title: Thermal flexibility is a repeatable mechanism to cope with environmental stressors in a passerine bird

abstract:
O_LIFor many vertebrates, urban environments are characterised by frequent environmental stressors. Coping with such stressors can demand that urban individuals activate energetically costly physiological pathways (e.g. the fight-or-flight response) more regularly than rural-living conspecifics. However, urban environments also commonly demand appreciable expenditure toward thermoregulation, owing to their often extreme climatic variations. To date, whether and how vertebrates can balance expenditure toward both the physiological stress response and thermoregulation, and thus persist in an urbanising world, remains an unanswered and urgent question among ecologists.
C_LIO_LIIn some species, changes in body surface temperature (Ts) and peripheral heat loss (qTot) that accompany the stress response are thought to balance energetic expenditure toward thermoregulation and responding to a stressor. Thus, augmentation of stressinduced thermal responses may be a mechanism by which urban individuals cope with simultaneously high thermoregulatory and stress-physiological demands.
C_LIO_LIWe tested whether stress-induced changes in Ts and qTot: (1) differed between urban- and rural-origin individuals, (2) reduce thermoregulatory demands in urban individuals relative to rural conspecifics, and (3) meet an essential first criterion for evolutionary responses to selection (variability among, and consistency within, individuals).
C_LIO_LIUsing the black-capped chickadee (Poecile atricapillus; n = 19), we show that neither rapid nor chronic changes in Ts and qTot following exposure to randomised stressors differed between urban- and rural-origin individuals (nurban = 9; nrural = 10). Nevertheless, we do find that stress-induced changes in Ts and qTot are highly repeatable across chronic time periods (RTs = 0.61; RqTot = 0.67) and display signatures of stabilising or directional selection (i.e. reduced variability and increase repeatability relative to controls).
C_LIO_LIOur findings suggest that, although urban individuals appear no more able to balance expenditure toward thermoregulation and the stress response than rural conspecifics, the capacity to do so may be subject to selection in some species. To our knowledge this is also the first study to report repeatability of any theorised stress-induced trade-off.
C_LI


title: Identifying nearshore nursery habitats for sharks in the Eastern Tropical Pacific from fisheries landings and interviews

abstract: The Eastern Tropical Pacific (ETP) comprising the coasts of Costa Rica, Panama, Colombia and Ecuador, represents an area of high marine biodiversity that supports productive fisheries and acts as an important migratory corridor for many marine species. Despite its biological importance, the ETP is understudied and lacks sufficient data for science-based fisheries management and conservation decision-making. This study aims to consolidate understanding of the current and historical distribution of sharks and mobulid rays in the ETP. We used interviews of coastal community stakeholders to document traditional knowledge of shark and mobulid ray species and distributions. We also analysed small-scale fisheries landings data, where available, to quantify local exploitation patterns and the importance of sharks and rays in small-scale fisheries catches. All shark species landed in the dominant nearshore gillnet fishery show very low mean individuals weights (<5 kg), indicating that the fisheries are dominated by juveniles, captured. Aside from smooth-hounds (Mustelus spp.), the scalloped hammerhead shark, Sphyrna lewini, is the most frequently landed shark species in the region by weight and number, with peaks in abundance between April - July. From 132 interviews in 51 communities across the three countries, and landings data from two small-scale fisheries sites, we identified 41 sites in 12 broad geographical zones as important shark nursery habitats. Of these sites, 68% were associated closely with large mangrove systems of the ETP, highlighting the importance of this habitat for shark life history. No patterns were seen in the occurrence or distribution of mobulid rays in coastal areas. Marine protected areas and responsible fishing zones cover 37% of identified nursery habitats in the ETP, 30% in Costa Rica, 48% in Panama and 30% in Colombia. These findings provide an important benchmark of the conservation status of sharks in the ETP and allow for the prioritisation of research and policy-making.

category: ecology


title: Horizontal transfer of microbial toxin genes to gall midge genomes

abstract:
A growing body of evidence points to a role for horizontal gene transfer (HGT) in the evolution of animal novelties. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (CdtB) from the Acyrthosiphon pisum Secondary Endosymbiont (APSE) bacteriophage to drosophilid and aphid genomes. Here, we report that cdtB is also found in the nuclear genome of the gall-forming  swede midge Contarinia nasturtii (Diptera: Cecidomyiidae). We subsequently searched genome sequences of all available cecidomyiid species for evidence of microbe-to-insect HGT events. We found evidence of pervasive transfer of APSE-like toxin genes to cecidomyiid nuclear genomes. Many of the toxins encoded by these horizontally transferred genes target eukaryotic cells, rather than prokaryotes. In insects, catalytic residues important for toxin function are conserved. Phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor of the toxin gene to cecidomyiid genomes, suggesting a broader pool of microbial donor lineages. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for HGT, which showed, that prokaryotic-to-insect HGT was more likely to occur between taxa in common environments. Our study highlights the horizontal transfer of genes encoding a new functional class of proteins in insects, toxins that target eukaryotic cells, which is potentially important in mediating interactions with eukaryotic pathogens and parasites.

Significance StatementThe diversity of genes encoded by phages infecting bacterial symbionts of eukaryotes represents an enormous, relatively unexplored pool of new eukaryotic genes through horizontal gene transfer (HGT). In this study, we discovered pervasive HGT of toxin genes encoded by Acyrthosiphon pisum secondary endosymbiont (APSE) bacteriophages and other microbes to the nuclear genomes of gall midges (Diptera: Cecidomyiidae). We found five toxin genes were transferred horizontally from phage, bacteria, or fungi into genomes of several cecidomyiid species. These genes were aip56, cdtB, lysozyme, rhs, and sltxB. Most of the toxins encoded by these genes antagonize eukaryotic cells, and we posit that they may play a protective role in the insect immune system.

category: evolutionary biology


title: Cryptic microgeographic variation in responses of larval Atlantic cod to warmer temperatures

abstract: Although temperature is known to drive species dynamics and distributions, our understanding of the extent to which thermal plasticity varies within species is poor. Differences in plasticity can arise through local adaptation to heterogeneous environments, hybridization, and the release of cryptic genetic variation in novel environments. Here, wild Atlantic cod (Gadus morhua) from contrasting environments inside and outside of a fjord system in southern Norway spawned freely in a semi-natural laboratory environment, generating pure crosses and reciprocal hybrids. A common-garden rearing experiment of the larvae at 6{degrees}C, 9.5{degrees}C, and 13{degrees}C revealed cryptic genetic variation in thermal responses of growth and survival at warmer temperatures. Variation in growth plasticity was greatest from 9.5{degrees}C to 13{degrees}C, the latter of which exceeds temperatures currently typical of larvae in their native environments. In contrast to our prediction of intermediate hybrid responses consistent with additive genetic effects, one reciprocal hybrid cross showed a 4% increase in size at the highest temperature, whereas most crosses exhibited 4-12% reductions in size. All crosses experienced severe (76-93%) reductions in survival from 9.5{degrees}C to 13{degrees}C. Variation in survival plasticity suggests a genetically variable basis for the severity with which survival declines with increasing temperature and the potential for an adaptive response to warming. Notably, we demonstrate the potential for hybridization between coexisting  fjord and  North Sea ecotypes that naturally inhabit the inner and outer fjord environments at contrasting frequencies. Yet, ecotype explained a minor (3-10%) component of growth reaction norm variation, suggesting it is insufficient for describing important biological variation. Current broad-scale management and lack of coastal monitoring impede the development of strategies to maintain the potential for adaptation to warming temperatures in systems with such phenotypic complexity resulting from cryptic genetic variation, coexisting ecotypes, and gene flow.


title: Two transcriptionally distinct pathways drive female development in a reptile with both genetic and temperature dependent sex determination

abstract:
How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox;  CaRe status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.

Author SummaryIn many reptiles and fish, environment can determine, or influence, the sex of developing embryos. How this happens at a molecular level that has eluded resolution for half a century of intensive research. We studied the bearded dragon, a lizard that has sex chromosomes (ZZ male and ZW female), but in which that temperature can override ZZ sex chromosomes to cause male to female sex reversal. This provides an unparalleled opportunity to disentangle, in the same species, the biochemical pathways required to make a female by these two different routes. We sequenced the transcriptomes of gonads from developing ZZ reversed and normal ZW dragon embryos and discovered that different sets of genes are active in ovary development driven by genotype or temperature. Females whose sex was initiated by temperature showed a transcriptional profile consistent with the recently-proposed Calcium-Redox hypotheses of cellular temperature sensing. These findings are an important for understanding how the environment influences the development of sex, and more generally how the environment can epigenetically modify the action of genes.

category: genetics


title: REHE: Fast Variance Components Estimation for Linear Mixed Models

abstract: AO_SCPLOWBSTRACTC_SCPLOWLinear mixed models are widely used in ecological and biological applications, especially in genetic studies. Reliable estimation of variance components is crucial for using linear mixed models. However, standard methods, such as the restricted maximum likelihood (REML), are computationally inefficient and may be unstable with small samples. Other commonly used methods, such as the Haseman-Elston (HE) regression, may yield negative estimates of variances. Utilizing regularized estimation strategies, we propose the restricted Haseman-Elston (REHE) regression and REHE with resampling (reREHE) estimators, along with an inference framework for REHE, as fast and robust alternatives that provide non-negative estimates with comparable accuracy to REML. The merits of REHE are illustrated using real data and benchmark simulation studies.


title: High-throughput selection of microalgae based on biomass accumulation rates in production environments using PicoShell Particles

abstract: Production of high-energy lipids by microalgae may provide a sustainable, renewable energy source that can help tackle climate change. However, microalgae engineered to produce more lipids usually grow slowly, leading to reduced overall yields. Unfortunately, tools that enable the selection of cells based on growth while maintaining high biomass production, such as well-plates, water-in-oil droplet emulsions, and nanowell arrays do not provide production-relevant environments that cells experience in scaled-up cultures (e.g. bioreactors or outdoor cultivation farms). As a result, strains that are developed in the lab often do not exhibit the same beneficial phenotypic behavior when transferred to industrial production. Here we introduce PicoShells, picoliter-scale porous hydrogel compartments, that can enable >100,000 individual cells to be compartmentalized, cultured in production-relevant environments, and selected based on growth and biomass accumulation traits using standard flow cytometers. PicoShells consist of a hollow inner cavity where cells are encapsulated, and a porous outer shell that allows for continuous solution exchange with the external environment so that nutrients, cell-communication factors, and cytotoxic cellular byproducts can transport freely in and out of the inner cavity. PicoShells can also be placed directly into shaking flasks, bioreactors, or other production-relevant environments. We experimentally demonstrate that Chlorella sp. and Saccharomyces cerevisiae grow to significantly larger colony sizes in PicoShells than in water-in-oil droplet emulsions (P < 0.05). We have also demonstrated that PicoShells containing faster biomass accumulating Chlorella clonal colonies can be selected using a fluorescence-activated cell sorter and re-grown. Using the PicoShell process, we select a Chlorella population that accumulates biomass 8% faster than does an un-selected population after a single selection cycle.

category: bioengineering


title: Automatic Identification and Avoidance of Axon Bundle Activation for Epiretinal Prosthesis

abstract:
Retinal prostheses must be able to activate cells in a selective way in order to restore high-fidelity vision. However, inadvertent activation of far-away retinal ganglion cells (RGCs) through electrical stimulation of axon bundles can produce irregular and poorly controlled percepts, limiting artificial vision. Therefore, the problem of axon bundle activation can be defined as the axonal stimulation of RGCs with unknown soma and receptive field locations, typically outside the electrode array. Here, a new algorithm is presented that utilizes electrical recordings to determine the stimulation current amplitudes above which bundle activation occurs. The method exploits several spatiotemporal characteristics of electrically-evoked spikes to overcome the challenge of detecting small axonal spikes in extracellular recordings. The algorithm was validated using large-scale ex vivo stimulation and recording experiments in macaque retina, by comparing algorithmically and manually identified bundle activation thresholds. The algorithm could be used in a closed-loop manner by a future epiretinal prosthesis to reduce poorly controlled visual percepts associated with bundle activation. The method may also be applicable to other types of retinal implants and to cortical implants.

ContributionsPT developed the algorithm and analyzed the data, with input from SMi and EJC. NB and NS helped with the analysis. SMa and LG performed dissections and collected the data. PT and VFH performed manual identification. PH, AS and AML developed and supported recording hardware and software. PT, EJC and SMi wrote the manuscript. NS and SMa edited it. EJC and SMi supervised the project.


title: Using de novo assembly to identify structural variation of complex immune system gene regions

abstract: Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data; application of these methods to larger samples would provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

category: bioinformatics


title: PIGNON: A protein-protein interaction-guided functional enrichment analysis for quantitative proteomics

abstract:
BackgroundQuantitative proteomics studies are often used to detect proteins that are differentially expressed across different experimental conditions. Functional enrichment analyses are then typically used to detect annotations, such as biological processes that are significantly enriched among such differentially expressed proteins to provide insights into the molecular impacts of the studied conditions. While common, this analytical pipeline heavily relies on arbitrary thresholds of significance. Indeed, a functional annotation may be dysregulated in a given experimental condition, while none or very few of its proteins may be individually considered to be significantly differentially expressed. Such an annotation would therefore be missed by standard approaches.

ResultsHerein, we propose a novel graph theory-based method, PIGNON, for the detection of differentially expressed functional annotations in different conditions. PIGNON does not assess the statistical significance of individual genes, but rather maps protein differential expression levels onto a protein-protein interaction network and measures the clustering of proteins from a given functional annotation within the network. This process allows the detection of functional annotations for which the proteins are differentially expressed and grouped in the network. A Monte-Carlo sampling approach is used to assess the clustering of proteins in an expression-weighted network. When applied to a quantitative proteomics analysis of different molecular subtypes of breast cancer, PIGNON detects Gene Ontology terms that are both significantly clustered in a protein-protein interaction network and differentially expressed across two breast cancer subtypes. PIGNON identified 168 breast cancer pathways dysregulated and clustered within the network between the HER2+ and triple negative subtypes, 203 breast cancer pathways shared by HER2+ and hormone receptor positive subtypes, 19 breast cancer pathways shared by hormone receptor positive and triple negative breast that are not detected by standard approaches. PIGNON identifies functional annotations that have been previously associated with specific breast cancer subtypes as well as novel annotations that may be implicated in the diseases.

ConclusionPIGNON provides an alternative to functional enrichment analyses and a more comprehensive characterization of quantitative datasets. Hence, it contributes to yielding a better understanding of dysregulated functions and processes in biological samples under different conditions.


title: SplicingFactory - Splicing diversity analysis for transcriptome data

abstract:
MotivationAlternative splicing contributes to the diversity of RNA found in biological samples. Current tools investigating patterns of alternative splicing check for coordinated changes in the expression or relative ratio of RNA isoforms where specific isoforms are up- or downregulated in a condition. However, the molecular process of splicing is stochastic and changes in RNA isoform diversity for a gene might arise between samples or conditions. A specific condition can be dominated by a single isoform, while multiple isoforms with similar expression levels can be present in a different condition. These changes might be the result of mutations, drug treatments or differences in the cellular or tissue environment. Here, we present a tool for the characterization and analysis of RNA isoform diversity using isoform level expression measurements.

ResultsWe developed an R package called SplicingFactory, to calculate various RNA isoform diversity metrics, and compare them across conditions. Using the package, we tested the effect of RNA-seq quantification tools, quantification uncertainty, gene expression levels, and isoform numbers on the isoform diversity calculation. We analyzed a set of CD34+ hematopoietic stem cells and myelodysplastic syndrome samples and found a set of genes whose isoform diversity change is associated with SF3B1 mutations.

Availability and implementationThe SplicingFactory package is freely available under the GPL-3.0 license from Bioconductor for the Windows, MacOS and Linux operating systems (https://www.bioconductor.org/packages/release/bioc/html/SplicingFactory.html).

Contactsebestyen.endre@med.semmelweis-univ.hu

Supplementary informationSupplementary data are available at Bioinformatics online.


title: RAD51 nuclear recruitment and inhibition towards innovative strategies against pancreatic cancer

abstract:
RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment.

Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a  domino effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug.

The present study sheds further light on the complexity of the HR pathway, paving the way for designing peptide and small organic molecule inhibitors of RAD51 as innovative anticancer and chemo/radiosensitizer compounds.

category: biophysics


title: Reciprocalspaceship: A Python Library for Crystallographic Data Analysis

abstract: AO_SCPLOWBSTRACTC_SCPLOWX-ray crystallography is an invaluable technique for studying the atomic structure of macromolecules. Much of crystallographys success is due to the software packages developed to enable the automated processing of diffraction data. However, the analysis of unconventional diffraction experiments can still pose significant challenges--many existing programs are closed-source, sparsely documented, or are challenging to integrate with modern libraries for scientific computing and machine learning. Here we describe reciprocalspaceship, a Python library for exploring reciprocal space. It provides a tabular representation for reflection data from diffraction experiments that extends the widely-used pandas library with built-in methods for handling space group, unit cell, and symmetry-based operations. As we illustrate, this library facilitates new modes of exploratory data analysis while supporting the prototyping, development, and release of new methods.


title: The C. elegans DAF-19M module: a shift from general ciliogenesis to ciliary and behavioral specialization

abstract: In animals, cilia are important for the interaction with environments and the proper function of tissues and organs. Understanding the distinctive identities of each type of ciliated cell is essential for therapeutic solutions for ciliopathies, complex disorders with impairments of various organs caused by defective cilia development and function. Here, we report a regulatory module consisting of a cascade of transcription factors and their target genes that confer the cell type-specific ciliary identities on the IL2 ciliated neurons in C. elegans. We found that DAF-19M, isoform of the sole C. elegans RFX transcription factor DAF-19, through X-box promoter motif variants, heads a regulatory module in IL2 neurons, comprising the core target genes klp-6 (kinesin), osm-9 (TRP channel), and cwp-4 (novel); under the overall control of terminal selector proteins UNC-86 and CFI-1. Considering the conservation of this DAF-19M module in IL2 neurons for nictation, a dauer larva-specific behavior, and in male-specific neurons for mating behavior, we propose the existence of an evolutionarily adaptable, hard-wired genetic module for distinct behaviors that share the feature "recognizing the environment."

category: molecular biology


title: Tig1 regulates proximo-distal identity during salamander limb regeneration

abstract: Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed  positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

category: developmental biology


title: Essential role of CFAP53 in sperm flagellum biogenesis

abstract: The sperm flagellum is essential for male fertility. Despite vigorous research progress towards understanding the pathogenesis of flagellum-related diseases, much remains unknown about the mechanisms underlying the flagellum biogenesis itself. Here, we show that the cilia and flagella associated protein 53 (Cfap53) gene is predominantly expressed in testes, and it is essential for sperm flagellum biogenesis. The knockout of this gene resulted in complete infertility in male mice but not in the females. CFAP53 localized to the manchette and sperm tail during spermiogenesis, the knockout of this gene impaired flagellum biogenesis. Furthermore, we identified two manchette and sperm tail-associated proteins that interacted with CFAP53 during spermiogenesis. The disruption of Cfap53 decreased the expression level of these two proteins and disrupted their localization in spermatids. Together, our results suggest that CFAP53 is an essential protein for sperm flagellum biogenesis, and its mutations might be associated with MMAF.


title: CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

abstract: AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

category: cancer biology


title: Epigenetic therapy to enhance therapeutic effects of PD-1 inhibition in uveal melanoma

abstract: Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but none of the treatments are approved for patients with metastatic uveal melanoma (UM). Here we hypothesized that the poor responses to immunotherapy of UM can be enhanced by epigenetic modulation using HDAC or BET inhibitors (BETi). Cultured uveal melanoma cells were treated with the HDAC inhibitor (HDACi) entinostat or BETi JQ1. Entinostat induced HLA expression and PD-L1, but JQ1 did not. A syngenic mouse model carrying B16-F10 melanoma cells were treated with PD-1 and CTLA-4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET-726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic UM is warranted.


title: DPSleep: Open-Source Longitudinal Sleep Analysis From Accelerometer Data

abstract: Wearable devices are now widely available to collect continuous objective behavioral data from individuals and to measure sleep. Here we introduce a pipeline to infer sleep onset, duration, and quality from raw accelerometer data and then quantify relationships between derived sleep metrics and other variables of interest. The pipeline released here for the deep phenotyping of sleep, as the "DPSleep" software package, uses (a) a stepwise algorithm to detect missing data; (b) within-individual, minute-based, spectral power percentiles of activity; and (c) iterative, forward- and backward-sliding windows to estimate the major Sleep Episode onset and offset. Software modules allow for manual quality control adjustment of derived sleep features and correction for time zone changes. In this report, we illustrate the pipeline with data from participants studied for more than 200 days each. Actigraphy-based measures of sleep duration are associated with self-report rating of sleep quality. Simultaneous measures of smartphone use and GPS location data support the validity of the sleep timing inferences and reveal how phone measures of sleep timing can differ from actigraphy data. We discuss the uses of DPSleep in relation to other available sleep estimation approaches and provide example use cases that include multi-dimensional, deep longitudinal phenotyping, extended measurement of dynamics associated with mental illness, and the possibility of combining wearable actigraphy and personal electronic device data (e.g., smartphone, tablet) to measure individual differences across a wide range of behavioral variation in health and disease.

category: neuroscience


title: Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to -lactam antibiotics

abstract: The Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded {beta}-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent {beta}-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased {beta}-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact {beta}-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased {beta}-lactam resistance. Mutation of lgt in an lspA background restored {beta}-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to {beta}-lactam antibiotics through impacts on cell envelope components other than PG.

category: microbiology


title: Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to beta-lactam antibiotics


title: Artificial Intelligence Approaches to Assessing Primary Cilia

abstract:
Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

category: cell biology


title: Living with relatives offsets the harm caused by pathogens in natural populations

abstract: Living with relatives can be highly beneficial, enhancing reproduction and survival. However, high relatedness can increase susceptibility to pathogens, a phenomenon known as the  monoculture effect. Here we examine if the benefits of living with relatives offsets the harm caused by pathogens, and if this depends if species typically live with kin. Using comparative meta-analysis across plants, animals and bacteria (nspecies= 56), we show that high within-group relatedness increases mortality when pathogens are present. Contrastingly, mortality decreased with relatedness when pathogens were rare, particularly in species that live with kin. Variation in pathogen abundances was lower across groups of relatives, but rates of mortality were more unpredictable. The effects of within-group relatedness were only evident when pathogens were manipulated, suggesting that the harm caused by pathogens is masked by the benefits of living with relatives. These results highlight the importance of kin selection for understanding disease spread in natural populations.


