title: Coccolith Sr/Ca is a Robust Temperature and Growth Rate Indicator that Withstands Dynamic Microbial Interactions

abstract:
Coccolithophores are a diverse group of calcifying microalgae that have left a prominent fossil record on Earth. Various coccolithophore relics, both organic and inorganic, serve as proxies for reconstruction of past oceanic conditions.

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

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

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

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


title: Actively growing cells are the predominant persisters in exponential phase of Escherichia coli

abstract: Bacterial persistence is a phenomenon in which a small fraction of isogenic bacterial cells survives a lethal dose of antibiotics. It is generally assumed that persistence is caused by cells with inactive growth generated prior to drug exposure. However, evidence from direct observation is scarce due to the extremely low frequencies of persisters. Here, we visualize the responses of more than 106 individual cells of wildtype Escherichia coli to lethal doses of antibiotics, sampling cells from different growth phases and culture media. We show that preexisting growth-arrested dormant persisters constitute only minor fractions of persistent cell lineages in populations sampled from exponential phase. Non-dormant persisters survive with radical morphological changes in response to drug exposure, including L-form-like morphologies or filamentation. Incubating cells under stationary phase conditions increases both the frequency and the probability of survival of dormant cells. While dormant cells in late stationary phase express a general stress response regulator, RpoS, at high levels, persistent cell lineages show lower levels of RpoS among the dormant cells. These results demonstrate that diverse survival pathways coexist within bacterial populations to achieve persistence and that persistence does not necessarily require dormant cells.

category: microbiology


title: Observation of non-dormant persister cells reveals diverse modes of survival in antibiotic persistence


title: Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

abstract: Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct non-overlapping binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1 and HIF-2 subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF- subunits and their dimerization partner HIF-1{beta} exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2 specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.

category: molecular biology


title: Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

abstract: The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding -catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of -catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of -catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that hypersensitive mutants affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive -catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that proper AJ plasticity requires appropriate tension sensitivity of -catenin.

category: cell biology


title: Polycomb Repressive Complexes occupancy reveals PRC2-independent PRC1 critical role in the control of limb development

abstract: The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1), in which PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidence pointed to cPRC1 and vPRC1 sharing a significant subset of targets. How the functional interplay between PRC2, cPRC1 and vPRC1 contributes to gene regulation remains partially understood. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, as the majority of PRC2- bound loci are bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of genes retaining CBX2 occupancy as well as a few genes with apparent gain of CBX2 binding. Importantly, among these genes, 56 of them correspond to developmental genes known for playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings emphasize the primary role of PRC2-independent PCR1 function in regulating developmental genes and questions the role of PRC2/cPRC1 in controlling developmental programs.

category: developmental biology


title: TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages

abstract:
During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

One-Sentence SummaryGaining sequential genetic access to vertebrate cell lineages.


title: Universal features shaping organelle gene retention

abstract: Mitochondria and plastids power complex life, and retain their own organelle DNA (oDNA) genomes, with highly reduced gene contents compared to their endosymbiont ancestors. Why some protein-coding genes are retained in oDNA and some lost remains a debated question. Here we harness over 15k oDNA sequences and over 300 whole genome sequences with tools from structural biology, bioinformatics, machine learning, and Bayesian model selection to reveal the properties of genes, and associated underlying mechanisms, that shape oDNA evolution. Striking symmetry exists between the two organelle types: gene retention patterns in both are predicted by the hydrophobicity of a protein product and its energetic centrality within its protein complex, with additional influences of nucleic acid and amino acid biochemistry. Remarkably, retention principles from one organelle type successfully and quantitatively predict retention in the other, supporting this universality; these principles also distinguish gene profiles in independent endosymbiotic relationships. The identification of these features shaping organelle gene retention both provides quantitative support for several existing evolutionary hypotheses, and suggests new biochemical and biophysical mechanisms influencing organelle genome evolution.

category: evolutionary biology


title: Transcriptome Dynamics of Floral Organs Approaching Blooming in the Flowering Cherry (Cerasus x yedoensis) Cultivar 'Somei-Yoshino'

abstract: To gain insights into the genetic mechanisms underlying blooming and petal movement in flowering cherry (Cerasus x yedoensis), we performed time-course RNA-seq analysis of the floral buds and open-flowers of the most popular flowering cherry cultivar,  Somei-Yoshino. Independent biological duplicate samples of floral buds and open-flowers were collected from  Somei-Yoshino trees grown at three different locations in Japan. RNA-seq reads obtained from floral bud and open-flower samples collected in the current study (in 2019) and in a previous study (in 2017) were aligned against the genome sequence of  Somei-Yoshino to quantify gene transcript levels. Clustering analysis of RNA-seq reads revealed dynamic changes in the transcriptome, with genes in seven modules predominantly expressed at specific time points, ranging from 5 weeks before flowering to 2 weeks after flowering. Based on the identified gene modules and Gene Ontology (GO) terms enriched at different floral stages, we speculate that the genetic mechanisms underlying petal movement and flower opening in cherry involve the processes of development, cell wall organization, reproduction, and metabolism, which are executed by genes encoding transcription factors, phytohormones, transporters, and polysaccharide metabolic enzymes. Furthermore, we propose a method for cherry bloom forecasting, based on gene expression levels at different time points before flowering as RNA markers.

category: plant biology


title: On stability of the Darwinian dynamics

abstract: Here we analyze Darwinian dynamics of cancer introduced in [1], extended by including a competition matrix, and evaluate (i) when the eco-evolutionary equilibrium is positive and (ii) when the eco-evolutionary equilibrium is asymptotically stable.

category: cancer biology


title: When inefficient speech-motor control affects speech comprehension: atypical electrophysiological correlates of language prediction in stuttering

abstract:
It is well attested that people predict forthcoming information during language comprehension. The literature presents different proposals on how this ability could be implemented. Here, we tested the hypothesis according to which language production mechanisms have a role in such predictive processing. To this aim, we studied two electroencephalographic correlates of predictability during speech comprehension - pretarget alpha-beta (8-30 Hz) power decrease and the post-target N400 event-related potential (ERP) effect, - in a population with impaired speech-motor control, i.e., adults who stutter (AWS), compared to typically fluent adults (TFA). Participants listened to sentences that could either constrain towards a target word or not, allowing or not to make predictions. We analyzed time-frequency modulations in a silent interval preceding the target and ERPs at the presentation of the target. Results showed that, compared to TFA, AWS display: i) a widespread and bilateral reduced power decrease in posterior temporal and parietal regions, and a power increase in anterior regions, especially in the left hemisphere (high vs. low constraining) and ii) a reduced N400 effect (non-predictable vs. predictable). The results suggest a reduced efficiency in generating predictions in AWS with respect to TFA. Additionally, the magnitude of the N400 effect in AWS is correlated with alpha power change in the right pre-motor and supplementary motor cortex, a key node in the dysfunctional network in stuttering. Overall, the results support the idea that processes and neural structures prominently devoted to speech planning and execution support prediction during language comprehension.

Significance StatementThe study contributes to the developing enterprise of investigating language production and comprehension not as separate systems, but as sets of processes which may be partly shared. We showed that a population with impaired speech-motor control, i.e., adults who stutter, are characterized by atypical electrophysiological patterns associated with prediction in speech comprehension. The results highlight that an underlying atypical function of neural structures supporting speech production also affects processes deployed during auditory comprehension. The implications are twofold: on the theoretical side, the study supports the need for a more integrated view of language comprehension and production as human capabilities, while on the applied and clinical side, these results might open new venues for efficient treatments of developmental stuttering.

category: neuroscience


title: Molecular transport and packing underlie increasing ribosome productivity in faster growing cells

abstract: Faster growing cells must make proteins more quickly. Increasing ribosome abundance only partly accounts for increased protein production; the productivity of individual ribosomes must also increase, almost doubling via an unknown mechanism. To investigate, we model translation elongation in Escherichia coli by explicitly representing spatially-resolved transport and reactions among individual molecules each occupying finite volume. We predict that physical transport of individual ternary complexes accounts for ~80% of elongation latency. We find crowding increases at faster growth, which predictably decreases diffusion of ternary complexes, but that crowding also improves proximity between ternary complexes and ribosomes. Despite slowed diffusion, we predict that improved proximity wins out, offering a simple mechanism for how individual elongating ribosomes become more productive. We speculate how crowding imposes a physical limit on growth rate and likely undergirds cellular behavior more broadly. Our work reveals a fundamental mechanism arising from physical transport at the colloidal scale that cannot be obtained via kinetics-only modeling.

category: biophysics


title: Ligand-induced transmembrane conformational coupling in monomeric EGFR

abstract: Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. While receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution from the single helix of a monomeric receptor has been challenging to isolate due to the complexity and ligand-dependence of the receptor-receptor interactions. By combining membrane nanodiscs produced wtih cell-free expression, single-molecule Forster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes are common to single-pass membrane proteins.


title: Altered circadian rhythms and sleep in a new Angelman Syndrome mouse model

abstract: Normal neurodevelopment requires precise expression of the key ubiquitin ligase gene Ube3a. Comparing newly generated mouse models for Ube3a down-regulation (models of Angelman syndrome) vs. Ube3a up-regulation (models for autism), we find reciprocal effects of Ube3a gene dosage on phenotypes associated with circadian rhythmicity, including the amount of locomotor activity. In contrast to previous reports, we find that Ube3a is imprinted in neurons of the suprachiasmatic nuclei, the pacemaking circadian brain locus. In addition, Ube3a-deficient mice lack the typical drop in wake late in the dark period and have blunted responses to sleep deprivation. Suppression of physical activity by light in Ube3a-deficient mice is not due to anxiety as measured by behavioral tests and stress hormones; quantification of stress hormones may serve as an easily measurable biomarker for evaluating potential therapeutic treatments for Angelman syndrome. We conclude that reduced Ube3a gene dosage affects not only neurodevelopment but also sleep patterns and circadian rhythms.


title: Parallel functional subnetworks embedded in the macaque face patch system

abstract:
During normal vision, our eyes provide the brain with a continuous stream of useful information about the world. How visually specialized areas of the cortex, such as face-selective patches, operate under natural modes of behavior is poorly understood. Here we report that, during the free viewing of videos, cohorts of face-selective neurons in the macaque cortex fractionate into distributed and parallel subnetworks that carry distinct information. We classified neurons into functional groups based on their video-driven coupling with fMRI time courses across the brain. Neurons from each group were distributed across multiple face patches but intermixed locally with other groups at each recording site. These findings challenge prevailing views about functional segregation in the cortex and underscore the importance of naturalistic paradigms for cognitive neuroscience.

One-Sentence SummaryNatural visual experience reveals parallel functional subnetworks of neurons embedded within the macaque face patch system


title: Experience-induced remodeling of the hippocampal post-synaptic proteome and phosphoproteome

abstract:
The post synaptic density (PSD) of excitatory synapses contains a highly organized protein network with thousands of proteins and is key node in the regulation of synaptic plasticity. To gain new mechanistic insight into experience-induced changes in the PSD, we examined the global dynamics of the PSD proteome and phosphoproteome in mice following various treatments. Mice were trained using an inhibitory avoidance (IA) task and hippocampal PSD fractions were isolated for quantitative proteomic and phosphoproteomics analysis. We used a sequential enrichment strategy to explore the concurrent events of protein expression and phosphorylation in the hippocampal PSD following IA training (IA) or immediate shock (Shock). We identified more than 6,200 proteins and 3,000 phosphoproteins in the sequential strategy covering a total of 7,429 proteins. On the phosphoproteins we identified a total of 9,589 phosphosites. Strikingly, of the significantly IA-regulated proteins and phosphoproteins, a large fraction of the proteins displayed an overall decrease in phosphorylation level. Bioinformatic analysis of proteins and phosphoproteins that were regulated by IA were annotated for an involvement in regulation of glutamate receptor functionality, calcium signaling, and synaptic plasticity. We also identified synaptic kinases, phosphatases and their respective phosphosites regulated by IA training or immediate shock. Furthermore, we found that AMPA receptor surface expression was regulated by protein phosphatase, Mg2+/Mn2+ dependent 1H (Ppm1h). Together, these results unravel the dynamic remodeling of the PSD upon IA learning or immediate shock and serve as a resource for elucidating the synaptic proteome dynamics induced by experience-dependent plasticity.

HighlightsO_LIThe proteome and phosphoproteome of mouse hippocampal PSD fractions were examined using quantitative phosphoproteomics and bioinformatics following inhibitory avoidance training or non-associative immediate shock.
C_LIO_LIApproximately 6,200 proteins and 3,000 phosphoproteins were identified and quantified in the hippocampal PSD fractions.
C_LIO_LIIA mediates widespread decreases in the abundance and phosphorylation of proteins in the hippocampal PSD fraction.
C_LIO_LIKinases, phosphatases and their phosphorylation status were dynamically and significantly regulated by IA and immediate shock.
C_LIO_LIFunctional validation shows that the protein phosphatase Ppm1h is linked to the regulation of synaptic plasticity in vitro and in vivo.
C_LI

In BriefQuantitative proteomics and phosphoproteomics combined with subcellular protein fractionation and bioinformatic analysis identifies a highly dynamic regulation of synaptic protein phosphorylation at the postsynaptic density following IA training and immediate shock.


title: Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

abstract: Chimeric antigen receptors (CARs) can re-direct T cells to target abnormal cells but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here we show that, while CARs have a >100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting-cells (APCs), they have nearly identical sensitivity when antigen is presented as purified protein on artificial surfaces. We next measured the impact of engaging accessory receptors (CD2, LFA-1, CD28, CD27, 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved TCR antigen sensitivity by 125 and 22-fold, respectively, but only improved CAR sensitivity by <5-fold. This differential effect of CD2 and LFA-1 engagement on TCR versus CAR sensitivity was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3{varepsilon} subunit (also known as a TRuC), and fully restored by exchanging the CAR variable domains with the TCR{beta} variable domains (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors, and suggest approaches to increase sensitivity.

category: immunology


title: IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

abstract: Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has nonredundant biological functions, we currently lack the tools to investigate the molecular basis for isoform-specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. We performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed {beta}-actin, we demonstrate that IntAct actins are properly expressed and that their filament incorporation is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin-binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed {gamma}-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for IntAct actins. Together, our data demonstrate that IntAct is a promising tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.


title: Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms to control cell motility and neurite outgrowth

abstract: Microtubule (MT) dynamics are modulated through the coordinated action of various MT-associated proteins (MAPs). However, the regulatory mechanisms underlying MT dynamics remain unclear. We show that the MAP7 family protein Map7D2 stabilizes MTs to control cell motility and neurite outgrowth. Map7D2 directly bound to MTs through its N-terminal half and stabilized MTs in vitro. Map7D2 localized prominently to the centrosome and partially on MTs in mouse N1-E115 neuronal cells, which expresses two of the four MAP7 family members, Map7D2 and Map7D1. Map7D2 loss decreased the resistance to the MT-destabilizing agent, nocodazole without affecting acetylated/detyrosinated stable MTs, suggesting that Map7D2 stabilizes MTs via direct binding. In addition, Map7D2 loss increased the rate of random cell migration and neurite outgrowth, presumably by disturbing the balance between MT stabilization and destabilization. Map7D1 exhibited similar subcellular localization and gene knock-down phenotypes to Map7D2. However, in contrast to Map7D2, Map7D1 was required for the maintenance of acetylated stable MTs. Taken together, our data suggest that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms in cell motility and neurite outgrowth.


title: Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms in neuronal cells


title: Clonal gametogenesis is triggered by intrinsic stimuli in the hybrids germ cells but is dependent on sex differentiation

abstract:
Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrids reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreduplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrids gonial cells or whether it is regulated by the surrounding gonadal tissue.

We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrids SSCs lead to sterility when transplanted into sexual males, but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females.

This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals.

Significance StatementAlthough sexual reproduction is a dominant trait among all eukaryotes, many taxa have evolved the ability to reproduce asexually. While asexuality appears to be linked to interspecific hybridization, it remains unknown how the coexistence of diverged genomes may initiate such a swap in reproduction. In our study, we transplanted germ cells between asexual hybrids and their parents. On one hand, the ability of clonal gametogenesis occurred exclusively in hybrid germ cells, suggesting that asexual development is directly triggered by the hybrid genomic constitution of the cell. On the other hand, clonality was observed only in cells transplanted into females, suggesting that the execution of clonal development is influenced by signals from the gonadal environment and regulated by somatic factors.


title: Maternal Ezh1/2 deficiency in oocyte delays H3K27me2/3 restoration and impairs epiblast development responsible for embryonic sub-lethality in mouse

abstract: Mammalian embryonic development is a complex process regulated by various epigenetic modifications. Recently, maternal histone H3 methylations were found to be inherited and reprogrammed in early embryos to regulate embryonic development. The enhancer of zest homolog 1 and 2 (Ezh1 and Ezh2) belong to the core components of Polycomb repressive complex 2 (PRC2) and are the histone methyltransferase of histone 3 lysine 27 (H3K27). How maternal Ezh1 and Ezh2 function on H3K27 methylation in in vivo preimplantation embryos and embryonic development are not clear. Here, we deleted Ezh1 or/and Ezh2 in growing oocytes using gene knockout mouse models, and found that H3K27me3 in oocytes was disappeared by loss of Ezh2 alone while H3K27me2 was absent upon deletion of both Ezh1 and Ezh2. The effects of Ezh1/2 were inherited in maternal knockout zygotes and early embryos, in which restoration of H3K27me3 was delayed until late blastocyte by loss of Ezh2 alone and H3K27me2 was reestablished until morulae by deletion of Ezh1 and Ezh2. However, the ablation of both Ezh1 and Ezh2, but not single Ezh1 or Ezh2, led to significantly decreased litter size due to growth retardation during post-implantation. Furthermore, maternal Ezh1/2 deficiency caused compromised H3K27me3 and pluripotent epiblast cells in late blastocyst, followed by defective development of epiblast. These results demonstrate that in oocytes, Ezh2 is indispensable for H3K27me3 while Ezh1 complements Ezh2 in H3K27me2. Also, maternal Ezh1/2-H3K27 methylation is inherited in descendant embryos and has a critical effect on fetus and placenta development. Thus, this work sheds light on maternal epigenetic modifications during embryonic development.


title: Early embryonic heat shock induces long-term epigenetic memory by affecting the transition to zygotic independence

abstract:
Early-life stress can generate persistent life-long effects that impact adult health and disease risk, but little is known of how such programming is established and maintained. Previous use of the Drosophila strain wm4h show that an early embryonic heat shock result in stable epigenetic alteration in the adult fly. To investigate the potential role of small non-coding RNA (sncRNA) in the initiation of such long-term epigenetic effects, we here generated a fine timeline of sncRNA expression during the first 5 stages of Drosophila embryogenesis in this strain. Building on this, we show that (1) miRNA is increased following early embryonic heat shock, and (2) the increased miRNA is coming from two separate sources, maternal and zygotic. By performing long RNA sequencing on the same single embryo, we found that a subgroup of miRNA with maternal origin, had a strong negative correlation with a group of early zygotic transcripts. Critically, we found evidence that one such early zygotic transcript, the insulator binding factor Elba1, is a Su(var) for wm4h. The findings provide insights of the dynamics and stress-sensitivity of sncRNA during the first embryonic stages in Drosophila and suggest an interplay between miRNA, Elba1 and long-term epigenetic alteration.

HIGHLIGHTSO_LIWe provide a high-resolution timeline for sncRNA for Drosophila stage 1-5 embryos
C_LIO_LIHeat shock before midblastula transition (MBT) results in a massive upregulation of miRNA at cellularization
C_LIO_LIHeat shock-induced miRNAs negatively associate with downregulation of a specific subset of pre-MBT genes
C_LIO_LIElba1 is a position-effect-variegation (PEV) modifier for wm4h
C_LIO_LIHeat shock-induces an "leaky" expression of genes that overlap with Elba 1-3 binding sites
C_LI


title: PHB3 Regulates Lateral Root Primordia Formation via NO-mediated Degradation of AUX/IAAs

abstract:
We previously showed that PHB3 regulates auxin-stimulated lateral root (LR) formation; however, the underlying molecular mechanism is unknown. Here, we demonstrate that PHB3 regulates LR development mainly through influencing lateral root primordia (LRP) initiation via affecting nitric oxide (NO) accumulation. The reduced LRP in phb3 was largely rescued by exogenous NO donor SNAP treatment. The decreased NO accumulation in phb3 caused a lower expression of GATA23 and LBD16 through inhibiting the degradation of IAA14/28. Overexpression of either GATA23 or LBD16 in phb3 mutant background recovered the reduced LRP number phenotype. These results indicate that PHB3 regulates LRP initiation via NO-mediated auxin signaling through regulating the degradation of IAA14/28.

HighlightPHB3 regulates IAA28 and IAA14 degradation by controlling NO accumulation, and thereby regulating LR founder cell identification and the transition to asymmetric division.


title: Single-cell profiling of transcriptome and histone modifications with EpiDamID

abstract: Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, including various combinations of transcriptome, genome and epigenome. However, comprehensive profiling of the histone post-translational modifications that influence gene expression at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of genetically engineered proteins. By fusing Dam to single-chain variable fragment antibodies, engineered chromatin reader domains, or endogenous chromatin-binding proteins, we render the DamID technology and all its implementations compatible with the genome-wide identification of histone post-translational modifications. Importantly, this enables the joint analysis of chromatin marks and transcriptome in a variety of biological systems at the single-cell level. In this study, we use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. We further demonstrate the applicability of this method to in vivo systems by mapping H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specifically in the notochord. Overall, EpiDamID is a new addition to a vast existing toolbox for obtaining systematic insights into the role of chromatin states during dynamic cellular processes.

category: genomics


title: Cost-effective epigenetic age profiling in shallow methylation sequencing data

abstract: There is a critical need for robust, high-throughput assays of biological aging trajectories. Among various approaches, epigenetic aging clocks emerged as reliable molecular trackers of the aging process. However, current methods for epigenetic age profiling are inherently costly and lack throughput. Here, we leverage the scAge framework for accurate prediction of biological age from very few bisulfite sequencing reads in bulk samples, thereby enabling drastic (100-1,000-fold) reduction in sequencing costs per sample. Our approach permits age assessment based on distinct assortments of CpG sites in different samples, without the need for targeted site enrichment or specialized reagents. We demonstrate the efficacy of this method to quantify the age of mouse blood samples across independent cohorts, identify the effect of calorie restriction as an attenuator of the aging process, and discern rejuvenation upon cellular reprogramming. We propose that this framework may be used for epigenetic age prediction in extremely high-throughput applications, enabling robust, large-scale and inexpensive interventions testing and age profiling.


title: Are reaching and grasping effector-independent? Similarities and differences in reaching and grasping kinematics between the hand and foot

abstract: While reaching and grasping are highly prevalent manual actions, neuroimaging studies provide evidence that their neural representations may be shared between different body parts, i.e. effectors. If these actions are guided by effector-independent mechanisms, similar kinematics should be observed when the action is performed by the hand or by a cortically remote and less experienced effector, such as the foot. We tested this hypothesis with two characteristic components of action: the initial ballistic stage of reaching, and the preshaping of the digits during grasping based on object size. We examined if these kinematic features reflect effector-independent mechanisms by asking participants to reach toward and to grasp objects of different widths with their hand and foot. First, during both reaching and grasping, the velocity profile up to peak velocity matched between the hand and the foot, indicating a shared ballistic acceleration phase. Secondly, maximum grip aperture and time of maximum grip aperture of grasping increased with object size for both effectors, indicating encoding of object size during transport. Differences between the hand and foot were found in the deceleration phase and time of maximum grip aperture, likely due to biomechanical differences and the participants inexperience with foot actions. These findings provide evidence for effector-independent visuomotor mechanisms of reaching and grasping that generalize across body parts.

category: animal behavior and cognition


title: Cryo-electron microscopy structure of the H3-H4 octasome without histones H2A and H2B

abstract: The canonical nucleosome, which represents the predominant packaging unit in eukaryotic chromatin, has an octameric core made up of two histone H2A-H2B and H3-H4 dimers with ~147 base-pair (bp) DNA wrapping around it. Non-nucleosome particles with alterative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Here we solved the structure of the H3-H4 octasome, which is a nucleosome-like particle with a core made up of four H3-H4 dimers. Two conformations, open and closed, are determined at 3.9 [A] and 3.6 [A] resolutions by cryo-electron microscopy, respectively. The H3-H4 octasome, made up of a di-tetrameric core, is wrapped by ~120 bp DNA in 1.5 negative superhelical turns. The symmetrical halves are connected by a unique H4-H4 interface along the dyad axis. In vivo crosslinking of cysteine probes placed at another unique H3-H3 interface demonstrated the existence of the H3-H4 octasome in cells.

category: biochemistry


title: Text Mining Drug-Protein Interactions using an Ensemble of BERT, Sentence BERT and T5 models

abstract: In this work, we trained an ensemble model for predicting drug-protein interactions within a sentence based on only its semantics. Our ensembled model was built using three separate models: 1) a classification model using a fine-tuned BERT model; 2) a fine-tuned sentence BERT model that embeds every sentence into a vector; and 3) another classification model using a fine-tuned T5 model. In all models, we further improved performance using data augmentation. For model 2, we predicted the label of a sentence using k-nearest neighbors with its embedded vector. We also explored ways to ensemble these 3 models: a) we used the majority vote method to ensemble these 3 models; and b) based on the HDBSCAN clustering algorithm, we trained another ensemble model using features from all the models to make decisions. Our best model achieved an F-1 score of 0.753 on the BioCreative VII Track 1 test dataset.

category: bioinformatics


title: Understanding the Genetics of Viral Drug Resistance by Integrating Clinical Data and Mining of the Scientific Literature

abstract: Drug resistance caused by mutations is a public health threat for existing and emerging viral diseases. A wealth of evidence about these mutations and their clinically-associated phenotypes is scattered across the literature, but a comprehensive perspective is usually lacking. This work aimed to produce a clinically-relevant view for the case of Hepatitis B virus (HBV) mutations by combining a chronic HBV clinical study with a compendium of genetic mutations systematically gathered from the scientific literature. We enriched clinical mutation data by systematically mining 2,472,725 scientific articles from PubMed Central in order to gather information about the HBV mutational landscape. By performing this analysis, we were able to identify mutational hotspots for each HBV genotype (A-E) and gene (C, X, P, S), as well as the location of disulfide bonds associated with these mutations. Through a modelling study, we also identified a mutational position common in both the clinical data and the literature that is located at the binding pocket for a known anti-HBV drug, namely entecavir. The results of this novel approach shows the potential of integrated analyses to assist in the development of new drugs for viral diseases that are more robust to resistance. Such analyses should be of particular interest due to the increasing importance of viral resistance in established and emerging viruses, such as for newly-developed drugs against SARS-CoV-2.


title: Chlamydomonas State Transition Is Quiet Around Pyrenoid and Independent from Thylakoid Deformation

abstract: Photosynthetic organisms have developed a rapid regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the ultrastructural dynamics of the thylakoid membrane to the ST mechanism, how ST is related to the ultrastructural dynamic of the thylakoid in Chlamydomonas remains elusive. To clarify the above-mentioned relation, here we used two specialized microscope techniques, observation via the excitation-spectral microscope (ESM) developed recently by us and the super-resolution imaging based on structured illumination microscopy (SIM). The ESM observation revealed a highly reversible rearrangement of LHCII-related fluorescence. More importantly, it clarified lower ST activity in the region surrounding the pyrenoid, which is the specific subcellular compartment associated with the carbon-fixation reaction. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations induced by the ST-inducing illumination. Fine irreversible thylakoid transformation was also observed for the Stt7-kinase-lacking mutant. This result, together with the nearly equal structural changes in the less active ST regions around the pyrenoid, suggested the independence of the observed fine structural changes from the LHCII phosphorylation.


title: Binding Affinity Estimation From Restrained Umbrella Sampling Simulations

abstract: The protein-ligand binding affinity quantifies the binding strength between a protein and its ligand. Computer modeling and simulations can be used to estimate the binding affinity or binding free energy using data- or physics-driven methods or a combination thereof. Here, we discuss a purely physics-based sampling approach based on biased molecular dynamics (MD) simulations, which in spirit is similar to the stratification strategy suggested previously by Woo and Roux. The proposed methodology uses umbrella sampling (US) simulations with additional restraints based on collective variables such as the orientation of the ligand. The novel extension of this strategy presented here uses a simplified and more general scheme that can be easily tailored for any system of interest. We estimate the binding affinity of human fibroblast growth factor 1 (hFGF1) to heparin hexasaccharide based on the available crystal structure of the complex as the initial model and four different variations of the proposed method to compare against the experimentally determined binding affinity obtained from isothermal calorimetry (ITC) experiments. Our results indicate that enhanced sampling methods that sample along the ligand-protein distance without restraining other degrees of freedom do not perform as well as those with additional restraint. In particular, restraining the orientation of the ligands plays a crucial role in reaching a reasonable estimate for binding affinity. The general framework presented here provides a flexible scheme for designing practical binding free energy estimation methods.


title: Event-triggered STED imaging

abstract: The observation of protein organization during cellular signalling calls for imaging methods with increased spatial and temporal resolution. STED nanoscopy can access dynamics of nanoscale structures in living cells. However, the available number of recordable frames is often limited by photo-bleaching. Here, we present an automated method, event-triggered STED, which instantly (< 40 ms) images synaptic proteins with high spatial and temporal resolution (~30 nm, 2.5 Hz) in small regions upon and at the site of local calcium sensing.


title: Cancer/Testis genes are predictive of breast tumor subtypes

abstract: Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis and no dedicated therapy, therefore new tools to understand, detect, and treat these tumors are needed. Certain germline genes are re-expressed in tumors, and constitute the Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here, we designed a new approach to examine Cancer/ Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the 2 Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells but not the microenvironment, and that they are not expressed by normal breast progenitors, in other words their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Basal-like tumors expressing both genes have a poorer outcome than tumors expressing either gene alone or neither gene. Therefore, these findings suggest a potential synergistic effect between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worse outcomes.


title: Estimating the Inter- and Intra-Rater Reliability for NASH Fibrosis Staging in the Presence of Bridge Ordinal Ratings with Hierarchical Bridge Category Models

abstract: The public health burden of non-alcoholic steatohepatitis (NASH), a liver condition characterized by excessive lipid accumulation and subsequent tissue inflammation and fibrosis, has burgeoned with the spread of western lifestyle habits. Progression of fibrosis into cirrhosis is assessed using histological staging scales (e.g., NASH Clinical Research Network (NASH CRN)). These scales are used to monitor disease progression as well as to evaluate the effectiveness of therapies. However, clinical drug trials for NASH are typically underpowered due to lower than expected inter-/intra-rater reliability, which impacts measurements at screening, baseline, and endpoint. Bridge ratings represent a phenomenon where pathologists assign two adjacent stages simultaneously during assessment and may further complicate these analyses when ad hoc procedures are applied. Statistical techniques, dubbed Bridge Category Models, have been developed to account for bridge ratings, but not for the scenario where multiple pathologists assess biopsies across time points. Here, we develop hierarchical Bayesian extensions for these statistical methods to account for repeat observations and use these methods to assess the impact of bridge ratings on the inter-/intra-rater reliability of the NASH CRN staging scale. We also report on how pathologists may differ in their assignment of bridge ratings to highlight different staging practices. Our findings suggest that Bridge Category Models can capture additional fibrosis staging heterogeneity with greater precision, which translates to potentially higher reliability estimates in contrast to the information lost through ad hoc approaches.

category: pathology


title: The hormone Fibroblast Growth Factor 19 stimulates water intake

abstract: Fibroblast growth factor 19 (FGF19) is a hormone with pleiotropic metabolic functions, leading to ongoing development of analogues for the treatment of metabolic disorders. On the other hand, FGF19 is overexpressed in a sub-group of hepatocellular carcinoma (HCC) patients and has oncogenic properties. It is therefore crucial to precisely define FGF19 effects, notably chronic exposure to elevated concentrations of the hormone. We used hydrodynamic gene transfer approach to generate a transgenic mouse model with long-term FGF19 hepatic overexpression. Here we describe a novel effect of FGF19, namely stimulation of water intake. This phenotype, lasting at least over a 6-month period, depends on signaling in the central nervous system and is independent of FGF21, although it mimics some of its features. We further show that HCC patients with high levels of circulating FGF19 have a reduced natremia, indicating dispogenic features. The present study provides evidence of a new activity for FGF19, which could be clinically relevant in the context of FGF19 overexpressing cancers and treatment of metabolic disorders by FGF19 analogues.

category: physiology


title: CaPti1/CaERF4 module positively regulates pepper resistance to bacterial wilt disease through coupling enhanced immunity and dehydration tolerance

abstract:
Bacterial wilt, a severe disease that affects over 250 plant species, is caused by Ralstonia solanacearum through vascular system blockade. Although both plant immunity and dehydration tolerance might contribute to disease resistance, whether and how they are related are still unclear. Herein, we provide evidence that immunity against R. solanacearum and dehydration tolerance are coupled and regulated by CaPti1-CaERF4 module. By expression profiling, virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana, both CaPti1 and CaERF4 were upregulated by R. solanacearum inoculation, dehydration stress and exogenously applied ABA. They in turn phenocopied with each other in promoting pepper resistance to bacterial wilt not only by activating HR cell death and SA-dependent CaPR1, but also by activating dehydration tolerance related CaOSM1 and CaOSR1, and stomata closure to reduce water loss in ABA signaling dependent manner. Yeast-two hybrid assay showed that CaERF4 interacts with CaPti1, which was confirmed by co-immunoprecipitation and pull-down assays. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that, upon R. solanacearum inoculation, CaPR1, CaOSM1 and CaOSR1 were directly targeted and positively regulated by CaERF4 via binding GCC-box or DRE-box, which was potentiated by CaPti1. In addition, our data indicate that CaPti1-CaERF4 complex might act downstream ABA signaling, since the exogenously applied ABA did not alter stomata aperture regulated by CaPti1-CaERF4 module. Importantly, CaPti1-CaERF4 module was found also acts positively in pepper growth and response to dehydration stress. Collectively, the results suggest that immunity and dehydration tolerance are coupled and positively regulated by CaPti1-CaERF4 in pepper plants to enhance resistance against R. solanacearum.

SummaryPepper immunity and dehydration tolerance are coupled and regulated by the CaPti1-CaERF4 module partially in a way related to ABA signaling and stomata closure.


title: Effects of Superhydrophobic Sand Mulching on Evapotranspiration and Phenotypic Responses in Tomatoes (Solanum lycopersicum) under Normal and Reduced Irrigation

abstract: Irrigated agriculture in arid and semi-arid regions is a vital contributor to the global food supply; however, these regions endure massive evaporative losses that are compensated by unsustainable freshwater withdrawals. Plastic mulches have been used to curtail evaporation, improve water-use efficiency, and ensure food-water security, but they are non-biodegradable and their disposal is unsustainable. We recently developed superhydrophobic sand (SHS), which comprises sand grains with a nanoscale wax coating that could offer a more sustainable mulching solution. Here, the effects of adding a 1.0 cm-thick layer of SHS mulch on the evapotranspiration and phenotypic responses of tomato (Solanum lycopersicum) plants are studied under normal and reduced irrigation. Under both irrigation regimes, SHS mulching suppressed evaporation and enhanced transpiration by 78% and 17%, respectively relative to the bare soil. Overall, SHS mulching enhanced root xylem vessel diameter, stomatal aperture, stomatal conductance, and chlorophyll content index by 21%, 25%, 28%, and 23%, respectively. Total fruit yields, total dry mass, and harvest index increased in SHS-mulched plants by 33%, 20%, and 16%, respectively than in bare soil. These findings demonstrate the potential of SHS to boost irrigation efficiency in water-limited environments and provide mechanistic insights behind yield enhancement by SHS mulching.


title: SCS313 Serrano, a big trefoil selected for flowering ability and seed production

abstract: SCS313 Serrano is a tetraploid cultivar of Lotus uliginosus developed by the Research and Rural Extension Company of Santa Catarina, Brazil, to improve flowering ability and seed production in low-latitude regions. SCS313 Serrano was developed from the selection of individual plants of the cultivar Grasslands Maku. Seeds from the initial breeding population was bulked and used to establish a field trial with spaced plants. The plants were selected and classified as late or early, regarding the beginning of flowering, through visual evaluations based on the time for the beginning of flowering. Selected plants were transplanted to crossing blocks and seed harvested on individual plants; a progeny test was conducted, with four replications, through the establishment of approximately 100 genotypes in a field. This process was repeated for three consecutive cycles of recurrent selection. Considering the mean time for the beginning of flowering and vigor performance, the best five genotypes were selected as parental lines for the synthetic cultivar SCS313 Serrano. A morphological difference between SCS313 Serrano and Grasslands Maku is that SCS313 Serrano has absence of hairs in stems whereas Grasslands Maku has a medium stem hair density. In addition, SCS313 Serrano exhibits profuse flowering ability while Grasslands Maku exhibits very sparse seedhead formation. SCS313 Serrano exhibited good persistence under wet conditions and similar forage yields compared to other lotus commercial cultivars. Thus, SCS313 Serrano is recommended to be used as pasture in mixtures with grass in livestock systems, mainly on wet soils.


title: Inferring Deep Brain Stimulation Induced Short-term Synaptic Plasticity Using Novel Dual Optimization Algorithm

abstract: Experimental evidence in both human and animal studies demonstrated that deep brain stimulation (DBS) can induce short-term synaptic plasticity (STP) in the stimulated nucleus. Given that DBS-induced STP may be connected to the therapeutic effects of DBS, we sought to develop an appropriate computational predictive model that infers the dynamics of STP in response to DBS at different frequencies. Existing methods for estimating STP - either model-based or model-free approaches - require access to pre-synaptic spiking activity. However, in the context of DBS, extracellular stimulation (e.g. DBS) can be used to elicit presynaptic activations directly. We present a model-based approach that integrates multiple individual frequencies of DBS-like electrical stimulation as pre-synaptic spikes and infers parameters of the Tsodyks-Markram (TM) model from post-synaptic currents of the stimulated nucleus. By distinguishing between the steady-state and transient responses of the TM model, we develop a novel dual optimization algorithm that infers the model parameters in two steps. First, the TM model parameters are calculated by integrating multiple frequencies of stimulation to estimate the steady state response of post-synaptic current through a closed-form analytical solution. The results of this step are utilized as the initial values for the second step in which a non-derivative optimization algorithm is used to track the transient response of the post-synaptic potential across different individual frequencies of stimulation. Moreover, we apply our algorithm to empirical data recorded from acute rodent brain slices of the subthalamic nucleus (STN) during DBS-like stimulation to infer dynamics of STP for inhibitory synaptic inputs.


title: Differential Vulnerability of Anterior Cingulate Cortex Cell-Types to Diseases and Drugs

abstract: In psychiatric disorders, mismatches between disease-states and therapeutic strategies are highly pronounced, largely because of unanswered questions regarding specific vulnerabilities of different cell-types and therapeutic responses. Which cellular events (housekeeping or salient) are most affected? Which cell-types succumb first to challenges, and which exhibit the strongest response to drugs? Are these events coordinated between cell-types? How does the disease-state and drug affect this coordination? To address these questions, we analyzed single-nucleus-RNAseq (sn-RNAseq) data from the human anterior cingulate cortex--a region involved in many psychiatric disorders. Density index, a metric for quantifying similarities and dissimilarities across functional profiles, was employed to identify common (housekeeping) or salient functional themes across all cell-types. Cell-specific signatures were integrated with existing disease and drug-specific signatures to determine cell-type-specific vulnerabilities, druggabilities, and responsiveness. Clustering of functional profiles revealed cell-types jointly participating in these events. SST and VIP interneurons were found to be most vulnerable, whereas pyramidal neurons were least vulnerable. Overall, the disease-state is superficial layer-centric, largely influences cell-specific salient themes, strongly impacts disinhibitory neurons, and influences astrocyte interaction with a subset of deep-layer pyramidal neurons. Drug activities, on the other hand, are deep layer-centric and involve activating a distinct subset of deep-layer pyramidal neurons to circumvent the disinhibitory circuit malfunctioning in the disease-state. These findings demonstrate a novel application of sn-RNAseq data to explain drug and disease action at a systems level, suggests a targeted drug development and reevaluate various postmortem-based findings.


title: Transcriptomic profiling of sex-specific olfactory neurons reveals subset-specific receptor expression in C. elegans

abstract: The nematode Caenorhabditis elegans utilizes chemosensation to navigate an ever-changing environment for its survival. A class of secreted small-molecule pheromones, termed ascarosides, play an important role in olfactory perception by affecting a host of biological function ranging from development to behavior. The ascaroside ascr#8 mediates sex-specific behaviors, driving avoidance in hermaphrodites and attraction in males. Males sense ascr#8 via the ciliated male-specific cephalic sensory (CEM) neurons, which exhibit radial symmetry along dorsal-ventral and left-right axes. Calcium imaging studies suggest a complex neural coding mechanism that translates stochastic physiological responses in these neurons to reliable behavioral outputs. To test the hypothesis that the neurophysiological complexity arises from differential expression of genes within subsets of these neurons, we performed cell-specific transcriptomic profiling of these sensory neurons. Expression profiling revealed between 20 and 639 genes enriched at least two-fold per CEM neuron and identified multiple G protein coupled receptor (GPCR) candidates enriched in non-overlapping subsets of CEM neurons. GFP reporter analysis confirmed that RNA expression of two of the GPCR genes, srw-97 and dmsr-12, is enriched in specific subsets of the CEM neurons. Single CRISPR-Cas9 knockouts of either srw-97 or dmsr-12 resulted in partial defects, while a double knockout of both srw-97 and dmsr-12 completely abolished the attractive response to ascr#8, suggesting that each receptor acts in a non-redundant manner in discrete olfactory neurons. Together, our results suggest that the evolutionarily distinct GPCRs SRW-97 and DMSR-12 act to facilitate male-specific sensation of ascr#8 through discrete subsets of CEM neurons.


title: Long-term stability of neural activity in the motor system

abstract: How an established behavior is retained and stably produced by a nervous system in constant flux remains a mystery. One possible solution is to fix the activity patterns of single neurons in the relevant circuits. Alternatively, activity in single cells could drift over time provided that the population dynamics are constrained to produce stable behavior. To arbitrate between these possibilities, we recorded single unit activity in motor cortex and striatum continuously for several weeks as rats performed stereotyped motor behaviors - both learned and innate. We found long-term stability in behaviorally-locked single neuron activity patterns across both brain regions. A small amount of neural drift observed over weeks of recording could be explained by concomitant changes in task-irrelevant behavioral output. These results suggest that stereotyped behaviors are generated by stable neural activity patterns.


title: Neural network models for spinal implementation of muscle synergies

abstract: Muscle synergies have been proposed as functional modules to simplify the complexity of body motor control; however, their neural implementation is still unclear. Converging evidence suggests that output projections of the spinal premotor interneurons (PreM-INs) underlie the formation of muscle synergies, but they exhibit a substantial variation across neurons and exclude standard models assuming a small number of unitary "modules" in the spinal cord. Here we compared neural network models for muscle synergies to seek a biologically plausible model that reconciles previous clinical and electrophysiological findings. We examined three neural network models: one with random connections (non-synergy model), one with a small number of spinal synergies (simple synergy model), and one with a large number of spinal neurons representing muscle synergies with a certain variation (population synergy model). We found that the simple and population synergy models emulate the robustness of muscle synergies against cortical stroke observed in human stroke patients. Furthermore, the size of the spinal variation of the population synergy matched well with the variation in spinal PreM-INs recorded in monkeys. These results suggest that a spinal population with moderate variation is a biologically plausible model for the neural implementation of muscle synergies.


title: Differential patterns of change in brain connectivity resulting from traumatic brain injury

abstract:
BackgroundTraumatic brain injury (TBI) damages white matter tracts, disrupting brain network structure and communication. There exists a wide heterogeneity in the pattern of structural damage associated with injury, as well as a large heterogeneity in behavioral outcomes. However, little is known about the relationship between changes in network connectivity and clinical outcomes.

MethodsWe utilize the rat lateral fluid-percussion injury (FPI) model of severe TBI to study differences in brain connectivity in 8 animals that received the insult and 11 animals that received only a craniectomy. Diffusion Tensor Imaging (DTI) is performed 5 weeks after the injury and network theory is used to investigate changes in white matter connectivity.

ResultsWe find that 1) global network measures are not able to distinguish between healthy and injured animals; 2) injury induced alterations predominantly exist in a subset of connections (subnetworks) distributed throughout the brain; and 3) injured animals can be divided into subgroups based on changes in network motifs - measures of local structural connectivity. Additionally, alterations in predicted functional connectivity indicate that the subgroups have different propensities to synchronize brain activity, which could relate to the heterogeneity of clinical outcomes such as the risk of developing post-traumatic epilepsy.

DiscussionThese results suggest that network measures can be used to quantify progressive changes in brain connectivity due to injury and differentiate among subpopulations with similar injuries but different pathological trajectories.

Impact StatementWhite matter tracts are important for efficient communication between brain regions and their connectivity pattern underlies proper brain function. Traumatic brain injury (TBI) damages white matter tracts and changes brain connectivity, but how specific changes relate to differences in clinical/behavioral outcomes is not known. Using network theory to study injury related changes in structural connectivity, we find that local measures of network structure can identify subgroups of injured rats with different types of changes in brain structure. Our results suggest that these different patterns of change could relate to differences in clinical outcomes such as the propensity to develop epilepsy.


title: A Locus Coeruleus- dorsal CA1 dopaminergic circuit modulates memory linking

abstract: Individual memories are often linked so that the recall of one triggers the recall of another. For example, contextual memories acquired close in time can be linked, and this is known to depend on temporary increase in excitability that drive the overlap between dorsal CA1 (dCA1) hippocampal ensembles encoding the linked memories. Here, we show that the Locus Coeruleus (LC) cells projecting to dCA1 have a key permissive role in contextual memory linking, without affecting contextual memory formation, and that this effect is mediated by dopamine and not by noradrenaline. Additionally, we found that LC to dCA1 projecting neurons modulate the excitability of dCA1 neurons, and the extent of overlap between dCA1 memory ensembles, as well as the stability of coactivity patterns within these ensembles. This discovery of a neuromodulatory system that specifically affects memory linking without affecting memory formation, reveals a fundamental separation between the brain mechanisms that modulate these two distinct processes.


title: Pharmacological impacts on schizophrenia functional analysis: a postmortem proteome study.

abstract: Schizophrenia (SCZ) is a severe and debilitating mental illness. Antipsychotic drugs (APDs) are used to treat both positive and negative SCZ symptoms, by influencing the cellular, subcellular-synaptic, and molecular processes. We posit that these effects influence our understanding of SCZ. To address this, we analyzed postmortem dorsolateral prefrontal cortex grey matter samples from control and SCZ subjects (n=10/group) using liquid-chromatography mass-spectrometry-based proteomics. We retrieved SCZ-altered and APD-influenced proteome-sets using linear and mixed linear models, respectively, and validated them experimentally using independent cohorts and insilico using published datasets. Functional analysis of proteome-sets was contrasted at the biological pathway, cell-type, subcellular-synaptic, and drug-target levels. The SCZ-altered proteome was conserved across several studies from DLPFC and other brain areas and was dependent on drug effect. At the pathway level, we observed an aberrant extracellular event and, except for homeostasis, signal-transduction, cytoskeleton, and dendrites associated downregulated changes, the APDs compensated for the majority of the SCZ-altered pathways. At the cell-type level, the up-and down-regulated SCZ-altered events were associated with two different subsets of striatum projecting layer-5 pyramidal-neurons regulating dopaminergic secretion. At the subcellular synaptic level, compensatory pre- and post-synaptic events were observed. At the drug target level, dopaminergic processes influence the SCZ-altered up-regulated proteome, whereas non-dopaminergic and a diverse array of non-neuromodulatory mechanisms influence the SCZ-altered down-regulated proteome. While these findings are dependent on pharmacological effects, they are also consistent with previous SCZ studies, implying the need to re-evaluate previous results. We discuss our findings in the context of cortico-striatal influence in SCZ-pathology.


title: Listening to speech in noisy scenes: Antithetical contribution of primary and non-primary auditory cortex

abstract: Invasive and non-invasive electrophysiological measurements during "cocktail-party"-like listening indicate that neural activity in human auditory cortex (AC) "tracks" the envelope of relevant speech. Due to the measurements limited coverage and/or spatial resolution, however, the distinct contribution of primary and non-primary auditory areas remains unclear. Using 7-Tesla fMRI, here we measured brain responses of participants attending to one speaker, without and with another concurrent speaker. Using voxel-wise modeling, we observed significant speech envelope tracking in bilateral Heschls gyrus (HG) and middle superior temporal sulcus (mSTS), despite the sluggish fMRI responses and slow temporal sampling. Neural activity was either positively (HG) or negatively (mSTS) correlated to the speech envelope. Spatial pattern analyses indicated that whereas tracking in HG reflected both relevant and (to a lesser extent) non-relevant speech, right mSTS selectively represented the relevant speech signal. These results indicate that primary and non-primary AC antithetically process ongoing speech suggesting a push-pull of acoustic and linguistic information.


title: A Riemannian approach to predicting brain function from the structural connectome

abstract: AO_SCPLOWBSTRACTC_SCPLOWOngoing brain function is largely determined by the underlying wiring of the brain, but the specific rules governing this relationship remain unknown. Emerging literature has suggested that functional interactions between brain regions emerge from the structural connections through mono-as well as polysynaptic mechanisms. Here, we propose a novel approach based on diffusion maps and Riemannian optimization to emulate this dynamic mechanism in the form of random walks on the structural connectome and predict functional interactions as a weighted combination of these random walks. Our proposed approach was evaluated in two different cohorts of healthy adults (Human Connectome Project, HCP; Microstructure-Informed Connectomics, MICs). Our approach outperformed existing approaches and showed that performance plateaus approximately around the third random walk. At macroscale, we found that the largest number of walks was required in nodes of the default mode and frontoparietal networks, underscoring an increasing relevance of polysynaptic communication mechanisms in transmodal cortical networks compared to primary and unimodal systems.


title: Gravity effects are better optimized by older than young adults when reaching with the non-dominant arm

abstract:
Motor lateralization refers to differences in the neural organization of cerebral hemispheres, resulting in different control specializations between the dominant and the non-dominant motor systems. Multiple studies proposed that the dominant hemisphere is specialized for open-loop optimization-like processes. Recently, comparing arm kinematics between upward and downward movements, we found that the dominant arm outperformed the non-dominant one regarding gravity-related motor optimization in healthy young subjects. The literature about aging effects on motor control presents several neurophysiological and behavioral evidences for an age-related reduction of motor lateralization. Here, we compare the lateralization of a well-known gravity-related optimal motor control process between young and older adults. Thirty healthy young (mean age = 24.1 {+/-} 3 years) and nineteen healthy older adults (mean age = 73.0 {+/-} 8) performed single degree-of-freedom vertical arm movements between two targets (upward and downward).

Participants alternatively reached with their dominant and non-dominant arms. We recorded arm kinematics and electromyographic activities of the prime movers (Anterior and Posterior Deltoids) and we analyzed parameters thought to represent the hallmark of the gravity-related optimization process (i.e directional asymmetries and negative epochs on the phasic EMG activity). We found no arm difference in older participants, such that parameters with both arms were similar to those of young participants with their dominant arm. With the non-dominant arm, these results suggest that older adults better optimize gravity effects than young adults.


title: Some rats take more time to make easier perceptual decisions

abstract: When subjects control the duration of sampling a sensory stimulus before making a decision, they generally take more time to make more difficult sensory discriminations. This has been found to be true of many rats performing visual tasks. But two rats performing visual motion discrimination were found to have inverted chronometric response functions: their average response time paradoxically increased with stimulus strength. We hypothesize that corrective decision reversals may underlie this unexpected observation.


title: Chronic stroke sensorimotor impairment is related to smaller hippocampal volumes: An ENIGMA analysis

abstract:
Persistent sensorimotor impairments after stroke can negatively impact quality of life. The hippocampus is involved in sensorimotor behavior but has not been widely studied within the context of post-stroke upper limb sensorimotor impairment. The hippocampus is vulnerable to secondary degeneration after stroke, and damage to this region could further weaken sensorimotor circuits, leading to greater chronic sensorimotor impairment. The purpose of this study was to investigate the cross-sectional association between non-lesioned hippocampal volume and upper limb sensorimotor impairment in people with chronic stroke. We hypothesized that smaller ipsilesional hippocampal volumes would be associated with worse upper-limb sensorimotor impairment.

Cross-sectional T1-weighted brain MRIs were pooled from 357 participants at the chronic stage after stroke (>180 days post-stroke) compiled from 18 research cohorts worldwide in the ENIGMA Stroke Recovery Working Group (age: median = 61 years, interquartile range = 18, range = 23-93; 135 women and 222 men). Sensorimotor impairment was estimated from the Fugl-Meyer Assessment of Upper Extremity scores. Robust mixed-effects linear models were used to test associations between post-stroke sensorimotor impairment and hippocampal volumes (ipsilesional and contralesional separately; Bonferroni-corrected, p-value < 0.025), controlling for age, sex, lesion volume, and lesioned hemisphere. We also performed an exploratory analysis to test whether sex differences influence the relationship between sensorimotor impairment and hippocampal volume.

Upper limb sensorimotor impairment was positively associated with ipsilesional (p = 0.005; d = 0.33) but not contralesional (p = 0.96; d = 0.01) hippocampal volume, such that impairment was worse for participants with smaller ipsilesional hippocampal volume. This association remained significant independent of lesion volume or other covariates (p = 0.001; d = 0.36). Evidence indicates an interaction between sensorimotor impairment and sex for both ipsilesional (p = 0.008; d = -0.29) and contralesional (p = 0.006; d = -0.30) hippocampal volumes, whereby women showed progressively worsening sensorimotor impairment with smaller hippocampal volumes compared to men.

The present study has identified a novel association between chronic post-stroke sensorimotor impairment and ipsilesional, but not contralesional, hippocampal volume. This finding was not due to lesion size and may be stronger in women. We also provide supporting evidence that smaller hippocampal volume post-stroke is likely a consequence of ipsilesional damage, which could provide a link between vascular disease and other disorders, such as dementia.


title: Increased alcohol self-administration following repeated Toll-like receptor 3 agonist treatment in male and female rats

abstract: Toll-like receptor (TLR) signaling may play an important role in the neuroimmune systems involvement in the development and maintenance of alcohol use disorder. In the present study we administered TLR3 agonist poly(I:C) in male and female Long-Evans rats to determine whether TLR3 agonism can increase alcohol consumption in a daily 15% alcohol operant self-administration paradigm. We found few effects when poly(I:C) was given every-other-day at 0.3 or 1.0 mg/kg, however when instead 1.0 mg/kg was given on consecutive days alcohol intake increased in the days following injections specifically in females. Furthermore, in a second experiment we found that this effect only emerged when rats had a history of multiple poly(I:C) injections. In the final experiment the dose was increased to 3.0 mg/kg on consecutive days which resulted in significant reductions on injection days in females that were not accompanied by subsequent increases. The dose was increased to 9 mg/kg for one final pair of injections which led to reductions in intake in both males and females but only increased subsequent alcohol consumption in males. Overall, poly(I:C) was able to increase subsequent alcohol consumption in both sexes, with females being sensitive to lower doses than males both in terms of changes in alcohol consumption and general locomotor reduction. These findings show that TLR3 agonism may be involved in driving increased alcohol consumption and add to the body of work identifying the neuroimmune system as a potential therapeutic target for AUD.


title: Dendritic cell ICAM-1 strengthens immune synapses but is dispensable for effector and memory responses

abstract:
Lymphocyte priming in lymph nodes (LNs) depends on the formation of functional TCR specific immune synapses (ISs) with antigen (Ag) presenting dendritic cells. The high affinity LFA-1 ligand ICAM-1 has been implicated in different ISs studied in vitro. The in vivo roles of DC ICAM-1 in Ag stimulated T cell differentiation have been unclear. In newly generated DC conditional ICAM-1 knockout mice, we report that under Th1 polarizing conditions, ICAM-1 deficient DCs could not engage in stable conjugates with newly generated CD8 blasts. Nevertheless, these DCs triggered normal lymphocyte priming, proliferation and differentiation into functional cytotoxic T cells (CTLs) and central memory lymphocytes (Tcm) in both vaccinated and virus infected skin. Single cell RNAseq analysis confirmed that Tcm were normally generated in these mice and gave rise to normal T effectors during a recall skin response. Our results suggest that although CD8 T cell blasts tightly bind DC-ICAM-1, strongly adhesive DC-T ISs are not necessary for functional TCR dependent DC mediated CD8 T cell proliferation and differentiation into productive effector and memory lymphocytes.

SummarySapoznikov et al generated a new genetic murine model deficient in dendritic cell expression of the key adhesion molecule ICAM-1 and found that CD8 lymphocytes do not require strong adhesion to dendritic cells for antigen-dependent differentiation into effector and memory T cells.


title: Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis

abstract: Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.


title: Single-cell RNA sequencing unveils intestinal eosinophil development and specialization

abstract: Eosinophils are an integral part of the gastrointestinal (GI) immune system that contribute to homeostatic and inflammatory processes. Here, we investigated the existence of functional subsets that carry out specialized tasks in health and disease. We used single-cell transcriptomics and high-dimensional flow cytometry to delineate murine eosinophil subpopulations and their ontogenetic relationship in the steady state and during infection and inflammation. Profiling of eosinophils from bone marrow, blood, spleen and several GI tissues revealed five distinct subsets representing consecutive developmental and maturation stages across organs, each controlled by a specific set of transcription factors. Furthermore, we discovered a highly adapted PD-L1+CD80+ eosinophil subset in the GI tract, characterized by its immune regulatory properties, bactericidal activity upon challenge infection and tissue-protective function during inflammation. Our data provide a framework for the characterization of eosinophil subsets in GI diseases and highlight their crucial contribution to homeostasis, immune regulation and host defense.


title: Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation

abstract: Robust and effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain energetically costly processes like growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin. By employing complementary genetic, epigenetic, and metabolic approaches, we revealed that tricarboxylic acid (TCA) cycle flux fuels biosynthetic processes while controlling the ratio of -ketoglutarate/succinate to modulate the activities of dioxygenases that are critical for driving T cell inflammation. In contrast to cancer cells, where succinate dehydrogenase (SDH)/complex II inactivation drives cell transformation and growth, SDH/complex II deficiency in T cells causes proliferation and survival defects when the TCA cycle is truncated, blocking carbon flux to support nucleosides biosynthesis. Accordingly, replenishing the intracellular nucleoside pool partially relieved the dependence of T cells on SDH/complex II for proliferation and survival. Conversely, SDH deficiency induces a pro-inflammatory gene signature in T cells and promotes T helper 1 (TH1) and T helper 17 (TH17) lineage differentiation. Mechanistically, the hypoxia-inducible factor 1 (HIF-1) is not required for succinate-induced inflammation in T cells. A reduced -ketoglutarate/succinate ratio in SDH deficient T cells promotes inflammation through changing the pattern of the transcriptional and chromatin-accessibility signatures and consequentially increasing the expression of the transcription factor, B lymphocyte-induced maturation protein-1 (Blimp-1). Collectively, our studies revealed a critical role of SDH/complex II in allocating carbon resources for anabolic processes and epigenetic regulation in T cell proliferation and inflammation.


title: Pufferfish (Tetraodon cutcutia) sampled from freshwater river serves an intermediate reservoir of sucrose nonfermenting variant of Vibrio cholerae PS-4.

abstract:
This study describes the genomic characteristics of Vibrio cholerae strain PS-4, which fails to ferment sucrose on thiosulfate{square}citrate{square}bile salt{square}sucrose (TCBS) agar medium. This bacterium was isolated from skin mucus of a freshwater pufferfish. In order to understand the sucrose nonfermenting phenotype, the genome of the strain PS-4 was sequenced. The gene encoding the sucrose specific phosphotransferase system IIB (sucR) was absent, resulting for the defective sucrose fermenting phenotype. In contrast, genes encoding glucose-specific transport system IIB (ptsG) and fructose specific transport system IIB (fruA) were present and showing acid production while growing with respective sugars. The overall genome relatedness indices (OGRI) such as in silico DDH, average nucleotide identity (ANI) and average amino acid identity (AAI) were above the threshold value, i.e., 70% and 95-96%, respectively. Phylogenomic analysis based on genome wide core genes and the non-recombinant core-genes, strain PS-4 clustered with Vibrio cholerae ATCC 14035T. Further, genes encoding ctx, zot, ace, tcp and rfb were absent. It showed hemolytic activity and reacted strongly to the R antibody. This is the first report on the existence of Vibrio cholerae from the pufferfish adds to our knowledge of the new ecological niche of this bacterium.

IMPORTANCEVibrio cholerae, the causative agent of cholera, is a natural inhabitant of aquatic environments. It ferments sucrose, producing characteristic yellow colonies on TCBS agar. Vibrio cholerae strain PS-4 described in this study was a sucrose nonfermenting variant associated with pufferfish skin and did not produce yellow colonies on TCBS agar. Genes encoding sucrose specific phosphotransferase system IIB (sucR) was absent. The observed phenotype in the characteristic metabolic pathway indicates niche specific adaptive evolution for this bacterium. Our study suggested that nonfermenting phenotype on TCBS agar may not be considered always for Vibrio cholerae species delineation.


title: Assessing long-read sequencing with Nanopore R9, R10, and PacBio CCS to obtain high-quality metagenome assembled genomes from complex microbial communities

abstract: Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-perfect genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-perfect microbial genomes from isolates or metagenomes without shortread or reference polishing.


title: Oxford Nanopore R10.4 long-read sequencing enables near-perfect bacterial genomes from pure cultures and metagenomes without short-read or reference polishing


title: Jaagsiekte sheep retrovirus infection induces changes in microRNA expression in the ovine lung

abstract:
Ovine pulmonary adenocarcinoma (OPA) is an infectious neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus. OPA is an important veterinary problem and is also a valuable large animal model for human lung adenocarcinoma. JSRV infects type 2 alveolar epithelial cells in the lung and induces the growth of tumors, but little is known about the molecular events that lead to the activation of oncogenic pathways in infected cells. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides with important roles in regulating gene expression in eukaryotes and with well-established roles in cancer. Here we used small-RNA sequencing to investigate the changes in miRNA expression that occur in JSRV-infected ovine lung. After filtering out low abundance miRNAs, we identified expression of 405 miRNAs, 32 of which were differentially expressed in JSRV-infected lung compared to mock-inoculated control lung. Highly upregulated miRNAs included miR-182, miR-183, miR-96 and miR-135b, which have also been associated with oncogenic changes in human lung cancer. Network analysis of genes potentially targeted by the deregulated miRNAs identified their involvement in pathways known to be dysregulated in OPA. We found no evidence to support the existence of miRNAs encoded by JSRV. This study provides the first information on miRNA expression in OPA and identifies a number of targets for future studies into the role of these molecules in the pathogenesis of this unique veterinary model for human lung adenocarcinoma.

IMPORTANCEOvine pulmonary adenocarcinoma is a neoplastic lung disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant welfare and economic concern for sheep producers and is a valuable large animal model for human lung adenocarcinoma. MicroRNAs are small RNA molecules of approximately 22 nucleotides with important functions in regulating gene expression in eukaryotes and with well-established roles in cancer. In this study, we examined the changes in microRNA expression that occur in the lung in response to JSRV infection. We identified differential expression of a number of host-encoded microRNAs in infected tissue, including microRNAs with roles in human cancer. We found no evidence that JSRV encodes a microRNA. This study provides new insights on the cellular response to JSRV infection in the ovine lung, which will inform future studies into the pathogenesis of OPA in sheep and its use as a model for human lung adenocarcinoma.


title: Xist spatially amplifies SHARP recruitment to balance chromosome-wide silencing and specificity to the X chromosome

abstract: Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes ([~]1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also called Spen) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. Our results suggest that this spatial amplification mechanism may be a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets.