title: Histone sample preparation for bottom-up mass spectrometry: a roadmap to informed decisions

abstract:
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment-between-runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols, while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX(R), Genovis), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues.

Raw data is available via ProteomeXchange with identifier PXD024423.


title: De novo transcriptome sequencing of the northern fowl mite, Ornithonyssus sylviarum, shed light into parasitiform poultry mites evolution and its chemoreceptor repertoires

abstract: The poultry red mite (PRM), Dermanyssus gallinae (De Geer), and the northern fowl mite (NFM), Ornithonyssus sylviarum (Canestrini and Fanzago), are the most serious pests of poultry, both of which have an expanding global prevalence. Research on NFM has been constrained by a lack of genomic and transcriptomic data. Here, we report and analyze the first transcriptome data for this species. A total of 28,999 unigenes were assembled, of which 19,750 (68.10%) were annotated using seven functional databases. The biological function of these unigenes was predicted using the GO, KOG, and KEGG databases. To gain insight into the chemosensory receptor-based system of parasitiform mites, we furthermore assessed the gene repertoire of gustatory receptors (GRs) and ionotropic receptors (IRs), both of which encode putative ligand-gated ion channel proteins. While these receptors are well characterized in insect model species, our understanding of chemosensory detection in mites and ticks is in its infancy. To address this paucity of data, we identified 9 IR/iGluRs and 2 GRs genes by analyzing transcriptome data in the NFM, while 9 GRs and 41 IR/iGluRs genes were annotated in the PRM genome. Taken together, the transcriptomic and genomic annotation of these two species provide a valuable reference for studies of parasitiform mites, and also helps to understand how chemosensory gene family expansion/contraction events may have been reshaped by an obligate parasitic lifestyle compared with their free-living closest relatives. Future studies should include additional species to validate this observation, and functional characterization of the identified proteins as a step forward in identifying tools for controlling these poultry pests.

category: molecular biology


title: Clathrin adaptors mediate two sequential pathwaysof intra-Golgi recycling

abstract: The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

category: cell biology


title: The single-cell epigenetic regulatory landscape in mammalian perinatal testis development

abstract: Spermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution data also revealed putative stem cells within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the  regulon of the mouse male germline and supporting somatic cells.

category: developmental biology


title: phenopype: a phenotyping pipeline for Python

abstract:
O_LIDigital images are an intuitive way to capture, store and analyze organismal phenotypes. Many biologists are taking images to collect high-dimensional phenotypic information from specimens, which are key to investigating complex ecological, evolutionary and developmental phenomena, such as relationships between trait diversity and ecosystem function, multivariate natural selection, or developmental plasticity. As a consequence, images are being collected at ever increasing rates, but extraction of the contained phenotypic information still poses a veritable analytical bottleneck.
C_LIO_LIphenopype is a high throughput phenotyping pipeline for the programming language Python that aims at alleviating this bottleneck. The package facilitates immediate extraction of high dimensional phenotypic data from laboratory grade digital images with low levels of background noise and complexity. At the core, phenopype provides functionality for rapid signal processing based image preprocessing and segmentation, followed by trait extraction, visualization and numerous options for data export. The functionality is provided by wrapping low-level Python computer vision libraries (e.g. OpenCV) into accessible functions, facilitating their incorporation into scientific image analysis workflows. In addition, phenopype provides a project management ecosystem to further simplify rapid data collection and to increase reproducibility.
C_LIO_LIphenopype offers two different workflows that support users during different stages of scientific image analysis. The low-throughput workflow uses regular Python syntax and has greater flexibility at the cost of reproducibility, which is suitable for prototyping during the initial stage of a research project. The high-throughput workflow allows users to specify and store image-specific settings for analysis in human-readable YAML format, and then execute all functions in one step by means of an interactive parser. This approach facilitates rapid program-user interactions during batch processing, and greatly increases scientific reproducibility.
C_LIO_LIOverall, phenopype intends to make the features of powerful but technically involved low-level CV libraries available to biologists with little or no Python coding experience. Therefore, phenopype is aiming to augment, rather than replace the utility of existing Python CV libraries, allowing biologists to focus on rapid and reproducible data collection. Furthermore, image annotations produced by phenopype can be used as training data, thus presenting a stepping stone towards the application of deep learning architectures.
C_LI

category: evolutionary biology


title: phenopype: A phenotyping pipeline for Python


title: Testing for evolutionary change in restoration: a genomic comparison between ex situ, native and commercial seed sources of Helianthus maximiliani

abstract: Globally imperiled ecosystems often depend upon collection, propagation, and storage of seed material for use in restoration. However, during the restoration process demographic changes, population bottlenecks, and selection can alter the genetic composition of seed material, with potential impacts for restoration success. The evolutionary outcomes associated with these processes have been demonstrated using theoretical and experimental frameworks, but no studies to date have examined the impact these processes have had on the seed material maintained for conservation and restoration. In this study, we compare genomic variation across seed sources used in conservation and restoration for the perennial prairie plant Helianthus maximiliani, a key component of restorations across North American grasslands. We compare individuals sourced from contemporary wild populations, ex situ conservation collections, commercially produced restoration material, and two populations selected for agronomic traits. Overall, we observed that ex situ and contemporary wild populations exhibited a similar genomic composition, while four of five commercial populations and selected lines were differentiated from each other and other seed source populations. Genomic differences across seed sources could not be explained solely by isolation by distance nor directional selection. We did find evidence of sampling effects for ex situ collections, which exhibited significantly increased coancestry relative to commercial populations, suggesting increased relatedness. Interestingly, commercially sourced seed appeared to maintain an increased number of rare alleles relative to ex situ and wild contemporary seed sources. However, while commercial seed populations were not genetically depauperate, the genomic distance between wild and commercially produced seed suggests differentiation in the genomic composition could impact restoration success. Our results point towards the importance of genetic monitoring of species used for conservation and restoration as they are expected to be influenced by the evolutionary processes that contribute to divergence during the restoration process.

category: genomics


title: A distinct assembly pathway of the human 39S late pre-mitoribosome

abstract: Assembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. However, little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solved cryo-EM structures of the human 39S large subunit pre-ribosomes, representing four distinct late state intermediates. Besides the MALSU1 complex, we identified several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involved rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we found deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

category: biochemistry


title: Gold nanocluster mediated delivery of siRNA to intact plant cells for efficient gene knockdown

abstract: RNA interference (RNAi), which involves the delivery of small interfering RNA molecules (siRNA), has been used to validate target genes in plants, to understand and control cellular metabolic pathways, and as a  green alternative for crop pest tolerance. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit limitations in host plant species range and their use can result in uncontrolled DNA integration into the plant host genome. Here, we synthesize polyethyleneimine functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plant cells and show these constructs enable efficient gene knockdown. We demonstrate that functionalized AuNCs protect siRNA from RNase degradation and are small enough (~2 nm) to bypass the plant cell wall which exhibits a size exclusion limit of 5-20 nm. These AuNCs in turn enable up to 76.5 {+/-} 5.9% GFP mRNA knockdown efficiency with no cellular toxicity. Our data suggest this simple and biocompatible platform for passive delivery of siRNA into intact plant cells could have broad applications in plant biotechnology.

category: bioengineering


title: High-content live-cell imaging reveals co-regulation of cardiomyocyte decisions of hypertrophy and apoptosis

abstract: Cardiomyocyte hypertrophy and apoptosis underlie cardiomyopathies and heart failure. While previous studies have described hypertrophy or apoptosis at the level of cell populations, how individual cells commit to these distinct yet co-regulated fates is unclear. We used high-content imaging to track single-cell hypertrophy and apoptosis dynamics, revealing new features and unique subpopulation responses. Catecholamines isoproterenol and norepinephrine induced heterogeneous analog hypertrophy and digital apoptosis, which support a "grow and/or die" conceptual model for cell decisions. Multinomial log-linear models indicated that a cells initial size and DNA content predict its susceptibility to hypertrophy and apoptosis. This work integrates dynamic morphological and biochemical cell profiling to reveal that cardiomyocyte hypertrophic and apoptotic responses to catecholamines represent an incoherent feedforward loop in which hypertrophy enhances survival.

