title: Eukaryotic initiation factor EIF-3.G augments mRNA translation efficiency to regulate neuronal activity.

abstract: The translation initiation complex eIF3 imparts specialized functions to regulate protein expression. However, understanding of eIF3 activities in neurons remains limited despite widespread dysregulation of eIF3 subunits in neurological disorders. Here, we report a selective role of the C. elegans RNA-binding subunit EIF-3.G in shaping the neuronal protein landscape. We identify a missense mutation in the conserved Zinc-Finger (ZF) of EIF-3.G that acts in a gain-of-function manner to dampen neuronal hyperexcitation. Using neuron type-specific seCLIP, we systematically mapped EIF-3.G-mRNA interactions and identified EIF-3.G occupancy on GC-rich 5'UTRs of a select set of mRNAs enriched in activity-dependent functions. We demonstrate that the ZF mutation in EIF-3.G alters translation in a 5'UTR dependent manner. Our study reveals an in vivo mechanism for eIF3 in governing neuronal protein levels to control activity states and offers insights into how eIF3 dysregulation contributes to neuronal disorders.


title: Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest, Drosophila suzukii

abstract: Originally from Asia, Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wing Drosophila (SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.

category: genetics


title: Identity and compatibility of reference genome resources

abstract:
Genome analysis relies on reference data like sequences, feature annotations, and aligner indexes. These data can be found in many versions from many sources, making it challenging to identify and assess compatibility among them. For example, how can you determine which indexes are derived from identical raw sequence files, or which annotations share a compatible coordinate system? Here, we describe a novel approach to establish identity and compatibility of reference genome resources. We approach this with three advances: First, we derive unique identifiers for each resource; second, we record parent-child relationships among resources; and third, we describe recursive identifiers that determine identity as well as compatibility of coordinate systems and sequence names. These advances facilitate portability, reproducibility, and re-use of genome reference data.

Availabilityhttps://refgenie.databio.org

category: genomics


title: Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

abstract: Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosiss pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

category: biochemistry


title: Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

abstract: Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.


title: Electrochemical measurements reveal reactive oxygen species in stress granules.

abstract: Stress granules (SGs) are membraneless organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arseniteinduced SGs, and H2O2 is the main species. The release kinetics of H2O2 from single SGs and the number of H2O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple end-point. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases, cancers and viral infections.


title: 3D genomic capture of regulatory immuno-genetic profiles in COVID-19 patients for prognosis of severe COVID disease outcome

abstract: Human infection with the SARS-CoV-2 virus leads to coronavirus disease (COVID-19). A striking characteristic of COVID-19 infection in humans is the highly variable host response and the diverse clinical outcomes, ranging from clinically asymptomatic to severe immune reactions leading to hospitalization and death. Here we used a 3D genomic approach to analyse blood samples at the time of COVID diagnosis, from a global cohort of 80 COVID-19 patients, with different degrees of clinical disease outcomes. Using 3D whole genome EpiSwitch(R) arrays to generate over 1 million data points per patient, we identified a distinct and measurable set of differences in genomic organization at immune-related loci that demonstrated prognostic power at baseline to stratify patients with mild forms of illness and those with severe forms that required hospitalization and intensive care unit (ICU) support. Further analysis revealed both well established and new COVID-related dysregulated pathways and loci, including innate and adaptive immunity; ACE2; olfactory, G{beta}{psi}, Ca2+ and nitric oxide (NO) signalling; prostaglandin E2 (PGE2), the acute inflammatory cytokine CCL3, and the T-cell derived chemotactic cytokine CCL5. We identified potential therapeutic agents for mitigation of severe disease outcome, with several already being tested independently, including mTOR inhibitors (rapamycin and tacrolimus) and general immunosuppressants (dexamethasone and hydrocortisone). Machine learning algorithms based on established EpiSwitch(R) methodology further identified a subset of 3D genomic changes that could be used as prognostic molecular biomarker leads for the development of a COVID-19 disease severity test.


title: Immunoinformatic Approach for the identification of T Cell and B Cell Epitopes in the Surface Glycoprotein and Designing a Potent Multiepitope Vaccine Construct Against SARS-CoV-2 including the new UK variant

abstract: The emergence of a novel coronavirus in China in late 2019 has turned into a SARS-CoV-2 pandemic affecting several millions of people worldwide in a short span of time with high fatality. The crisis is further aggravated by the emergence and evolution of new variant SARS-CoV-2 strains in UK during December, 2020 followed by their transmission to other countries. A major concern is that prophylaxis and therapeutics are not available yet to control and prevent the virus which is spreading at an alarming rate, though several vaccine trials are in the final stage. As vaccines are developed through various strategies, their immunogenic potential may drastically vary and thus pose several challenges in offering both arms of immunity such as humoral and cell-mediated immune responses against the virus. In this study, we adopted an immunoinformatics-aided identification of B cell and T cell epitopes in the Spike protein, which is a surface glycoprotein of SARS-CoV-2, for developing a new Multiepitope vaccine construct (MEVC). MEVC has 575 amino acids and comprises adjuvants and various cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes that possess the highest affinity for the respective HLA alleles, assembled and joined by linkers. The computational data suggest that the MEVC is non-toxic, non-allergenic and thermostable with the capability to elicit both humoral and cell-mediated immune responses. The population coverage of various countries affected by COVID-19 with respect to the selected B and T cell epitopes in MEVC was also investigated. Subsequently, the biological activity of MEVC was assessed by bioinformatic tools using the interaction between the vaccine candidate and the innate immune system receptors TLR3 and TLR4. The epitopes of the construct were analyzed with that of the strains belonging to various clades including the new variant UK strain having multiple unique mutations in S protein. Due to the advantageous features, the MEVC can be tested in vitro for more practical validation and the study offers immense scope for developing a potential vaccine candidate against SARS-CoV-2 in view of the public health emergency associated with COVID-19 disease caused by SARS-CoV-2.

category: bioinformatics


title: phastSim: efficient simulation of sequence evolution for pandemic-scale datasets

abstract:
Sequence simulators are fundamental tools in bioinformatics, as they allow us to test data processing and inference tools, as well as being part of some inference methods. The ongoing surge in available sequence data is however testing the limits of our bioinformatics software. One example is the large number of SARS-CoV-2 genomes available, which are beyond the processing power of many methods, and simulating such large datasets is also proving difficult. Here we present a new algorithm and software for efficiently simulating sequence evolution along extremely large trees (e.g. < 100, 000 tips) when the branches of the tree are short, as is typical in genomic epidemiology. Our algorithm is based on the Gillespie approach, and implements an efficient multi-layered search tree structure that provides high computational efficiency by taking advantage of the fact that only a small proportion of the genome is likely to mutate at each branch of the considered phylogeny. Our open source software is available from https://github.com/NicolaDM/phastSim and allows easy integration with other Python packages as well as a variety of evolutionary models, including indel models and new hypermutatability models that we developed to more realistically represent SARS-CoV-2 genome evolution.

Author summaryOne of the most influential responses to the SARS-CoV-2 pandemic has been the widespread adoption of genome sequencing to keep track of viral spread and evolution. This has resulted in vast availability of genomic sequence data, that, while extremely useful and promising, is also increasingly hard to store and process efficiently. An important task in the processing of this genetic data is simulation, that is, recreating potential histories of past and future virus evolution, to benchmark data analysis methods and make statistical inference. Here, we address the problem of efficiently simulating large numbers of closely related genomes, similar to those sequenced during SARS-CoV-2 pandemic, or indeed to most scenarios in genomic epidemiology. We develop a new algorithm to perform this task, that provides not only computational efficiency, but also extreme flexibility in terms of possible evolutionary models, allowing variation in mutation rates, non-stationary evolution, and indels; all phenomena that play an important role in SARS-CoV-2 evolution, as well as many other real-life epidemiological scenarios.


title: Role of bacterial persistence in spatial population expansion

abstract: Bacterial persistence, tolerance to antibiotics via stochastic phenotype switching provides a survival strategy and a fitness advantage in temporally fluctuating environments. Here we study its possible benefit in spatially varying environments using a Fisher wave approach. We study the spatial expansion of a population with stochastic switching between two phenotypes in spatially homogeneous conditions and in the presence of an antibiotic barrier. Our analytical results show that the expansion speed in growth-supporting conditions depends on the fraction of persister cells at the leading edge of the population wave. The leading edge contains a small fraction of persister cells, keeping the effect on the expansion speed minimal. The fraction of persisters increases gradually in the interior of the wave. This persister pool benefits the population when it is stalled by an antibiotic environment. In that case, the presence of persister enables the population to spread deeper into the antibiotic region and to cross an antibiotic region more rapidly. The interplay of population dynamics at the interface separating the two environments and phenotype switching in the antibiotic region results in a optimal switching rate. Overall, our results show that stochastic switching can promote population expansion in the presence of antibiotic barriers or other stressful environments.

category: biophysics


title: Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

abstract: The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 {micro}M. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 {micro}M, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 {micro}M). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

category: pharmacology and toxicology


title: The phosphoproteome of rice leaves responds to water and nitrogen supply

abstract: The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is both costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants have processes for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, on plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

category: plant biology


title: Inflammatory signals are sufficient to elicit TOX expression in mouse and human CD8 T cells

abstract: T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which induces a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in specific pathogen-free laboratory mice typically do not express TOX, functional human Tmem show heterogeneous TOX expression levels. Whether TCR-independent mechanisms can alter TOX expression in human and murine Tmem has not been defined. We report that human and mouse Tmem increase TOX expression following stimulation with inflammatory cytokines IL-12, IL-15, and IL-18. TOX and PD-1 expression patterns often appear to be directly correlated, however, we found that TOX is not necessary for cytokine-driven expression of PD-1. Together, these observations highlight that inflammation is sufficient to alter TOX and PD-1 expression and that the signals regulating TOX expression appear well conserved in human and murine Tmem.

category: immunology


title: Killed whole genome-reduced bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model

abstract:
As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reduced E. coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-{gamma} responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Significance StatementWe report a new vaccine platform to express vaccine antigens on surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrated the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface of E.coli to produce killed whole cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates IFN-{gamma} responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly-protective coronavirus vaccine since a Betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against Alphacoronavirus PEDV. When using a vaccine appropriate bacteria vector, this inexpensive new vaccine platform offers the potential for use in developing countries.

category: microbiology


title: Replication kinetic, cell tropism and associated immune responses in SARS-CoV-2 and H5N1 virus infected human iPSC derived neural models

abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite ACE2, TMPRSS2 and NPR1 expression in all cultures. However, this sparse infection did result in the production of type-III-interferons and IL-8. In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS.


title: Establishment of Developmental Gene Silencing by Ordered Polycomb Complex Recruitment in Early Zebrafish Embryos

abstract:
Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal how developmental gene poising is established de novo in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with  Placeholder nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of Polycomb Repressive Complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

Impact StatementDe novo polycomb domains are formed in zebrafish early embryos by focal histone H2Aub1 deposition by Rnf2-PRC1 - which imposes transcription silencing - followed by subsequent recruitment of Aebp2-PRC2 and H3K27me3 deposition.

category: molecular biology


title: The arrested state of processing bodies supports mRNA regulation in early development

abstract: Biomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states for in vivo biological function remains limited. Here, we investigated the physical properties of in vivo processing bodies (P bodies) and their impact on mRNA storage in mature Drosophila oocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release of bicoid (bcd) mRNA, a body axis determinant, and that P body dissolution leads to bcd release. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

category: cell biology


title: Metagenomic analysis reveals the signature of gut microbiota associated with human chronotypes

abstract: Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of opposite chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chronotype information for precision medicine, those factors that shape chronotypes remain poorly understood. Here, we assessed whether the various chronotypes are associated with different gut microbiome compositions. Using metagenomic sequencing analysis, we established a distinct signature associated with chronotype based on two bacterial genera, Alistipes (elevated in "larks") and Lachnospira (elevated in "owls"). We identified three metabolic pathways associated with the early chronotype, and linked distinct dietary patterns with different chronotypes. Our work demonstrates an association between the gut microbiome and chronotype and may represent the first step towards developing dietary interventions aimed at ameliorating the deleterious health correlates of the late chronotype.


title: A novel heteromeric pantothenate kinase complex in apicomplexan parasites

abstract: Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.


title: A Comprehensive Analysis of Allostery in 14-3-3 ζ Docking Proteins using the Spatial Convolution Model (SCM)

abstract:
The Spatial Convolution Model (SCM) analyzes allostery based on the spatial evolution of the docking protein elastic media, whereby convolution of the media in response to wave propagation is solved as a function of Z fluctuations and backbone vibration modes. We show that although the elastic media is a complex three-dimensional structure allostery behaves as if it occurs along a stretched oscillating string, where inhomogeneities along the string effect local entropies responsible for ligand binding and transduction of allosteric waves. To identify inhomogeneities along the string, we ignored local density and tension changes during wave propagation and resolved helix wave and physical properties by applying molecular string and beam bending theories. Importantly, we show that allostery occurs at three major scales and that propagation of standing waves create a rolling entropy which drives entropy transfers between fields. Conversion of resonance energy to quantum harmonic oscillators allowed us to consider effects of damping and interactions with the surrounding media as well as to model effects of residue interaction strength on entropy transfer.

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title: Towards the NMR solution Structure and the Dynamics of the C-terminal Region of APOL1 and its G1, G2 Variants with a Membrane Mimetic

abstract: Secreted apolipoprotein L1 (APOL1) is well known as an innate immune factor, protecting against African trypanosomiasis. The intracellular form has multiple functions, including regulating autophagy, intracellular vesicle trafficking, and ion channel activity. The APOL1 protein (G0) has two common variants (denoted G1 and G2) in the C-terminal region and are associated with a high risk of chronic kidney disease (CKD) and progression to end-stage kidney disease. Our previous studies using molecular modeling suggested that APOL1 G1 and G2 stabilize an autoinhibited state of the C-terminus, leading to impaired intracellular interactions with SNARE proteins. To characterize the structural consequence of kidney disease-associated APOL1 variants further, we assigned the C-terminal region proteins using 1H, 13C, 15N multidimensional nuclear magnetic resonance (NMR) spectra in solution in the presence of membrane mimetic dodecylphosphocholine micelles. We then derived models for the three-dimensional structure of APOL1-G0, and -G1 and -G2 variant C-terminal regions using the chemical shifts of the main chain nuclei followed by NMR relaxation measurements. The data suggest that changes in the three-dimensional structure of APOL1 C-terminal region induced by kidney disease-associated variants, not least the alteration of key sidechains and their interactions, could disrupt membrane association and the yet to be characterized protein-protein interactions including its binding partners, such as SNARE proteins. Such interactions could underlie the intracellular mechanisms that mediate the pathogenesis of CKD. In the future, one may try to reverse such structural and dynamics changes in the protein by designing agents that may bind and then mitigate APOL1 variant-associated CKD.


title: Noncanonical mRNA decay by the endoplasmic-reticulum stress sensor IRE1α promotes cancer-cell survival

abstract: Eukaryotic IRE1 mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, mammalian RIDD seemingly targets only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1-controlled mRNAs in human breast cancer cells, we discovered not only canonical endomotif-containing RIDD substrates, but also many targets lacking recognizable motifs--degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1 displayed two basic endoribonuclease modalities: endomotif-specific cleavage, minimally requiring dimers; and endomotif-independent promiscuous processing, requiring phospho-oligomers. An oligomer-deficient mutant that did not support RIDDLE failed to rescue cancer-cell viability. These results link IRE1 oligomers, RIDDLE, and cell survival, advancing mechanistic understanding of the UPR.

category: cancer biology


title: An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines

abstract: Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.


title: CISH targeting in NK cells activates natural cytotoxicity receptor signaling and reduce cell exhaustion to unsilence primary anti-tumor response

abstract:
Cytokine inducible SH2-containing protein (CISH) is a natural killer (NK) cell negative regulator of cytokine signaling pathway. To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). We detected no developmental or homeostatic difference in NK cells.

However, global gene expression of Cishfl/flNcr1Ki/+ NK cells compared to Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways. Indeed, CISH protein expression level increases upon NCR triggering. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis. Remarkably, we found that Cishfl/flNcr1Ki/+ mice were also more resistant to primary breast cancer growth in addition to superior control of spontaneous tumor metastasis. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, we argue that specifically enhancing NK cell function is sufficient to boost anti-tumor response to both primary and secondary tumor models. Using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. According to the results in our mouse model, CISH deletion favors NCR signaling and anti-tumor functions in human NK cells. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.


title: Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity


title: Rationally designed immunogens enable immune focusing to the SARS-CoV-2 receptor binding motif

abstract:
Eliciting antibodies to surface-exposed viral glycoproteins can lead to protective responses that ultimately control and prevent future infections. Targeting functionally conserved epitopes may help reduce the likelihood of viral escape and aid in preventing the spread of related viruses with pandemic potential. One such functionally conserved viral epitope is the site to which a receptor must bind to facilitate viral entry. Here, we leveraged rational immunogen design strategies to focus humoral responses to the receptor binding motif (RBM) on the SARS-CoV-2 spike. Using glycan engineering and epitope scaffolding, we find an improved targeting of the serum response to the RBM in context of SARS-CoV-2 spike imprinting. Furthermore, we observed a robust SARS-CoV-2-neutralizing serum response with increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses and represents an adaptable design approach for targeting conserved epitopes on other viral glycoproteins.

One Sentence SummarySARS-CoV-2 immune focusing with engineered immunogens


title: AC2P20 selectively kills M. tuberculosis  at acidic pH by depleting free thiols

abstract: Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a [~]220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.


title: HIV-1 Vpr Induces Degradation of Nucleolar Protein CCDC137 as a Consequence of Cell Cycle Arrest

abstract: Expression of HIV-1 accessory proteins Vif and Vpr results in G2/M cell cycle arrest by hijacking the host ubiquitin-proteasome system. Vif directs cell cycle arrest by targeting protein phosphatase 2, regulatory subunit B alpha (PP2AB56) for degradation. However, the ubiquitination target(s) of Vpr that is directly responsible for G2/M arrest has remained elusive. Recently, Vpr directed degradation of nucleolar protein coiled-coil domain containing 137 (CCDC137), also known as retinoic acid resistance factor (RaRF), has been implicated as the proximal event leading to G2/M cell cycle arrest. In this study we aimed to further investigate this finding. We confirm that CCDC137 is targeted for degradation in the presence of Vpr with a requirement for the CUL4ADDB1.DCAF1 E3 ligase complex. However, degradation of CCDC137 is a general consequence, rather than a trigger, of G2/M arrest. Thus, whether induced by Vpr expression or pharmacologically via CDK1 inhibition, G2/M blockade results in degradation of CCDC137. Furthermore, siRNA-mediated depletion of CCDC137 failed to induce G2/M arrest.


title: Parasitic wasps do not lack lipogenesis

abstract: Fatty acids are crucial primary metabolites for virtually any creature on earth. Therefore, most organisms do not rely exclusively on nutritional supply with fatty acids but have the ability to synthesize fatty acids and triacylglycerides de novo from carbohydrates, a process called lipogenesis. The ubiquity of lipogenesis has been questioned by a series of studies reporting that many parasitic wasps (parasitoids) do not accumulate lipid mass despite having unlimited access to sugar. This has been interpreted as an evolutionary metabolic trait loss in parasitoids. Here, we demonstrate de novo biosynthesis of fatty acids from 13C-labeled -D-glucose in eleven species of parasitoids from six families. We furthermore show with the model organism Nasonia vitripennis that lipogenesis occurs even when lipid reserves are still intact, but relative 13C-incorporation rates increase in females with widely depleted fat reserves. Therefore, we conclude that the presumed "lack of lipogenesis" in parasitoids needs to be re-evaluated.


