title: Nanopore callers for epigenetics from limited supervised data

abstract: Nanopore sequencing platforms combined with supervised machine learning (ML) have been effective at detecting base modifications in DNA such as 5mC and 6mA. These ML-based nanopore callers have typically been trained on data that span all modifications on all possible DNA k-mer backgrounds--a complete training dataset. However, as nanopore technology is pushed to more and more epigenetic modifications, such complete training data will not be feasible to obtain. Nanopore calling has historically been performed with Hidden Markov Models (HMMs) that cannot make successful calls for k-mer contexts not seen during training because of their independent emission distributions. However, deep neural networks (DNNs), which share parameters across contexts, are increasingly being used as callers, often outperforming their HMM cousins. It stands to reason that a DNN approach should be able to better generalize to unseen k-mer contexts. Indeed, herein we demonstrate that a common DNN approach (DeepSignal) outperforms a common HMM approach (Nanopolish) in the incomplete data setting. Furthermore, we propose a novel hybrid HMM-DNN approach, Amortized-HMM, that outperforms both the pure HMM and DNN approaches on 5mC calling when the training data are incomplete. Such an approach is expected to be useful for calling 5hmC and combinations of cytosine modifications, where complete training data are not likely to be available.


title: Automated compound testing in zebrafish xenografts identifies combined MCL-1 and BCL-XL inhibition to be effective against Ewing sarcoma

abstract: Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.

category: cancer biology


title: Fyn kinase inhibition using AZD0530 improves recognition memory and reduces depressive-like behaviour in an experimental model of Parkinson's disease

abstract:
Fyn kinase has recently been established as a major upstream regulator of neuroinflammation in PD. This study aimed to determine if inhibition of Fyn kinase could lead to reduced neuroinflammation and improvements in motor and non-motor impairments in an early-stage model of PD. An experimental model of PD was produced using intra-striatal injection (4{micro}l) of the neurotoxin 6-OHDA (5{micro}g/{micro}l). Sprague Dawley rats (n=42) were given either vehicle, 6mg/kg or 12mg/kg of Fyn kinase inhibitor (AZD0530) daily for 32 days via oral gavage and tested on a battery of tasks assessing motor, cognitive and neuropsychiatric outcomes. AZD 0530 administration led to improvement in volitional locomotion and recognition memory, as well as a reduction in depressive-like behaviour. Pathologically, an inflammatory response was observed; however, there were no significant differences in markers of neuroinflammation between treatment groups. Taken together, results indicate a potential therapeutic benefit for use of Fyn kinase inhibition to treat non-motor symptoms of PD, although mechanisms remain to be elucidated.

HIGHLIGHTSO_LIFyn kinase has recently been proposed as a major upstream regulator of microglial activation in Parkinsons disease (PD).
C_LIO_LIThis study was the first to evaluate the effects of Fyn kinase inhibition in a rodent model of PD.
C_LIO_LIFyn kinase inhibition using the Fyn kinase inhibitor AZD 0530 was capable of improving volitional locomotion and recognition memory and reducing depressive-like behaviour in a rodent model of PD.
C_LIO_LIInterestingly, while increases in microglial activation were observed in this rodent model of PD, AZD 0530 did not significantly reduce this activation.
C_LIO_LIThis suggests that the behavioural improvements associated with Fyn kinase inhibition may occur independently of neuroinflammation and may be attributable to other brain mechanisms, including actions on NMDA or 5-HT6 receptors.
C_LI

category: neuroscience


title: Psychophysiological Evidence for Fear Extinction Learning via Mental Imagery

abstract: Imagery-based extinction procedures have long been used in the treatments of fear-related conditions. The assumption is that imagery can substitute for the perceptual stimuli in the extinction process. Yet, experimental validations of this assumption have been limited in number and some have relied exclusively on measures of autonomic reactivity without consideration of conscious feelings of fear. The current investigation sought to assess whether imagery-based exposure could lead to extinction of conditioned fear to the corresponding perceptual stimulus. Conditioned fear responses were measured by both a physiological (i.e., skin conductance response/SCR) and a subjective (i.e., self-reported fear) measure. Participants (N = 56) first underwent perceptual differential fear conditioning, then imagery extinction, then perceptual extinction. SCR evidence was found for successful fear conditioning, generalization of fear from viewing to imagery, and most importantly, the absence of differential fear after imagery extinction upon re-exposure to the conditioned perceptual stimulus. Self-reported fear confirmed the acquisition and generalization of fear and provided evidence of a significant reduction in differential fear conditioning across extinction. Consistent with clinical evidence of the efficacy of imagery extinction and the existing limited experimental literature, the current study offers support for fear extinction to perceptual stimuli via imagery exposure.


title: Glial AP1 promotes early TBI recovery but chronically drives tauopathy

abstract: The emergence of degenerative disease after traumatic brain injury is often described as an acceleration of normal age-related processes. Whether similar molecular processes occur in injury and in age is unclear. Here we identify a functionally dynamic and lasting transcriptional response in glia, mediated by the conserved transcription factor AP1. In the early post-TBI period, glial AP1 is essential for recovery, ensuring brain integrity and animal survival. In sharp contrast, chronic AP1 activation promotes human tau pathology, tissue loss, and mortality. We show a similar process activates in healthy brains with age. Importantly, AP1 activity is present in moderate human TBI, decades after injury, and associates with microglial activation and tauopathy. Our data provide key molecular insight into glia, highlighting that the same molecular process drives dynamic and contradictory glia behavior in TBI, first acting to protect but chronically promoting disease.


title: Evaluation of RNAlater™ as a field-compatible preservation method for metaproteomic analyses of bacteria-animal symbioses

abstract:
Field studies are central to environmental microbiology and microbial ecology as they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows to study these communities  in situ which requires protein preservation directly in the field as protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is non-hazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability in field conditions when targeted for metaproteomics is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater storage after 1 day, 1 week and 4 weeks at room temperature (22-23 {degrees}C). We evaluated protein preservation using one dimensional liquid chromatography tandem mass spectrometry (1D-LC-MS/MS). We found that RNAlater and flash freezing preserved proteins equally well in terms of total number of identified proteins or relative abundances of individual proteins and none of the test time points were altered compared to t0. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomics data and the logistical requirements for field deployment we recommend RNAlater for protein preservation of field-collected samples when targeted for metaproteomcis.

ImportanceMetaproteomics, the large-scale identification and quantification of proteins from microbial communities, provides direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory set-ups samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations due to its hazardous nature and transport restrictions. Our study shows that RNAlater can serve as a low hazard, easy to transport alternative to flash freezing for field preservation of samples for metaproteomics. We show that RNAlater preserves the metaproteome equally well as compared to flash freezing and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.

category: microbiology


title: Pressure and curvature control of contact inhibition in epithelia growing under spherical confinement

abstract: Morphogenesis requires spatiotemporal regulation of cell shape and proliferation, both regulated by biochemical and mechanical cues. In epithelia, this regulation is called contact inhibition, but disentangling biochemical from mechanical cues remains challenging. Here, we show that epithelia growing under confinement accumulate pressure that inhibits proliferation above a threshold value, which depends on the {beta}-catenin pathway. Before inhibition of proliferation, cell aspect ratio abruptly increased upon reaching confluency. This shape transition occurred at low, constant pressure and was mainly controlled by cell density and contractility, correlating with YAP/TAZ pathway inhibition. In our system, epithelia spontaneously buckle: we observed that folding transiently reactivates both the YAP/TAZ pathway and cell proliferation. Altogether, our results support that different mechanical cues part of contact inhibition regulate cell proliferation through different mechanosensing pathways. Proliferation is regulated by sustained, tissue-level pressure through the {beta}-catenin pathway, and by local curvature and pressure changes through the YAP/TAZ pathway.

category: cell biology


title: Analysis of conditional colocalization relationships and hierarchies from three-color microscopy images

abstract: Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within various subcellular compartments and allows the interrogation of molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images. This limits their applicability and the type of information that they reveal, leading to underutilization of multicolor microscopy images. Here we describe an approach, termed "conditional colocalization analysis," for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We showcase two applications of conditional colocalization analysis, one addressing the question of compartmentalization of molecular interactions, and one investigating the hierarchy of molecular interactions in a multimolecular complex. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.


title: Mapping QTLs for phenotypic and morpho-physiological traits related to grain yield under late sown conditions in wheat (Triticum aestivum L.)

abstract: The elevating temperature makes heat stress one of the major issues for wheat production globally. To elucidate genetic basis and map heat tolerance traits, a set of 166 doubled haploid lines (DHLs) derived from the cross between PBW3438/IC252874 was used. The population was evaluated under Normal sown (NS) and late sown (LS) conditions, by exposing to heat stress during rabi season. The canopy temperature (CT) showed positive correlations with grain yield, whereas Soil plant analysis development (SPAD) was not significantly correlated and associated with GY in both the normal and late sown conditions. Composite interval mapping (CIM) identified total 12 Quantitative trait loci (QTLs) viz., 2 (Normal sown), 10 (late sown) mapped on linkage groups 1A, 1D, 2B, 2D, 3B, 4D, 5B and 6D, during both the crop seasons 2017-18 and 2018-19. Combining the results of these QTLs revealed a major stable QTL for grain yield (GY) on chromosome 3B with 11.84% to 21.24% explaining phenotypic variance under both sowing conditions. QTL for CT and SPAD was detected on chromosome 1A while QTL for GY on chromosome 3B and 5B. The identified QTLs in the genomic regions could be targeted for genetic improvement and marker assisted selection for heat tolerance in wheat. The tools like SPAD and CT could be exploited to screen the large number of breeding lines.

category: plant biology


title: Post-Infection Entry Mechanism of Ricin A Chain-Pokeweed Antiviral Proteins (RTA-PAPs) Chimeras is Mediated by Viroporins

abstract: The limitations of virus-specific antiviral drugs became apparent during the current COVID-19 pandemic. The search for broad range antiviral proteins of a new kind to answer current and future pandemics has become an even more pressing matter. Here, the author further describes the expected anti-SARS-CoV-2 mechanisms of a novel broad range antiviral chimeric protein constructed between ricin A chain and pokeweed antiviral proteins. The latest in protein-ligand docking software were used to determine binding affinity of RTA-PAPs to SARS-CoV-2 frameshift stimulation element and elucidate the preferential post-infection entry mechanisms of RTA-PAPs into virus infected cells over non-infected ones, by doing a comparative analysis between in vitro and in silico results on numerous viruses. The results obtained strongly suggest that the post-infection preferential entry of RTA-PAPs into infected cells is mediated by the presence of viroporins integrated into the host cell membrane. The discovery of this mechanism revealed RTA-PAPs, and proteins like them, to be a new class of broad range antivirals that target with high specificity viroporin producing viruses, and with gain of functions in antiviral activities, post-infection.

category: synthetic biology


title: Oligomeric α-Synuclein induces skin degeneration in a reconstructed human epidermis model

abstract: Cell senescence may promote epidermal inflammation and degeneration, termed as inflammaging, which is accompanied by keratinocyte loss, resulting in fine lines of wrinkles. Recent findings showed that healthy elderly skin expresses age- and neuron-related amyloidogenic proteins, such as tau, {beta}-Amyloid34, and -synuclein (-Syn), typically found in patients with neurodegenerative diseases. These proteins form toxic aggregates that trigger inflammatory signals. Herein, we investigated the impact of oligomeric -Syn (O-Syn) on the neurosphere (NP) and the reconstructed human epidermis (RHE) 3D models. First, we found the expression of -Syn, {beta}-Amyloid, and amyloid precursor protein (APP) in the RHE. Second, we challenged the RHE and NP with O-Syn, which decreased RHE regeneration, measured by the percentage of cell proliferation and thickness of the stratum basale, but did not affect NP neurite outgrowth. O-Syn did not decrease the number of human neonatal epidermal keratinocytes (HEKn) but, as seen for the RHE, it also decreased the proliferation of HEKn. We confirmed that the oligomeric, and not the monomeric -Syn species, accounted for the proliferation-decreasing effect. O-Syn also increased the NF-kB nuclear translocation in HEKn analyzed by nucleus/cytoplasm NF-{kappa}B fluorescence intensity. In addition, O-Syn triggered inflammation in the RHE, by increasing the mRNA levels of IL-1{beta} and tumor necrosis factor-alpha (TNF-), and the release of TNF- in a time-dependent manner. These findings show that O-Syn does not affect neurite outgrowth but induces a decrease in keratinocyte proliferation along with epidermal inflammation. With our tridimensional models, we demonstrated that the neurodegenerative protein O-Syn also degenerates the epidermis, drawing attention to the need of target-based screening to prevent and treat the effects of skin aging.


title: The predicted bZIP transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans

abstract: Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts. For example, almost nothing is known about the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans. Two types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is a positive-sense RNA virus, and microsporidia, which are fungal pathogens. Surprisingly, these molecularly distinct pathogens induce a common host transcriptional response called the Intracellular Pathogen Response (IPR). Here we describe zip-1 as an IPR regulator that functions downstream of all known IPR activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor of previously unknown function, and we show how zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for all triggers of the IPR, and that this transcription factor plays a protective role against intracellular pathogen infection in C. elegans.

category: immunology


title: Memory B cells control SARS-CoV-2 variants upon mRNA vaccination of naive and COVID-19 recovered individuals.

abstract: How a previous SARS-CoV-2 infection may amplify and model the memory B cell (MBC) response elicited by mRNA vaccines was addressed by a comparative longitudinal study of two cohorts, naive individuals and disease-recovered patients, up to 2 months after vaccination. The quality of the memory response was assessed by analysis of the VDJ repertoire, affinity and neutralization against variants of concerns (VOC), using unbiased cultures of 2452 MBCs. Upon boost, the MBC pool of recovered patients selectively expanded, further matured and harbored potent neutralizers against VOC. Maturation of the MBC response in naive individuals was much less pronounced. Nevertheless, and as opposed to their weaker neutralizing serum response, half of their RBD-specific MBCs displayed high affinity towards multiple VOC and one-third retained neutralizing potency against B.1.351. Thus, repeated vaccine challenges could reduce these differences by recall of affinity-matured MBCs and allow naive vaccinees to cope efficiently with VOC.


title: Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated with Anti-Tumor Immunity

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


title: Metagenome-based comparisons of decay rates and host-specificity of fecal microbial communities for improved microbial source tracking

abstract:
Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST.

SYNOPSISWe track cow, pig, and human fecal pollution in lake water over time with metagenomics and benchmark these novel protocols against standard culture-based and qPCR tests for water quality monitoring.


title: SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

abstract: During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and currently, four variants of concerns (VOCs) are considered as the hazardous SARS-CoV-2 variants to the human society1. The newly emerging VOC, the B.1.617.2/Delta variant, closely associates with a huge COVID-19 surge in India in Spring 20212. However, its virological property remains unclear. Here, we show that the B.1.617.2/Delta variant is highly fusogenic, and notably, more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates the spike protein cleavage and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity than the parental virus. Our data suggest that the P681R mutation is a hallmark that characterizes the virological phenotype of the B.1.617.2/Delta variant and is closely associated with enhanced pathogenicity.


title: SARS-CoV-2 spike P681R mutation, a hallmark of the Delta variant, enhances viral fusogenicity and pathogenicity


title: The membrane associated accessory protein is an adeno-associated viral egress factor

abstract: Adeno-associated viruses (AAV) rely on helper viruses to transition from latency to lytic infection. Some recombinant AAV serotypes are secreted in a pre-lytic manner as extracellular vesicle (EV)-associated particles, although mechanisms underlying such are unknown. Here, we discover that the membrane-associated accessory protein (MAAP), expressed from a (+1) frameshifted open reading frame (ORF) in the AAV capsid (cap) gene, is a novel viral egress factor. MAAP contains a highly conserved, cationic amphipathic domain critical for AAV secretion. Wild type or recombinant AAV with a mutated MAAP start site (MAAP{Delta}) show markedly attenuated secretion and correspondingly, increased intracellular retention. Trans-complementation with recombinant MAAP restored extracellular secretion of multiple AAV/MAAP{Delta} serotypes. MAAP is sorted into recycling Rab11+ vesicles and strongly associates with EV markers upon fractionation. In addition to characterizing a novel viral egress factor, these studies highlight a prospective engineering platform to modulate secretion of AAV vectors or other EV-associated cargo.

category: molecular biology


title: Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules

abstract: We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.


title: Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1

abstract: Andersen-Tawil Syndrome (ATS) is associated with life threatening arrhythmias of unknown mechanism. We report on a mouse model carrying the trafficking-deficient mutant Kir2.1{Delta}314-315. The mouse recapitulates the electrophysiological phenotype of type 1 (ATS1), with slower conduction velocities in response to flecainide, QT prolongation exacerbated by isoproterenol, and increased vulnerability to calcium-mediated arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1{Delta}314-315 expression significantly reduced inward rectifier K+ and Na+ inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel sarcoplasmic reticulum (SR) microdomain of functional Kir2.1 channels contributing to intracellular Ca2+ homeostasis. Kir2.1{Delta}314-315 cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca2+ release events. This is the first in-vivo demonstration of a dual arrhythmogenic mechanism of ATS1 defects in Kir2.1 channel function at the sarcolemma and the SR, with overlap between ATS1 and CPVT.


title: Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

abstract: During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes [1]. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity [2-7]. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force [8]. Instead, the kinesin-12 family motor KLP-18 performs this function [9-12]. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles [12]. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.


title: Whole Genome Sequence and CAZyme distribution of the cellulase hyper producing filamentous fungus Penicillium janthinellum NCIM 1366

abstract: Penicillium janthinellum NCIM 1366, capable of secreting cellulases that are highly efficient in the hydrolysis of lignocellulosic biomass, was sequenced to understand its cellulolytic machinery. De novo sequencing and assembly revealed a 37.6 Mb genome encoding 11,848 putative proteins, 93% of which had significant BLAST-P hits. The majority of the top hits (those with over 60% UniProt identity) belonged to P. brasilianum. Carbohydrate active enzymes (CAZymes) and other enzymes involved in lignocellulose degradation were also predicted from this strain and compared with those of the industrial workhorse of cellulase production-Trichoderma reesei RUT-C30. The comparison showed that the fungus encodes a far higher number of CAZYmes (422) as compared to T. reesei RUT-C30 (244), which gives a plausible explanation for its overall effectiveness in biomass hydrolysis. An analysis of the secreted CAZymes and annotated ligninases identified 216 predicted proteins which may be directly involved in the breakdown of lignocellulose.

category: genomics


title: Benchmarking UMI-based single cell RNA-sequencing preprocessing workflows

abstract:
Single-cell RNA sequencing (scRNA-seq) technologies and associated analysis methods have undergone rapid development in recent years. This includes methods for data preprocessing, which assign sequencing reads to genes to create count matrices for downstream analysis. Several packaged preprocessing workflows have been developed that aim to provide users with convenient tools for handling this process. How different preprocessing workflows compare to one another and influence downstream analysis has been less well studied.

Here, we systematically benchmark the performance of 9 end-to-end preprocessing workflows (Cell Ranger, Optimus, salmon alevin, kallisto bustools, dropSeqPipe, scPipe, zUMIs, celseq2 and scruff) using datasets with varying levels of biological complexity generated on the CEL-Seq2 and 10x Chromium platforms. We compare these workflows in terms of their quantification properties directly and their impact on normalization and clustering by evaluating the performance of different method combinations. We find that lowly expressed genes are discordant between workflows and observe that some workflows have systematic biases towards particular classes of genomics features. While the scRNA-seq preprocessing workflows compared varied in their detection and quantification of genes across datasets, after downstream analysis with performant normalization and clustering methods, almost all combinations produced clustering results that agreed well with the known cell type labels that provided the ground truth in our analysis.

In summary, the choice of preprocessing method was found to be less influential than other steps in the scRNA-seq analysis process. Our study comprehensively compares common scRNA-seq preprocessing workflows and summarizes their characteristics to guide workflow users.

category: bioinformatics


title: mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation.

abstract: mRNA 5' cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F-directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F- mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E-cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E- mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E-cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F-mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5' end, and suggest a model for how eIF4F-mRNA dynamics establish mRNA sensitivity to translational control processes.

category: biophysics


title: Characterization of endogenous Rubus yellow net virus in raspberries

abstract: Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnavirids are found in plants as endogenous, inactive sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV infections, we sequenced the genomes of various Rubus cultivars and mined eight additional published whole genome sequencing datasets. Sequence analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, small sequence fragments. We developed assays to genotype the six main endoRYNV lineages as well as the only known episomal lineage in commercial Rubus. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding programs have been using a limited pool of germplasm that harbored endoRYNVs.


title: Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

abstract: Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome, might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant bryophytes. Whereas angiosperms repair DNA double strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in bryophytes homologous recombination (HR) is the main DNA-DSB repair pathway facilitating the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in the moss Physcomitrella using haploid plants and autodiploid plants, generated via an artificial duplication of the whole genome. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the whole genome duplication event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p<0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.


title: Light-induction of endocannabinoids and activation of Drosophila TRPC channels

abstract: Drosophila phototransduction represents a classical model for signaling cascades that culminate with activation of TRP channels. TRP and TRPL are the canonical TRP (TRPC) channels, which are gated by light stimulation of rhodopsin and engagement of Gq and phospholipase C{beta} (PLC). Despite decades of investigation, the mechanism of TRP activation in photoreceptor cells is unresolved. Here, using a combination of genetics, lipidomics and Ca2+ imaging, we found that light increased the levels of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG) in vivo. The elevation in 2-LG strictly depended on the PLC encoded by norpA. Moreover, this endocannabinoid upregulated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.


title: No evidence for changes in GABA concentration, functional connectivity, or working memory following continuous theta burst stimulation over dorsolateral prefrontal cortex

abstract: Continuous theta burst stimulation (cTBS) is thought to reduce cortical excitability and modulate functional connectivity, possibly by altering cortical inhibition at the site of stimulation. However, most evidence comes from the motor cortex and it remains unclear whether similar effects occur following stimulation over other brain regions. We assessed whether cTBS over left dorsolateral prefrontal cortex altered gamma aminobutyric acid (GABA) concentration, functional connectivity and brain dynamics at rest, and brain activation and memory performance during a working memory task. Seventeen healthy individuals participated in a randomised, sham-controlled, cross-over experiment. Before and after either real or sham cTBS, magnetic resonance spectroscopy was obtained at rest to measure GABA concentrations. Functional magnetic resonance imaging (fMRI) was also recorded at rest and during an n-back working memory task to measure functional connectivity, regional brain activity (low-frequency fluctuations), and task-related patterns of brain activity. We could not find evidence for changes in GABA concentration (P=0.66, Bayes factor [BF10]=0.07), resting-state functional connectivity (P(FWE)>0.05), resting-state low-frequency fluctuations (P=0.88, BF10=0.04), blood-oxygen level dependent activity during the n-back task (P(FWE) >0.05), or working memory performance (P=0.13, BF10=0.05) following real or sham cTBS. Our findings add to a growing body of literature suggesting the effects of cTBS are highly variable between individuals and question the notion that cTBS is a universal  inhibitory paradigm.


title: Predicting microbiome compositions through deep learning.

abstract: Microbes can form complex communities that perform critical functions in maintaining the integrity of their environment or their hosts well-being. Rationally managing these microbial communities requires improving our ability to predict how different species assemblages affect the final species composition of the community. However, making such a prediction remains challenging because of our limited knowledge of the diverse physical, biochemical, and ecological processes governing microbial dynamics. To overcome this challenge, here we present a deep learning framework that automatically learns the map between species assemblages and community compositions from training data only, without knowledge of any of the above processes. First, we systematically validate our framework using synthetic data generated by classical population dynamics models. Then, we apply it to experimental data of both in vitro and in vivo communities, including ocean and soil microbial communities, Drosophila melanogaster gut microbiota, and human gut and oral microbiota. In particular, we show how our framework learns to perform accurate out-of-sample predictions of complex community compositions from a small number of training samples. Our results demonstrate how deep learning can enable us to understand better and potentially manage complex microbial communities.


category: systems biology


title: Predicting microbiome compositions from species assemblages through deep learning.


title: Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

abstract: RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.


title: Demographic Inferences and Loci Under Selection in a Recently Expanded Coral Population

abstract: Coral poleward range expansions in response to warming oceans have been historically observed, however contemporary expansion rates of some coral species have become more rapid as global temperatures rise at unprecedented rates. Range expansion can lead to reduced genetic diversity and surfing of deleterious mutations in expanding populations, potentially limiting the ability for adaption and persistence in novel environments. Successful expansions that overcome these founder effects and colonize new habitat have been attributed to multiple introductions from different sources, hybridization with native populations, or rapid adaptive evolution. Here, we investigate population genomic patterns of the reef-building coral Acropora hyacinthus along a latitudinal cline that includes a well-established range expansion front in Japan using 2b-RAD sequencing. A total of 184 coral samples were collected across seven sites spanning from [~]24{degrees}N to near its northern range front at [~]33{degrees}N. We uncover the presence of three cryptic lineages of A. hyacinthus, which occupy discrete areas within this region. Only one lineage is present at the expansion front and we find evidence of its historical occupation of marginal habitats. Within this lineage we also find evidence of bottleneck pressures associated with expansion events including higher clonality, increased linkage disequilibrium, and lower genetic diversity in range edge populations compared to core populations. Asymmetric migration between populations was also detected with lower migration from edge sites. Lastly, we describe genomic signatures of local adaptation potentially attributed to lower winter temperatures experienced at the more recently expanded northern populations. Together these data illuminate the genomic consequences of range expansion in a coral and highlight how adaptation to colder temperatures along the expansion front may facilitate further range expansion in this coral lineage.

category: evolutionary biology


title: Quantitative Analysis of Genomic Sequences of Virus RNAs Using a Metric-Based Algorithm

abstract: This work aims to study the virus RNAs using a novel accelerated algorithm on exploring any-length genomic fragments in sequences using Hamming distance between the binary-expressed RNA symbols and explored pattern characters. The found repetitive genomic sub-sequences of different lengths were placed on one plot as genomic trajectories (walks) to increase the effectiveness of geometrical multi-scale genomic studies. Primary attention was paid to the building and analysis of the atg-triplet walks composing the schemes or skeletons of the viral RNAs. The 1-D distributions of these codon-starting atg-triplets were built with the single-symbol walks for full-scale analyses. The visual examination was followed by calculating statistical parameters of genomic sequences, including the estimation of geometry deviation and fractal properties of inter-atg distances. This approach was applied to the SARS CoV-2, MERS CoV, Dengue and Ebola viruses, whose complete genomic sequences are taken from GenBank and GISAID databases. Relative stability of these distributions for SARS CoV-2 and MERS CoV viruses was found, unlike the Dengue and Ebola distributions that showed increased deviation of their geometrical and fractal characteristics of atg-distributions.


title: The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei

abstract: The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.

category: biochemistry


title: Astrovirus Disrupts Intestinal Barrier Function by Activating Epithelial-Mesenchymal Transition

abstract: Human astroviruses (HAstV), positive sense single-stranded RNA viruses, are one of the leading causes of diarrhea worldwide. Despite their high prevalence, the cellular mechanisms of astrovirus pathogenesis remain ill-defined. Previous studies showed HAstV increased epithelial barrier permeability by causing a relocalization of the tight junction protein, occludin. In these studies, we demonstrate that HAstV infection induces epithelial-mesenchymal transition (EMT), by upregulation the transcription of EMT-related genes within 8 hours post-infection (hpi), followed by the loss of cell-cell contacts and disruption of polarity by 24 hpi. Unlike the relocalization of tight junction proteins, HAstV-induced EMT requires productive replication and is independent of cellular factors including transforming growth factor-{beta} (TGF-{beta}). While multiple classical HAstV serotypes, including clinical isolates, induce EMT, the non-classical genotype HAstV-VA1 and two strains of reovirus are incapable of inducing EMT. This finding puts classical strains of HAstV-1 in an exclusive group of non-oncogenic viruses triggering EMT.


title: Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication

abstract:
Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonellas intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting SPI-1 T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still restricted intracellular Salmonella replication. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.

Author summarySalmonella enterica serovar Typhimurium is a gastrointestinal bacterial pathogen that causes diarrheal disease and is a major cause of morbidity and mortality worldwide. Salmonella uses molecular syringe-like machines called type III secretion systems (T3SSs) to inject virulence factors into host cells. These T3SSs enable Salmonella to infect and survive within host cells such as macrophages. However, host cells contain a family of cytosolic immune receptors, termed NAIPs, that recognize T3SS and flagellin components. Upon detecting these components, NAIPs recruit the adaptor protein NLRC4 to form signaling complexes called inflammasomes. Inflammasomes activate host proteases called caspases that mount robust immune responses against the invading pathogen. While mice encode multiple NAIPs that have been extensively studied, much remains unknown about how the single human NAIP mediates inflammasome responses to Salmonella in macrophages. Our study reveals that while NAIP is necessary to detect individual T3SS ligands in human macrophages, it is only partially required for inflammasome responses to Salmonella infection. We found that the NLRP3 and CASP4/5 inflammasomes are also activated, and the combination of NAIP- and NLRP3-mediated recognition limits intracellular Salmonella replication in human macrophages. Our results demonstrate that human macrophages employ multiple inflammasomes to mount robust host defense against Salmonella infection.


title: Stiffness of fluid and gel phase lipid nanovesicles: weighting the contributions of membrane bending modulus and luminal pressurization

abstract: The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available Force Spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the contributions of membrane rigidity and luminal pressure to the overall vesicle stiffness are described as a series of springs; this approach allows estimating the two contributions for both gel and fluid phase liposomes. Atomic Force Microscopy-based FS (AFM-FS), performed on both vesicles and Supported Lipid Bilayers (SLBs), is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. Results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The herein presented results offer a simple way to quantify two of the most important parameters in vesicular nanomechanics, and as such represent a first step towards a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.


title: IUCNN - deep learning approaches to approximate species' extinction risk

abstract:
AimThe global Red List (RL) from the International Union for the Conservation of Nature is the most comprehensive global quantification of extinction risk, and widely used in applied conservation as well as in biogeographic and ecological research. Yet, due to the time- consuming assessment process, the RL is biased taxonomically and geographically, which limits its application on large scales, in particular for understudied areas such as the tropics, or understudied taxa, such as most plants and invertebrates. Here we present IUCNN, an R- package implementing deep learning models to predict species RL status from publicly available geographic occurrence records (and other traits if available).

InnovationWe implement a user-friendly workflow to train and validate neural network models, and subsequently use them to predict species RL status. IUCNN contains functions to address specific issues related to the RL framework, including a regression-based approach to account for the ordinal nature of RL categories and class imbalance in the training data, a Bayesian approach for improved uncertainty quantification, and a target accuracy threshold approach that limits predictions to only those species whose RL status can be predicted with high confidence. Most analyses can be run with few lines of code, without prior knowledge of neural network models. We demonstrate the use of IUCNN on an empirical dataset of [~]14,000 orchid species, for which IUCNN models can predict extinction risk within minutes, while outperforming comparable methods.

Main conclusionsIUCNN harnesses innovative methodology to estimate the RL status of large numbers of species. By providing estimates of the number and identity of threatened species in custom geographic or taxonomic datasets, IUCNN enables large-scale analyses on the extinction risk of species so far not well represented on the official RL.

category: ecology


title: vWCluster: A Network Based Clustering of Multi-omics Breast Cancer Data Based on Vector-Valued Optimal Transport

abstract: In this paper, we present a network-based clustering method based on the vector-valued Wasserstein distance derived from optimal mass transport (OMT) theory. This distance allows for the natural integration of multi-layer representations of data in a given network from which one derives clusters via a hierarchical clustering approach. In this study, we applied the methodology, called vector Wasserstein clustering (vW-cluster), to multi-omics data from the two largest breast cancer studies. The resultant clusters showed significantly different survival rates in Kaplan-Meier analysis in both datasets. CIBERSORT scores were compared among the identified clusters. Out of the 22 CIBERSORT immune cell types, 9 were commonly significantly different in both datasets, suggesting the difference of tumor immune microenvironment in the cluster. vWCluster can aggregate multi-omics data represented as a vectorial form in a network with multiple layers, taking into account the concordant effect of heterogeneous data, and further identify subtypes of tumors with different survival rates.


title: LongStitch: High-quality genome assembly correction and scaffolding using long reads

abstract:
BackgroundGenerating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads.

ResultsLongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 2.0-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently runs in under five hours using less than 23GB of RAM.

ConclusionsDue to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.


title: Simple models highlight differences in the walking biomechanics of young children and adults

abstract: Adults conserve metabolic energy during walking by minimizing the step-to-step transition work performed by the legs during double support and by utilizing spring-like mechanisms in their legs, but little is known as to whether children utilize these same mechanisms. To gain a better understanding, we studied how children (5-6 years) and adults modulate the mechanical and metabolic demands of walking at their preferred speed, across slow (75%), preferred (100%), and fast (125%) step frequencies. We quantified the 1) positive mass-specific work done by the trailing leg during step-to-step transitions and 2) the legs spring-likebehavior during single support. On average, children walked with a 36% greater net cost of transport (COT; J/kg/m) than adults (p=0.03), yet both groups increased their net COT at varying step frequencies. After scaling for speed, children generated [~]2-fold less trailing limb positive scaled mechanical work during the step-to-step transition (p=0.02). Unlike adults, children did not modulate their trailing limb positive work to meet the demands of walking at 75% and 125% of their preferred step frequency. In single support, young children operated their stance limb with much greater compliance than adults [Formula] vs. 11.35; p=.023). Our observations suggest that the mechanics of walking in children 5-6 years are fundamentally distinct from the mechanics of walking in adults and may help to explain a childs higher net COT. These insights have implications for the design of assistive devices for children and suggest that children cannot be simply treated as scaled down versions of adults.


title: Inertia triggers nonergodicity of fractional Brownian motion

abstract: How related are the ergodic properties of the over- and underdamped Langevin equations driven by fractional Gaussian noise? We here find that for massive particles performing fractional Brownian motion (FBM) inertial effects not only destroy the stylized fact of the equivalence of the ensemble-averaged mean-squared displacement (MSD) to the time-averaged MSD (TAMSD) of overdamped or massless FBM, but also concurrently dramatically alter the values of the ergodicity breaking parameter (EB). Our theoretical results for the behavior of EB for underdamped ot massive FBM for varying particle mass m, Hurst exponent H, and trace length T are in excellent agreement with the findings of extensive stochastic computer simulations. The current results can be of interest for the experimental community employing various single-particle-tracking techniques and aiming at assessing the degree of nonergodicity for the recorded time series (studying e.g. the behavior of EB versus lag time). To infer FBM as a realizable model of anomalous diffusion for a set single-particle-tracking data when massive particles are being tracked, the EBs from the data should be compared to EBs of massive (rather than massless) FBM.


title: The contributions of extracellular matrix and sarcomere properties to passive muscle stiffness in cerebral palsy

abstract: Cerebral palsy results from an upper motor neuron lesion and has significant effects on skeletal muscle stiffness throughout the body. The increased stiffness that occurs is partly a result of changes in the microstructural components of muscle. In particular, alterations in extracellular matrix, sarcomere length, fibre diameter, and fat content have been reported; however, experimental studies have shown wide variability in the degree to which each component is altered. Many studies have reported alterations in the extracellular matrix, while others have reported no changes. A consistent finding throughout the literature is increased sarcomere length in cerebral palsy muscle. Often more than one component is altered, making it difficult to determine the individual effects on stiffness. The purpose of this study is to use a modeling approach to isolate individual effects of microstructural alterations that typically occur during cerebral palsy on whole muscle behavior; in particular, the extracellular matrix volume fraction, stiffness, and sarcomere length. These microstructural effects can be captured using a three dimensional model of muscle. We found that the extracellular matrix volume fraction has a larger effect on stiffness compared to sarcomere length, even when coupled with decreased extracellular matrix stiffness. Additionally, the effects of sarcomere length in passive stiffness are mitigated by the increased extracellular matrix volume fraction. Using this model, we can achieve a better understanding of the possible combinations of microstructural changes that can occur during cerebral palsy. Developing these insights into diseased muscle tissue will help to direct future clinical and experimental procedures.

category: physiology


title: A personalized network framework reveals predictive axis of anti-TNF response across diseases

abstract: Personalized treatment of complex diseases has been mostly predicated on biomarker identification of one drug-disease combination at a time. Here, we used a novel computational approach termed Disruption Networks to generate a new data type, contextualized by cell-centered individual-level networks, that captures biology otherwise overlooked when performing standard statistics. The new data-type extends beyond the  feature level space, to the  relations space, by quantifying individual-level breaking or rewiring of cross-feature relations. Applying disruption network to dissect high-dimensional blood data, we discover and validate that the RAC1-PAK1 axis is predictive of anti-TNF response in inflammatory bowel disease. Intermediate monocytes, which correlate with the inflammatory state, play a key role in the RAC1-PAK1 responses, supporting their modulation as a therapeutic target. This axis also predicts response in rheumatoid arthritis, validated in three public cohorts. Our findings support blood-based drug response diagnostics across immune-mediated diseases, implicating common mechanisms of non-response.


title: Increased brain growth in escaped rainbow trout

abstract: Recent examples of rapid brain size plasticity in response to novel laboratory environments suggest that fish brain size is a flexible trait, allowing growth or shrinkage of brain tissue based on short term needs. Nevertheless, it remains to be seen if plasticity of fish brain size is relevant to natural environmental conditions. Here, using rainbow trout escaped from a farming operation as a natural experiment, we demonstrate that adult fish brain size can change rapidly in response to life in a natural lake environment. Specifically, escaped trout had on average 15% heavier brains relative to body size than captive trout after living for about 7 months in the lake. Because relative brain size of most escaped trout fell above the range of variation seen within the captive trout population, we conclude that increased brain size was achieved by plasticity after escape. Brain morphology analysis showed that the most anterior regions (olfactory bulbs and rest of telencephalon) contributed most to the increase in overall brain size in escaped trout. Relative size of the heart ventricle, another organ which can be subject to plastic changes under variable environmental conditions in fish, did not differ between escaped and captive trout. Massive and selective brain growth under the changed environmental conditions associated with escape from holding pens highlighted the plastic potential of fish brain size and suggests that a shift to increased complexity of life in the wild setting of a lake imposed greatly increased cognitive requirements on escaped trout.

category: zoology


title: Towards more accurate preclinical glioblastoma modelling: reverse translation of clinical standard of care in a glioblastoma mouse model.

abstract:
IntroductionContrarily to clinical trials, where glioblastoma (GBM) patients are usually heavily pre-treated (surgery, radiotherapy (RT) and temozolomide (TMZ)) before receiving the experimental therapies, in preclinical GBM studies experimental treatments have mostly been administered as monotherapy or combined with a very limited part of the clinical standard of care. This discrepancy can explain the failed clinical translation of preclinically successful treatments. This study is aimed at evaluating the feasibility and survival impact of the clinical standard of care in glioma-bearing mice. Methods. Neurospheres CT-2A tumor-bearing mice were treated with fluorescence-guided tumor resection, focal RT and oral TMZ.

ResultsThe implementation of postoperative intensive care treatment reduced surgical mortality by approximately 50% and increased experimental cost-effectiveness. Partial and total tumor resection significantly prolonged survival, the latter showing the strongest effect. Mice treated with surgery combined with RT or with RT and TMZ survived significantly longer than untreated controls.

ConclusionsImplementing the current GBM clinical standard of care in preclinical research is feasible and it leads to results in line with those obtained in patients. Systematic preclinical evaluation of the efficacy of experimental therapies in combination with standard of care treatments could improve the translational impact of next generation GBM animal studies.


title: Whole-genome sequencing and analysis of two azaleas, Rhododendron ripense and Rhododendron kiyosumense

abstract: To enhance the genomics and genetics of azalea, the whole-genome sequences of two species of Rhododendron were determined and analyzed in this study: Rhododendron ripense, the cytoplasmic donor and ancestral species of large-flowered and evergreen azalea cultivars, respectively; and Rhododendron kiyosumense, a native of Chiba prefecture (Japan) seldomly bred and cultivated. A chromosome-level genome sequence assembly of R. ripense was constructed by single-molecule real-time (SMRT) sequencing and genetic mapping, while the genome sequence of R. kiyosumense was assembled using the single-tube long fragment read (stLFR) sequencing technology. The R. ripense genome assembly contained 319 contigs (506.7 Mb; N50 length: 2.5 Mb) and was assigned to the genetic map to establish 13 pseudomolecule sequences. On the other hand, the genome of R. kiyosumense was assembled into 32,308 contigs (601.9 Mb; N50 length: 245.7 kb). A total of 34,606 genes were predicted in the R. ripense genome, while 35,785 flower and 48,041 leaf transcript isoforms were identified in R. kiyosumense through Iso-Seq analysis. Overall, the genome sequence information generated in this study enhances our understanding of genome evolution in the Ericales and reveals the phylogenetic relationship of closely-related species. This information will also facilitate the development of phenotypically attractive azalea cultivars.


title: Fast and powerful statistical method for context-specific QTL mapping in multi-context genomic studies

abstract: Recent studies suggest that context-specific eQTLs underlie genetic risk factors for complex diseases. However, methods for identifying them are still nascent, limiting their comprehensive characterization and downstream interpretation of disease-associated variants. Here, we introduce FastGxC, a method to efficiently and powerfully map context-specific eQTLs by leveraging the correlation structure of multi-context studies. We first show via simulations that FastGxC is orders of magnitude more powerful and computationally efficient than previous approaches, making previously year-long computations possible in minutes. We next apply FastGxC to bulk multi-tissue and single-cell RNA-seq data sets to produce the most comprehensive tissue- and cell-type-specific eQTL maps to date. We then validate these maps by establishing that context-specific eQTLs are enriched in corresponding functional genomic annotations. Finally, we examine the relationship between context-specific eQTLs and human disease and show that FastGxC context-specific eQTLs provide a three-fold increase in precision to identify relevant tissues and cell types for GWAS variants than standard eQTLs. In summary, FastGxC enables the construction of context-specific eQTL maps that can be used to understand the context-specific gene regulatory mechanisms underlying complex human diseases.


title: Habitat size, health and saturation do not alter movement decisions or the preference for familiarity in a social coral-reef fish

abstract: While habitat is often a limiting resource for group-living animals, we have yet to understand what aspects of habitat are particularly important for the maintenance of sociality. As anthropogenic disturbances rapidly degrade the quality of many habitats, site-attached animals are facing additional stressors that may alter the trade-offs of moving or remaining philopatric. Here we examined how habitat health, size and saturation affect movement decisions of a coral-dwelling goby, Gobiodon quinquestrigatus, that resides within bleaching-susceptible Acropora coral hosts. To assess effects of habitat health, we translocated individuals far from their home corals into dead corals with the choice of adjacent healthy corals. To assess effects of habitat size and saturation, we manipulated coral sizes and the number of residents in healthy corals. Remarkably, 55% of gobies returned home regardless of treatment, 7% stayed in the new coral, and the rest were not found. Contrary to expectations, habitat factors did not affect how costs of movement influence group-living decisions in this species. These site-attached fishes preferred to home instead of choosing alternative habitat, which suggests a surprising awareness of their ecological surroundings. However, disregarding alternative high-quality habitat is concerning as it may affect population persistence under conditions of rapid habitat degradation.

category: animal behavior and cognition


title: Electrodetection of small molecules by conformation-mediated signal enhancement

abstract: Electrochemical biosensors allow the rapid, selective, and sensitive transduction of critical biological parameters into measurable signals. However, current electrochemical biosensors often fail to selectively and sensitively detect small molecules due to their small size and low molecular complexity. We have developed an electrochemical biosensing platform that harnesses the analyte-dependent conformational change of highly selective solute-binding proteins to amplify the signal generated by analyte binding. Using this platform, we constructed and characterized two biosensors that can sense leucine and glycine, respectively. We show that these biosensors can selectively and sensitively detect their targets over a wide range of concentrations - up to seven orders of magnitude - and that the selectivity of these sensors can be readily altered by switching the bioreceptors binding domain. Our work represents a new paradigm for the design of a family of modular electrochemical biosensors, where access to electrode surfaces can be controlled by protein conformational change.


title: Probabilistic modeling methods for cell-free DNA methylation based cancer classification

abstract:
BackgroundcfMeDIP-seq is a low-cost method for determining the DNA methylation status of cell-free DNA and it has been successfully combined with statistical methods for accurate cancer diagnostics. We investigate the diagnostic classification aspect by applying statistical tests and dimension reduction techniques for feature selection and probabilistic modeling for the cancer type classification, and we also study the effect of sequencing depth.

MethodsWe experiment with a variety of statistical methods that use different feature selection and feature extraction methods as well as probabilistic classifiers for diagnostic decision making. We test the (moderated) t-tests and the Fishers exact test for feature selection, principal component analysis (PCA) as well as iterative supervised PCA (ISPCA) for feature generation, and GLMnet and logistic regression methods with sparsity promoting priors for classification. Probabilistic programming language Stan is used to implement Bayesian inference for the probabilistic models.

Results and conclusionsWe compare overlaps of differentially methylated genomic regions as chosen by different feature selection methods, and evaluate probabilistic classifiers by evaluating the area under the receiver operating characteristic (AUROC) scores on discovery and validation cohorts. While we observe that many methods perform equally well as, and occasionally considerably better than, GLMnet that was originally proposed for cfMeDIP-seq based cancer classification, we also observed that performance of different methods vary across sequencing depths, cancer types and study cohorts. Overall, methods that seem robust and promising include Fishers exact test and ISPCA for feature selection as well as a simple logistic regression model with the number of hyper and hypomethylated regions as features.


title: X-DPI: A structure-aware multi-modal deep learning model for drug-protein interactions prediction

abstract:
MotivationIdentifying the drug-protein interactions (DPIs) is crucial in drug discovery, and a number of machine learning methods have been developed to predict DPIs. Existing methods usually use unrealistic datasets with hidden bias, which will limit the accuracy of virtual screening methods. Meanwhile, most DPIs prediction methods pay more attention to molecular representation but lack effective research on protein representation and high-level associations between different instances. To this end, we presented here a novel structure-aware multi-modal DPIs prediction model, X-DPI, performing on a curated industry-scale benchmark dataset.

ResultsWe built a high-quality benchmark dataset named GalaxyDB for DPIs prediction. This industry-scale dataset along with an unbiased training procedure resulted in a more robust benchmark study. For informative protein representation, we constructed a structure-aware graph neural network method from the protein sequence by combining predicted contact maps and graph neural networks. Through further integration of structure-based representation and high-level pre-trained embeddings for molecules and proteins, our model captured more effectively the feature representation of the interactions between them. As a result, X-DPI outperformed state-of-the-art DPIs prediction methods and obtained 5.30% Mean Square Error (MSE) improved in the DAVIS dataset and 8.89% area under the curve (AUC) improved in GalaxyDB dataset. Moreover, our model is an interpretable model with the transformer-based interaction mechanism, which can accurately reveal the binding sites between molecule and protein.


title: Teaching Python for Data Science: Collaborative development of a modular & interactive curriculum

abstract: We are bioinformatics trainees at the University of Michigan who started a local chapter of Girls Who Code to provide a fun and supportive environment for high school women to learn the power of coding. Our goal was to cover basic coding topics and data science concepts through live coding and hands-on practice. However, we could not find a resource that exactly met our needs. Therefore, over the past three years, we have developed a curriculum and instructional format using Jupyter notebooks to effectively teach introductory Python for data science. This method, inspired by The Carpentries organization, uses bite-sized lessons followed by independent practice time to reinforce coding concepts, and culminates in a data science capstone project using real-world data. We believe our open curriculum is a valuable resource to the wider education community and hope that educators will use and improve our lessons, practice problems, and teaching best practices. Anyone can contribute to our educational materials on GitHub.

category: scientific communication and education


title: Mental practice modulates functional connectivity between the cerebellum and the primary motor cortex.

abstract: Our brain has the extraordinary capacity to improve motor skills through mental practice. Conceptually, this ability is attributed to internal forward models, which are neural networks that can predict the sensory consequences of motor commands. While the cerebellum is considered as a potential locus of internal forward models, evidence for its involvement in mental practice is missing. In our study, we employed single and dual transcranial magnetic stimulation technique to probe the level of corticospinal excitability and of cerebellar-brain inhibition, respectively, before and after a mental practice session or a control session. Motor skills (i.e., accuracy and speed) were measured using a sequential finger tapping-task. Here, we show that mental practice enhances both speed and accuracy. In parallel, the functional connectivity between the cerebellum and the primary motor cortex changes, with less inhibition from the first to the second, expressing the existence of neuroplastic changes within the cerebellum after mental practice. These findings reveal that the corticocerebellar loop is a major neural circuit for skill improvement after mental practice.

category: neuroscience


title: Imaging transcriptomics: Convergent cellular, transcriptomic, and molecular neuroimaging signatures in the healthy adult human brain

abstract: The expansion of neuroimaging techniques over the last decades has opened a wide range of new possibilities to characterize brain dysfunction in several neurological and psychiatric disorders. However, the lack of specificity of most of these techniques, such as magnetic resonance imaging (MRI)-derived measures, to the underlying molecular and cellular properties of the brain tissue poses limitations to the amount of information one can extract to inform precise models of brain disease. The integration of transcriptomic and neuroimaging data, known as  imaging transcriptomics, has recently emerged as an indirect way forward to test and/or generate hypotheses about potential cellular and transcriptomic pathways that might underly specific changes in neuroimaging MRI biomarkers. However, the validity of this approach is yet to be examined in-depth. Here, we sought to bridge this gap by performing imaging transcriptomic analyses of the regional distribution of well-known molecular markers, assessed by positron emission tomography (PET), in the healthy human brain. We focused on tracers spanning different elements of the biology of the brain, including neuroreceptors, synaptic proteins, metabolism, and glia. Using transcriptome-wide data from the Allen Brain Atlas, we applied partial least square regression to rank genes according to their level of spatial alignment with the regional distribution of these neuroimaging markers in the brain. Then, we performed gene set enrichment analyses to explore the enrichment for specific biological and cell-type pathways among the genes most strongly associated with each neuroimaging marker. Overall, our findings show that imaging transcriptomics can recover plausible transcriptomic and cellular correlates of the regional distribution of benchmark molecular imaging markers, independently of the type of parcellation used to map gene expression and neuroimaging data. Our data support the plausibility and robustness of imaging transcriptomics as an indirect approach for bridging gene expression, cells and macroscopical neuroimaging and improving our understanding of the biological pathways underlying regional variability in neuroimaging features.


title: Two distinct networks containing position-tolerant representations of actions in the human brain

abstract: Humans can recognize other peoples actions in the social environment. This action recognition ability is rarely hindered by the movement of people in the environment. The neural basis of this tolerance to changes in the position of observed actions is not fully understood. Here, we aimed to identify brain regions capable of generalizing representations of actions across different positions and investigate the representational content of these regions. fMRI data were recorded from twenty-two subjects while they were watching video clips of ten different human actions in Point Light Display format. Each stimulus was presented in either the upper or the lower visual fields. Multivoxel pattern analysis and a searchlight technique were employed to identify brain regions that contain position-tolerant action representation: linear support vector machine classifiers were trained with fMRI patterns in response to stimuli presented in one position and tested with stimuli presented in another position. Results of this generalization test showed above-chance classification in the left and right lateral occipitotemporal cortex, right intraparietal sulcus, and right post-central gyrus. To explore the representational content of these regions, we constructed models based on the objective measures of movements and human subjective judgments about actions. We then evaluated the brain similarity matrix from the cross-position classification analysis based on these models. Results showed cross-position classifications in the lateral occipito-temporal ROIs were more strongly related to the subjective judgments, while those in the dorsal parietal ROIs were more strongly related to the objective movements. An ROI representational similarity analysis further confirmed the separation of the dorsal and lateral regions. These results provide evidence for two networks that contain abstract representations of human actions with distinct representational content.


title: Rotavirus NSP1 contributes to intestinal viral replication, pathogenesis, and transmission

abstract:
Rotavirus (RV)-encoded non-structural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1 knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development.

IMPORTANCERotavirus remains one of the most important causes of severe diarrhea and dehydration in young children worldwide. Although NSP1 is dispensable for rotavirus replication in cell culture, its exact role in virus infection in vivo remains unclear. In this study, we demonstrate that in the context of a fully replication-competent, pathogenic, and transmissible murine rotavirus, loss of NSP1 expression substantially attenuated virus replication in the gastrointestinal tract, diarrheal disease, and virus transmission in suckling mice. Notably, the NSP1-deficient murine rotavirus also replicated poorly in mice lacking host interferon signaling. Our data provide the first piece of evidence that NSP1 is essential for murine rotavirus replication in vivo, making it an attractable target for developing improved next-generation rotavirus vaccines better suited for socioeconomically disadvantaged and immunocompromised individuals.

category: microbiology


title: Detection of a novel enterotropic Mycoplasma gallisepticum-like in European Starling (Sturnus vulgaris) around poultry farms in France

abstract:
Recent outbreaks of highly pathogenic avian influenza in southwest France have raised questions regarding the role of commensal wild birds in the introduction and dissemination of pathogens between poultry farms. To assess possible infectious contacts at the wild-domestic bird interface, the presence of Mycoplasma gallisepticum (MG) was studied in the two sympatric compartments in southwest France. Among various peridomestic wild birds (n=385), standard PCR primers targeting the 16S rRNA of MG showed a high apparent prevalence (up to 45%) in cloacal swabs of European starlings (Sturnus vulgaris, n=108), while the MG-specific mgc2 gene was not detected. No tracheal swab of these birds tested positive, and no clinical sign was observed in positive birds, suggesting commensalism in the digestive tract of starlings. A mycoplasma strain was then isolated from a starling swab and its whole genome was sequenced using both Illumina and Nanopore technologies. Phylogenetic analysis showed that it was closely related to MG and M. tullyi, although it was a distinct species.

A pair of specific PCR primers targeting the mgc2-like gene of this MG-like strain was designed and used to screen again the same avian populations and a wintering urban population of starlings (n=50). Previous PCR results obtained in starlings were confirmed to be mostly due to this strain (20/22 positive pools). In contrast, the strain was not detected in fresh faeces of urban starlings. Furthermore, it was detected in one tracheal pool of cattle egrets and one cloacal pool of white wagtails, suggesting infectious transmissions between synanthropic birds with similar feeding behaviour. As the new starling mycoplasma was not detected in free-range ducks (n=80) in close contact with positive starlings, nor in backyard (n=320) and free-range commercial (n=720) chickens of the area, it might not infect poultry. However, it could be involved in mycoplasma gene transfer in such multi-species contexts.


title: Functional Redundancy in Ocean Microbiomes Controls Trait Stability

abstract:
Theory predicts that functional redundancy in microbial communities increases trait stability, meaning that traits or functions are less likely to be lost from the community when species go extinct. However, few experiments have empirically tested this prediction, especially in the context of microbial communities and at the landscape scale. In part, the lack of metrics for functional redundancy in microbial ecosystems has prevented addressing this question. In a companion manuscript we proposed a quantitative metric for functional redundancy called Contribution Evenness (CE) that is optimized to reflect trait stability. Here, we use CE to predict the stability of marine microbial functions to species and transcription loss. Using transcriptomes deposited in the Ocean Microbial Reference Gene Catalog (OM-RGC.v2), a catalog of genes and transcripts sequenced by the TARA Ocean expedition, we quantified the functional redundancy for 4,314 KEGG Orthologs (KOs) across 124 marine sites. Functional redundancy was highly correlated with a latent variable consisting of four ocean physiochemical parameters: oxygen and chlorophyll a concentrations, depth, and salinity. Functional redundancy was higher at the poles than in non-polar regions. Simultaneously, regional {beta}-diversity for individual functions was higher for functions with higher functional redundancy. These observations provide evidence that higher functional redundancy indicates increased stability of microbial ecosystem functions on spatiotemporal scales consistent with surface ocean mixing. We suggest that future changes in ocean physiochemistry could likely influence this stability for functions with lower functional redundancy.

ImportanceFunctional redundancy describes the state of multiple species performing the same function. Theory suggests functional redundancy stabilizes microbial community functions from disturbances leading to species loss or other changes to the microbiome. Previous work suggests that functional redundancy is common in ocean microbiomes which implies traits should be more stable among metacommunities. Some laboratory experiments demonstrate this idea, but it is difficult to test in the natural world. In a companion manuscript, we proposed a functional redundancy metric sensitive to trait stability. Here, we used this metric to show that functional redundancy varied substantially among ocean microbiomes and that regions with higher functional redundancy had higher regional trait stability. Last, we noted that variations in functional redundancy strongly correlated to ocean physiochemistry. Thus, changes in ocean physiochemistry via climate change may alter community traits to become more or less resistant to disturbance relative to contemporary conditions.


title: LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

abstract:
The productions of antibiotics are usually regulated by cluster-situated regulators (CSRs), which can directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). However, few studies have looked into the regulation of CSRs on the targets outside the BGC. Here, we screened the targets of LmbU in the whole genome of S. lincolnensis, and found 14 candidate targets, among of which, 8 targets can bind to LmbU by EMSAs. Reporter assays in vivo revealed that LmbU repressed transcription of SLINC_RS02575 and SLINC_RS05540, while activated transcription of SLINC_RS42780. In addition, disruptions of SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 promoted the production of lincomycin, and qRT-PCR showed that SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. Whats more, the homologues of LmbU and its targets SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 are widely found in actinomycetes, while the distributions of DNA-binding sites (DBS) of LmbU are diverse, indicating the regulatory mechanisms of LmbU homologues in various strains are different and complicated.

IMPORTANCELincomycin is widely used in clinic treatment and animal husbandry. Our previous study firstly demonstrated that LmbU, a novel transcriptional regulator family, functions as a CSR and positively regulates lincomycin biosynthesis. Here, we revealed that LmbU may act as a pleiotropic transcriptional regulator, and directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 which are located outside the lmb cluster and negatively regulate lincomycin biosynthesis. Interestingly, the homologues of LmbU and its targets are widely found in actinomycetes, indicating the regulatory patterns of LmbU to the targets may exist in a variety of strains. Collectively, our findings elucidated the molecular mechanism with which LmbU regulates the target genes outside the lmb culster, and draw a network diagram of LmbU regulation on lincomycin biosynthesis. This lays a solid foundation for the realization of high-yield of lincomycin in industry, and provides the theoretical basis for the functional research of LmbU family proteins.