category: bioinformatics


title: Tree canopy influences ground level atmospheric electrical and biogeochemical variability

abstract: Static electric fields in the atmosphere are increasingly recognized to interact with various organisms over several levels of biological organization. Recently, a link between atmospheric electrical variations and biogeochemical processes has been established in the context of open fields, yet biological structures like trees produce substantial alterations in atmospheric electric properties. Here, we assess whether these structural changes affect the dynamics of both biogenic and abiotic electrical landscapes and their relation to geochemical processes. To this end, we theoretically assess how trees alter their surrounding electric fields and empirically compare the temporal dynamics of atmospheric potential gradients, positive ions in the near-ground level atmosphere and soil electrochemical properties in an open field and under a tree. The developed model of electric fields around trees provides insight into the extent to which trees shield underlying electric landscape, revealing that a substantial increase in atmospheric potential gradient only marginally affects the electric field under the canopy. We further show that soil electrochemical properties are tied to temporal dynamics of positive ions in the near-ground level atmosphere, and that the presence of a tree reduces the temporal variability in both ground level positive ions concentrations and soil redox potential. This suggests that a tree can have a stabilizing effect on drivers of temporal variability in atmospheric electricity and soil electro-chemistry, thereby likely indirectly influencing soil microorganisms and processes as well as electro-sensitive organisms that perceive and utilize atmospheric electric fields.

category: biophysics


title: Structural basis of anti-SARS-CoV-2 activity of hydroxychloroquine: specific binding to NTD/CTD and disruption of LLPS of N protein

abstract: Great efforts have led to successfully developing the spike-based vaccines but challenges still exist to completely terminate the SARS-CoV-2 pandemic. SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in almost all key steps of the viral life cycle, thus representing a top drug target. Almost all key functions of N protein including liquid-liquid phase separation (LLPS) depend on its capacity in interacting with nucleic acids. Therefore, only the variants with their N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Very recently, hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of SARS-CoV-2 N protein with Kd of 112.1 and 57.1 M respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS essential for the viral life cycle. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to them all. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex decodes a promising strategy for further design of better anti-SARS-CoV-2 drugs from HCQ. Therefore, HCQ is a promising candidate to help terminate the pandemic.


title: Design of anti-SARS-CoV-2 molecules from hydroxychloroquine (HCQ)for better binding SARS-CoV-2 N protein


title: Structural basis of anti-SARS-CoV-2 activity of HCQ: specific binding to N protein to disrupt its interaction with nucleic acids and LLPS


title: Single-stranded nucleic acid sensing and coacervation by linker histone H1

abstract: The linker histone H1 is the most abundant group of eukaryotic chromatin-binding proteins. The mechanism underlying the diverse physiological functions of H1 remains unclear. Here we used single-molecule fluorescence and force microscopy to observe the behavior of H1 on DNA under different tensions. Unexpectedly, we found that H1 coalesces around nascent ssDNA. Molecular dynamics simulations revealed that multivalent and transient interactions between H1 and ssDNA mediate their phase separation. We further showed that longer and unpaired nucleic acids result in more viscous, gel-like H1 droplets. Finally, we imaged H1 puncta in cells under normal and stressed conditions and observed that RPA and H1 occupy separate nuclear regions. Overall, our results provide a new perspective to understanding the role of H1 in genome organization and maintenance.


title: Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors

abstract: Angiogenesis is essential for sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Thus, understanding mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1 in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1 at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases (PHDs) to bind and hydroxylate HIF-1, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Overexpression of PIM1 is sufficient to stabilize HIF-1 in normoxia and stimulate angiogenesis in a HIF-1-dependent manner in vivo. CRISPR mutants of HIF-1 (Thr455D) showed increased tumor growth, proliferation and angiogenesis. Moreover, T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

category: cancer biology


title: Molecular characterization reveals genomic and transcriptomic subtypes of metastatic urothelial carcinoma

abstract:
BackgroundMolecular characterization of primary urothelial carcinoma (UC) revealed molecular subtypes with different genomic, transcriptomic, and clinicopathological characteristics, which might guide therapeutic decision making. A comprehensive molecular characterization of metastatic UC (mUC), however, is currently lacking in the literature. Because of the lethality of mUC, with few therapeutic options available for patients, a multi-omics characterization of mUC could aid to improve patient selection for new and existing therapies.

MethodsTo define the molecular landscape of mUC and to identify potential targets for therapy, we performed whole genome DNA sequencing on fresh-frozen metastatic tumor biopsies of 116 mUC patients, and mRNA sequencing on 90 matched biopsies.

ResultsHierarchical clustering based on mutational signatures revealed two major genomic subtypes. The most prevalent subtype (67%) consisted almost exclusively of tumors with high APOBEC mutagenesis. APOBEC mutagenesis was detected in 91% of the samples, and appeared to be an ongoing process in mUC based on analysis of eight patients from whom serial biopsies were obtained during treatment. Contrary to the overall distribution of mutations, APOBEC associated mutations occurred throughout the genome, and independently of predicted accessible or transcribed genomic regions, suggesting that these mutations were generated during replication. Transcriptomic analysis revealed five mRNA-based subtypes: two luminal subtypes (40%), a stroma-rich (24%), basal/squamous (23%), and non-specified subtype (12%). The transcriptomic subtypes were different regarding driver gene alterations (e.g. ELF3 and TSC1), gene amplifications (NECTIN4 and PPARG), pathway activity, and immune cell infiltration. By integrating the genomic and transcriptomic data, potential therapeutic options per transcriptomic subtype and individual patient were proposed.

ConclusionsThis study expands our knowledge on the molecular landscape of mUC, and serves as a reference for subtype-oriented and patient-specific research on the etiology of mUC, and for novel drug development.

Trial registrationThe mUC cohort studied here is part of the Netherlands nationwide study of the center for personalized cancer treatment consortium (CPCT-02 Biopsy Protocol, NCT01855477), and the Drug Rediscovery Protocol (DRUP Trial, NCT02925234).


title: 3'-Sulfated Lewis A is a Biomarker for Metaplastic and Oncogenic Transformation of Several Gastrointestinal Epithelia

abstract:
IntroductionMultiple previous studies have shown the monoclonal antibody Das-1 (formerly called 7E12H12) specifically recognizes metaplastic and carcinomatous lesions in multiple organs of the gastrointestinal system (e.g. Barretts esophagus, intestinal-type metaplasia of the stomach, gastric adenocarcinoma, high-grade pancreatic intraepithelial neoplasm, and pancreatic ductal adenocarcinoma) as well as in other organs (bladder and lung carcinomas). Beyond being a useful biomarker in tissue, mAb Das-1 has recently proven to be more accurate than current paradigms for identifying cysts harboring advanced neoplasia. Though this antibody has been used extensively for clinical, basic science, and translational applications for decades, its epitope has remained elusive.

MethodsIn this study, we chemically deglycosylated a standard source of antigen, which resulted in near complete loss of the signal as measured by western blot analysis. The epitope recognized by mAb Das-1 was determined by affinity to a comprehensive glycan array and validated by inhibition of a direct ELISA.

ResultsThe epitope recognized by mAb Das-1 is 3-Sulfo-Lewis A (3-Sulfo-LeA). 3-Sulfo-LeA is broadly reexpressed across numerous GI epithelia and elsewhere only after metaplastic and carcinomatous transformation.

Discussion3-Sulfo-LeA is a clinically important antigen that can be detected both intracellularly in tissue using immunohistochemistry and extracellularly in cyst fluid and serum by ELISA. The results open new avenues for tumorigenic risk stratification of various gastrointestinal lesions.


title: Context dependent isoform specific PI3K inhibition confers drugresistance in Hepatocellular carcinoma cells

abstract:
BackgroundTargeted therapies for Primary liver cancer (HCC) is limited to the multi-kinase inhibitors, and not fully effective due to the resistance to these agents because of the heterogeneous molecular nature of HCC developed during chronic liver disease stages and cirrhosis. Although combinatorial therapy can increase the efficiency of targeted therapies through synergistic activities, isoform specific effects of the inhibitors are usually ignored. This study concentrated on PI3K/Akt/mTOR pathway and the differential combinatory bioactivities of isoform specific PI3K- inhibitor (PIK-75) or PI3K-{beta} inhibitor (TGX-221) with Sorafenib dependent on PTEN context.

MethodsThe bioactivities of inhibitors on PTEN adequate Huh7 and deficient Mahlavu cells were investigated with real time cell growth, cell cycle and cell migration assays. Differentially expressed genes from RNA-Seq were identified by edgeR tool. Systems level network analysis of treatment specific pathways were performed with Prize Collecting Steiner Tree (PCST) on human interactome and enriched networks were visualized with Cytoscape platform.

ResultsOur data from combinatory treatment of Sorafenib and PIK-75 and TGX-221 showed opposite effects; while PIK-75 displays synergistic effects on Huh7 cells leading to apoptotic cell death, Sorafenib with TGX-221 display antagonistic effects and significantly promotes cell growth in PTEN deficient Mahlavu cells. Transcriptomic states of combinatory treatments with PIK-75 and TGX-221 inhibitors were identified in PTEN deficient and adequate cells. Molecular interactions and cell signaling pathways were reconstructed and analyzed in-depth to understand mechanism of differential synergistic or antagonistic effects of PI3K- (PIK-75) and PI3K-{beta} (TGX-221) inhibitors with Sorafenib.

ConclusionsSimultaneously constructed and analyzed differentially expressed cellular networks presented in this study, revealed distinct consequences of isoform specific PI3K inhibition in PTEN adequate and deficient liver cancer cells. We demonstrated the importance of context dependent and isoform specific PI3K/Akt/mTOR signaling inhibition in drug resistance during combination therapies. (https://github.com/cansyl/Isoform-spesific-PI3K-inhibitor-analysis).


title: RABL6A promotes pancreatic neuroendocrine tumor angiogenesis and progression in vivo

abstract: Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet {beta} cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.


title: Self-ubiquitination of a pathogen type-III effector traps and blocks the autophagy machinery to promote disease

abstract: Beyond its role in cellular homeostasis, autophagy plays anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One prominent role of anti-microbial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in depletion of a bacterial virulence factor and unravels an unprecedented pathogen strategy to counteract defense-related autophagy.

category: plant biology


title: A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component


title: The radish Ogura fertility restorer impedes translation elongation along its cognate CMS-causing mRNA

abstract: The control of mRNA translation has been increasingly recognized as a key regulatory step for gene control but clear examples in eukaryotes are still scarce. Nucleo-cytoplasmic male sterilities (CMS) represent ideal genetic models to dissect genetic interactions between the mitochondria and the nucleus in plants. This trait is determined by specific mitochondrial genes and is associated with a pollen sterility phenotype that can be suppressed by nuclear genes known as restorer-of-fertility (Rf) genes. In the study, we focused on the Ogura CMS system in rapeseed and showed that the suppression to male sterility by the PPR-B fertility restorer (also called Rfo) occurs through a specific inhibition of the translation of the mitochondria-encoded CMS-causing mRNA orf138. We also demonstrate that PPR-B binds within the coding sequence of orf138 and acts as a ribosome blocker to specifically impede translation elongation along the orf138 mRNA. Rfo is the first recognized fertility restorer shown to act this way. These observations will certainly facilitate the development of synthetic fertility restorers for CMS systems in which efficient natural Rfs are lacking.


title: 3D models of Alzheimer's disease patient microglia recapitulate disease phenotype and show differential drug responses compared to 2D

abstract: Alzheimers disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brains resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that faithfully recapitulates patient-specific microglial characteristics. To address this shortcoming, we developed novel 3D models of monocyte-derived microglia-like cells (MDMi) from AD patients. MDMi in 3D exhibited mature microglial features, including a highly branched morphology and enhanced bonafide microglial marker expression compared to 2D. Moreover, AD MDMi in 3D co-cultures with neuro-glial cells showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-{beta}. Drug screening assays in 3D AD MDMi revealed different cytokine responses compared to 2D. Our study demonstrates disease- and drug-specific responses in 3D MDMi models that are not apparent in 2D and presents a new 3D platform for more effective and personalised drug testing.

category: neuroscience


title: Antibody repertoire analysis of tumor-infiltrating B cells reveals distinct signatures and distributions across tissues

abstract: The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.

category: immunology


title: The transcriptional repressor REST is a major negative regulator of endocrine differentiation during pancreas organogenesis

abstract: Understanding genomic regulatory mechanisms of pancreas differentiation is relevant to the pathophysiology of diabetes mellitus, and to the development of replacement therapies. Numerous transcription factors promote {beta} cell differentiation, although less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrine gene programs in the embryonic pancreas. However, pancreatic Rest knock-out mice failed to show increased numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we now observe a marked increase in the formation of pancreatic endocrine cells. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts, and induced {beta}-cell specific genes in human adult duct-derived organoids. Finally, we define REST genomic programs that suppress pancreatic endocrine differentiation. These results establish a crucial role of REST as a negative regulator of pancreatic endocrine differentiation.


title: Differences in orthodenticle expression promote ommatidial size variation between 	Drosophila species

abstract: The compound eyes of insects exhibit extensive variation in ommatidia number and size, which affects how they see and underlies adaptations in their vision to different environments and lifestyles. However, very little is known about the genetic and developmental bases underlying differences in compound eye size. We previously showed that the larger eyes of Drosophila mauritiana compared to D. simulans is caused by differences in ommatidia size rather than number. Furthermore, we identified an X-linked chromosomal region in D. mauritiana that results in larger eyes when introgressed into D. simulans. Here, we used a combination of fine-scale mapping and gene expression analysis to further investigate positional candidate genes on the X chromosome. We found that orthodenticle is expressed earlier in D. mauritiana than in D. simulans during ommatidial maturation in third instar larvae, and we further show that this gene is required for the correct organisation and size of ommatidia in D. melanogaster. Using ATAC-seq, we have identified several candidate eye enhancers of otd as well as potential direct targets of this transcription factor that are differentially expressed between D. mauritiana and D. simulans. Taken together, our results suggest that differential timing of otd expression contributes to natural variation in ommatidia size between D. mauritiana and D. simulans, which provides new insights into the mechanisms underlying the regulation and evolution of compound eye size in insects.


title: Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency

abstract: Current technologies for acquiring spatial transcript information from tissue sections rely on either RNA probes or spatial barcodes. The former methods require a priori knowledge for probeset formulation; the latter have yet to achieve single cell resolution and/or transcript capture efficiencies approaching dissociative, single-cell methods. Here, we describe a novel spatial transcriptome assay called polony (or DNA cluster)-indexed library-sequencing (PIXEL-seq). It improves upon other spatial barcoding methods by employing "continuous" polony oligos arrayed across a customized gel surface. In terms of assay performance, PIXEL-seq attains [≤] 1 {micro}m resolution and captures >1,000 unique molecular identifiers/10x10 {micro}m2. In other words, this global, naive platform achieves subcellular spatial transcriptome mapping while maintaining high transcript capture efficiencies.


title: Mapping origins of variation in neural trajectories of human pluripotent stem cells

abstract:
Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. We identify differences in the spontaneous self-organization of individual hPSC lines during self-renewal that lie along a fundamental axis of in vivo development. Distinct stable and dynamic transcriptional elements were revealed by decomposition of RNA-seq data in pluripotency and early lineage emergence. Stable differences within pluripotency predicted regional bias in the dynamics of neural differentiation that were also observed in large collections of hPSC lines. Using replicate human induced PSC (hiPSC) lines and paired adult tissue, we demonstrate that cells from individual humans expressed unique transcriptional signatures that were maintained throughout life. In addition, replicate hiPSC lines from one donor showed divergent expression phenotypes driven by distinct chromatin states. These stable transcriptional states are under both genetic and epigenetic control and predict bias in subsequent forebrain and hindbrain neural trajectories. These results define mechanisms controlling transcription in pluripotency to first establish human neural diversity. Data availability: GSE164055; https://nemoanalytics.org/p?l=KimEtAL2021&g=GBX2.