title: The role of vimentin-nuclear interactions in persistent cell motility through confined spaces

abstract: The ability of cells to move through small spaces depends on the mechanical properties of the cellular cytoskeleton and on nuclear deformability. In mammalian cells, the cytoskeleton is comprised of three interacting, semi-flexible polymer networks: actin, microtubules, and intermediate filaments (IF). Recent experiments of mouse embryonic fibroblasts with and without vimentin have shown that the IF vimentin plays a role in confined cell motility. We, therefore, develop a minimal model of cells moving through confined geometries that effectively includes all three types of cytoskeletal filaments with a cell consisting of an actomyosin cortex and a deformable cell nucleus and mechanical connections between the two cortices--the outer actomyosin one and the inner nuclear one. By decreasing the amount of vimentin, we find that the cell speed is typically faster for vimentin-null cells as compared to cells with vimentin. Vimentin-null cells also contain more deformed nuclei in confinement. Finally, vimentin affects nucleus positioning within the cell. By positing that as the nucleus position deviates further from the center of mass of the cell, microtubules become more oriented in a particular direction to enhance cell persistence or polarity, we show that vimentin-nulls are more persistent than vimentin-full cells. The enhanced persistence indicates that the vimentin-null cells are more subjugated by the confinement since their internal polarization mechanism that depends on cross-talk of the centrosome with the nucleus and other cytoskeletal connections is diminished. In other words, the vimentin-null cells rely more heavily on external cues. Our modeling results present a quantitative interpretation for recent experiments and have implications for understanding the role of vimentin in the epithelial-mesenchymal transition.


title: Birdsong Phrase Verification and Classification Using Siamese Neural Networks

abstract: The process of learning good features to discriminate among numerous and different bird phrases is computationally expensive. Moreover, it might be impossible to achieve acceptable performance in cases where training data is scarce and classes are unbalanced. To address this issue, we propose a few-shot learning task in which an algorithm must make predictions given only a few instances of each class. We compared the performance of different Siamese Neural Networks at metric learning over the set of Cassinis Vireo syllables. Then, the network features were reused for the few-shot classification task. With this approach we overcame the limitations of data scarcity and class imbalance while achieving state-of-the-art performance.


title: Single-cell transcriptome profiling simulation reveals the impact of sequencing parameters and algorithms on clustering

abstract: Despite of scRNA-seq analytic algorithms developed, their performance for cell clustering cannot be quantified due to the unknown "true" clusters. Referencing the transcriptomic heterogeneity of cell clusters, a "true" mRNA number matrix of cell individuals was defined as ground truth. Based on the matrix and real data generation procedure, a simulation program (SSCRNA) for raw data was developed. Subsequently, the consistence between simulated data and real data was evaluated. Furthermore, the impact of sequencing depth, and algorithms for analyses on cluster accuracy was quantified. As a result, the simulation result is highly consistent with that of the real data. It is found that mis-classification rate can be attributed to multiple reasons on current scRNA platforms, and clustering accuracy is not only sensitive to sequencing depth increasement, but can also be reflected by the position of the cluster on TSNE plot. Among the clustering algorithms, Gaussian normalization method is more appropriate for current workflows. In the clustering algorithms, k-means&louvain clustering method performs better in dimension reduced data than full data, while k-means clustering method is stable under both situations. In conclusion, the scRNA simulation algorithm developed restores the real data generation process, discovered impact of parameters on mis-clustering, compared the normalization/clustering algorithms and provided novel insight into scRNA analyses.


title: A graph-based cell tracking algorithm with few manually tunable parameters and automated segmentation error correction

abstract: Automatic cell segmentation and tracking enables to gain quantitative insights into the processes driving cell migration. To investigate new data with minimal manual effort, cell tracking algorithms should be easy to apply and reduce manual curation time by providing automatic correction of segmentation errors. Current cell tracking algorithms, however, are either easy to apply to new data sets but lack automatic segmentation error correction, or have a vast set of parameters that needs either manual tuning or annotated data for parameter tuning. In this work, we propose a tracking algorithm with only few manually tunable parameters and automatic segmentation error correction. Moreover, no training data is needed. We compare the performance of our approach to three well-performing tracking algorithms from the Cell Tracking Challenge on data sets with simulated, degraded segmentation - including false negatives, over- and under-segmentation errors. Our tracking algorithm can correct false negatives, over- and under-segmentation errors as well as a mixture of the aforementioned segmentation errors. On data sets with under-segmentation errors or a mixture of segmentation errors our approach performs best. Moreover, without requiring additional manual tuning, our approach ranks several times in the top 3 on the 6th edition of the Cell Tracking Challenge.


abstract:
The inference of phylogenetic trees from sequence data has become a staple in evolutionary research. Bayesian inference of such trees is predominantly based on the Metropolis-Hastings algorithm. For high dimensional and correlated data this algorithm is known to be inefficient. There are gradient based algorithms to speed up such inference. Building on recent research which uses gradient based approaches for the inference of phylogenetic trees in a Bayesian framework, I present an algorithm which is capable of performing No-U-Turn sampling for phylogenetic trees. As an extension to Hamiltonian Monte Carlo methods, No-U-Turn sampling comes with the same benefits, such as proposing distant new states with a high acceptance probability, but eliminates the need to manually tune hyper parameters. Evaluated on real data sets, the new sampler shows that it converges faster to the target distribution. The results also indicate that a higher number of topologies are traversed during sampling by the new algorithm in comparison to traditional Markov Chain Monte Carlo approaches. This new algorithm leads to a more efficient exploration of the posterior distribution of phylogenetic tree topologies.

Author summaryPhylogentic trees are important for our understanding of evolutionary relationships. But even for only a small number of entities the number of possible trees is immense. In order to efficiently search through this large space and analyze the distributions of these trees, different algorithmic solutions have been proposed. A phylogenetic tree is not only defined by the way it groups the entities but also by the length of the branches. This nature of the phylogentic trees complicates the exploration of these distributions. Building on research and algorithmic ideas for non tree like data and recent developments on the mathematics of trees, I propose a new method which is able to traverse the space of possible trees more rapidly. By using a search strategy which is guided by the data and the underlying evolutionary model, the algorithm is able to better sample from the desired distribution. I am able to show that the new algorithm can indeed analyze the correct distribution but can do so with a higher efficiency than other algorithms.


title: Multicellular Spatial Model of RNA Virus Replication and Interferon Responses Reveals Factors Controlling Plaque Growth Dynamics

abstract:
Respiratory viruses present major health challenges, as evidenced by the 2009 influenza pandemic and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestation at the cell and tissue levels are vital to understanding the mechanisms of immunopathology and developing improved, strain independent treatments. Here, we present a novel spatialized multicellular spatial computational model of two principal components of tissue infection and response: RNA virus replication and type-I interferon mediated antiviral response to infection within lung epithelial cells. The model is parameterized using data from influenza virus infected cell cultures and, consistent with experimental observations, exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. Modulating the phosphorylation of STAT or altering the ratio of the diffusion constants of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of arrest on diffusion constants highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro experiments to measure these diffusion constants. Sensitivity analyses were performed under conditions creating both continuous plaque growth and arrested plaque growth. Findings suggest that plaque growth and cytokine assay measurements should be collected during arrested plaque growth, as the model parameters are significantly more sensitive and more likely to be identifiable. The models metrics replicate experimental immunostaining imaging and titer based sampling assays. The model is easy to extend to include SARS-CoV-2-specific mechanisms as they are discovered or to include as a component linking epithelial cell signaling to systemic immune models.

