during practice at running that simultaneously provides a means to measure local neuronal activity using photometry. We analyzed body position and paw movement to evaluate changes in motor coordination over practice sessions on the treadmill using DeepLabCut and custom-built code. By correlating improvements in motor coordination during training with simultaneous neuronal calcium activity in the striatum, we found that DMS direct pathway neurons exhibited decreased activity as the mouse gains proficiency at running. In contrast, direct pathway activity in the DLS was similar throughout training and did not correlate with learned skill proficiency. Pharmacological blockade of D1 dopamine receptors in these subregions during task performance confirmed that dopamine neurotransmission in the DMS direct pathway activity is necessary for efficient motor coordination learning, while dopamine signalling in the DLS is important for both coordination learning and maintenance of the acquired skill. These results provide new tools to measure changes in fine motor skills during simultaneous recordings of brain activity, revealing fundamental features of the neuronal substrates of motor learning.

sentences: Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

sentences: Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals receiving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a "Hybrid" vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specific RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs.

sentences: Effects of cover crops on beetle assemblages in tea plantations Beetles are visible members of food webs in tea plantations, with high species richness and abundance. Many tea pests, as well as natural enemies, are members of this order, so a knowledge of how groundcovers affect beetles can aid pest management. We collected beetles in a replicated field experiment in the Wuyi Mountains, Fujian Province China. Tea was intercropped with Paspalum notatum or Chamaecrista rotundifolia, or rows were cleared to bare ground, or in the control they were left unmanaged to allow weeds to grow naturally. Sampling, done by sweep netting and vegetation beating, was conducted monthly, between May 2006 and April 2008, and Coleoptera abundance, biomass, species richness and assemblage structures were compared between groundcover treatments. Total beetle abundance and species richness were significantly higher in tea intercropped with C. rotundifolia and bare ground than in naturally grown weedy control. Whilst there was no difference between predator assemblages among treatments for any measure, herbivores were more abundant, weighed more, and were more diverse in C. rotundifolia treatments than in weedy control. Biomass and species richness were also greater in plots with P. notatum groundcover than those in weedy control. We found that beetle assemblages varied both seasonally and with ground cover treatment, but the potential pest control impact of more species-rich beetle assemblages was mixed, and further work is needed to gain information on trophic groups with potential benefits for use in non-insecticidal pest management.

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A quadratic analytical solution of root pressure generation provides insights about bamboo and other species We derived a steady-state model of whole root pressure generation through the combined action of all parallel segments of fine roots. This may be the first complete analytical solution for root pressure, which can be applied to complex roots/shoots.

The osmotic volume of a single root is equal to that of the vessel lumen in fine roots and adjacent apoplastic spaces. Water uptake occurs via passive osmosis and active solute uptake ([Formula], osmol s-1), resulting in the osmolal concentration Cr (mol{middle dot}kg-1 of water) at a fixed osmotic volume. Solute loss occurs via two passive processes: radial diffusion of solute Km (Cr-Csoil), where Km is the diffusional constant and Csoil is the soil-solute concentration) from fine roots to soil and mass flow of solute and water into the whole plant from the end of the fine roots.

The proposed model predicts the quadratic function of root pressure [Formula], where b and c are the functions of plant hydraulic resistance, soil water potential, solute flux, and gravitational potential.

The present study investigates the theoretical dependencies of Pr on the factors detailed above and demonstrates the root pressure-mediated distribution of water through the hydraulic architecture of a 6.8-m-tall bamboo shoot.

One sentence SummaryAn analytical solution for root pressure is derived and illustrated by applying it to the measured hydraulic architecture of a 7-meter bamboo shoot.

sentences: Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.

sentences: Circulating CD138 (syndecan-1) enhances APRIL-mediated autoreactive B cell survival and differentiation in MRL/Lpr mice High levels of serum CD138, a heparan sulfate-bearing proteoglycan, correlates with increased disease activity in systemic lupus erythematosus (SLE) patients. Mechanisms responsible for serum CD138 production and its biological function in SLE disease remain poorly understood. In this study, corroborating patient data, we detected an increase in serum CD138 in MRL/Lpr mice parallel to disease activity. Although TCR{beta}+CD138+ T cells expand in MRL/Lpr mice as the disease progresses, surprisingly, TCR{beta}+CD138- cells were the primary source of circulating CD138 as the transfer of TCR{beta}+CD138- cells to young MRL/Lpr mice, but not TCR{beta}+CD138+ cells, resulted with higher serum CD138 in the recipient mice. We found that elevated trypsin, expressed by TCR{beta}+CD138- cells, was able to cleave CD138 from T cells. Moreover, suggesting the contribution of cleaved CD138 to the increase in blood CD138, trypsin inhibitors  defined trypsin inhibitor (DTI) or leupeptin increased CD138 expression on TCR{beta}+CD138- cells. Furthermore, soluble CD138 was able to bind  a proliferation inducing ligand (APRIL) and enhanced APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular-signal-regulated kinase (ERK) in B cells. APRIL receptor,  transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated on TACI deficient B cells. These findings indicate a regulatory role for soluble CD138 in B cell differentiation and autoreactive antibody secretion in SLE disease.

sentences: A Facile Immunopeptidomics Workflow for Capturing the HLA-I Ligandome with PEAKS XPro Identifying antigens displayed specifically on tumour cell surfaces by human leukocyte antigen (HLA) proteins is important for the development of immunotherapies and cancer vaccines. The difficulty in capturing an HLA ligandome stems from the fact that many HLA ligands are derived from splicing events or contain mutations, hindering their identification in a standard database search. To address this challenge, we developed an immunopeptidomics workflow with PEAKS XPro that uses de novo sequencing to uncover such peptides and identifies mutations for neoantigen discovery. We demonstrate the utility of this workflow by re-analyzing HLA-I ligandome datasets and reveal a vast diversity in peptide sequences among clones derived from a colorectal cancer tumour. Over 8000 peptides predicted to bind HLA-I molecules were identified by de novo sequencing only (not found in the UniProt database) and make up over 50% of identified peptides from each sample. Lastly, tumour-specific mutations and consensus sequence motif characteristics are defined. This workflow is widely applicable to any immunopeptidomic mass spectrometry dataset and does not require custom database generation for neoantigen discovery.

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The fungal root endophyte Serendipita vermifera displays inter-kingdom synergistic beneficial effects with the microbiota in Arabidopsis thaliana and barley Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen in Arabidopsis thaliana and barley. We additionally show early inter-kingdom growth promotion benefits which are host and microbiota composition dependent.

HighlightsO_LIThe root endophytic fungus Serendipita vermifera can functionally replace core bacterial microbiota members in mitigating pathogen infection and disease symptoms.
C_LIO_LIS. vermifera additionally stabilizes and potentiates the protective activities of root-associated bacteria and mitigates the negative effects of a non-native bacterial community in A. thaliana.
C_LIO_LIInter-kingdom synergistic beneficial effects do not require extensive host transcriptional reprogramming nor high levels of S. vermifera colonisation.
C_LIO_LIInter-kingdom protective benefits are largely independent of the host while synergism leading to early inter-kingdom growth promotion is driven by host species and microbiota composition.
C_LI

sentences: Runaway evolution from male-male competition Wondrously elaborate weapons and displays that appear to be counter to ecological optima are widespread features of male contests for mates across the animal kingdom. To understand how such diverse traits evolve, here we develop a quantitative genetic model of sexual selection for a male signaling trait that mediates aggression in male-male contests and show that an honest indicator of aggression can generate selection on itself by altering the social environment. This can cause selection to accelerate as the trait is elaborated, leading to runaway evolution. Thus, an evolving source of selection provided by the social environment is the fundamental unifying feature of runaway sexual selection driven by either male-male competition or female mate choice. However, a key difference is that runaway driven by male-male competition requires signal honesty. Our model identifies simple conditions that provide clear, testable predictions for empirical studies using standard quantitative genetic methods.

sentences: Model-based hearing-restoration strategies for cochlear synaptopathy pathologies It is well known that ageing and noise exposure are important causes of sensorineural hearing loss, and can result in damage of the outer hair cells or other structures of the inner ear, including synaptic damage to the auditory nerve (AN), i.e., cochlear synaptopathy (CS). Despite the suspected high prevalence of CS among people with self-reported hearing difficulties but seemingly normal hearing, conventional hearing-aid algorithms do not compensate for the functional deficits associated with CS. Here, we present and evaluate a number of auditory signal-processing strategies designed to maximally restore AN coding for listeners with CS pathologies. We evaluated our algorithms in subjects with and without suspected age-related CS to assess whether physiological and behavioural markers associated with CS can be improved. Our data show that after applying our algorithms, envelope-following responses and perceptual amplitude-modulation sensitivity were consistently enhanced in both young and older listeners. Speech intelligibility showed small improvements across participants, with the young group benefitting the most from processed speech. Our proposed hearing-restoration algorithms can be rapidly executed and can thus extend the application range of current hearing aids and hearables, while leaving sound amplification unaffected.

sentences: Nasal BCG Exposure Accelerates Dural Lymphatics Development via Macrophages' Role in Newborn Mice The dural lymphatics develop mainly during the first postnatal month. Lymphatics may be shaped by immune activation when bacterial infection happens. BCG, a strong immune activator, is widely injected to newborns. Moreover, human beings are nasally exposed in daily life to bacterial stimuli. These background prompted us to investigate whether neonatal BCG injection combined with nasally exposure exerts an influence on dural lymphatics develop. Here, mice received a single dose intracutaneous (i.c.) BCG injection immediately after birth followed by repeated nasal BCG challenge once a day (i.c./nasal group). These mice had an accelerated dural lymphatics growth and increased levels of several cytokines and VEGFR-3. Furthermore, macrophages were identified as a key mediator of these alterations. Mice that received mere one dose i.c. BCG injection showed no significant alterations in these indexes. Additionally, mere repeated nasal BCG challenge induced similar effects to i.c./nasal challenge but with a slighter extent. Taken together, these findings show that repeated nasal BCG vaccination accelerates dural lymphatics development in neonatal mice, especially in the presence of neonatal i.c. BCG injection.

sentences: Idea Paper: Effects of gonad type and body mass on the time required for sex change in fishes Sex change is a well-known phenomenon in teleost fishes, and it takes several days to a few months depending on the species and direction of sex change. However, the underlying factors influencing the time required for sex change (TS) remain unclear. Given that the time for producing a new gonad largely determines TS, the gonad type (i.e., whether fish retain the gonad of opposite sex or not [delimited or non-delimited]) and metabolic rate are the ultimate determinants of TS. This study sought to test two hypotheses: (1) the delimited gonad shortens TS; and (2) TS scales with mass0.1-0.2, because the metabolic scaling exponent ({beta}) in fishes is 0.8-0.9 and biological times scale with mass1-{beta} in general. We compiled data on TS for 12 female-to-male and 14 male-to-female sex-changing species from the literature. Results of individual examinations of the effects of gonad type and mass were consistent with our hypotheses. However, upon simultaneous examination of the effects of gonad type and mass, these effects became unclear because of their strong multicollinearity. The compiled data for delimited and non-delimited gonads were biased toward the smaller and larger species, respectively, precluding us from being able to statistically distinguish between these effects. Small species with non-delimited gonads and large species with delimited gonads exist; however, their TS has not been measured with high temporal resolution thus far. Therefore, additional experiments on these species are required to statistically distinguish between, as well as to better understand, the effects of gonad type and mass on TS.

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Multiple routes of functional diversification of the plant BAHD acyltransferase family revealed by comparative biochemical and genomic analyses Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Here, we addressed this question using BAHD acyltransferases as a model, and identified the routes by which duplication, promiscuity and sequence changes influenced BAHD diversification. This fast-evolving family expanded drastically during land plant evolution from 1-5 copies in algae to [~]100 copies in diploid angiosperm genomes. In vitro characterization of fourteen BAHDs against a substrate panel and compilation of >160 published activities revealed the wide prevalence of promiscuity among BAHDs. Using phylogenetic analysis, we predicted the substrate classes that the ancestral enzymes were likely capable of using prior to land plant origins. While the anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms, in vitro testing of BAHDs from non-seed plant lineages suggested that the ability to acylate anthocyanins likely existed promiscuously millions of years prior to its fixation. Motif enrichment analysis in anthocyanin-acylating BAHDs identified two motifs fixed in the largest anthocyanin acylating clade. Molecular dynamic simulations and enzyme kinetics revealed the important role of an active site tryptophan, whose bulkiness, hydrophobicity and aromaticity are critical for anthocyanin acylation. Our results thus describe the molecular processes in robust, evolvable enzymes that drive emergence of functional diversity in enzyme families.

One sentence summaryUsing a combination of phylogenetics, biochemistry and protein structure analysis, we investigated how the BAHD acyltransferase family evolved to use a structurally diverse array of substrates.

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Reciprocal interactions between the gut microbiome and mammary tissue mast cells promote metastatic dissemination of HR+ breast tumors Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, prior to breast tumor initiation, enhances early dissemination of hormone-receptor positive (HR+) mammary tumor cells. Here, we sought to define mammary tissue mediators of dysbiosis-induced tumor dissemination. We found that commensal dysbiosis increased both the frequency and profibrogenicity of mast cells in the mammary tissue, a phenotypic change that persisted after tumor implantation. Fibroblast activation and tissue remodeling associate with enhanced breast tumor metastasis. We employed pharmacological and adoptive transfer approaches to demonstrate that mammary tissue mast cells from dysbiotic animals enhances dissemination of HR+ tumor cells. Collagen levels in mammary tissues from HR+ breast cancer patients correlated with mast cell abundance, suggesting clinical relevance of mast cell-mediated fibroblast activation. Together, these data demonstrate that a gut-mast cell axis exists that induces fibroblast activation and orchestrates early dissemination of HR+ breast tumors.

SignificanceOur study defines the mechanism by which an inflammatory gut microbiome facilitates HR+ breast tumor cell dissemination. We establish that gut commensal dysbiosis triggers mammary tissue mast cells to facilitate early metastatic dissemination. These findings highlight a novel gut microbiome-innate immune cell axis involved in negative breast cancer outcomes.