title: The mutation profile of SARS-CoV-2 is primarily shaped by the host antiviral defense

abstract:
Understanding SARS-CoV-2 evolution is a fundamental effort in coping with the COVID-19 pandemic. The virus genomes have been broadly evolving due to the high number of infected hosts world-wide. Mutagenesis and selection are the two inter-dependent mechanisms of virus diversification. However, which mechanisms contribute to the mutation profiles of SARS-CoV-2 remain under-explored. Here, we delineate the contribution of mutagenesis and selection to the genome diversity of SARS-CoV-2 isolates. We generated a comprehensive phylogenetic tree with representative genomes. Instead of counting mutations relative to the reference genome, we identified each mutation event at the nodes of the phylogenetic tree. With this approach, we obtained the mutation events that are independent of each other and generated the mutation profile of SARS-CoV-2 genomes. The results suggest that the heterogeneous mutation patterns are mainly reflections of host (i) antiviral mechanisms that are achieved through APOBEC, ADAR, and ZAP proteins and (ii) probable adaptation against reactive oxygen species.

ImportanceSARS-CoV-2 genomes are evolving worldwide. Revealing the evolutionary characteristics of SARS-CoV-2 is essential to understand host-virus interactions. Here, we aim to understand whether mutagenesis or selection is the primary driver of SARS-CoV-2 evolution. This study provides an unbiased computational method for profiling and analyzing independently occurring SARS-CoV-2 mutations. The results point out three host antiviral mechanisms shaping the mutational profile of SARS-CoV-2 through APOBEC, ADAR, and ZAP proteins. Besides, reactive oxygen species might have an impact on the SARS-CoV-2 mutagenesis.


title: S-acylation targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by T cell receptors at the immune synapse

abstract: Efficient immune responses require Ca2+ fluxes across ORAI1 channels during engagement of T cell receptors (TCR) at the immune synapse (IS) between T cells and antigen presenting cells. Here, we show that ZDHHC20-mediated S-acylation of the ORAI1 channel at residue Cys143 is required for TCR assembly and signaling at the IS. Cys143 mutations reduced ORAI1 currents and store-operated Ca2+ entry in HEK-293 cells and nearly abrogated long-lasting Ca2+ elevations, NFATC1 translocation, and IL-2 secretion evoked by TCR engagement in Jurkat T cells. The acylation-deficient channel had reduced mobility in lipids, accumulated in cholesterol-poor domains, formed tiny clusters, failed to reach the IS and unexpectedly also prevented TCR recruitment to the IS. Our results establish S-acylation as a critical regulator of ORAI1 channel assembly and function at the IS and reveal that local Ca2+ fluxes are required for TCR recruitment to the synapse.

category: biochemistry


title: The yeast eIF2 kinase Gcn2 facilitates H2O2-mediated feedback inhibition of both protein synthesis and ER oxidative folding during recombinant protein production

abstract:
Recombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, micro-cultivation and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak -amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find Gcn2-dependent phosphorylation of eIF2 to increase alongside translational attenuation in strains engineered to produce large amounts of -amylase. Gcn2 removal significantly improved -amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H2O2 levels and the unfolded protein response whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H2O2.

ImportanceReactive oxygen species (ROS) accumulate during recombinant protein production both in yeast and Chinese hamster ovary cells, two of the most popular organisms used in the multi-million dollar protein production industry. Here we document increased H2O2 in the cytosol of yeast cells producing -amylase. Since H2O2 predominantly targets the protein synthesis machinery and activates the translation initiation factor kinase Gcn2, we removed Gcn2, resulting in increased recombinant -amylase production in two different previously engineered high-producing protein production strains. Removal of this negative feed-back loop thus represents a complementary strategy for improving recombinant protein production efforts currently used in yeast. Gcn2-deficiency also increased the expression of antioxidant genes and the ER-foldase PDI1, suggesting that protein synthesis and ER oxidative folding are linked and feed-back regulated via H2O2. Identification of additional components in this complex regulation may further improve protein production and contribute to the development of novel protein-based therapeutic strategies.


title: A protein-based biosensor for detecting calcium by magnetic resonance imaging

abstract: Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer an attractive approach to noninvasively image neural activity with wide coverage in deep brain regions. However, current MRI sensors for calcium are based on synthetic architectures fundamentally incompatible with genetic technologies for in vivo delivery and targeting. Here, we present a protein-based MRI sensor for calcium, derived from a calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 weighted contrast in response to biologically relevant calcium concentrations. Corresponding changes in relaxation times are comparable to synthetic calcium sensors and exceed those of previous protein-based MRI sensors for other neurochemical targets. The biological applicability of calprotectin was established by detecting calcium in lysates prepared from a neuronal cell line. Calprotectin thus represents a promising path towards imaging neural activity by combining the benefits of MRI and protein sensors.


title: Single molecule tracking of AMPA receptors shows the role of synaptic insertion during maintenance of chemical LTP

abstract: Long term potentiation (LTP) likely contributes to memory formation. Early expression of LTP involves insertion of AMPA receptors (AMPARs) to the extrasynaptic membrane followed by their lateral diffusion into the synaptic membrane. However, whether a similar mechanism mediates the maintenance of LTP is unclear. Using single-molecule microscopy, we quantified that 6 GluA1- and 11 GluA2-containing endogenous AMPARs were added per synapse in cultured hippocampal neurons at 20 min following chemical LTP (cLTP) induction for 10 min, resulting in a 54% increase for both subunits. Single molecular tracking of transfected subunits revealed that the number of exocytosed subunits at the synapse increased by 15-18% from 5 to 20 min following cLTP induction, but their lateral exchange between synaptic and extrasynaptic membranes was minimal. These findings suggest that cLTP maintenance is contributed largely by synaptic insertion of AMPARs rather than the surface diffusion of exocytosed AMPARs from extrasynaptic to synaptic regions.


title: Photosynthesis dynamics and regulation sensed in the frequency domain

abstract:
Foundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after a dark acclimation or light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation before time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between the dark- and light-acclimation.

We present experimental data and mathematical models demonstrating that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency-domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts, and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects non-linear phenomena, including photosynthetic regulation. To support these claims, we present a frequency- and time-domain response in chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in the frequency range 1000 - 0.001 Hz. Based on these experimental data and numerical as well as analytical mathematical models, we propose that frequency-domain measurements can become a versatile new tool in plant sensing.

One sentence summaryIt is proposed to characterize photosynthesis in the frequency domain without the need for dark adaptation and, thus, without assumptions about the dark-to-light transition.

category: plant biology


title: Effects of tDCS Dose and Electrode Montage on regional cerebral blood flow and motor behavior

abstract:
We used three dose levels (Sham, 2mA, and 4mA) and two different electrode montages (unihemispheric or bihemispheric) to examine DOSE and MONTAGE effects on regional cerebral blood flow (rCBF) as a surrogate marker of neural activity, and on a finger sequence task, as a surrogate behavioral measure drawing on brain regions targeted by transcranial direct current stimulation (tDCS). We placed the anodal electrode over the right motor region (C4) while the cathodal or return electrode was placed either over a left supraorbital region (unihemispheric montage) or over the left motor region (C3 in the bihemispheric montage). Performance changes in the finger sequence task for both hands (left hand: p = 0.0026, and right hand: p = 0.0002) showed a linear tDCS dose response but no montage effect. rCBF in the right hemispheric perirolandic area increased with dose under the anodal electrode (p = 0.027). In contrast, in the perirolandic ROI in the left hemisphere, rCBF showed a trend to increase with dose (p = 0.053) and a significant effect of montage (p = 0.00004). The bihemispheric montage showed additional rCBF increases in frontomesial regions in the 4mA condition but not in the 2mA condition. Furthermore, we found correlations between rCBF changes in the right perirolandic region and improvements in the finger sequence task performance (FSP) for the left and right hand. Our data support not only a strong direct tDCS dose effect for rCBF and FSP as surrogate measures of targeted brain regions but also indirect effects on rCBF in functionally connected regions (e.g., frontomesial regions), particularly in the higher dose condition and on FSP of the ipsilateral hand (to the anodal electrode). At a higher dose and irrespective of polarity, a wider network of sensorimotor regions is positively affected by tDCS.