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title: High prevalence and diversity of beta-lactamase-encoding bacteria in cryosoils and ancient permafrost

abstract:
BackgroundAntimicrobial resistance is one of the major challenges affecting public health. It is mostly due to the continuous emergence of extended-spectrum {beta}-lactamase from various environments followed by their rapid dissemination and selection in clinical settings. The warming of Earths climate is the other global threat facing human society, in particular with the Arctic regions experiencing a twice faster warming than the global average and permafrost affected by widespread thawing. A potentially dreadful combination of these two threats would be the release and dispersion of harmful microbes that have remained confined to largely uninhabited Arctic regions, or are stored dormant in permafrost.

MethodsEnvironmental DNA was isolated from 12 soil samples from various Arctic and subarctic pristine regions in Siberia (Yakutia and Kamchatka), including nine permafrost samples collected at various depths. The large datasets obtained from high throughput sequencing was assembled in contigs and their protein-gene contents predicted. We used exhaustive similarity searches to perform taxonomical assignments of bacterial, archaeal, and eukaryotic organisms, as well as DNA viruses. In addition, we specifically identified {beta}-lactamase genes and their prevalence per bacterial genome estimated through the detection of two universal single copy genes.

FindingsA total of 9.217 1011 bp were exploited, leading to a total of 525,313 contigs at least 5kb in size. The DNA content of the various samples was found to be highly variable, not strictly correlated with the depth or radio-carbon-based deposit age, and most likely linked to the global density of microbes trapped in the corresponding permafrost layers. Bacteria account for more than 90% of the contigs in most samples, followed by Eukaryotes and Archaea (always lower than 10%). Viruses represented less than 2% of all contigs in all samples. The taxonomic profiles of surface cryosoils and deep permafrost samples exhibited a high diversity, including between permafrost samples originating from various depths in the same borehole. In all samples, bacterial contigs carrying different {beta}-lactamases from class A to D were identified.

InterpretationNo clear common taxonomic feature could be found shared by surface cryosoils or ancient permafrost layers. However, most samples (9/12) exhibited a high frequency of {beta}-lactamase genes, with an estimated average close to 1 copy/bacterial genome. In addition to the well-documented reactivation of infectious ancient pathogens (bacteria, viruses, protozoa), we show now that global warming could contribute to the emergence of new antibiotic resistances through the mobilization by contemporary bacteria of ancient DNA released from thawing permafrost.

FundingCNRS PRC research grant (PRC1484-2018) to C.A. E C-F was supported by a PhD grant (DGA/DS/MRIS) #2017 60 0004. GG and JS were funded by ERC PETA-CARB (#338335) and the HGF Impulse and Networking Fund (ERC-0013).

category: microbiology


title: A Cdc42-Borg4-Septin 7 axis regulates HSCs polarity and function

abstract:
Aging of hematopoietic stem cells (HSCs) is caused by an elevated activity of the small RhoGTPase Cdc42 and an apolar distribution of proteins. Mechanisms by which Cdc42 activity controls polarity of HSCs are not known. Binder of RhoGTPases proteins (borgs) are known effector proteins of Cdc42 that are able to regulate the cytoskeletal septin network. Here we show that Cdc42 interacts with borg4, which in turn interacts with septin 7 to regulate the polar distribution of Cdc42, borg4 and septin 7 within HSCs. Genetic deletion of either borg4 or septin 7 in HSCs resulted in a reduced frequency of HSCs polar for Cdc42 or borg4 or septin 7 and a reduced engraftment potential and decreased lymphoid-primed multipotent progenitors (LMPPs) frequency in the bone marrow. In aggregation our data identify a Cdc42-borg4-septin 7 axis to be essential for maintenance of polarity within HSCs and for HSC function and provide rationale for further investigating the role of borgs and septins for the regulation of compartmentalization within stem cells.

Graphical Abstract

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title: Default Mode Network spatial configuration varies across task domains

abstract: Recent developments in network neuroscience suggest reconsidering what we thought we knew about the Default Mode Network (DMN). Although this network has always been seen as unitary and associated with the resting state, a new deconstructive line of research is pointing out that the DMN could be divided into multiple subsystems supporting different functions. By now, it is well known that the DMN is not only deactivated by tasks, but also involved in affective, mnestic, and social paradigms, among others. Nonetheless, it is starting to become clear that the array of activities in which it is involved, might also be extended to more extrinsic functions. The present meta-analytic study is meant to push this boundary a bit further. The BrainMap database was searched for all experimental paradigms activating the DMN, and their activation maps were then computed. An additional map of task-induced deactivations was also created. A Multidimensional Scaling indicated that such maps could be arranged along an anatomo-psychological gradient, which goes from midline core activations, associated with the most internal functions, to the involvement of lateral cortices in more external tasks. Further investigations suggested that such extrinsic mode is especially related to reward, semantic, and emotional functions. However, an important finding was that the variability of task-induced DMN anatomic redistribution was hard to recapitulate, as none of the maps, or any linear combination of them, could represent the whole space of its dynamical reconfiguration. Altogether, our findings suggest that the DMN may be characterized by a richer functional diversity and a more spatial complexity than previously suggested.


title: MLL3/MLL4 Histone Methyltranferase Activity Dependent Chromatin Organization at Enhancers during Embryonic Stem Cell Differentiation

abstract: MLL3 (KMT2C) and MLL4 (KMT2D), the major mono-methyltransferases of histone H3 lysine 4 (H3K4), are required for cellular differentiation and embryonic development in mammals. We previously observed that MLL3/4 promote long-range chromatin interactions at enhancers, however, it is still unclear how their catalytic activities contribute to enhancer-dependent gene activation in mammalian cell differentiation. To address this question, we mapped histone modifications, long-range chromatin contacts as well as gene expression in MLL3/4 catalytically deficient mouse embryonic stem (ES) cells undergoing differentiation toward neural precursor cells. We showed that MLL3/4 activities are responsible for deposition of H3K4me1 modification and formation of long-range enhancer-promoter contacts at a majority of putative enhancers gained during cell differentiation, but are dispensable for most candidate enhancers found in undifferentiated ES cells that persist through differentiation. While transcriptional induction at most genes is unaltered in the MLL3/4 catalytically deficient cells, genes making more contacts with MLL3/4-dependent putative enhancers are disproportionately affected. These results support that MLL3/4 contributes to cellular differentiation through histone-methyltransferase-activity dependent induction of enhancer-promoter contacts and transcriptional activation at a subset of lineage-specific genes.


title: HIV-1 Vpu promotes phagocytosis of infected CD4+ T cells by macrophages through downregulation of CD47

abstract:
Human immunodeficiency virus (HIV) remodels the cell surface of infected cells to facilitate viral dissemination and promote immune evasion. The membrane-associated Vpu accessory protein encoded by HIV-1 plays a key role in this process by altering cell surface levels of multiple host proteins. Using an unbiased quantitative plasma membrane profiling approach, we previously identified CD47 as a putative host target downregulated by Vpu. CD47 is a ubiquitously-expressed cell surface protein that interacts with the myeloid cell inhibitory receptor SIRP to deliver a "dont-eat-me" signal, thus protecting cells from phagocytosis. In this study, we investigate whether CD47 modulation by HIV-1 Vpu might promote the susceptibility of macrophages to viral infection via phagocytosis of infected CD4+ T cells. Indeed, we find that Vpu downregulates CD47 expression on infected CD4+ T cells leading to an enhanced capture and phagocytosis by macrophages. Interestingly, it is through this process that a CCR5-tropic transmitted/founder (T/F) virus, which otherwise poorly infects macrophages in its cell-free form, becomes infectious in macrophages. Importantly, we show that HIV-1-infected cells expressing a Vpu-resistant CD47 mutant are less prone to infect macrophages through phagocytosis. Mechanistically, Vpu forms a physical complex with CD47 through its transmembrane domain and targets the latter for lysosomal degradation. These results reveal a novel role of Vpu in modulating macrophage infection, which has important implications for HIV-1 transmission in early stages of infection and the establishment of viral reservoir.

IMPORTANCEMacrophages play critical roles in HIV transmission, viral spread early in infection, and as a reservoir of virus. Selective capture and engulfment of HIV-1 infected T cells was shown to drive efficient macrophage infection suggesting that this mechanism represents an important mode of infection notably for weakly macrophage-tropic T/F viruses. In this study, we provide insight into the signals that regulate this process. We show that the HIV-1 accessory protein Vpu downregulates cell surface levels of CD47, a host protein that interacts with the inhibitory receptor SIRP to deliver a "dont-eat-me" signal to macrophages. This allows for enhanced capture and phagocytosis of infected T cells by macrophages, ultimately leading to their productive infection even with T/F virus. These findings provide new insights into the mechanisms governing the intercellular transmission of HIV-1 to macrophages with implications for the establishment of the macrophage reservoir and early HIV-1 dissemination in vivo.


title: Burrowing crabs and physical factors hasten marsh recovery at panne edges

abstract: Salt marsh loss is projected to increase as sea-level rise accelerates with global climate change. Salt marsh loss occurs along both lateral creek and channel edges and in the marsh interior, when pannes expand and coalesce. Often, edge loss is attributed to erosive processes whereas dieback in the marsh interior is linked to excessive inundation or deposition of wrack. We conducted a two-year field experiment (2016-2018) in a central California estuary, where, immediately preceding our study, marsh dieback at creek edges and panne expansion occurred during a period of severe drought and an overlapping warm water event. Our study explored how an abundant burrowing crab, shown to have strong negative effects on marsh biomass near creek edges, affects panne dynamics. We also explored which panne attributes best predicted their dynamics. Overall, we found that pannes contracted during the study period, but with variable rates of marsh recovery across pannes. Our model incorporating both physical and biological factors explained 86% of the variation in panne contraction. The model revealed a positive effect of crab activity, sediment accretion, and a composite of depth and elevation on panne contraction, and a negative effect of panne size and distance to nearest panne. The positive crab effects detected in pannes contrast with negative effects we had earlier detected near creek edges, highlighting the context-dependence of top-down and bioturbation effects in marshes. As global change continues and the magnitude and frequency of disturbances increases, understanding the dynamics of marsh loss in the marsh interior as well as creek banks will be critical for the management of these coastal habitats.

category: ecology


title: Deep learning-based pan-cancer classification model reveals cancer-specific gene expression signatures

abstract: The identification of cancer-specific biomarkers and therapeutic targets is one of the primary goals of cancer genomics. Thousands of cancer genomes, exomes, and transcriptomes have been sequenced to date. In this study, we conducted a pan-cancer analysis of transcriptome datasets from 37 cancer types provided by The Cancer Genome Atlas (TCGA) in an effort to identify cancer-specific gene expression signatures. We employed deep neural networks to train a model on the transcriptome profile datasets for all cancer types. The model was validated, and its predictive accuracy was determined using an independent dataset, achieving > 97% prediction accuracy across cancer types. This strongly suggests that there are distinct gene expression signatures associated with various cancer types. We interpreted the model using SHapley Additive exPlanations (SHAP) to identify specific gene signatures that significantly contributed to the classification of cancer types. In addition to known biomarkers, we identified several novel biomarkers in different cancer types. These cancer-specific gene signatures are valuable candidates for future studies of their potential utility as cancer biomarkers and putative therapeutic targets.


title: Parallel representation of context and multiple context-dependent values in ventro-medial prefrontal cortex

abstract: Value representations in ventromedial prefrontal-cortex (vmPFC) are known to guide the choice between options. But the value of an option can be different in different task contexts. Goal-directed behavior therefore requires to know the current context and associated values of options, and to flexibly switch between value representations in a task-dependent manner. We tested whether task-relevant and -irrelevant values influence behavior and asked whether both values are represented together with context signals in vmPFC. Thirty-five participants alternated between tasks in which stimulus color or motion predicted rewards. As expected, neural activity in vmPFC and choices were largely driven by task-relevant values. Yet, behavioral and neural analyses indicate that participants also retrieved the values of irrelevant features, and computed which option would have been best in the alternative context. Investigating the probability distributions over values and contexts encoded in multivariate fMRI signals, we find that vmPFC maintains representations of the current context, i.e. task state, the value associated with it, and the hypothetical value of the alternative task state. Crucially, we show that evidence for irrelevant value signals in vmPFC relates to behavior on multiple levels, competes with expected value signals, and interacts with task state representations. Our results thus suggest that different value representations are represented in parallel and imply a link between neural representations of task states, their associated values and their influence on behavior. This sheds new light on vmPFCs role in decision making, bridging between a hypothesized role in mapping observations onto the task states of a mental map, and computing value expectations for alternative states.


title: Representations of context and context-dependent values in vmPFC compete for guiding behavior


title: A comparison of the Mouse Song Analyzer and DeepSqueak ultrasonic vocalization analysis systems in C57BL/6J, FVB.129, and FVB mice

abstract: Vocal communication is an essential behavior in mammals and is relevant to human neurodevelopmental conditions. Mice produce communicative vocalizations, known as ultrasonic vocalizations (USVs), that can be recorded with various programs. The Mouse Song Analyzer is an automated analysis system, while DeepSqueak is a semi-automated system. We used data from C57BL/6J, FVB.129, and FVB mice to compare whether the DeepSqueak and Mouse Song Analyzer systems measure a similar total number, duration, and fundamental frequency of USVs. We found that the two systems detected a similar quantity of USVs for FVB.129 mice (r= .90, p< .001), but displayed lower correlations for C57BL/6J (r= .76, p< .001) and FVB mice (r= .60, p< .001). We also found that DeepSqueak detected significantly more USVs for C57BL/6J mice than the Mouse Song Analyzer. The two systems detected a similar duration of USVs for C57BL/6J (r= .82, p< .001), but lower correlations for FVB.129 (r= .13, p< .001) and FVB mice (r= .51, p< .01) were found, with DeepSqueak detecting significantly more USVs per each strain. We found lower than acceptable correlations for fundamental frequency in C57BL/6J (r= .54, p< .01), FVB.129 (r= .76, p< .001), and FVB mice (r= .07, p= .76), with the Mouse Song Analyzer detecting a significantly higher fundamental frequency for FVB.129 mice. These findings demonstrate that the strain of mouse used significantly affects the number, duration, and fundamental frequency of USVs that are detected between programs. Overall, we found that DeepSqueak is more accurate than the Mouse Song Analyzer.

category: animal behavior and cognition


title: The fungal root endophyte Serendipita vermifera displays inter-kingdom synergistic beneficial effects with the microbiota in Arabidopsis thaliana and barley

abstract:
Plant root-associated bacteria can confer protection against pathogen infection. By contrast, the beneficial effects of root endophytic fungi and their synergistic interactions with bacteria remain poorly defined. We demonstrate that the combined action of a fungal root endophyte from a widespread taxon with core bacterial microbiota members provides synergistic protection against an aggressive soil-borne pathogen in Arabidopsis thaliana and barley. We additionally show early inter-kingdom growth promotion benefits which are host and microbiota composition dependent.