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The role of interference between vectors in control of plant diseases Aphids are the primary vector of plant viruses. Transient aphids, which probe several plants per day, are considered to be the principal vectors of non-persistently transmitted (NPT) viruses. However, resident aphids, which can complete their life cycle on a single host and are affected by agronomic practices, can transmit NPT viruses as well. Moreover, they can interfere both directly and indirectly with transient aphids, eventually shaping plant disease dynamics. By mean of an epidemiological model, originally accounting for ecological principles and agronomic practices, we explore the consequences of fertilization and irrigation, pesticide deployment and roguing of infected plants on the spread of viral diseases in crops. Our results indicate that the spread of NPT viruses can be i) both reduced or increased by fertilization and irrigation, depending on whether the interference is direct or indirect; ii) counter-intuitively increased by pesticide application and iii) reduced by roguing infected plants. We show that a better understanding of vectors interactions would enhance our understanding of disease transmission, supporting the development of disease management strategies.

Author summaryA range of both experimental and theoretical studies show that the behaviour and population dynamics of insects depend strongly upon interactions with other insect species. These interactions have the potential to greatly affect the dynamics of insect-vectored plant disease, as transmission of viruses is intimately dependent on the local density of vectors, as well as how they select and move between potential host plants. Surprisingly, the effects of interaction between vector species on epidemics remains little studied and even worse understood, probably because experimentation is costly and difficult. Here, we present a model which permits to investigate the effect of interaction between a virus, two vector species, and the host plant on the spread of viral disease in crops. In this study, our model is used to explore the consequences of common agronomic practices on epidemics. Our study highlights the importance of exploring vectors interactions to enhance the understanding of disease transmission, supporting the development of disease management strategies.

sentences: Sequence Determinants of Human Junctophilin-2 Protein Nuclear Localization and Phase Separation Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data for the first time revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains and residues encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

sentences: Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response Antifreeze proteins (AFPs) from the model crop, Brachypodium distachyon, allow freeze survival and attenuate pathogen-mediated ice nucleation. Intriguingly, each Brachypodium AFP gene encodes two proteins, an autonomous AFP and a leucine-rich repeat (LRR). We present structural models suggesting that ice-binding motifs on the ~13 kDa AFPs can "spoil" nucleating arrays on the ~120 kDa bacterial ice nucleating proteins that form ice at high sub-zero temperatures, consistent with decreases in ice nucleating activity by lysates from wildtype compared to transgenic Brachypodium lines. Strikingly, the expression of Brachypodium LRRs in transgenic Arabidopsis inhibited an immune response to pathogen flagella peptides (flg22), with structural modelling suggesting this was due to affinity of the LRR domains to flg22. Thus, these genes play distinctive roles in connecting freeze survival and anti-pathogenic systems via their encoded proteins ability to adsorb to ice as well as attenuating bacterial ice nucleation and the host immune response.

sentences: Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

sentences: Divergent encoding of active avoidance behavior in corticostriatal and corticolimbic projections Active avoidance behavior, in which an animal performs an action to avoid a stressor, is crucial for survival and may provide insight into avoidance behaviors seen in anxiety disorders. Active avoidance requires the dorsomedial prefrontal cortex (dmPFC), which is thought to regulate avoidance via downstream projections to the striatum and amygdala. However, the endogenous activity of projection-defined dmPFC subpopulations during active avoidance learning remains unexplored. Here we utilized fiber photometry to record from the dmPFC and its downstream projections to the dorsomedial striatum (DMS) and the basolateral amygdala (BLA) during active avoidance learning in mice. We examined neural activity during conditioned stimulus (CS) presentations, active avoidance, and cued freezing. Both prefrontal projections showed learning-related increases in activity during CS onset throughout active avoidance training. The dmPFC as a whole showed increased activity during avoidance and decreased activity during cued freezing. Finally, dmPFC-DMS and dmPFC-BLA projections showed divergent encoding of active avoidance behavior, with the dmPFC-DMS projection showing increased activity and the dmPFC-BLA showing decreased activity during active avoidance. Our results identify differential prefrontal encoding of active and passive coping behaviors in the same behavioral paradigm and demonstrate divergent encoding of active avoidance in projection-specific dmPFC subpopulations.

sentences: Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids Direct capture and sequencing revealed a new DNA population in biofluids, named ultra-short single-stranded DNA (ussDNA), which was neglected by conventional DNA extraction and sequencing approaches. Evaluation of the size distribution and abundance of ussDNA in biofluids manifested generality of its presence in human, animal, and plants. Red blood cells were found to contain abundant ussDNA with enriched functional elements, and thus possesses great potential as a novel DNA biomarker.

sentences: In Silico Analysis and Characterization of Differentially Expressed Genes to Distinguish Glioma Stem Cells from Normal Neural Stem Cells Glioblastoma multiforme (GBM) is a heterogeneous, invasive primary brain tumor that develops chemoresistance post therapy. Theories regarding the aetiology of GBM focus on transformation of normal neural stem cells (NSCs) to a cancerous phenotype or tumorigenesis driven via glioma stem cells (GSCs). Comparative RNA-Seq analysis of GSCs and NSCs can provide a better understanding of the origin of GBM. Thus, in the current study, we performed various bioinformatics analyses on transcriptional profiles of a total 40 RNA-seq samples including 20 NSC and 20 GSC, that were obtained from the NCBI-SRA (SRP200400). First, differential gene expression (DGE) analysis using DESeq2 revealed 348 significantly differentially expressed genes between GSCs and NSCs (padj. value <0.05, log2fold change [≥] 3.0 (for GSCs) and [≤] -3.0 (for NSCs)) with 192 upregulated and 156 downregulated genes in GSCs in comparison to NSCs. Subsequently, exploratory data analysis using principal component analysis (PCA) based on key significant genes depicted the clear separation between both the groups. Further, Hierarchical clustering confirmed the distinct clusters of GSC and NSC samples. Eventually, the biological enrichment analysis of the significant genes showed their enrichment in tumorigenesis pathways such as Wnt-signalling, VEGF-signalling and TGF-{beta}-signalling pathways. Conclusively, our study depicted significant differences in the gene expression patterns between NSCs and GSCs. Besides, we also identified novel genes and genes previously unassociated with gliomagenesis that may prove to be valuable in establishing diagnostic, prognostic biomarkers and therapeutic targets for GBM.

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Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8.

Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs.

sentences: Zebrafish Neuromesodermal Progenitors Undergo a Critical State Transition in vivo The transition state model of cell differentiation proposes that a transient window of gene expression stochasticity precedes entry into a differentiated state. As this has been assessed primarily in vitro, we sought to explore whether it can also be observed in vivo. Zebrafish neuromesodermal progenitors (NMps) differentiate into spinal cord and paraxial mesoderm at the late somitogenesis stages. We observed an increase in gene expression variability at the 24 somite stage (24ss) prior to their differentiation. From our analysis of a published 18ss scRNA-seq dataset, we showed that the NMp population possesses a signature consistent with a population undergoing a critical transition. By building in silico composite gene expression maps from our image data, we were able to assign an  NM index to each in silico NMp based on the cumulative expression of its neural and mesodermal markers. With the NM index distributions, we demonstrated that cell population heterogeneity of the NMps peaked at 24ss. We then incorporated stochasticity and non-autonomy into a genetic toggle switch model and uncovered the existence of rebellious cells, which we then confirmed by reexamining the composite maps. Taken together, our work supports the transition state model within an endogenous cell fate decision making event.

sentences: Inter-Individual Variability in Dorsal Stream Dynamics During Word Production The current standard model of language production involves a sensorimotor dorsal stream connecting areas in the temporo-parietal junction with those in the inferior frontal gyrus and lateral premotor cortex. These regions have been linked to various aspects of word production such as phonological processing or articulatory programming, primarily through neuropsychological and functional imaging group studies. Most if not all the theoretical descriptions of this model imply that the same network should be identifiable across individual speakers. We tested this hypothesis by quantifying the variability of activation observed across individuals within each dorsal stream anatomical region. This estimate was based on electrical activity recorded directly from the cerebral cortex with millisecond accuracy in awake epileptic patients clinically implanted with intracerebral depth electrodes for pre-surgical diagnosis. Each regions activity was quantified using two different metrics, intra-cerebral evoked related potentials and high gamma activity, at the level of the group, the individual, and the recording contact. Using picture naming task, the two metrics show simultaneous activation of parietal and frontal regions in line with models that posit interactive processing during word retrieval. They also reveal different levels of variability across brain regions and patients except in auditory and motor regions. The independence and non-uniformity of cortical activity according to the two metrics push the current model towards sub-second and sub-region explorations focused on individualized language speech production. Several hypotheses are considered for this within-region heterogeneity.

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The CCL2 Chemokine Promotes Early Seeding of the Latent HIV Reservoir HIV infects long-lived CD4 memory T cells establishing a latent viral reservoir that necessitates lifelong anti-retroviral therapy (ART). How this reservoir is formed so swiftly remains unknown. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, which can drive recruitment of cells expressing the CCR2 receptor including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find 1) treatment of humanized mice with anti-CCL2 antibodies during HIV infection decreases reservoir seeding and 2) CCR2/5+ cells from the blood of HIV-infected individuals on long term ART contain significantly more provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the hosts innate inflammatory CCL2 response to HIV infection recruits CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation likely contributing to rapid formation of the latent HIV reservoir.

GRAPHICAL ABSTRACTWhy is the latent HIV reservoir established so early following infection? An innate immune response occurs during acute infection that establishes a "zone of inflammation" (step 1). The CCL2 chemokine is produced in part through IFI16 sensing of HIV DNA in abortively infected cells. CCL2 promotes rapid recruitment of CCR2/5+ memory CD4 T cells (step 2). Many of these cells become productively infected (step 3) and a fraction become latently infected (step 4). Thus, HIV hijacks the host inflammatory response to rapidly establish the latent reservoir. In support of this model, we find HIV reservoir reduction in humanized mice treated with anti-CCL2 antibodies during early infection. Further, we find that CCR2/5+ CD4 T cells harbor a substantial fraction of detectable proviruses in the blood of HIV-infected individuals on long-term suppressive ART.

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sentences: Multi-color 4D superresolution light-sheet microscopy reveals organelle interactions at isotropic 100-nm resolution and sub-second timescales Long-term visualization of the dynamic organelle-organelle or protein-organelle interactions throughout the three-dimensional space of whole live cells is essential to better understand their functions, but this task remains challenging due to the limitations of existing three-dimensional fluorescence microscopy techniques, such as an insufficient axial resolution, low volumetric imaging rate, and photobleaching. Here, we present the combination of a progressive deep-learning superresolution strategy with a dual-ring-modulated SPIM design capable of visualizing the dynamics of intracellular organelles in live cells for hours at an isotropic spatial resolution of [~]100 nm in three dimensions and a temporal resolution up to [~]17 Hz. With a compelling spatiotemporal resolution, we substantially reveal the complex spatial relationships and interactions between the endoplasmic reticulum (ER) and mitochondria throughout live cells, providing new insights into ER-mediated mitochondrial division. We also localized the motion of Drp1 oligomers in three dimensions and observed Drp1-mediated mitochondrial branching for the first time.

sentences: Absence of postsynaptic activity on developing neurons alters gene expression profiles without preventing the refinement of inputs Synaptic activity plays several roles as developing neurons make connections with their targets. It acts locally at synapses to influence the expression of genes needed to establish and maintain synaptic contacts. And, downstream it provides the necessary activity to strengthen and refine connections. Many studies have demonstrated how synaptic activity alters synaptic strength and increases synapse numbers. Much less is known, however, about the long-term consequences when a circuit develops without synaptic activity. To address this, we developed a mosaic model of sympathetic ganglia where synaptically-active and synaptically-inactive sympathetic neurons develop side-by-side in vivo. This model allowed us to address two issues. One is the relationship between activity and the refinement of converging inputs; the second is how synaptic activity contributes to a neurons gene expression profile. Our results indicate that converging presynaptic inputs to synaptically-silent neurons do not require postsynaptic activity to refine, provided these neurons share targets with synaptically-active neurons. Second, we demonstrate with single-cell RNA sequencing experiments that the expression of many genes by sympathetic neurons is independent of endogenous activity or local signals immediately downstream of excitatory postsynaptic potentials. An exception are genes required for neurotransmitter metabolism: We found that for a large sub-population of sympathetic neurons, synaptic activity increases the expression of adrenergic genes and supresses the expression of cholinergic genes. We conclude that signals generated locally at synapses do not initiate refinement of converging inputs, and that synaptic activitys influence on a neurons gene expression profiles is complex and depends on context.

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The effect of biohanin A isolated from the callus culture of meadow clover (Trifolium pratense L.) on the life expectancy and survival of Caenorhabditis elegans Oxidative stress, in which healthy aging is limited, is a problem, since in the process there is an uncontrolled production of reactive radicals that negatively affect cells. Heat stress is closely related to oxidative stress, as it stimulates the production of radicals. Meadow clover (Trifolium pratense L.) is a promising source of isoflavonoids that have various positive effects on the body. The aim of the work is to study the effect of biohanin A, isolated from Trifolium pratense L. callus cultures, on the life expectancy of the C. elegans model organism, as well as its survival under oxidative and thermal stress.

It was found that biohanin A increased the lifespan of C. elegans worms. The best concentration was determined - 100 microns, at which the survival rate of nematodes after 61 days of incubation was 16.0%. After 48 hours, the survival rate of worms was the highest (87.1%) at a concentration of biohanin A of 200 microns. Other concentrations 10, 50, 100 (81,2, 83,0, 82,4 %) they also showed a higher survival rate compared to the control (74.0%). In addition, the survival rate under heat stress of C. elegans was higher by 12.0; 8.4; 4.0% compared with the control. Thus, the results show the antioxidant potential of biohanin A from plant material under oxidative and thermal stress. The substance also had a positive effect on the lifespan of C. elegans.

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Force-induced changes of PilY1 drive surface sensing by Pseudomonas aeruginosa During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1.

ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.

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Shape Dimensionality Metrics for Landmark Data This paper is concerned with rank deficiency in phenotypic covariance matrices: first to establish it is a problem by measuring it, and then proposing methods to treat for it. Significant rank deficiency can mislead current measures of whole-shape phenotypic integration, because they rely on eigenvalues of the covariance matrix, and highly rank deficient matrices will have a large percentage of meaningless eigenvalues. This paper has three goals. The first is to examine a typical geometric morphometric data set and establish that its covariance matrix is rank deficient. We employ the concept of information, or Shannon, entropy to demonstrate that a sample of dire wolf jaws is highly rank deficient. The different sources of rank deficiency are identified, and include the Generalized Procrustes analysis itself, use of the correlation matrix, insufficient sample size, and phenotypic covariance. Only the last of these is of biological interest.

Our second goal is to examine a test case where a change in integration is known, allowing us to document how rank deficiency affects two measures of whole shape integration (eigenvalue standard deviation and standardized generalized variance). This test case utilizes the dire wolf data set from Part 1, and introduces another population that is 5000 years older. Modularity models are generated and tested for both populations, showing that one population is more integrated than the other. We demonstrate that eigenvalue variance characterizes the integration change incorrectly, while the standardized generalized variance lacks sensitivity. Both metrics are impacted by the inclusion of many small eigenvalues arising from rank deficiency of the covariance matrix. We propose a modification of the standardized generalized variance, again based on information entropy, that considers only the eigenvalues carrying non-redundant information. We demonstrate that this metric is successful in identifying the integration change in the test case.

The third goal of this paper is to generalize the new metric to the case of arbitrary sample size. This is done by normalizing the new metric to the amount of information present in a permuted covariance matrix. We term the resulting metric the  relative dispersion, and it is sample size corrected. As a proof of concept we us the new metric to compare the dire wolf data set from the first part of this paper to a third data set comprising jaws of Smilodon fatalis. We demonstrate that the Smilodon jaw is much more integrated than the dire wolf jaw. Finally, this information entropy-based measures of integration allows comparison of whole shape integration in dense semilandmark environments, allowing characterization of the information content of any given shape, a quantity we term  latent dispersion.

sentences: Fatty acids produced by the gut microbiota dampen host inflammatory responses by modulating intestinal SUMOylation The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. These metabolites regulate intestinal cell activities by modulating either gene transcription or post-translational modifications of gut proteins. The effect of gut commensal bacteria on SUMOylation, an essential ubiquitin-like modification in intestinal physiology, remains however unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in different intestinal cell lines in a pH-dependent manner. We demonstrate that these metabolites induce an oxidative stress which inactivates intestinal deSUMOylases and promotes the hyperSUMOylation of chromatin-bound proteins. In order to determine the impact of these modifications on intestinal physiology, we focused on the NF-{kappa}B signaling pathway, a key player in inflammation known to be regulated by SUMOylation. We demonstrated that the hyperSUMOylation induced by SCFAs/BCFAs inhibits the activation of the NF-{kappa}B pathway in intestinal cells by blocking the degradation of the inhibitory factor I{kappa}B in response to TNF. This results in a decrease in pro-inflammatory cytokines expression, such as IL8 or CCL20, as well as a decrease in intestinal epithelial permeability in response to TNF. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses by the gut microbiota.

sentences: Helping those who help others for indirect fitness benefits not indirect reciprocity Helping those who help others appears to be a widespread phenomenon. It is typically framed as indirect reciprocity in which individuals who are seen to help later receive returns from third parties. However, indirect reciprocity only works when individuals condition their help not just on how their recipient has behaved in the past but also on whether their recipient was justified in behaving that way. It also requires sufficient repeated interactions of this type among other individuals for a benefit to be reciprocated. These factors limit the scope of indirect reciprocity to explain cases where people do help those who help others. Here, I propose instead that helping can be explained by the indirect fitness benefits (or  relatedness) that result from helping other helpers in groups. This means that when individuals help other helpers, they may not make any returns via indirect reciprocity, but rather they may be helping a strategy of helping those who help. In this way, the helping strategy can spread even when helping has no net benefit to the individual helper. This is a form of relatedness in which individuals help their kin that are recognized by their helping behaviour. As such, conditional helping is likely to be found where population structure promotes relatedness through non-random association. The analysis suggests indirect reciprocity may not have played the decisive role in the evolution of human cooperation that is often thought, but paradoxically that the use of image scores deserves renewed attention as a strategy of helping those with the same behaviour.

sentences: Hierarchical progressive learning of cell identities in single-cell data Supervised methods are increasingly used to identify cell populations in single-cell data. Yet, current methods are limited in their ability to learn from multiple datasets simultaneously, are hampered by the annotation of datasets at different resolutions, and do not preserve annotations when retrained on new datasets. The latter point is especially important as researchers cannot rely on downstream analysis performed using earlier versions of the dataset. Here, we present scHPL, a hierarchical progressive learning method which allows continuous learning from single-cell data by leveraging the different resolutions of annotations across multiple datasets to learn and continuously update a classification tree. We evaluate the classification and tree learning performance using simulated as well as real datasets and show that scHPL can successfully learn known cellular hierarchies from multiple datasets while preserving the original annotations. scHPL is available at https://github.com/lcmmichielsen/hierarchicalprogressivelearning.

sentences: A meta-analysis of the effect of organic and mineral fertilizers on soil microbial diversity Organic agriculture, employing manures or composts, has been proposed as a way of mitigating undesirable impacts of mineral fertilizer use. Of particular interest is the effect of fertilizer regime on soil microbes, which are key to nutrient cycling, plant health and soil structure. However, the effect of fertilizers on soil microbial diversity remains poorly understood. Since biological diversity is an important determinant of ecosystem function and a fundamental metric in community ecology, the effects of fertilizer regimes on soil microbial diversity are of theoretical and applied interest. Here, we conduct a meta-analysis of 37 studies reporting microbial diversity metrics in mineral fertilized (NPK), organically fertilized (ORG) and unfertilized control (CON) soils. Of these studies, 32 reported taxonomic diversity derived from sequencing, gradient gel electrophoresis, or RFLP. Functional diversity, derived from Biolog Ecoplate measures of carbon substrate metabolism, was reported in 8 studies, with 3 studies reporting both diversity metrics. Bacterial and archaeal diversity was reported in 28 taxonomic studies, and fungal diversity in 8 taxonomic studies. We found that functional diversity was 2.8 % greater in NPK compared with CON, 7.0 % greater in ORG vs CON, and 3.8 % greater in ORG vs NPK. Bacterial and archaeal taxonomic diversity was not significantly different between NPK and CON, but on average 2.9% greater in ORG vs CON, and 2.4 % greater in ORG vs. NPK. Fungal taxonomic diversity was not significantly different between any treatment pairs. There was very high residual heterogeneity in all meta-analyses of soil diversity, suggesting that a large amount of further research is required to fully understand the influence of fertilizer regimes on microbial diversity and ecosystem function.

sentences: SPARClink: an interactive tool to visualize the impact of the SPARC program The NIH SPARC program seeks to accelerate the development of therapeutic devices that modulate electrical activity in nerves to improve organ function. SPARC-funded researchers are generating rich datasets from neuromodulation research that are curated and shared according to FAIR (Findable, Accessible, Interoperable, and Reusable) guidelines and are accessible to the public on the SPARC data portal. Keeping track of the utilization of these datasets within the larger research community is a feature that will benefit data generating researchers in showcasing the impact of their SPARC outcomes. This will also allow the SPARC program to display the impact of the FAIR data curation and sharing practices that have been implemented. This manuscript provides the methods and outcomes of SPARClink, our web tool for visualizing the impact of SPARC, which won the 2nd prize at the 2021 SPARC FAIR Codeathon. With SPARClink, we built a system that automatically and continuously finds new published SPARC scientific outputs (datasets, publications, protocols) and the external resources referring to them. SPARC datasets and protocols are queried using publicly accessible REST APIs (provided by Pennsieve and Protocols.io) and stored in a publicly accessible database. Citation information for these resources is retrieved using the NIH reporter API and NCBI Entrez system. A novel knowledge-graph-based structure was created to visualize the results of these queries and showcase the impact that the FAIR data principles can have on the research landscape when they are adopted by a consortium.

sentences: A graph-based cell tracking algorithm with few manually tunable parameters and automated segmentation error correction Automatic cell segmentation and tracking enables to gain quantitative insights into the processes driving cell migration. To investigate new data with minimal manual effort, cell tracking algorithms should be easy to apply and reduce manual curation time by providing automatic correction of segmentation errors. Current cell tracking algorithms, however, are either easy to apply to new data sets but lack automatic segmentation error correction, or have a vast set of parameters that needs either manual tuning or annotated data for parameter tuning. In this work, we propose a tracking algorithm with only few manually tunable parameters and automatic segmentation error correction. Moreover, no training data is needed. We compare the performance of our approach to three well-performing tracking algorithms from the Cell Tracking Challenge on data sets with simulated, degraded segmentation - including false negatives, over- and under-segmentation errors. Our tracking algorithm can correct false negatives, over- and under-segmentation errors as well as a mixture of the aforementioned segmentation errors. On data sets with under-segmentation errors or a mixture of segmentation errors our approach performs best. Moreover, without requiring additional manual tuning, our approach ranks several times in the top 3 on the 6th edition of the Cell Tracking Challenge.

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Transcriptional variability accelerates pre-leukemia by cell diversification and perturbation of protein synthesis Transcriptional variability facilitates stochastic cell diversification and can in turn underpin adaptation to stress or injury. We hypothesize that it may analogously facilitate progression of pre-malignancy to cancer. To investigate this, we initiated pre-leukemia in mouse cells with enhanced transcriptional variability due to conditional disruption of the histone lysine acetyltransferase gene Kat2a. By combining single-cell RNA-sequencing of pre-leukemia with functional analysis of transformation, we show that Kat2a loss results in global variegation of cell identity and accumulation of pre-leukemic cells. Leukemia progression is subsequently facilitated by destabilization of ribosome biogenesis and protein synthesis, which confer a transient transformation advantage. The contribution of transcriptional variability to early cancer evolution reflects a generic role in promoting cell fate transitions, which, in the case of well-adapted malignancies, contrastingly differentiates and depletes cancer stem cells. In other words, transcriptional variability confers forward momentum to cell fate systems, with differential multi-stage impact throughout cancer evolution.

One-sentence summaryLoss of Kat2a enhances transcriptional variability of ribosome biosynthetic programs and transiently accelerates pre-leukemia

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Independent Genomic Sources of Brain Structure and Function IntroductionThe last decade has seen a surge in well powered genome-wide association studies (GWASs) of complex behavioural traits, disorders, and more recently, of brain structural and functional neuroimaging features. However, the extreme polygenicity of these complex traits makes it difficult to translate the GWAS signal into mechanistic biological insights. We postulate that the covariance of SNP-effects across many brain features, as be captured by latent genomic components of SNP effect sizes. These may partly reflect the concerted multi-locus genomic effects through known molecular pathways and protein-protein interactions. Here, we test the feasibility of a new data-driven method to derive such latent components of genome-wide effects on more than thousand neuroimaging derived traits, and investigate their utility in interpreting the complex biological processes that shape the GWAS signal.

MethodsWe downloaded the GWAS summary statistics of 3,143 brain imaging-derived phenotypes (IDPs) from the UK Biobank, provided by the Oxford Brain Imaging Genetics (BIG) Server (Elliott et al. 2018). Probabilistic independent component analysis (ICA) was used to extract two hundred independent genomic components from the matrix of SNP-effect sizes. We qualitatively describe the distribution of the latent components loadings in the neuroimaging and the genomic dimensions. Gene-wide statistics were calculated for each genomic component. We tested the genomic components enrichment for molecular pathways using MSigDB, and for single-cell RNA-sequencing of adult and foetal brain cells.

Results200 components explained 80% of the variance in SNP-effects sizes. Each MRI modality and data processing method projected the imaging data into a clearly distinct cluster in the genomic component embedded space. Among the 200 genomic components, 157 were clearly driven by a single locus, while 39 were highly polygenic. Together, these 39 components were significantly enriched for 2,274 MSigDB gene sets (fully corrected for multiple testing across gene-sets and components). Several components were sensitive to molecular pathways, single cell expression profiles, and brain traits in patterns consistent with knowledge across these biological levels. To illustrate this, we highlight a component that implicated axonal regeneration pathways, which was specifically enriched for gene expression in oligodendrocyte precursors, microglia and astrocytes, and loaded highly on white matter neuroimaging traits. We highlight a second component that implicated synaptic function and neuron projection organization pathways that was specifically enriched for neuronal cell transcriptomes.

ConclusionWe propose genomic ICA as a new method to identify latent genetic factors influencing brain structure and function by multimodal MRI. The derived latent genomic dimensions are highly sensitive to known molecular pathways and cell-specific gene expression profiles. Genomic ICA may help to disentangle the many different biological routes by which the genome defines the inter-individual variation of the brain. Future research is aimed at using this method to profile individual subjects genomic data along the new latent dimensions and evaluating the utility of these dimensions in stratifying heterogeneous patient populations.

sentences: pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

sentences: A nesprin-4/kinesin-1 cargo model for nucleus positioning in cochlear outer hair cells Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

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Mitochondrial OPA1 deficiency causes reversible defects in adult neurogenesis-associated spatial memory in mice Mitochondria are integrative hubs central to cellular adaptive pathways. Such pathways are critical in highly differentiated post-mitotic neurons, the plasticity of which sustains brain function. Consequently, defects in mitochondrial dynamics and quality control appear instrumental in neurodegenerative diseases and may also participate in cognitive impairments. To directly test this hypothesis, we analyzed cognitive performances in a mouse mitochondria-based disease model, due to haploinsufficiency in the mitochondrial optic-atrophy-type-1 (OPA1) protein. While in Dominant Optic Atrophy (DOA) models, the known main symptoms are late onset visual deficits, we discovered early impairments in hippocampus-dependent spatial memory attributable to defects in adult neurogenesis. Moreover, less connected hippocampal adult-born neurons showed a decrease in mitochondrial content. Remarkably, modulating mitochondrial function through voluntary exercise or pharmacological treatment restored spatial memory.

Altogether, our study identifies a crucial role for OPA1-dependent mitochondrial functions in adult neurogenesis, and thus in hippocampal-dependent cognitive functions. More generally, our findings show that adult neurogenesis is highly sensitive to mild mitochondrial defects, generating impairments in spatial memory that can be detected at an early stage and counterbalanced by physical exercise and pharmacological targeting of mitochondrial dynamics. Thus, early amplification of mitochondrial function appears beneficial for late-onset neurodegenerative diseases.

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Base-resolution prediction of transcription factor binding signals by a deep learning framework Transcription factors (TFs) play an important role in regulating gene expression, thus the identification of the sites bound by them has become a fundamental step for molecular and cellular biology. In this paper, we developed a deep learning framework leveraging existing fully convolutional neural networks (FCN) to predict TF-DNA binding signals at the base-resolution level, called FCNsignal. The proposed FCNsignal can simultaneously achieve the following tasks: (i) modeling the base-resolution signals of binding regions; (ii) discriminating binding or non-binding regions; (iii) locating TF-DNA binding regions; (iv) predicting binding motifs. The experimental results on 53 TF ChIP-seq datasets and 6 chromatin accessibility ATAC-seq datasets show that our proposed framework outperforms some existing state-of-the-art methods. In addition, we explored to use the trained FCNsignal to locate all potential TF-DNA binding regions on a whole chromosome and predict DNA sequences of arbitrary length, and the results show that our framework can find most of the known binding regions and accept sequences of arbitrary length. Furthermore, we demonstrated the potential ability of our framework in discovering causal disease-associated single-nucleotide polymorphisms (SNPs) through a series of experiments.

Author summaryIdentification of transcription factor binding sites (TFBSs) is fundamental to study gene regulatory networks in biological systems, as TFs activate or suppress the transcription of genes by binding to specific TFBSs. With the development of high-throughput sequencing technologies and deep learning (DL), several DL-based approaches have been developed for systematically studying TFBSs, achieving impressive performance. Nevertheless, these methods either excessively focus on discriminating binding or non-binding sequences or individually accomplish multiple TFBSs-associated tasks. In this work, we provide an integrated framework, which utilizes the FCN architecture to predict TF-DNA binding signals at the base-resolution level, to simultaneously study multiple TFBSs-associated tasks. More importantly, we also demonstrate that our proposed framework has the ability to locate all potential TF-DNA binding regions from DNA sequences of arbitrary length. We hope that our framework can provide a new perspective on studying the mechanism of TF-DNA binding and its related tasks.

sentences: NGSpop: A desktop software that supports population studies by identifying sequence variations from next-generation sequencing data Next-generation sequencing (NGS) is widely used in all areas of genetic research, such as for genetic disease diagnosis and breeding, and it can produce massive amounts of data. The identification of sequence variants is an important step when processing large NGS datasets; however, currently, the process is complicated, repetitive, and requires concentration, which can be taxing on the researcher. Therefore, to support researchers who are not familiar with bioinformatics in identifying sequence variations regularly from large datasets, we have developed a fully automated desktop software, NGSpop. NGSpop includes functionalities for all the variant calling and visualization procedures used when processing NGS data, such as quality control, mapping, filtering details, and variant calling. In the variant calling step, the user can select the GATK or DeepVariant algorithm for variant calling. These algorithms can be executed using pre-set pipelines and options or customized with the user-specified options. NGSpop is implemented using JavaFX (version 1.8) and can thus be run on Unix like operating systems such as Ubuntu Linux (version 16.04, 18.0.4). Although there are several pipelines and visualization tools available for NGS data analysis, most integrated environments do not support batch processes; thus, variant detection cannot be automated for population-level studies. The NGSpop software, developed in this study, has an easy-to-use interface and helps in rapid analysis of multiple NGS data from population studies.

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Potential therapy for progressive vision loss due to PCDH15-associated Usher syndrome developed in an orthologous Usher mouse Usher syndrome type I (USH1) is characterized by congenital deafness, vestibular areflexia, and progressive retinal degeneration with age. The protein-truncating p.Arg245* founder variant of PCDH15 has an ~2% carrier frequency among Ashkenazi Jews, accounting for nearly 60% of their USH1 cases. Here, longitudinal ocular phenotyping in thirteen USH1F individuals harboring the p.Arg245* variant revealed progressive retinal degeneration, leading to severe loss of vision with macular atrophy by the sixth decade. Half of the affected individuals met either the visual acuity or visual field loss definition for legal blindness by the middle of their fifth decade of life. Mice homozygous for p.Arg250* (Pcdh15R250X; equivalent to human p.Arg245*) also have early visual deficits evaluated using electroretinography. Light-dependent translocation of phototransduction cascade proteins, arrestin and transducin, was found to be impaired in Pcdh15R250X mice. Retinal pigment epithelium-(RPE) specific visual retinoid cycle proteins, RPE65 which converts all-trans retinoids to 11-cis retinoids and CRALBP that transports retinoids, and key retinoid levels were also reduced in Pcdh15R250X mice, suggesting a dual role for protocadherin-15 in photoreceptors and RPE. Administration of exogenous 9-cis retinal, an analog of the naturally occurring 11-cis retinal, improved ERG amplitudes in these mutant mice, suggesting a basis for a clinical trial of exogenous FDA approved retinoids to preserve vision in USH1F patients.

SummaryIn a preclinical setting studying exogenous retinoids using a novel Usher syndrome mouse model, we describe a potential therapy to treat PCDH15-mediated visual dysfunction.

sentences: Phyletic distribution and diversification of the Phage Shock Protein stress response system in bacteria and archaea The bacterial cell envelope is an essential structure that protects the cell from environmental threats, while simultaneously serving as communication interface and diffusion barrier. Therefore, maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protection networks that connect stress perception, transmembrane signal transduction and mediation of cellular responses upon cell envelope stress. The phage shock protein (PSP) stress response is one of such conserved protection networks. Most of the knowledge about the Psp response comes from studies in the Gram-negative model bacterium, Escherichia coli where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of Proteobacteria, Actinobacteria, and Firmicutes; however, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and demonstrated that the PSP system deviates dramatically from the proteobacterial prototype. Two of its core proteins, PspA and PspC, have been integrated in various (often phylum-specifically) conserved protein networks during evolution. Based on protein sequence and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system of PSP networks in bacteria and archaea. We performed a comprehensive in vivo protein interaction screen for the PSP network newly identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected PSP response system, illustrating the validity of our approach. Our study highlights the diversity of PSP organization and function across many bacterial and archaeal phyla and will serve as foundation for future studies of this envelope stress response beyond model organisms.

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Chronic Stress Alters Astrocyte Morphology in Mouse Prefrontal Cortex BackgroundNeuromorphological changes are consistently reported in the prefrontal cortex (PFC) of patients with stress-related disorders and in rodent stress models, but the effects of stress on astrocyte morphology and potential link to behavioral deficits are relatively unknown.

MethodsTo answer these questions, transgenic mice expressing green fluorescent protein (GFP) under the glial fibrillary acid protein (GFAP) promotor were subjected to 7, 21 or 35 days of chronic restraint stress (CRS). CRS induced behavioral effects on anhedonia- and anxiety-like behaviors were measured using the sucrose intake and the PhenoTyper tests, respectively. PFC GFP+ or GFAP+ cells morphology was assessed using Sholl analysis and associations with behavior were determined using correlation analysis.

ResultsCRS-exposed male and female mice displayed anxiety-like behavior at 7, 21 and 35 days and anhedonia-like behavior at 35 days. Analysis of GFAP+ cell morphology revealed significant atrophy of distal processes following 21 and 35 days of CRS. CRS induced similar decreases in intersections at distal radii for GFP+ cells, accompanied by increased proximal processes. In males, the number of intersections at the most distal radius step significantly correlated with anhedonia-like behavior (r=0.622, p<0.05) for GFP+ cells and with behavioral emotionality calculated by z-scoring all behavioral measured deficits (r=-0.667, p<0.05). Similar but not significant correlations were observed in females. No correlation between GFP+ cell atrophy with anxiety-like behavior was found.

ConclusionChronic stress exposure induces a progressive atrophy of cortical astroglial cells, potentially contributing to maladaptive neuroplastic and behavioral changes associated with stress-related disorders.

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Somite morphogenesis is required for axial blood vessel formation Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate towards the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.

Summary statementRetinoic acid induced somite morphogenesis generates a midline cavity that accommodates migrating angioblasts, which form the axial blood vessels.

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Mechanism of a COVID-19 nanoparticle vaccine candidate that elicits a broadly neutralizing antibody response to SARS-CoV-2 variants Vaccines that induce potent neutralizing antibody (NAb) responses against emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for combating the coronavirus disease 2019 (COVID-19) pandemic. We demonstrated that mouse plasma induced by self-assembling protein nanoparticles (SApNPs) that present 20 rationally designed S2G{Delta}HR2 spikes of the ancestral Wuhan-Hu-1 strain can neutralize the B.1.1.7, B.1.351, P.1, and B.1.617 variants with the same potency. The adjuvant effect on vaccine-induced immunity was investigated by testing 16 formulations for the multilayered I3-01v9 SApNP. Using single-cell sorting, monoclonal antibodies (mAbs) with diverse neutralization breadth and potency were isolated from mice immunized with the receptor binding domain (RBD), S2G{Delta}HR2 spike, and SApNP vaccines. The mechanism of vaccine-induced immunity was examined in mice. Compared with the soluble spike, the I3-01v9 SApNP showed 6-fold longer retention, 4-fold greater presentation on follicular dendritic cell dendrites, and 5-fold stronger germinal center reactions in lymph node follicles.

ONE-SENTENCE SUMMARYWith a well-defined mechanism, spike nanoparticle vaccines can effectively counter SARS-CoV-2 variants.

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NestedBD: Bayesian Inference of Phylogenetic Trees From Single-Cell DNA Copy Number Profile Data Under a Birth-Death Model Copy number aberrations (CNAs) are ubiquitous in many types of cancer. Inferring CNAs from cancer genomic data could help shed light on the initiation, progression, and potential treatment of cancer. While such data have traditionally been available via "bulk sequencing", the more recently introduced techniques for single-cell DNA sequencing (scDNAseq) provide the type of data that makes CNA inference possible at the single-cell resolution.

In this paper, we introduce a new birth-death evolutionary model of CNAs as well as a Bayesian method, NestedBD, for the inference of evolutionary trees (topologies and branch lengths with relative mutation rates) from single-cell data under this model. We assessed the accuracy of our method on both simulated and biological data and compared it to the accuracy of two standard phylogenetic tools, namely neighbor-joining and maximum parsimony (MP). We show through simulations that our method infers more accurate topologies and branch lengths. We also studied the ancestral state reconstruction accuracy with the birth-death evolutionary model and found it outperformed MP. Finally, running all three methods on a colorectal cancer data set, we observed that among all three methods, only the phylogeny inferred by NestedBD clearly separated the primary tumor cells from the metastatic ones, providing a more plausible history of the tumor cells.

sentences: Predictors of Taxonomic Inflation and its Role in Primate Conservation Species are the main unit used to measure biodiversity, but different preferred diagnostic criteria can lead to very different delineations. For instance, named primate species have more than doubled since 1982. Such increases have been termed "taxonomic inflation" and have been attributed to the widespread adoption of the  phylogenetic species concept (PSC) in preference to the previously popular  biological species concept (BSC). Criticisms of the PSC have suggested taxonomic inflation may be biased toward particular taxa and have unfavourable consequences for conservation. Here, we explore predictors of taxonomic inflation across primate taxa since the initial application of the PSC nearly 40 years ago. We do not find evidence that diversification rate, the rate of lineage formation over evolutionary time, is linked to inflation, contrary to expectations if the PSC identifies incipient species. We also do not find evidence of research effort in fields where work has been suggested to motivate splitting being associated with increases in species numbers among genera. To test the suggestion that splitting groups is likely to increase their perceived risk of extinction, we test whether genera that have undergone more splitting have also observed a greater increase in their proportion of threatened species since the introduction of the PSC. We find no cohesive signal of inflation leading to higher threat probabilities across primate genera. Overall, this analysis sends a positive message that threat statuses of primate species are not being overwhelmingly affected by splitting in line with what has recently been reported for birds. Regardless, we echo warnings that it is unwise for conservation to be reliant on taxonomic stability. Species (however defined) are not independent from one another, thus, monitoring and managing them as such may not meet the overarching goal of conserving biodiversity.

sentences: MutSigCVsyn: Identification of Thirty Synonymous Cancer Drivers Synonymous mutations, which change only the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations can act as cancer drivers has been explored in several recent studies. However, none of these studies control for all three levels (patient, histology, and gene) of mutational heterogeneity that are known to affect the accurate identification of non-synonymous cancer drivers. Here, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify synonymous cancer drivers based on a novel non-coding background model that takes into account the mutational heterogeneity across these levels. Examining 2,572 PCAWG cancer whole-genome sequences, MutSigCVsyn identifies 30 novel synonymous drivers that include mutations in promising candidates like BCL-2. By bringing the best practices in non-synonymous driver identification to the analysis of synonymous drivers, these are promising candidates for future experimental study.

sentences: Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yor1, a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1{Delta} strains revealed defects in non-lytic exocytosis and capsule size, when compared to wild-type strains. We detected no difference in growth rates, cell body size and vesicle secretion. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

sentences: Impairment of SARS-CoV-2 spike-glycoprotein maturation and fusion-activity by nitazoxanide: an effect independent of spike variants emergence SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion-glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike-variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike biogenesis, hampering its maturation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

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MeSSII interplays the amylose and amylopectin biosynthesis by protein interactions in cassava storage root Starch is a glucose polymer synthesized by green plants for energy storage, and is crucial for plant growth and reproduction. The biosynthesis of starch polysaccharides is mediated by members of the large starch synthase (SS) protein superfamily. Here, we showed that in cassava storage roots, soluble starch synthase II (MeSSII) plays an important role in starch biosynthesis via forming protein complexes with other starch biosynthetic enzymes by directly interacting with MeSSI, MeSBEII and MeISAII. The MeSSII-RNAi cassava lines showed increased amylose content and reduced intermediate chain of amylopectin (B1 type) biosynthesis in their storage roots, leading to altered starch physico-chemical properties. Further gel permeation chromatography analysis of starch biosynthetic enzymes between the wild type and MeSSII-RNAi lines confirmed the key rule of MeSSII in the organization of heteromeric starch synthetic protein complexes, including the MeSSII-MeSSI-MeGBSSI and MeSBEII-MeSSI-MeSSII-MeISAII-MeISAI complexes. The lack of MeSSII in cassava also reduced the binding capacity of the MeSSI, MeSBEII, MeISAI, and MeISAII to starch granules. Our results show a close coordination between granule-bound starch synthase and amylopectin biosynthetic enzymes, which implies that the processes of amylose synthesis and amylopectin synthesis are directly interrelated. These findings shed light on the key components of the starch biosynthesis machinery in root crops.

One Sentence SummaryHere, by focusing on cassava SSII, we elucidated its function and the molecular mechanism by which it chaperones the starch synthase complex to regulate starch biosynthesis in cassava storage roots.

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