HighlightsO_LItDCS-DOSE had a linear effect on finger sequence performance for both hands
C_LIO_LIrCBF changes in both perirolandic ROIs demonstrated tDCS-DOSE effects, and left perirolandic ROI demonstrated tDCS-MONTAGE effects.
C_LIO_LISimulated current intensity in the left and right perirolandic ROI strongly correlated with the contralateral hands finger sequence performance.
C_LIO_LItDCS-Tolerability scores did not correlate with change in rCBF or finger sequence performance of the left hand.
C_LI

Graphical Abstract

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title: Myelin biogenesis is associated with pathological ultrastructure that is resolved by microglia during development

abstract: To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, in a process thought to be error-free, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in CNS myelin during development by combining ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia in a phosphatidylserine-dependent manner. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that requires substantial refinement.


title: Systematic measurement of combination drug landscapes to predict in vivo treatment outcomes for tuberculosis

abstract:
A lengthy multidrug chemotherapy is required to achieve a durable cure in tuberculosis. Variation in Mycobacterium tuberculosis drug response is created by the differing microenvironments in lesions, which generate different bacterial drug susceptibilities. To better realize the potential of combination therapy to shorten treatment duration, multidrug therapy design should deliberately explore the vast combination space. We face a significant scaling challenge in making systematic drug combination measurements because it is not practical to use animal models for comprehensive drug combination studies, nor are there well-validated high-throughput in vitro models that predict animal outcomes. We hypothesized that we could both prioritize combination therapies and quantify the predictive power of various in vitro models for drug development using a dataset of drug combination dose responses measured in multiple in vitro models. We systematically measured M. tuberculosis response to all 2- and 3-drug combinations among ten antibiotics in eight conditions that reproduce lesion microenvironments. Applying machine learning to this comprehensive dataset, we developed classifiers predictive of multidrug treatment outcome in a mouse model of disease relapse. We trained classifiers on multiple mouse models and identified ensembles of in vitro models that best describe in vivo treatment outcomes. Furthermore, we found that combination synergies are less important for predicting outcome than metrics of potency. Here, we map a path forward to rationally prioritize combinations for animal and clinical studies using systematic drug combination measurements with validated in vitro models. Our pipeline is generalizable to other difficult-to-treat diseases requiring combination therapies.

One Sentence SummarySignatures of in vitro potency and drug interaction measurements predict combination therapy outcomes in mouse models of tuberculosis.


title: DMRscaler: A Scale-Aware Method to Identify Regions of Differential DNA Methylation Spanning Basepair to Multi-Megabase Features

abstract:
BackgroundPathogenic mutations in genes that control chromatin function have been implicated in rare genetic syndromes. These chromatin modifiers exhibit extraordinary diversity in the scale of the epigenetic changes they affect, from single basepair modifications by DNMT1 to whole genome structural changes by PRM1/2. Patterns of DNA methylation are related to a diverse set of epigenetic features across this full range of epigenetic scale, making DNA methylation valuable for mapping regions of general epigenetic dysregulation. However,existing methods are unable to accurately identify regions of differential methylation across this full range of epigenetic scale directly from DNA methylation data.

ResultsTo address this, we developed DMRscaler, a novel method that uses an iterative windowing procedure to capture regions of differential DNA methylation (DMRs) ranging in size from single basepairs to whole chromosomes. We benchmarked DMRscaler against several DMR callers in simulated and natural data comparing XX and XY peripheral blood samples. DMRscaler was the only method that accurately called DMRs ranging in size from 100 bp to 1 Mb (pearsons r = 0.96) and up to 152 Mb on the X-chromosome. We then analyzed methylation data from rare-disease cohorts that harbor chromatin modifier gene mutations in NSD1, EZH2, and KAT6A where DMRscaler identified novel DMRs spanning gene clusters involved in development.

ConclusionTaken together, our results show DMRscaler is uniquely able to capture the size of DMR features across the full range of epigenetic scale and identify novel, co-regulated regions that drive epigenetic dysregulation in human disease.

category: genomics


title: The excitatory/inhibitory balance of synaptic transmission is impaired at higher extracellular Ca2+ concentrations in Scn1a+/- mouse model of Dravet syndrome

abstract: Dravet syndrome (DS) is an intractable form of childhood epilepsy that occurs in infancy. More than 80% of all patients have a heterozygous abnormality in the SCN1A gene, which encodes a subunit of Na+ channels in the brain. However, the detailed pathogenesis of DS remains unclear. This study investigated the synaptic pathogenesis of this disease in terms of excitatory/inhibitory balance using a mouse model of DS. We show that excitatory postsynaptic currents were similar between Scn1a knock-in neurons (Scn1a+/- neurons) and wild-type neurons, but inhibitory postsynaptic currents were significantly lower in Scn1a+/- neurons. Moreover, both the vesicular release probability and the number of inhibitory synapses were significantly lower in Scn1a+/- neurons compared with wild-type neurons. There was no proportional increase in inhibitory postsynaptic current amplitude in response to increased extracellular Ca2+ concentrations. Our study revealed that the number of inhibitory synapses is significantly reduced in Scn1a+/- neurons, while the sensitivity of inhibitory synapses to extracellular Ca2+ concentrations is markedly increased. These data suggest that Ca2+ tethering in inhibitory nerve terminals may be disturbed following the synaptic burst, likely leading to epileptic symptoms.


title: Inhibitory synaptic transmission is impaired at higher extracellular Ca2+ concentrations in Scn1a+/- mouse model of Dravet syndrome


title: De novo inter-regional coactivations of pre-configured local ensembles support memory

abstract: Neuronal ensembles in the amygdala, ventral hippocampus, and prefrontal cortex are involved in fear memory; however, how the inter-regional ensemble interactions support memory remains elusive. Using multi-regional large-scale electrophysiology in the afore-mentioned structures of fear-conditioned rats, we demonstrated that local ensembles activated during fear memory acquisition were inter-regionally coactivated during subsequent sleep, which relied on brief bouts of fast network oscillations. During memory retrieval, coactivations reappeared, accompanying fast oscillations. Ensembles contributing to inter-regional coactivation were configured prior to memory acquisition in the amygdala and prefrontal cortex but developed through experience in the hippocampus. Our observation suggests that elements of a given memory are instantly encoded within various brain regions in a pre-configured manner, whereas hippocampal ensembles and the network for inter-regional integration of the distributed information develop in an experience-dependent manner to form a new memory, which is consistent with hippocampal memory index hypothesis.


title: Individual variability in structural brain development from late childhood to young adulthood

abstract: A fundamental task in neuroscience is to characterize the brains developmental course. While replicable group-level models of structural brain development from childhood to adulthood have recently been identified, we have yet to quantify and understand individual differences in structural brain development. The present study examined individual variability and sex differences in changes in brain structure, as assessed by anatomical MRI, across ages 8.0-26.0 years in 269 participants (149 females) with three time points of data (807 scans), drawn from three longitudinal datasets collected in the Netherlands, Norway, and USA. We further investigated the relationship between overall brain size and developmental changes, as well as how females and males differed in change variability across development. There was considerable individual variability in the magnitude of changes observed for all included brain measures. However, distinct developmental patterns of change were observed for total brain and cortical gray matter, cortical thickness, and white matter surface area, with individuals demonstrating either stability or decreases in early adolescence, then almost universal decreases during mid-to-late adolescence, before returning to more variable patterns in early adulthood. White matter volume demonstrated a similar developmental pattern of variability, but with individuals shifting from increases to a majority stabilizing during mid-to-late adolescence. We observed sex differences in these patterns, and also an association between an individuals brain size and their overall rate of change. The present study provides new insight as to the amount of individual variance in changes in structural morphometrics from late childhood to early adulthood in order to obtain a more nuanced picture of brain development. The observed individual- and sex-differences in brain changes also highlight the importance of further studying individual variation in developmental patterns in healthy, at-risk, and clinical populations.


title: Slow phase-locked endogenous modulations support selective attention to sound

abstract: To make sense of complex soundscapes, listeners must select and attend to task-relevant streams while ignoring uninformative sounds. One possible neural mechanism underlying this process is alignment of endogenous oscillations with the temporal structure of the target sound stream. Such a mechanism has been suggested to mediate attentional modulation of neural phase-locking to the rhythms of attended sounds. However, such modulations are compatible with an alternate framework, where attention acts as a filter that enhances exogenously-driven neural auditory responses. Here we attempted to adjudicate between theoretical accounts by playing two tone steams varying across condition in tone duration and presentation rate; participants attended to one stream or listened passively. Attentional modulation of the evoked waveform was roughly sinusoidal and scaled with rate, while the passive response did not. This suggests that auditory attentional selection is carried out via phase-locking of slow endogenous neural rhythms.


title: Habitat complexity in organic olive orchards modulates the abundance of natural enemies but not the attraction to plant species

abstract:
Semi-natural habitat complexity and organic management could affect the abundance and diversity of natural enemies and pollinators in olive orchards. Nonetheless, in such agroecosystems the effect of plant structure, plant richness, and plant attraction on the arthropod fauna has been poorly documented. Here we evaluate the influence of those effects jointly as an expression of arthropod abundance and richness in olive trees, ground cover, and adjacent vegetation within organic olive orchards. For this, we used generalized linear models and non-metric multidimensional scaling (NMDS) integrating generalized additive models. Our results suggest that natural enemies and pollinators are mainly attracted to A. radiatus, D. catholica, and L. longirrostris within ground cover and G. cinerea speciosa, Q. rotundifolia, R. officinalis, T. zygis gracilis, and U. parviflorus within adjacent vegetation. Accordingly, habitat complexity showed a positive relationship with the abundance of key families of natural enemies and pollinators but not with the number of taxa. NMDS showed that plant richness and plant arrangement and scattering affected the key families differently, suggesting that each key family responds to their individual needs for plant resources but forming groups modulated by complexity. This pattern was especially seeing in predators and omnivores. Our findings support that the higher the plant richness and structure of a semi natural-habitat within an olive orchard, the higher the abundance and richness of a given arthropod community (a pattern found in natural ecosystems). The information presented here can be used by producers and technicians to increase the presence and abundance of natural enemies and pollinators within organic olive orchards, and thus improve the ecosystem services provided by semi-natural habitats.

Graphical abstract

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title: Selection on growth rate and local adaptation drive genomic adaptation during experimental range expansions in the protist Tetrahymena thermophila

abstract:
O_LIPopulations that expand their range can undergo rapid evolutionary adaptation of life-history traits, dispersal behaviour, and adaptation to the local environment. Such adaptation may be aided or hindered by sexual reproduction, depending on the context.
C_LIO_LIHowever, few studies have investigated the genomic causes and consequences or genetic architecture of such adaptation during range expansions.
C_LIO_LIWe here studied genomic adaptation during experimental range expansions of the protist Tetrahymena thermophila in landscapes with a uniform environment or a pH-gradient. Specifically, we investigated two aspects of genomic adaptation during range expansion. Firstly, we investigated the genetic architecture of adaptation in terms of the underlying numbers of allele frequency changes from standing genetic variation and de novo variants. We focused on how sexual reproduction may alter this genetic architecture. Secondly, identified genes subject to selection caused by the expanding range itself, and directional selection due to the presence or absence of the pH-gradient. We focused this analysis on alleles with large frequency changes that occurred in parallel in more than one population to identify the most likely candidate targets of selection.
C_LIO_LIWe found that sexual reproduction altered genetic architecture both in terms of de novo variants and standing genetic variation. However, sexual reproduction affected allele frequency changes in standing genetic variation only in the absence of long-distance gene flow. Adaptation to the range expansion affected genes involved in cell divisions and DNA repair, whereas adaptation to the pH-gradient additionally affected genes involved in ion balance, and oxidoreductase reactions. These genetic changes may result from selection on growth and adaptation to low pH.
C_LIO_LIOur results suggest that the evolution of life-history and the adaptation to the local environment has a genetic basis during our range expansion experiment. In the absence of gene flow, sexual reproduction may have aided genetic adaptation. Gene flow may have swamped expanding populations with maladapted alleles, thus reducing the extent of evolutionary adaptation during range expansion. Sexual reproduction also altered the genetic architecture of our evolving populations via de novo variants, possibly by purging deleterious mutations or by revealing fitness benefits of rare genetic variants.
C_LI


title: Electric fish use electrocommunication signals to fine tune relative dominance and access to resources

abstract: Animals across species compete for limited resources. While in some species competition behavior is solely based on own abilities, others assess their opponents to facilitate these interactions. Using cues and communication signals, contestants gather information about their opponent, adjust their behavior accordingly, and can thereby avoid high costs of escalating fights. We tracked electrocommunication signals, in particular "rises", and agonistic behaviors of the gymnotiform electric fish Apteronotus leptorhynchus in staged competition experiments. A larger body size relative to the opponent was the sole significant predictor for winners. Sex and the frequency of the continuously emitted electric field were only mildly influencing competition outcome. In males, correlations of body-size and winning were stronger than in females and, especially when losing against females, communication and agonistic interactions were enhanced, hinting towards males being more motivated to compete. Fish that lost competitions emitted the majority of rises in dependence on the competitors relative size and sex. The emission of rises was costly since it provoked ritualized biting or chasing behaviors by the other fish. Despite winners being accurately predictable based on rise numbers already after the initial 25 minutes, losers continued to emit rises. The number of rises emitted by losers and the duration of chasing behaviors depended in similar ways on physical attributes of contestants. The detailed evaluation of these correlations hint towards A. leptorhynchus adjusting their competition behavior according to mutual assessment, where rises could signal a losers motivation to continue assessment through ritualized fighting.

category: animal behavior and cognition


title: Electrocommunication signals indicate motivation to compete during dyadic interactions of an electric fish


title: Analytical Performance of a Standardized Kit for Mass Spectrometry-based Measurements of Human Glycosaminoglycans

abstract:
Glycosaminoglycans (GAGs) are long linear sulfated polysaccharides implicated in processes linked to disease development such as mucopolysaccharidosis, respiratory failure, cancer, and viral infections, thereby serving as potential biomarkers. A successful clinical translation of GAGs as biomarkers depends on the availability of standardized GAG measurements. However, owing to the analytical complexity associated with the quantification of GAG concentration and structural composition, a standardized method to simultaneously measure multiple GAGs is missing. In this study, we sought to characterize the analytical performance of a ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-MS/MS)-based kit for the quantification of 17 GAG disaccharides. The kit showed acceptable linearity, selectivity and specificity, accuracy and precision, and analyte stability in the absolute quantification of 15 GAG disaccharides. In native human samples, here using urine as a reference matrix, the analytical performance of the kit was acceptable for the quantification of CS disaccharides. Intra- and inter-laboratory tests performed in an external laboratory demonstrated robust reproducibility of GAG measurements showing that the kit was acceptably standardized. In conclusion, these results indicated that the UHPLC-MS/MS kit was standardized for the simultaneous measurement of GAG disaccharides allowing for comparability of measurements and enabling translational research.

SummaryAnalytical performance of a kit for standardized GAG measurements, based on an established UHPLC-MS/MS method


title: Cold-acclimation induces life stage-specific responses in the cardiac proteome of Western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance

abstract: Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3{degrees}C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. Since protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold-acclimation would evoke preparatory changes in protein expression that would support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20{degrees}C (warm) or 3{degrees}C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis using labeled isobaric tags and mass spectrometry. The relative abundances of 1316 identified proteins were compared between temperatures and developmental stages. The effect of cold-acclimation on the cardiac proteome was most evident when life stage was included as a covariable, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart from its hatchling to adult form and, only after this maturation occurs, will cold-acclimation induce protein expression changes appropriate for supporting heart function during prolonged anoxia. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of life stage- and temperature-induced changes to the cardiac proteome, including reduced complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stagespecific anoxia tolerance in turtles.

category: physiology


title: Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

abstract: The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescence in situ hybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.


title: In-depth characterization of HIV-1 reservoirs reveals links to viral rebound during treatment interruption

abstract: The HIV-1 reservoir is composed of cells harboring latent proviruses that are capable of contributing to viremia upon antiretroviral treatment (ART) interruption. Although this reservoir is known to be maintained by clonal expansion, the contribution of large, infected cell clones to residual viremia and viral rebound remains underexplored. Here, we conducted an extensive analysis on four ART-treated individuals who underwent an analytical treatment interruption (ATI). We performed subgenomic (V1-V3 env), near full-length proviral and integration site sequencing, and used multiple displacement amplification to sequence both the integration site and provirus from single HIV-infected cells. We found eight proviruses that could phylogenetically be linked to plasma virus obtained before or during the ATI. This study highlights a role for HIV-infected cell clones in the maintenance of the replication-competent reservoir and suggests that infected cell clones can directly contribute to rebound viremia upon ATI.


title: Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption


title: Speciation of the Mycobacterium tuberculosis complex using mass spectrometry: a proof-of-concept.

abstract: Mycobacteria that form the Mycobacterium tuberculosis complex are responsible for deadly tuberculosis in animals and patients. Identification of these pathogens at the species level is of primary importance for treatment and source tracing, and currently relies on DNA analysis, including whole genome sequencing (WGS), which takes a whole day. In this study, we report on the unprecedented identification of the M. tuberculosis complex species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), with WGS as the comparative gold standard. In a first step, an optimised peptide extraction applied to 24 isolates otherwise identified in three of the 11 M. tuberculosis complex species by WGS, yielded 94 MALDI-TOF spectra which clustered according to WGS identification. In a second step, 70/74 (95%) other isolates were correctly identified at the species level by this clustering method. This study is the first to report a MALDI-TOF-MS method of identification of M. tuberculosis complex mycobacteria at the species level and is easily implantable in clinical microbiology laboratories.


title: A novel capsid protein network allows the characteristic inner membrane structure of Marseilleviridae giant viruses

abstract: Marseilleviridae is a family of the new order of giant viruses, which exhibit a characteristic inner membrane. Here, we investigated the entire structure of tokyovirus, a species of Marseillevirus at 7.7 [A] resolution using 1 MV high-voltage cryo-EM and single particle analysis. The minor capsid lattice formed by five proteins, shows a novel structure compared to other icosahedral giant viruses. Under the minor capsid proteins, scaffold proteins connect two five-fold vertices and interact with the inner membrane. Previously reported giant viruses utilise "tape measure" proteins, proposed to control its capsid size, which could not be identified in tokyovirus, but scaffold proteins appear to perform a similar role. A density on top of the major capsid protein was identified, which suggested to be a 14kDa glycoprotein. Our observations suggest that the icosahedral particle of Marseilleviridae is constructed with a novel capsid protein network, which allows the characteristic inner membrane structure.


title: Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

abstract: Phosphatidylinositol (PI) is an essential phospholipid and critical component of membrane bilayers. The complete deacylation of PI by phospholipases of the B-type leads to the production of intracellular and extracellular glycerophosphoinositol (GPI), a water-soluble glycerophosphodiester. Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters that resides at the plasma membrane in yeast. Once internalized, GPI can be degraded to produce inositol, phosphate and glycerol, thereby contributing to reserves of these building blocks. Not surprisingly, GIT1 gene expression is controlled by nutrient balance, with limitation for phosphate or inositol each increasing GIT1 expression to facilitate GPI uptake. Less is known about how Git1 protein levels or localization are controlled. Here we show that the -arrestins, an important class of protein trafficking adaptor, regulate the localization of Git1 in a manner dependent upon their association with the ubiquitin ligase Rsp5. Specifically, -arrestin Aly2 is needed for effective Git1 internalization from the plasma membrane under basal conditions. However, in response to GPI-treatment of cells, either Aly1 or Aly2 can promote Git1 trafficking to the vacuole. Retention of Git1 at the cell surface, as occurs in aly1{Delta} aly2{Delta} cells, results in impaired growth in the presences of excess exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of this metabolite or its downstream products leads to cellular toxicity. We further show that regulation of -arrestin Aly1 by the protein phosphatase calcineurin improves both steady-state and ligand-induced trafficking of Git1 when a mutant allele of Aly1 that mimics the dephosphorylated state at calcineurin-regulated residues is employed. Thus, calcineurin regulation of Aly1 is important for the GPI-ligand induced trafficking of Git1 by this -arrestin, however, the role of calcineurin in regulating Git1 trafficking is much broader than can simply be explained by regulation of the -arrestins. Finally, we find that loss of Aly1 and Aly2 leads to an increase in phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole and this alteration is further exacerbated by addition of GPI, suggesting that the effect is at least partially linked to Git1 function. Indeed, loss of Aly1 and Aly2 leads to increased incorporation of inositol label from 3H-inositol-labelled GPI into PI, confirming that internalized GPI influences PI synthesis and indicating a role for the -arrestins in regulating the process.


title: Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

abstract: Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.


title: Modulation of SARS-CoV-2 Spike-induced Unfolded Protein Response (UPR) in HEK293T cells by selected small chemical molecules

abstract: Coronaviruses (CoV) exploits the endoplasmic reticulum (ER) of the host cells for replication and in doing so, increases ER stress. evokes Unfolded Protein Response (UPR) and possibly autophagy, which could all attribute to the pathophysiology of the viral infections. To date, little is known about the roles of ER stress, UPR, and autophagy in SARS-CoV-2 infection. Here we over-expressed the viral Spike (S) protein in cultured HEK293T cells, as it has been shown that such protein is largely responsible for UPR activation in other CoV-infected cells. We noticed, in the transfected cells, heightened ER stress, activation of the PERK-eIF2 arm of the UPR, induction of autophagy and cell death. When we treated the transfected cells with Tauroursodeoxycholic acid (TUDCA), 4-phenyl butyric acid (PBA), Salubrinal, Trazadone hydrochloride, and Dibenzoylmethane (DBM), we saw reduced the BiP/GRP78 levels, but only PBA and TUDCA could significantly diminish the levels of peIF2 and autophagy expression.


title: Age-related susceptibility to insulin resistance is due to a combination of CPT1B decline and lipid overload

abstract:
BACKGROUNDAdvanced age increases the susceptibility to diet-induced insulin resistance (IR). A key driver of this phenomenon is lipid accumulation in the skeletal muscle. It is debated, however, whether this is due to dietary lipid overload or decline of mitochondrial function. To address the interplay of diet and age in the flexibility of muscle lipid and glucose handling, we put young and aged mice on a low- or high-fat diet (HFD).

RESULTSAs expected, aged mice were more susceptible to IR when given a HFD than young mice. The HFD induced intramuscular lipid accumulation specifically in aged mice, including C18:0-containing ceramides and diacylglycerols. This was reflected by the mitochondrial {beta}-oxidation capacity, which was upregulated by the HFD in young, but not in old mice. Conspicuously, most {beta}-oxidation proteins were upregulated by the HFD in both groups, but carnitine palmitoyltransferase 1B (CPT1B) declined in aged animals. Computational modelling traced the flux control mostly to CPT1B, suggesting a CPT1B-driven loss of flexibility to the HFD with age. Finally, in old animals glycolytic protein levels were reduced and less flexible to the diet.

CONCLUSIONWe conclude that intramuscular lipid accumulation and decreased insulin sensitivity are not due to age-related mitochondrial dysfunction or nutritional overload alone, but rather to their interaction. Moreover, we identify CPT1B as a potential target to counteract age-dependent intramuscular lipid accumulation and thereby IR.


title: The protein modifier SUMO is critical for Arabidopsis shoot meristem maintenance at warmer ambient temperatures

abstract:
Short heat waves (>37{degrees}C) are extremely damaging to non-acclimated plants and their capacity to recover from heat stress is key for their survival. To acclimate, the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1) subfamily activates a transcriptional response that resolves incurred damages. In contrast, little is known how plants acclimate to sustained non-detrimental warm periods at 27-28{degrees}C. Plants respond to this condition with a thermomorphogenesis response. In addition, HSFA1 is critical for plant survival during these warm periods. We find that SUMO, a protein modification whose conjugate levels rise sharply during acute heat stress in eukaryotes, is critical too for plant longevity during warm periods, in particular for normal shoot meristem development. The known SUMO ligases were not essential to endure these warm periods, alone or in combination. Thermo-lethality was also not seen when plants lacked certain SUMO proteases or when SUMO chain formation was blocked. The SUMO-dependent thermo-resilience was as well independent of the autoimmune phenotype of the SUMO mutants. As acquired thermotolerance was normal in the sumo1/2 knockdown mutant, our data thus reveal a role for SUMO in heat acclimation that differs from HSFA1 and SIZ1. We conclude that SUMO is critical for shoot meristem integrity during warm periods.

HighlightThe protein modifier SUMO governs shoot meristem maintenance in Arabidopsis allowing sustained rosette development when plants endure a sustained warm non-detrimental period of 28 degrees Celsius.


title: Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

abstract: Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutations in genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.


title: Low doses of the organic insecticide spinosad trigger lysosomal defects, ROS driven lipid dysregulation and neurodegeneration in flies

abstract: The plight of insect populations around the world and the threats it poses to agriculture and ecosystems has thrown insecticide use into the spotlight. Spinosad is an organic insecticide, considered less harmful to beneficial insects than synthetic insecticides, but its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad reduce cholinergic response in neurons by antagonizing D6 nAChRs. D6 nAChRs are transported to lysosomes that become enlarged and accumulate upon spinosad treatment. Oxidative stress is initiated in the central nervous system, and spreads to midgut and disturbs lipid storage in metabolic tissues in a D6-dependent manner. Spinosad toxicity was ameliorated with the antioxidant N-Acetylcysteine amide (NACA). Chronic exposures lead to mitochondrial defects, severe neurodegeneration and blindness in adult animals. The many deleterious effects of low doses of this insecticide reported here point to an urgent need for rigorous investigation of its impacts on beneficial insects.


title: Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

abstract: Microglia, the main immunocompetent cells of the brain regulate neuronal function in health and disease, but their contribution to cerebral blood flow (CBF) remained elusive. Here we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. We show that microglia establish direct purinergic contacts with cells in the neurovascular unit that shape cerebral perfusion in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in the barrel cortex after whisker stimulation. We also reveal that hypercapnia, which is associated with acidification, induces microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Furthermore, the absence or dysfunction of microglia markedly impairs adaptation to hypoperfusion via P2Y12R after transient unilateral common carotid artery occlusion, which is also influenced by CX3CR1-mediated actions. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation with broad implications for common neurological diseases.


title: CVTree: A Parallel Alignment-free Phylogeny and Taxonomy Tool based on Composition Vectors of Genomes

abstract:
CVTree is an alignment-free algorithm to infer phylogenetic relationships from genome sequences. It had been successfully applied to study phylogeny and taxonomy of viruses, prokaryotes, and fungi based on the whole genomes, as well as chloroplasts, mitochondria, and metagenomes. Here we presented the standalone software for the CVTree algorithm. In the software, a parallel workflow for the CVTree algorithm was designed. Based on the workflow, new alignment-free methods were also implemented. And by examining the phylogeny and taxonomy of 13903 prokaryotes based on 16S rRNA sequences, we showed that CVTree software is an efficient and effective tool for the studying of phylogeny and taxonomy based on genome sequences.

Availabilityhttps://github.com/ghzuo/cvtree


title: Sensory cilia act as a specialized venue for regulated EV biogenesis and signaling

abstract: Extracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study signaling EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.


title: Population genetics as a tool to elucidate pathogen reservoirs: Lessons from Pseudogymnoascus destructans, the causative agent of white-nose disease in bats

abstract: Emerging infectious diseases pose a major threat to human, animal, and plant health. The risk of species-extinctions increases when pathogens can survive in the absence of the host, for example in environmental reservoirs. However, identifying such reservoirs and modes of infection is often highly challenging. In this study, we investigated the presence and nature of an environmental reservoir for the ascomycete fungus Pseudogymnoascus destructans, the causative agent of white-nose disease. We also characterised the modes and timing of transmission of the pathogen; key elements to better understand the disease dynamics. Using 18 microsatellite markers, we determined the genotypic and genic (based on allele frequencies) differentiation between 1,497 P. destructans isolates collected from nine closely situated hibernacula in North-Eastern Germany. One hibernaculum was the focus of intensive sampling in which both the bats and walls of the site were sampled at regular intervals over five consecutive winter seasons (1,062 isolates). We found significant genic differentiation between sites and few multi-locus genotypes shared across hibernacula (genotypic differentiation). This demonstrates that each hibernaculum has an essentially unique population of the fungus. This would be expected if bats purge viable P. destructans over the summer, preventing the mixing and exchange of the pathogen in maternity colonies, where bats from all of the studied hibernacula meet. Results from the intensively sampled site show higher measures of genotypic richness on walls compared to bats, the absence of genic differentiation between bats and walls, and stable relative abundance of multi-locus genotypes over multiple winter seasons. This clearly implicates hibernacula walls as the main environmental reservoir of the pathogen, from which bats become re-infected annually.

category: ecology


title: Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates

abstract:
Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it.

We analyzed 146,917 SARS-CoV-2 genome assemblies and 2,393 NGS datasets from GISAID, NCBI Virus and NCBI SRA archives focusing on non-synonymous mutations in the spike protein.

Only around 13.8% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.98% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 2,592 distinct variants were found in total. The majority of the observed variants were recurrent, but only nine and 23 recurrent variants were found in at least 0.5% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 15.1% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition.

These findings demonstrate the increasing importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.