HighlightsO_LIThe root endophytic fungus Serendipita vermifera can functionally replace core bacterial microbiota members in mitigating pathogen infection and disease symptoms.
C_LIO_LIS. vermifera additionally stabilizes and potentiates the protective activities of root-associated bacteria and mitigates the negative effects of a non-native bacterial community in A. thaliana.
C_LIO_LIInter-kingdom synergistic beneficial effects do not require extensive host transcriptional reprogramming nor high levels of S. vermifera colonisation.
C_LIO_LIInter-kingdom protective benefits are largely independent of the host while synergism leading to early inter-kingdom growth promotion is driven by host species and microbiota composition.
C_LI


title: Engineering the AEG1 promoter from cotton to develop male sterile lines

abstract: This work reports on modifying the Upstream Regulatory Module (URM, 1.5 Kb region upstream of the open reading frame) of Anther Expressing Gene 1 (AEG1) from cotton to achieve anther specific activity. AEG1 was identified in a previous study aimed to isolate a promoter with tapetum specific activity. Such a promoter could then be used to express barnase and barstar genes for developing male sterile and restorer lines for hybrid seed production in cotton. The AEG1 URM was observed to be active in tapetum as well as in roots making it unusable to drive the expression of barnase gene. Analysis of the URM showed the presence of several root specific motifs. Two modified AEG1 URMs were developed, by removing or mutating these motifs and its activity checked in tobacco. The activity of one of the modified URMs, AEG1({Delta}Bmut) was restricted to the anther tissue as observed using the reporter gene {beta}-glucuronidase. The study also demonstrates that male sterile lines could be developed in tobacco using the AEG1({Delta}Bmut) URM to express the barnase gene. This work thus shows the possibility of engineering promoters to achieve tissue specificity and to develop male sterile lines in cotton.


title: Fronto-medial theta coordinates posterior maintenance of working memory content

abstract:
How does the human brain manage multiple bits of information to guide goal-directed behaviour? Successful working memory (WM) functioning has consistently been linked to oscillatory power in the theta frequency band (4-8 Hz) over fronto-medial cortex (fronto-medial theta, FMT). Specifically, FMT is thought to reflect the mechanism of an executive sub-system that coordinates maintenance of memory contents in posterior regions. However, direct evidence for the role of FMT in controlling specific WM content is lacking. Here we collected high-density Electroencephalography (EEG) data whilst participants engaged in load-varying WM tasks and then used multivariate decoding methods to examine WM content during the maintenance period. Higher WM load elicited a focal increase in FMT. Importantly, decoding of WM content was driven by posterior/parietal sites, which in turn showed load-induced functional theta coupling with fronto-medial cortex. Finally, we observed a significant slowing of FMT frequency with increasing WM load, consistent with the hypothesised broadening of a theta  duty cycle to accommodate additional WM items. Together these findings demonstrate that frontal theta orchestrates posterior maintenance of WM content. Moreover, the observed frequency slowing elucidates the function of FMT oscillations by specifically supporting phase-coding accounts of WM.

Significance StatementHow does the brain juggle the maintenance of multiple items in working memory (WM)? Here we show that increased WM demands increase theta power (4-8 Hz) in fronto-medial cortex. Interestingly, using a machine learning approach, we found that the content held in WM could be read out not from frontal, but from posterior areas. These areas were in turn functionally coupled with fronto-medial cortex, consistent with the idea that frontal cortex orchestrates WM representations in posterior regions. Finally, we observed that holding an additional item in WM leads to significant slowing of the frontal theta rhythm, supporting computational models that postulate longer  duty cycles to accommodate additional WM demands.


title: The molecular logic of the self-organization of primitive streak and neuroectoderm fates

abstract: The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we reassessed the contribution of retinoic acid (RA) signaling at early stages of neural commitment and its cross-talk with TGF{beta} and Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification from epiblast and primitive streak-like cells up to the emergence of anterior and posterior neural fates. In conditions thought to lack RA synthesis, we discovered a hitherto unidentified residual RA production via a sensitive RA reporter. Genetic perturbations proved that the RA-degrading enzyme Cyp26a1 safeguard the developmental capabilities of the PS-like cells, limiting neural differentiation in wild type and in Chrd-/-Nog-/- or Dkk1-/- cells. Finally, the knockout of the three RAR receptors highlighted their function as negative regulators of loci critical for neural induction. Overall, we identified two mechanisms whereby components of the RA pathway can control the formation of neural progenitors in our PS-like context: a RA-dependent neural induction gated by RARs, and a receptor-mediated repression in the absence of ligand.


title: Multi-layered regulation of neuroectoderm differentiation by retinoic acid in a primitive streak-like context


title: Geographic Confounding in Genome-Wide Association Studies

abstract: Gene-environment correlations can bias associations between genetic variants and complex traits in genome-wide association studies (GWASs). Here, we control for geographic sources of gene-environment correlation in GWASs on 56 complex traits (N=69,772-271,457). Controlling for geographic region significantly decreases heritability signals for SES-related traits, most strongly for educational attainment and income, indicating that socio-economic differences between regions induce gene-environment correlations that become part of the polygenic signal. For most other complex traits investigated, genetic correlations with educational attainment and income are significantly reduced, most significantly for traits related to BMI, sedentary behavior, and substance use. Controlling for current address has greater impact on the polygenic signal than birth place, suggesting both active and passive sources of gene-environment correlations. Our results show that societal sources of social stratification that extend beyond families introduce regional-level gene-environment correlations that affect GWAS results.

category: genetics


title: Sequence determinants of human gene regulatory elements

abstract: DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. To obtain a global and unbiased view of the relative importance of different sequence determinants in gene expression, we measured transcriptional activity of DNA sequences that are in aggregate [~]100 times longer than the human genome in three different cell types. We show that enhancers can be classified to three main types: classical enhancers1, closed chromatin enhancers and chromatin-dependent enhancers, which act via different mechanisms and differ in motif content. Transcription factors (TFs) act generally in an additive manner with weak grammar, with classical enhancers increasing expression from promoters by a mechanism that does not involve specific TF-TF interactions. Few TFs are strongly active in a cell, with most activities similar between cell types. Chromatin-dependent enhancers are enriched in forkhead motifs, whereas classical enhancers contain motifs for TFs with strong transactivator domains such as ETS and bZIP; these motifs are also found at transcription start site (TSS)-proximal positions. However, some TFs, such as NRF1 only activate transcription when placed close to the TSS, and others such as YY1 display positional preference with respect to the TSS. TFs can thus be classified into four non-exclusive subtypes based on their transcriptional activity: chromatin opening, enhancing, promoting and TSS determining factors - consistent with the view that the binding motif is the only atomic unit of gene expression.


title: Data Augmentation Through Monte Carlo Arithmetic Leads to More Generalizable Classification in Connectomics

abstract:
Machine learning models are commonly applied to human brain imaging datasets in an effort to associate function or structure with behaviour, health, or other individual phenotypes. Such models often rely on low-dimensional maps generated by complex processing pipelines. However, the numerical instabilities inherent to pipelines limit the fidelity of these maps and introduce computational bias. Monte Carlo Arithmetic, a technique for introducing controlled amounts of numerical noise, was used to perturb a structural connectome estimation pipeline, ultimately producing a range of plausible networks for each sample. The variability in the perturbed networks was captured in an augmented dataset, which was then used for an age classification task. We found that resampling brain networks across a series of such numerically perturbed outcomes led to improved performance in all tested classifiers, preprocessing strategies, and dimensionality reduction techniques. Importantly, we find that this benefit does not hinge on a large number of perturbations, suggesting that even minimally perturbing a dataset adds meaningful variance which can be captured in the subsequently designed models.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/423084v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@bccd23org.highwire.dtl.DTLVardef@13fcfe1org.highwire.dtl.DTLVardef@68e987org.highwire.dtl.DTLVardef@1bbada9_HPS_FORMAT_FIGEXP  M_FIG This paper demonstrates how Monte Carlo Arithmetic, a dataagnostic perturbation technique, can be used for dataset augmentation during the generation of structural connectomes and improve downstream phenotypic prediction.