Author SummaryCOVID-19 is possibly the defining healthcare crisis of the current generation, with tens of millions of global cases and more than a million reported deaths. Respiratory lung infections form lesions in the lungs, whose number and size correlate with severity of illness. In some severe cases, the disease triggers a severe inflammatory condition known as cytokine storm. Given the complexity of the immune system, computational modeling is needed to link molecular signaling at the site of inflection to the signaling impact on the overall immune system, ultimately revealing how severe inflammatory conditions may emerge. Here, we created a computational model of the early stages of infection that simulates lung cells infected with RNA viruses, such those responsible for COVID-19 and influenza, to help explore how the disease forms viral plaques, an in vitro analog to lesion growth in the lung. Our model recapitulates in vitro observations that pretreatment of biological signaling molecules called with type-I interferons, which are currently being evaluated for treatment of COVID-19. Analyzing the model, we, can stop viral plaque growth. We found that enhancing certain aspects of the innate immune system, such as the JAK/STAT pathway, may be able to stop viral plaque growth, suggesting molecules involved in this pathway as possible drug candidates. Quantifying the parameters needed to model interferon signaling and viral replication, experiments should be performed under conditions that inhibit viral growth, such as pretreating cells with interferon. We present a computational framework that is essential to constructing larger models of respiratory infection induced immune responses, can be used to evaluate drugs and other medical interventions quickly, cheaply, and without the need for animal testing during the initial phase, and that defines experiments needed to improve our fundamental understanding of the mechanisms regulating the immune response.

category: systems biology


title: Causal-inference regulates audiovisual spatial recalibration via its influence on audiovisual perception

abstract:
To obtain a coherent perception of the world, our senses need to be in alignment. When we encounter misaligned cues from two sensory modalities, the brain must infer which cue is faulty and recalibrate the corresponding sense. We examined whether and how the brain uses cue reliability to identify the miscalibrated sense by measuring the audiovisual ventriloquism aftereffect for stimuli of varying reliability. Visual spatial reliability was smaller, comparable to and greater than that of auditory stimuli. To adjust for modality-specific biases, visual stimulus locations were chosen based on perceived alignment with auditory stimulus locations for each participant. During audiovisual recalibration, participants were presented with bimodal stimuli with a fixed perceptual spatial discrepancy; they localized one modality, cued after stimulus presentation. Unimodal auditory and visual localization was measured before and after the recalibration phase. We compared participants behavior to the predictions of three models of recalibration: (a) Reliability-based: each modality is recalibrated based on its relative reliability--less reliable cues are recalibrated more; (b) Fixed-ratio: the degree of recalibration for each modality is fixed; (c) Causal-inference: recalibration is directly determined by the discrepancy between a cue and its final estimate, which in turn depends on the reliability of both cues, and inference about how likely the two cues derive from a common source. Vision was hardly recalibrated by audition. Auditory recalibration by vision changed idiosyncratically as visual reliability decreased: the extent of auditory recalibration either decreased monotonically, first increased and then decreased, or increased monotonically. The latter two patterns cannot be explained by either the reliability-based or fixed-ratio models. Only the causal-inference model of recalibration captures the idiosyncratic influences of cue reliability on recalibration. We conclude that cue reliability, causal inference, and modality-specific biases guide cross-modal recalibration indirectly by determining the perception of audiovisual stimuli.

Author summaryAudiovisual recalibration of spatial perception occurs when we receive audiovisual stimuli with a systematic spatial discrepancy. The brain must determine to which extent both modalities should be recalibrated. In this study, we scrutinized the mechanisms the brain employs to do so. To this aim, we conducted a classical recalibration task in which participants were adapted to spatially discrepant audiovisual stimuli. The visual component of the bimodal stimulus was either less, equally, or more reliable than the auditory component. We measured the amount of recalibration by computing the difference between participants unimodal localization responses before and after the recalibration task. Across participants, the influence of visual reliability on auditory recalibration varied fundamentally. We compared three models of recalibration. Only a causal-inference model of recalibration captured the diverse influences of cue reliability on recalibration found in our study, and this model is able to replicate contradictory results found in previous studies. In this model, recalibration depends on the discrepancy between a cue and its final estimate. Cue reliability, perceptual biases, and the degree to which participants infer that the two cues come from a common source govern audiovisual perception and therefore audiovisual recalibration.

category: neuroscience


title: The neural mechanisms of active removal from working memory

abstract: The ability to frequently update the contents working memory (WM) is vital for the flexible control of behavior. Whether there even exists a mechanism for the active removal of information from working memory, however, remains poorly understood. In this Registered Report we will test the predictions of models for two different (and not mutually exclusive) mechanisms of active removal: adaptation-hijacking and mental-context shifting. We will collect functional magnetic resonance imaging (fMRI) data while subjects perform a novel "ABC-retrocuing" task designed to elicit two modes of removal, active or passive (Shan & Postle, Registered Report). The adaptation-hijacking model posits an adaptation-like modification of perceptual circuits combined with a weak activation of the to-be-removed item. Its predictions will be assessed by using multivariate inverted encoding modeling (IEM) and photic "pings" to assay the state of feature-selective encoding channels and of activity-silent representations under active-removal versus passive-removal conditions. A second model - "working memory episodic memory" (WMEM) - posits that interference from no-longer-relevant information is minimized by making the mental context associated with new information dissimilar from that associated with the to-be-"removed" information. This will be tested by using representational similarity analysis (RSA) to compare the rate of contextual drift under active-removal versus passive-removal conditions.


title: Micrometer-resolution reconstruction and analysis of whole mouse brain vasculature by synchrotron-based phase-contrast tomographic microscopy

abstract: Nervous tissue metabolism is mainly supported by the dense thread of blood vessels which mainly provides fast supplies of oxygen and glucose. Recently, the supplying role of the brain vascular system has been examined in major neurological conditions such as the Alzheimers and Parkinsons diseases. However, to date, fast and reliable methods for the fine level microstructural extraction of whole brain vascular systems are still unavailable. We present a methodological framework suitable for reconstruction of the whole mouse brain cerebral microvasculature by X-ray tomography with the unprecedented pixel size of 0.65 m. Our measurements suggest that the resolving power of the technique is better than in many previous studies, and therefore it allows for a refinement of current measurements of blood vessel properties. Relevant insights emerged from analyses characterizing the regional morphology and topology of blood vessels. Specifically, vascular diameter and density appeared non-homogeneously distributed among the brain regions suggesting preferential sites for high-demanding metabolic requirements. Also, topological features such as the vessel branching points were non-uniformly distributed among the brain districts indicating that specific architectural schemes are required to serve the distinct functional specialization of the nervous tissue. In conclusion, here we propose a combination of experimental and computational method for efficient and fast investigations of the vascular system of entire organs with submicrometric precision.


title: High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

abstract:
1P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consist of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10-4 and 10-2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses, and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naive state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanitransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.

2 Author SummaryATP-sensitive purinergic receptors comprise a network of cell-surface receptors that activate upon ATP binding, allowing them to transmit information in a tissue- and context-dependent manner. In bone, mechanically-stimulated osteoblasts release ATP that stimulates low- and high-affinity P2 receptors in neighboring cellular populations, inducing appropriate physiological responses. P2 receptor signaling is characterized by elevations in intracellular calcium levels. When simultaneously stimulated by their common ligand, ATP, the contribution of each P2 receptor subtype gives rise to a complex calcium response, exhibiting oscillatory characteristics and biphasic dose-dependent behaviours. Here we used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts. The latter was done by developing a mathematical model that was comprised of a subset of low-(P2X7) and high-(P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models. We demonstrated that this model recapitulates experimental recordings of ATP-induced calcium signaling in osteoblasts and describes the dynamic interplay between P2Y2 and P2X7 receptors in the P2 receptor network.


title: Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

abstract:
Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements.

We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.


title: DNA Methylation Networks Underlying Mammalian Traits

abstract: Epigenetics has hitherto been studied and understood largely at the level of individual organisms. Here, we report a multi-faceted investigation of DNA methylation across 11,117 samples from 176 different species. We performed an unbiased clustering of individual cytosines into 55 modules and identified 31 modules related to primary traits including age, species lifespan, sex, adult species weight, tissue type and phylogenetic order. Analysis of the correlation between DNA methylation and species allowed us to construct phyloepigenetic trees for different tissues that parallel the phylogenetic tree. In addition, while some stable cytosines reflect phylogenetic signatures, others relate to age and lifespan, and in many cases responding to anti-aging interventions in mice such as caloric restriction and ablation of growth hormone receptors. Insights uncovered by this investigation have important implications for our understanding of the role of epigenetics in mammalian evolution, aging and lifespan.

category: evolutionary biology


title: Bloom syndrome helicase contributes to germ line development and longevity in zebrafish

abstract: RecQ helicases - also known as the  guardians of the genome - play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish (Danio rerio) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51, however, blm appears to effect its function independent of tp53. Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.


title: The salmon louse genome: copepod features and parasitic adaptations.

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


title: Using machine learning and big data to explore the drug resistance landscape in HIV

abstract:
Drug resistance mutations (DRMs) appear in HIV under treatment pressure. DRMs are commonly transmitted to naive patients. The standard approach to reveal new DRMs is to test for significant frequency differences of mutations between treated and naive patients. However, we then consider each mutation individually and cannot hope to study interactions between several mutations. Here, we aim to leverage the ever-growing quantity of high-quality sequence data and machine learning methods to study such interactions (i.e. epistasis), as well as try to find new DRMs.

We trained classifiers to discriminate between Reverse Transcriptase Inhibitor (RTI)-experienced and RTI-naive samples on a large HIV-1 reverse transcriptase (RT) sequence dataset from the UK (n {approx} 55, 000), using all observed mutations as binary representation features. To assess the robustness of our findings, our classifiers were evaluated on independent data sets, both from the UK and Africa. Important representation features for each classifier were then extracted as potential DRMs. To find novel DRMs, we repeated this process by removing either features or samples associated to known DRMs.

When keeping all known resistance signal, we detected sufficiently prevalent known DRMs, thus validating the approach. When removing features corresponding to known DRMs, our classifiers retained some prediction accuracy, and six new mutations significantly associated with resistance were identified. These six mutations have a low genetic barrier, are correlated to known DRMs, and are spatially close to either the RT active site or the regulatory binding pocket. When removing both known DRM features and sequences containing at least one known DRM, our classifiers lose all prediction accuracy. These results likely indicate that all mutations directly conferring resistance have been found, and that our newly discovered DRMs are accessory or compensatory mutations. Moreover, we did not find any significant signal of epistasis, beyond the standard resistance scheme associating major DRMs to auxiliary mutations.

Author summaryAlmost all drugs to treat HIV target the Reverse Transcriptase (RT) and Drug resistance mutations (DRMs) appear in HIV under treatment pressure. Resistant strains can be transmitted and limit treatment options at the population level. Classically, multiple statistical testing is used to find DRMs, by comparing virus sequences of treated and naive populations. However, with this method, each mutation is considered individually and we cannot hope to reveal any interaction (epistasis) between them. Here, we used machine learning to discover new DRMs and study potential epistasis effects. We applied this approach to a very large UK dataset comprising{approx} 55, 000 RT sequences. Results robustness was checked on different UK and African datasets.

Six new mutations associated to resistance were found. All six have a low genetic barrier and show high correlations with known DRMs. Moreover, all these mutations are close to either the active site or the regulatory binding pocket of RT. Thus, they are good candidates for further wet experiments to establish their role in drug resistance. Importantly, our results indicate that epistasis seems to be limited to the classical scheme where primary DRMs confer resistance and associated mutations modulate the strength of the resistance and/or compensate for the fitness cost induced by DRMs.


title: Myelin contributes to microstructural growth in human sensory cortex during early infancy.

abstract: Development of cortical tissue during infancy is critical for the emergence of typical brain functions in cortex. However, how cortical microstructure develops during infancy remains unknown. We measured the longitudinal development of cortex from newborns to six-months-old infants using multimodal quantitative imaging of cortical microstructure. Here we show that infants cortex undergoes profound microstructural tissue growth during the first six months of human life. Comparison of postnatal to prenatal transcriptomic gene expression data demonstrates that myelination and synaptic processes are dominant contributors to this postnatal microstructural tissue growth. Using visual cortex as a model system, we find hierarchical microstructural growth: higher-level visual areas have less mature tissue at birth than earlier visual areas but grow at faster rates. This overturns the prevailing view that visual areas that are most mature at birth develop fastest. Together, in vivo, longitudinal, and quantitative measurements, which we validated with ex vivo transcriptomic data, shed new light on the rate, sequence, and specific biological mechanisms of developing cortical systems. Importantly, our findings propose a new hypothesis that cortical myelination is a key factor in cortical development during early infancy, which has significant implications for diagnosis of neurodevelopmental disorders and delays in infants.


title: Infants' cortex undergoes microstructural growth coupled with myelination


title: Decoding the Information Structure Underlying the Neural Representation of Concepts

abstract: The nature of the representational code underlying conceptual knowledge remains a major unsolved problem in cognitive neuroscience. We assessed the extent to which different representational systems contribute to the instantiation of lexical concepts in high-level, heteromodal cortical areas previously associated with semantic cognition. We found that lexical semantic information can be reliably decoded from a wide range of heteromodal cortical areas in frontal, parietal, and temporal cortex. In most of these areas, we found a striking advantage for experience-based representational structures (i.e., encoding information about sensory-motor, affective, and other features of phenomenal experience), with little evidence for independent taxonomic or distributional organization. These results were found independently for object and event concepts. Our findings indicate that concept representations in heteromodal cortex are based, at least in part, on experiential information. They also reveal that, in most heteromodal areas, event concepts have more heterogeneous representations (i.e., they are more easily decodable) than object concepts, and that other areas beyond the traditional "semantic hubs" contribute to semantic cognition, particularly the posterior cingulate gyrus and the precuneus.


title: Single Prolonged Stress Reduces Intrinsic Excitability and Excitatory Synaptic Drive onto Pyramidal Neurons in the Infralimbic Prefrontal Cortex of Adult Rats

abstract:
Post-traumatic stress disorder (PTSD) is a chronic, debilitating mental illness marked by abnormal fear responses and deficits in extinction of fear memories. The pathophysiology of PTSD is linked to decreased activation of the ventromedial prefrontal cortex (vmPFC). This study aims to investigate underlying functional changes in synaptic drive and intrinsic excitability of pyramidal neurons in the rodent homolog of the vmPFC, the infralimbic cortex (IL), following exposure to single prolonged stress (SPS), a paradigm that mimics core symptoms of PTSD in rats. Rats were exposed to SPS and allowed one week of recovery following which brain slices containing the PFC were prepared for whole-cell patch clamp recordings from layer V pyramidal neurons in the IL. Our results indicate that SPS reduces spontaneous excitatory synaptic drive to pyramidal neurons. In addition, SPS decreases the intrinsic membrane excitability of IL PFC pyramidal cells, as indicated by an increase in rheobase, decrease in input resistance, hyperpolarization of resting membrane potential, and a reduction in repetitive firing rate. Our results suggest that SPS causes a lasting reduction in PFC activity, supporting a body of evidence linking traumatic stress with prefrontal hypoactivity.

Graphical AbstractSPS causes a decrease in excitatory synaptic drive and intrinsic excitability of IL pyramidal neurons.

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org.highwire.dtl.DTLVardef@135ad04org.highwire.dtl.DTLVardef@f1ea6forg.highwire.dtl.DTLVardef@564c0eorg.highwire.dtl.DTLVardef@19b4c21_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: The long-term effects of genomic selection: Response to selection, additive genetic variance and genetic architecture

abstract: Genomic selection has revolutionized genetic improvement in animals and plants, but little is known of its long term effects. Here we investigate the long-term effects of genomic selection on the change in the genetic architecture of traits over generations. We defined the genetic architecture as the subset, allele frequencies and statistical additive effects of causal loci. We simulated a livestock population under 50 generations of phenotypic, pedigree, or genomic selection for a single trait, controlled by either only additive, additive and dominance, or additive, dominance and epistatic effects. The simulated epistasis was based on yeast data. The observed change in genetic architecture over generations was similar for genomic and pedigree selection, and slightly smaller for phenotypic selection. Short-term response was highest with genomic selection, while long-term response was highest with phenotypic selection, especially when non-additive effects were present. This was mainly because the loss in genetic variance and in segregating loci was much greater with genomic selection. Compared to pedigree selection, genomic selection lost a similar amount of the genetic variance but maintained more segregating loci, which on average had lower minor allele frequencies. For all selection methods, the presence of epistasis limited the changes in allele frequency and the fixation of causal loci, and substantially changed the statistical additive effects over generations. Our results show that non-additive effects can have a substantial impact on the change in genetic architecture. Therefore, non-additive effects can substantially impact the accuracy and future genetic gain of genomic selection.


title: BCL-XL blockage in TNBC models confers vulnerability to inhibition of specific cell cycle regulators

abstract:
Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments.

MethodsBioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MMTV-R26Met mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drugs mechanisms of action.

ResultsWe report that high expression levels of BCL-XL and specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MMTV-R26Met and human TNBC cell lines tested. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell growth by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis.

ConclusionsOur studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies.


title: Prolonged development of long-term potentiation at lateral entorhinal cortex synapses onto adult-born neurons

abstract: Critical period plasticity at adult-born neuron synapses is widely believed to contribute to the learning and memory functions of the hippocampus. Experience regulates circuit integration and for a transient interval, until cells are ~6 weeks old, new neurons display enhanced long-term potentiation (LTP) at afferent and efferent synapses. Since neurogenesis declines substantially with age, this raises questions about the extent of lasting plasticity offered by adult-born neurons. Notably, however, the hippocampus receives sensory information from two major cortical pathways. Broadly speaking, the medial entorhinal cortex conveys spatial information to the hippocampus via the medial perforant path (MPP), and the lateral entorhinal cortex, via the lateral perforant path (LPP), codes for the cues and items that make experiences unique. While enhanced critical period plasticity at MPP synapses is relatively well characterized, no studies have examined long-term plasticity at LPP synapses onto adult-born neurons, even though the lateral entorhinal cortex is uniquely vulnerable to aging and Alzheimers pathology. We therefore investigated LTP at LPP inputs both within (4-6 weeks) and beyond (8+ weeks) the traditional critical period. At immature stages, adult-born neurons did not undergo significant LTP at LPP synapses, and often displayed long-term depression after theta burst stimulation. However, over the course of 3-4 months, adult-born neurons displayed increasingly greater amounts of LTP. Analyses of short-term plasticity point towards a presynaptic mechanism, where transmitter release probability declines as cells mature, providing a greater dynamic range for strengthening synapses. Collectively, our findings identify a novel form of new neuron plasticity that develops over an extended interval, and may therefore be relevant for maintaining cognitive function in aging.


title: Rap-Phr systems in B. subtilis 3610 affect matrix gene expression and play a role in biofilm formation in vitro and on the plant root

abstract:
Natural isolates of the soil-dwelling bacterium Bacillus subtilis form robust biofilms under laboratory conditions and colonize plant roots. B. subtilis biofilm gene expression displays phenotypic heterogeneity that is influenced by a family of Rap-Phr regulatory systems. Most Rap-Phr systems in B. subtilis have been studied independently, in different genetic backgrounds and under distinct conditions, hampering true comparison of the Rap-Phr systems impact on bacterial differentiation. Here, we investigated each of the 12 Rap-Phr systems of B. subtilis NCIB 3610 for their effect on biofilm formation. By studying single {Delta}rap-phr mutants, we show that despite redundancy between the cell-cell communication systems, deletion of each of the 12 Rap-Phr systems influences matrix gene expression, which could possibly enable fine-tuning of the timing and level of matrix production in response to specific conditions. Furthermore, some of the {Delta}rap-phr mutants demonstrated altered biofilm formation in vitro and colonization of Arabidopsis thaliana roots, but not necessarily similarly in both processes, indicating that the pathways regulating matrix gene expression and other factors important for biofilm formation may be differently regulated under these distinct conditions.

Significance StatementNatural isolates of Bacillus subtilis form robust biofilms in vitro and on plant roots. The formation of these heterogeneous populations is regulated by diverse Rap-Phr systems. However, most Rap-Phr systems in B. subtilis have been studied independently and in different genetic backgrounds. Here, we report that all 12 Rap-Phr systems affect matrix gene expression, while some of them affect development of in vitro biofilms and plant root colonization. Our study highlights the importance of the Rap-Phr systems in environmental adaptation of B. subtilis, specifically during biofilm formation in the rhizosphere.


title: Deletion of Rap-Phr systems in Bacillus subtilis influences in vitro biofilm formation and plant root colonization


title: Toward quantifying the adaptive role of bacterial pangenomes during environmental perturbations

abstract:
Metagenomic surveys have revealed that natural microbial communities are predominantly composed of sequence-discrete, species-like populations but the genetic and/or ecological mechanisms that maintain such populations remain speculative, limiting our understanding of population speciation and adaptation to environmental perturbations. To address this knowledge gap, we sequenced 112 Salinibacter ruber isolates and 12 companion metagenomes recovered from four adjacent saltern ponds in Mallorca, Spain that were experimentally manipulated to dramatically alter salinity and light intensity, the two major drivers of these ecosystems. Our analyses showed that the pangenome of the local Sal. ruber population is open and similar in size ([~]15,000 genes) to that of randomly sampled Escherichia coli genomes. While most of the accessory (non-core) genes showed low in situ coverage based on the metagenomes compared to the core genes, indicating that they were functionally unimportant and/or ephemeral, 3.49% of them became abundant when salinity (but not light intensity) conditions changed and encoded for functions related to osmoregulation. Nonetheless, the ecological advantage of these genes, while significant, was apparently not strong enough to purge diversity within the population. Collectively, our results revealed a possible mechanism for how this immense gene diversity is maintained, which has implications for the prokaryotic species concept.

Significance StatementThe pangenomes of bacterial species, i.e., the number of non-redundant genes carried by members of the species, can be enormous based on the genome sequencing of isolates from various sites around the globe and different years. However, to what extent this pattern of gene diversity applies to natural bacterial populations, i.e., strains co-occurring in the same site, and the value of this diversity for population adaptation during environmental transition remains unclear. This study showed that while the pangenome of a natural population can be similarly large, only a small fraction of the pangenome appears to be functionally important when conditions change. Taken together, these results provided quantitative insights into the extent and functional significance of the accessory pangenome of a natural, species-like population.


title: Low pathogenic avian influenza virus infection retards colon microbiome diversification in two different chicken lines

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


title: Atomistic Dynamics of a Viral Infection Process: Release of Membrane Lytic Peptides from a Non-Enveloped Virus

abstract: Molecular simulations have played an instrumental role in uncovering the structural dynamics and physical properties of virus capsids. In this work we move beyond equilibrium physicochemical characterization of a virus system to study a stage of the infection process which is required for viral proliferation. Despite many biochemical and functional studies, the molecular mechanism of host cell entry by non-enveloped viruses remains largely unresolved. Flock House Virus (FHV) is model system for non-enveloped viruses and is the subject of the current study. FHV infects through the acid-dependent endocytic pathway, where low pH triggers externalization of membrane disrupting ({gamma}) peptides from the capsid interior. Employing all-atom equilibrium and enhanced sampling simulations, the mechanism and energetics of {gamma} peptide liberation and the effect of pH on this process is investigated. Our computations agree with experimental findings and reveal nanoscopic details regarding the pH control mechanism which are not readily accessible in experiments.


title: Social Reactivation of Fear Engrams Enhances Recall and Reinstatement

abstract:
For group-living species such as humans and rodents, conspecific interactions pervasively shape emotion (1-3), attention (4), and cognitive ability (5-8). Higher-order cognitive processes such as memory within a social brain are thus interlaced with social influences. Traditional laboratory rodent cages offer a limited but nonetheless rich multi-modal landscape of communication, including auditory calls (9-12), chemical signaling (13, 14), and tactile stimulation (15, 16). The absence of such social encounters in singly housed animals results in cognitive impairments and depression-like phenotypes (17), likely obscuring how the social brain has evolved to function. It is thus important to understand the relationship between social context and how individuals process memories. As social interaction recruits hippocampal (18) and amygdalar (19) circuitry that also serves as hubs for non-social memory traces(20-24), we hypothesized that pre-existing ensembles in these regions can be modulated by social experiences and lead to changes in memory expression. Here we show that stressful social experiences enhance the recall of previously acquired fear memories in male but not female mice. Activity-dependent tagging of cells in the dentate gyrus (DG) during fear learning revealed that these ensembles were endogenously reactivated during the social experiences in males. These reactivated cells were shown to be functional components of engrams, as optogenetic stimulation of the cells active during the social experience in previously fear conditioned animals was sufficient to drive fear-related behaviors. Our findings suggest that social encounters can reactivate pre-existing DG engrams and thereby strengthen discrete memories.

Significance StatementSocial environments can bolster and protect cognitive abilities. However, the relationship between social stimuli and individually learned memories remains enigmatic. Our work reveals that exposure to a stressed, naive non-familiar conspecific or to the ambient olfactory-auditory cues of a recently stressed familiar conspecific induces reactivation of the cellular ensembles associated with a fear memory in the hippocampus. Artificially activating the hippocampal ensemble active during the social experience induces fearful behaviors only in animals that have previously acquired a negative memory, suggesting a fear-driving function of the reactivated ensembles and demonstrating the interaction between individual history and social experience. The neural resurgence of fear-driving ensembles during social experiences leads to a context-specific enhancement of fear recall. Our findings provide evidence that unlike directly physical stressors, ambient social stimuli can reactivate and amplify an individuals memories.


title: Social Reactivation of Fear Engrams Enhances Memory Recall


title: Modulated termination of non-coding transcription partakes in the regulation of gene expression

abstract:
Pervasive transcription is a universal phenomenon leading to the production of a plethora of non-coding RNAs. If left uncontrolled, pervasive transcription can be harmful for genome expression and stability. However, non-coding transcription can also play important regulatory roles, for instance by promoting the repression of specific genes by a mechanism of transcriptional interference. The efficiency of transcription termination can strongly influence the regulatory capacity of non-coding transcription events, yet very little is known about the mechanisms modulating the termination of non-coding transcription in response to environmental cues.

Here, we address this question by investigating the mechanisms that regulate the activity of the main actor in termination of non-coding transcription in budding yeast, the helicase Sen1. We identify a phosphorylation at a conserved threonine of the catalytic domain of Sen1 and we provide evidence that phosphorylation at this site reduces the efficiency of Sen1-mediated termination. Interestingly, we find that this phosphorylation impairs termination at an unannotated non-coding gene, thus repressing the expression of a downstream gene encoding the master regulator of Zn homeostasis, Zap1. Consequently, many additional genes exhibit an expression pattern mimicking conditions of Zn excess, where ZAP1 is naturally repressed.

Our findings provide a novel paradigm of gene regulatory mechanism relying on the direct modulation of non-coding transcription termination.