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title: Dynamic assembly of the calcium hemostasis modulator 1 channel gates ATP permeation

abstract: Calcium hemostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. The currently reported CALHM structures are all in an ATP-conducting state, and the gating mechanism of ATP permeation remains elusive. Here, we report three cryo-EM reconstructions of Danio rerio heptameric CALHM1s with ordered or flexible long C-terminal helices as well as Danio rerio octameric CALHM1 with flexible long C-terminal helices at resolutions of 3.2 [A], 2.9 [A], and 3.5 [A]. Structural analysis revealed that the heptameric CALHM1s are in an ATP nonconducting state in which the pore diameter in the middle is approximately 6.6 [A]. Compared with those inside the octameric CALHM1s, the N- helices inside heptameric CALHM1s are in the "down" position to avoid steric clash with neighboring TM1 helices. Molecular dynamic simulation shows that the pore size is significantly increased for ATP molecule permeation during the movement of the N- helix from the "down" position to the "up" position. Therefore, we proposed a mechanism in which the "piston-like" motion of the N-helix drives the dynamic assembly of the CALHM1 channel for ATP molecule permeation. Our results provide insights into the ATP permeation mechanism of the CALHM1 channel.


title: Microscope-Cockpit: Python-based bespoke microscopy for bio-medical science

abstract:
We have developed "Microscope-Cockpit" (Cockpit), a highly adaptable open source user-friendly Python-based GUI environment for precision control of both simple and elaborate bespoke microscope systems. The user environment allows next-generation near-instantaneous navigation of the entire slide landscape for efficient selection of specimens of interest and automated acquisition without the use of eyepieces. Cockpit uses "Python-Microscope" (Microscope) for high-performance coordinated control of a wide range of hardware devices using open source software. Microscope also controls complex hardware devices such as deformable mirrors for aberration correction and spatial light modulators for structured illumination via abstracted device models. We demonstrate the advantages of the Cockpit platform using several bespoke microscopes, including a simple widefield system and a complex system with adaptive optics and structured illumination. A key strength of Cockpit is its use of Python, which means that any microscope built with Cockpit is ready for future customisation by simply adding new libraries, for example machine learning algorithms to enable automated microscopy decision making while imaging.

HighlightsO_LIUser-friendly setup and use for simple to complex bespoke microscopes.
C_LIO_LIFacilitates collaborations between biomedical scientists and microscope technologists.
C_LIO_LITouchscreen for near-instantaneous navigation of specimen landscape.
C_LIO_LIUses Python-Microscope, for abstracted open source hardware device control.
C_LIO_LIWell-suited for user training of AI-algorithms for automated microscopy.
C_LI


title: A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

abstract: The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.

category: molecular biology


title: An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay


title: Plasma amyloid beta ratios in autosomal dominant Alzheimers disease: the influence of genotype

abstract: In-vitro studies of autosomal dominant Alzheimers disease (ADAD) implicate longer A{beta} peptides in pathogenesis, however less is known about the behaviour of ADAD mutations in-vivo. In this cross-sectional cohort study, we used liquid chromatography-tandem mass spectrometry to analyse 66 plasma samples from ADAD family members who were at-risk of inheriting a mutation or were already symptomatic. We tested for differences in plasma A{beta}42:38, 38:40 and 42:40 ratios between Presenilin1 (PSEN1) and Amyloid Precursor Protein (APP) carriers. We examined the relationship between plasma and in-vitro models of A{beta} processing and, among PSEN1 carriers, tested for associations with parental age at onset (AAO). 39 participants were mutation carriers (28 PSEN1 and 11 APP). Age- and sex-adjusted models showed marked differences in plasma A{beta} between APP and PSEN1: higher A{beta}42:38 in PSEN1 versus APP (p<0.001) and non-carriers (p<0.001); higher A{beta}38:40 in APP versus PSEN1 (p<0.001) and non-carriers (p<0.001), while A{beta}42:40 was higher in APP and PSEN1 compared to non-carriers (both p<0.001). A{beta} profiles were reasonably consistent in plasma and cell lines. Within PSEN1, sex-adjusted models demonstrated negative associations between (i)A{beta}42:40 (ii)A{beta}42:38 and parental AAO. In-vivo differences in A{beta} processing between APP and PSEN1 provide insights into ADAD pathophysiology which can inform therapy development.


title: A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

abstract: The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.


title: Myocarditis in naturally infected pets with the British variant of COVID-19

abstract: Domestic pets can contract SARS-CoV-2 infection but, based on the limited information available to date, it is unknown whether the new British B.1.1.7 variant can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by the British B.1.1.7 variant of SARS-CoV-2 diagnosed at a specialist veterinary hospital in the South-East of England. Furthermore, we discovered that many owners and handlers of these pets had developed Covid-19 respiratory symptoms 3-6 weeks before their pets became ill and had also tested PCR positive for Covid-19. Interestingly, all these B.1.1.7 infected pets developed atypical clinical manifestations, including severe cardiac abnormalities secondary to myocarditis and a profound impairment of the general health status of the patient but without any primary respiratory signs. Together, our findings demonstrate for the first time the ability for companion animals to be infected by the B.1.1.7 variant of SARS-CoV-2 and raise questions regarding its pathogenicity in these animals. Moreover, given the enhanced infectivity and transmissibility of B.1.1.7 variant for humans, these findings also highlights more than ever the risk that companion animals may potentially play a significant role in SARS-CoV-2 outbreak dynamics than previously appreciated.


title: Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies

abstract: Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.


title: Semaphorin 3C attracts MGE-derived cortical interneurons in the deepmigratory stream guiding them into the developing neocortex

abstract:
While it is known that Semaphorin 3C acts as a guidance cue for axons during brain development, their potential role during interneuron migration is largely unknown. One striking observation is that Sema3C demarcates the pallial/subpallial border and the intracortical pathway of cortical interneurons in the dorsal telencephalon. Moreover, migrating cortical interneurons express Neuropilin1 and Neuropilin2, described receptors for Semaphorin 3A, 3F and 3C. All these reasons prompt us to examine possible roles for Sema3C on cortical interneuron migration.

Using several in vitro approaches, we showed that Nrp1-expressing MGE-derived interneurons from the deep migratory stream migrate towards the increasing Sema3C gradients. In contrast, inhibitory neurons from the superficial migratory stream that express Nrp2, do not respond to this guidance cue. In the present study, we proposed that diffusible Sema3C expressed in the Pallium provides a permissive corridor that attracts the Nrp1-expressing interneurons from the DMS into the dorsal telencephalon.


title: A novel SARS-CoV-2 related virus with complex recombination isolated from bats in Yunnan province, China

abstract: A novel beta-coronavirus, SARS-CoV-2, emerged in late 2019 and rapidly spread throughout the world, causing the COVID-19 pandemic. However, the origin and direct viral ancestors of SARS-CoV-2 remain elusive. Here, we discovered a new SARS-CoV-2-related virus in Yunnan province, in 2018, provisionally named PrC31, which shares 90.7% and 92.0% nucleotide identities with SARS-CoV-2 and the bat SARSr-CoV ZC45, respectively. Sequence alignment revealed that several genomic regions shared strong identity with SARS-CoV-2, phylogenetic analysis supported that PrC31 shares a common ancestor with SARS-CoV-2. The receptor binding domain of PrC31 showed only 64.2% amino acid identity with SARS-CoV-2. Recombination analysis revealed that PrC31 underwent multiple complex recombination events within the SARS-CoV and SARS-CoV-2 sub-lineages, indicating the evolution of PrC31 from yet-to-be-identified intermediate recombination strains. Combination with previous studies revealed that the beta-CoVs may possess more complicated recombination mechanism. The discovery of PrC31 supports that bats are the natural hosts of SARS-CoV-2.

category: zoology


title: Proprioception-based movement goals support imitation and are disrupted in apraxia

abstract: The ability to imitate observed actions serves as an efficient method for learning novel movements and is specifically impaired (without concomitant gross motor impairments) in the neurological disorder of limb apraxia, a disorder common after left hemisphere stroke. Research with apraxic patients has advanced our understanding of how people imitate. However, the role of proprioception in imitation has been rarely assessed directly. Prior work has proposed that proprioceptively sensed body position is transformed into a visual format, supporting the attainment of a desired imitation goal represented visually (i.e., how the movement should look when performed). In contrast, we hypothesized a more direct role for proprioception: we suggest that movement goals are also represented proprioceptively (i.e., how a desired movement should feel when performed), and the ability to represent or access such proprioceptive goals is deficient in apraxia. Using a novel imitation task in which a robot cued meaningless trajectories proprioceptively or visually, we probed the role of each sensory modality. We found that patients with left hemisphere stroke were disproportionately worse than controls at imitating when cued proprioceptively versus visually. This proprioceptive versus visual disparity was associated with apraxia severity as assessed by a traditional imitation task, but could not be explained by general proprioceptive impairment or speed-accuracy trade-offs. These data suggest that successful imitation depends in part on the ability to represent movement goals in terms of how those movements should feel, and that deficits in this ability contribute to imitation impairments in patients with apraxia.


title: C-Jun N-terminal kinase post-translational regulation of pain-related Acid-Sensing Ion Channels 1b and 3

abstract: Neuronal proton-gated Acid-Sensing Ion Channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathological diseases. Such conditions often involve in parallel the activation of various signaling pathways such as the Mitogen Activated Protein Kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal Kinase (JNK), as a new post-translational positive regulator of ASIC channels in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit and species dependent manner, targeting both rat and human ASIC1b and ASIC3 subunits but only mouse ASIC1b subunit. The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost upon mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNF. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons, and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.


title: Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

abstract:
SARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5 capping of viral mRNAs. The formation of the 5 cap-1 structure is known to require a guanylyltransferase (GTase) as well as 5 triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5 end of viral RNA via a 5 to 5 triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

ImportanceSARS-CoV-2 is a respiratory RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Coronaviruses encode an RNA polymerase which, in combination with other viral proteins, is responsible for synthesising capped viral mRNA. mRNA cap synthesis requires a guanylyltransferase enzyme; here we show that the SARS-CoV-2 guanylyltransferase is located in the viral RNA polymerase, and we identify the protein domain responsible for guanylyltransferase activity. Furthermore we demonstrate that remdesivir triphosphate, the active metabolite of remdesivir, inhibits both the guanylyltransferase and RNA polymerase functions of the SARS-CoV-2 RNA polymerase. These findings improve understanding of the coronavirus mRNA cap synthesis mechanism, in addition to highlighting a new target for the development of therapeutics to treat SARS-CoV-2 infection.


title: The mechanosensitive Ca2+-permeable ion channel PIEZO1 promotes satellite cell function in skeletal muscle regeneration

abstract: Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscle, play an essential role in muscle regeneration. During the regeneration process, cues from the surrounding microenvironment are critical for the proliferation and function of MuSCs. However, the mechanism by which mechanical stimuli from the MuSCs niche is converted into biochemical signals to promote muscle regeneration is yet to be determined. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel that is activated by membrane tension, mediates the spontaneous Ca2+ influx to controls the regenerative function of MuSCs. Our genetically engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs, and the conditional deletion of Piezo1 in MuSCs delays myofiber regeneration after myofiber injury, which is at least in part due to the growth defect in MuSCs via the reduction in RhoA-mediated actomyosin formation. Thus, we provide the first evidence in MuSCs that PIEZO1, a bona fide mechanosensitive ion channel, promotes the proliferative and regenerative function during skeletal muscle regeneration.

category: cell biology


title: Stonewall prevents expression of testis-enriched genes and binds to insulator elements in D. melanogaster

abstract:
Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered that testis-enriched genes, including the differentiation factor bgcn and a large testis-specific cluster on chromosome 2, are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these genes.

Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also find that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries.


title: Genetic variability associated with OAS1 expression in myeloid cells increases the risk of Alzheimer's disease and severe COVID-19 outcomes

abstract: Genome-wide association studies of late-onset Alzheimers disease (AD) have highlighted the importance of variants associated with genes expressed by the innate immune system in determining risk for AD. Recently, we and others have shown that genes associated with variants that confer risk for AD are significantly enriched in transcriptional networks expressed by amyloid-responsive microglia. This allowed us to predict new risk genes for AD, including the interferon-responsive oligoadenylate synthetase 1 (OAS1). However, the function of OAS1 within microglia and its genetic pathway are not known. Using genotyping from 1,313 individuals with sporadic AD and 1,234 control individuals, we confirm that the OAS1 variant, rs1131454, is associated with increased risk for AD and decreased OAS1 expression. Moreover, we note that the same locus was recently associated with critical illness in response to COVID-19, linking variants that are associated with AD and a severe response to COVID-19. By analysing single-cell RNA-sequencing (scRNA-seq) data of isolated microglia from APPNL-G-F knock-in and wild-type C57BL/6J mice, we identify a transcriptional network that is significantly upregulated with age and amyloid deposition, and contains the mouse orthologue Oas1a, providing evidence that Oas1a plays an age-dependent function in the innate immune system. We identify a similar interferon-related transcriptional network containing OAS1 by analysing scRNA-seq data from human microglia isolated from individuals with AD. Finally, using human iPSC-derived microglial cells (h-iPSC-Mg), we see that OAS1 is required to limit the pro-inflammatory response of microglia. When stimulated with interferon-gamma (IFN-{gamma}), we note that cells with lower OAS1 expression show an exaggerated pro-inflammatory response, with increased expression and secretion of TNF-. Collectively, our data support a link between genetic risk for AD and susceptibility to critical illness with COVID-19 centred on OAS1 and interferon signalling, a finding with potential implications for future treatments of both AD and COVID-19, and the development of biomarkers to track disease progression.


title: The Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape

abstract: Considerable number of works have reported alkaline phosphatase (ALP) enabled intracellular targeting by peptide assemblies, but little is known how these substrates of ALP enters cells. Here we show that the nanoscale assemblies of phosphopeptides, as a dynamic continuum, cluster ALP to enable caveolae mediated endocytosis (CME) and eventual endosomal escape. Specifically, fluorescent phosphopeptides, as substrates of tissue nonspecific alkaline phosphatase (TNAP), undergo enzyme catalyzed self-assembly to form nanofibers. As shown by live cell imaging, the nanoparticles of phosphopeptides, being incubated with HEK293 cells overexpressing red fluorescent protein-tagged TNAP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME, further dephosphorylation of the phosphopeptides form the peptide nanofibers for endosomal escape inside cells. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Moreover, decreasing the formation of peptide nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of supramolecular assemblies for cellular uptake, this work not only illustrates an effective design for enzyme responsive supramolecular therapeutics, but also provides mechanism insights for understanding the dynamics of cellular uptakes of proteins or exogenous peptide aggregates at nanoscale.


title: Time-lagged Independent Component Analysis of Random Walks and Protein Dynamics

abstract: Time-lagged independent component analysis (tICA) is a widely used dimension reduction method for the analysis of molecular dynamics (MD) trajectories and has proven particularly useful for the construction of protein dynamics Markov models. It identifies those  slow collective degrees of freedom onto which the projections of a given trajectory show maximal autocorrelation for a given lag time. Here we ask how much information on the actual protein dynamics and, in particular, the free energy landscape that governs these dynamics the tICA-projections of MD-trajectories contain, as opposed to noise due to the inherently stochastic nature of each trajectory. To answer this question, we have analyzed the tICA-projections of high dimensional random walks using a combination of analytical and numerical methods. We find that the projections resemble cosine functions and strongly depend on the lag time, exhibiting strikingly complex behaviour. In particular, and contrary to previous studies of principal component projections, the projections change non-continuously with increasing lag time. The tICA-projections of selected 1 s protein trajectories and those of random walks are strikingly similar, particularly for larger proteins, suggesting that these trajectories contain only little information on the energy landscape that governs the actual protein dynamics. Further the tICA-projections of random walks show clusters very similar to those observed for the protein trajectories, suggesting that clusters in the tICA-projections of protein trajectories do not necessarily reflect local minima in the free energy landscape. We also conclude that, in addition to the previous finding that certain ensemble properties of non-converged protein trajectories resemble those of random walks, this is also true for their time correlations. Due to the higher complexity of the latter, this result also suggests tICA analyses as a more sensitive tool to test MD simulations for proper convergence.


title: PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy data

abstract:
As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microcopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized.

HighlightsO_LIWe present PySTACHIO, a refined version of our spot tracking algorithm
C_LIO_LIWe demonstrate highly improved performance over previous MATLAB versions
C_LIO_LIPySTACHIO can accurately estimate stoichiometries and 2D diffusion coefficients
C_LIO_LIPerformance is comparable to state-of-the-art packages on challenge data
C_LIO_LIPySTACHIO has both GUI and command line interfaces and can be hosted as a web app
C_LI

Graphical abstract

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title: PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy