title: Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose)

abstract: AO_SCPLOWBSTRACTC_SCPLOWADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally-occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as "readers" for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts) - optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems.

category: synthetic biology


title: Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo


title: Cytokinin- microbiome interactions regulate developmental functions

abstract: The interaction of plants with the complex microbial networks that inhabit them is important for plant health. While the reliance of plants on their microbial inhabitants for defense against invading pathogens is well documented, the acquisition of data concerning the relationships between plant developmental stage or aging, and microbiome assembly, is still underway. In this work, we observed developmental-age dependent changes in the phyllopshere microbiome of tomato. The plant hormone cytokinin (CK) regulates various plant growth and developmental processes. Here, we show that age-related shifts in microbiome content vary based on content of, or sensitivity to, CK. We observed a developmental age associated decline in microbial richness and diversity, accompanied by a decline in the presence of growth promoting and resistance inducing bacilli in the phyllosphere. This decline was absent from CK-rich or CK-hypersensitive genotypes. Bacillus isolates we obtained from CK rich genotypes were found to re-program the transcriptome to support morphogenesis and alter the leaf developmental program when applied to seedlings, and enhance yield and agricultural productivity when applied to mature plants. Our results support the notion that CK-dependent effects on microbiome content support developmental functions, suggesting that these are mediated by CK in part via the bacterial community.

category: plant biology


title: The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB

abstract: Vascular endothelial cells (EC) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, the hallmarks of the severe disease. However, the mechanisms by which EC are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin 5{beta}1 signaling. Incubation of human umbilical vein EC with whole spike, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-{kappa}B and enhanced the expression of leukocyte adhesion molecules VCAM1 and ICAM1, the adhesion of peripheral blood leukocytes, and the permeability of the monolayer. Inhibitors of integrin 5{beta}1 activation prevented these effects. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin 5{beta}1 in EC to activate Rho GTPases, eNOS pathways, and the NF-{kappa}B gene expression program responsible for vascular leakage and leukocyte infiltration, respectively. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin 5{beta}1 as a promising target for treating vascular inflammation in COVID-19.

category: cell biology


title: Regional Variant Analysis of Spike Glycoprotein Mutations of SARS-CoV-2 and Its Implications in COVID-19 Pandemic Control

abstract: Mutations in the spike glycoprotein have various impacts on the receptor binding, antibody interaction, and host range of SARS-CoV-2. As the interaction of spike glycoprotein with the human ACE2 receptor is the entry point of SARS-CoV-2 in human cells, mutations in the spike protein itself contain numerous impacts on the pandemic. Here, we analysed all the mutations in the spike glycoprotein from123 strains isolated from Kerala, India. We also predicted the possible structural relevance of the unique mutations based on topological analysis of the residue interaction network of the spike glycoprotein structure.

category: genetics


title: Allosteric perspective on the mutability and druggability of the SARS-CoV-2 Spike protein

abstract: Recent developments in the SARS-CoV-2 pandemic point to its inevitable transformation into an endemic disease, urging both diagnostics of emerging variants of concern (VOCs) and design of the variant-specific drugs in addition to vaccine adjustments. Exploring the structure and dynamics of the SARS-CoV-2 Spike protein, we argue that the high mutability characteristic of RNA viruses coupled with the remarkable flexibility and dynamics of viral proteins result in a substantial involvement of allosteric mechanisms. While allosteric effects of mutations should be considered in predictions and diagnostics of new VOCs, allosteric drugs advantageously avoid escaping mutations via non-competitive inhibition originating from many alternative distal locations. The exhaustive allosteric signalling and probing maps provide a comprehensive picture of allostery in the Spike protein, making it possible to locate sites of potential mutations that could work as new VOCs "drivers", and to determine binding patches that may be targeted by newly developed allosteric drugs.

category: bioinformatics


title: Analysis of B-cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences

abstract: Analyzing B cell receptor (BCR) repertoires is immensely useful in evaluating ones immunological status. Conventionally, repertoire analysis methods have focused on comprehensive assessments of clonal compositions, including V(D)J segment usage, nucleotide insertions/deletions, and amino acid distributions. Here, we introduce a novel computational approach that applies deep-learning-based protein embedding techniques to analyze BCR repertoires. By selecting the most frequently occurring BCR sequences in a given repertoire and computing the sum of the vector representations of these sequences, we represent an entire repertoire as a 100-dimensional vector and eventually as a single data point in vector space. We demonstrate that this new approach enables us to not only accurately cluster BCR repertoires of coronavirus disease 2019 (COVID-19) patients and healthy subjects but also efficiently track minute changes in immune status over time as patients undergo treatment. Furthermore, using the distributed representations, we successfully trained an XGBoost classification model that achieved a mean accuracy rate of over 87% given a repertoire of CDR3 sequences.


title: Computational analysis of B cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences


title: Langerhans cells and cDC1s play redundant roles in mRNA-LNP induced protective anti-influenza and anti-SARS-CoV-2 responses

abstract:
Nucleoside modified mRNA combined with Acuitas Therapeutics lipid nanoparticles (LNP) have been shown to support robust humoral immune responses in many preclinical animal vaccine studies and later in humans with the SARS-CoV-2 vaccination. We recently showed that this platform is highly inflammatory due to the LNPs ionizable lipid component. The inflammatory property is key to support the development of potent humoral immune responses. However, the mechanism by which this platform drives T follicular helper cells (Tfh) and humoral immune responses remains unknown. Here we show that lack of Langerhans cells or cDC1s neither significantly affected the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cells and humoral immune responses, nor susceptibility towards the lethal challenge of influenza and SARS-CoV-2. However, the combined deletion of these two DC subsets led to a significant decrease in the induction of PR8 HA and SARS-CoV-2 RBD-specific Tfh cell and humoral immune responses. Despite these observed defects, the still high antibody titers were sufficient to confer protection towards lethal viral challenges. We further found that IL-6, but not neutrophils, was required to generate Tfh cells and antibody responses.

In summary, here we bring evidence that the mRNA-LNP platform can support protective adaptive immune responses in the absence of specific DC subsets through an IL-6 dependent and neutrophil independent mechanism.

category: immunology


title: SARS-CoV-2 fears green: the chlorophyll catabolite Pheophorbide a is a potent antiviral

abstract: SARS-CoV-2 pandemic is having devastating consequences worldwide. Although vaccination advances at good pace, effectiveness against emerging variants is unpredictable. The virus has displayed a remarkable resistance to treatments and no drugs have been proved fully effective against Covid-19. Thus, despite the international efforts, there is still an urgent need for new potent and safe antivirals against SARS-CoV-2. Here we exploited the enormous potential of plant metabolism using the bryophyte Marchantia polymorpha and identified a potent SARS-CoV-2 antiviral, following a bioactivity-guided fractionation and mass-spectrometry approach. We found that the chlorophyll derivative Pheophorbide a (PheoA), a porphyrin compound similar to animal Protoporphyrin IX, has an extraordinary antiviral activity against SARS-CoV-2 preventing infection of cultured monkey and human cells, without noticeable cytotoxicity. We also show that PheoA prevents coronavirus entry into the cells by directly targeting the viral particle. Besides SARS-CoV-2, PheoA also displayed a broad-spectrum antiviral activity against (+) strand RNA viral pathogens such as HCV, West Nile, and other coronaviruses, but not against (-) strand RNA viruses, such as VSV. Our results indicate that PheoA displays a remarkable potency and a satisfactory therapeutic index, which together with its previous use in photoactivable cancer therapy in humans, suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

category: molecular biology


title: DNA-controlled Spatio-temporal Patterning of Active Matter

abstract: Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton; an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here, we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel comprised of microtubules: a DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties.

category: biochemistry


title: DNA-controlled Spatio-temporal Patterning of a Cytoskeletal Active Gel


title: Model-guided engineering of DNA sequences with predictable site-specific recombination rates

abstract:
Site-specific recombination (SSR) is an important tool in genome editing and gene circuit design. However, its applications are limited by the inability to simply and predictably tune SSR reaction rates across orders of magnitude. Facile rate manipulation can in principle be achieved by modifying the nucleotide sequence of the DNA substrate of the recombinase, but the design principles for rationally doing so have not been elucidated. To enable predictable tuning of SSR reaction kinetics via DNA sequence, we developed an integrated experimental and computational method to parse individual nucleotide contributions to the overall reaction rate, which we used to analyze and engineer the DNA attachment sequence attP for the inversion reaction mediated by the serine recombinase Bxb1. A quantitative PCR method was developed to measure the Bxb1 reaction rate in vitro. Then, attP sequence libraries were designed, selected, and sequenced to inform a machine-learning model, which revealed that the Bxb1 reaction rate can be accurately represented assuming independent contributions of nucleotides at key positions. Next, we used the model to predict the performance of DNA site variants in reaction rate assays both in vitro and in Escherichia coli, with flipping rates ranging from 0.01- to 10-fold that of the wild-type attP sequence. Finally, we demonstrate that attP variants with predictable DNA recombination rates can be used in concert to achieve kinetic control in gene circuit design by coordinating the coexpression of two proteins in both their relative proportion and their total amount. Our high-throughput, data-driven method for rationally tuning SSR reaction rates through DNA sequence modification enhances our understanding of recombinase function and expands the synthetic biology toolbox.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

abstract: Next generation sequencing has revealed the presence of many RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, many of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Recombinant LLOV (rLLOV) displays typical filovirus features, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal against pandemic preparedness.

category: microbiology


title: Unique features of magic spot metabolism in Clostridioides difficile

abstract: The  magic spot alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by CdRSH and CdRelQ. These enzymes are transcriptionally activated by diverse environmental stresses, but both exclusively synthesize pGpp rather than ppGpp as has previously been reported. While direct synthesis of pGpp from a GMP substrate and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5 phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes magic spot signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms.


title: Multi-plasmid clash in a bacterial community: plasmid viability depends on the ecological setting of hosts

abstract: Plasmids are genetic elements that disperse horizontally between different strains and species of bacteria and a major factor in the dissemination of virulence factors and antibiotic resistance. Understanding the ecology of plasmids has a notable anthropocentric value and therefore the interactions between bacterial hosts and individual plasmids have been studied in detail. However, bacterial systems often carry multiple genetically distinct plasmids, but dynamics of these multiplasmid "clashes" has remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids in five different ecological settings. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are trade-offs in plasmid fitness that stem from the ecological situation of their initial hosts. Growth rates and maximum population densities increased in all communities and treatments over the 42-day evolution experiment although plasmid contents at the end varied notably. We show that large multiresistance conferring plasmids are unfit when the community also contains smaller plasmids with fewer resistance genes. This suggests that restraining the use to few antibiotics can make bacterial communities sensitive to others. The hosts also appear to react to the presence of multiple genetically different plasmids by enhancing fimbriae production instead of alleviating costs of individual plasmids. In general, the survivors of the here-studied multi-plasmid clash are significantly affected by the presence or absence of antibiotic selection and plasmid-free hosts of varying fitness. Therefore, these trade-offs in different settings can explain for example why some resistance plasmids have an advantage during a rapid proliferation of antibiotic sensitive pathogen whereas others dominate in alternative situations.


title: Genomic remnants of ancestral hydrogen and methane metabolism in Archaea drive anaerobic carbon cycling

abstract: Methane metabolism is among the hallmarks of Archaea, originating very early in their evolution. Other than its two main complexes, methyl-CoM reductase (Mcr) and tetrahydromethanopterin-CoM methyltransferase (Mtr), there exist other genes called "methanogenesis markers" that are believed to participate in methane metabolism. Many of them are Domains of Unknown Function. Here we show that these markers emerged together with methanogenesis. Even if Mcr is lost, the markers and Mtr can persist resulting in intermediate metabolic states related to the Wood-Ljungdahl pathway. Beyond the markers, the methanogenic ancestor was hydrogenotrophic, employing the anaplerotic hydrogenases Eha and Ehb. The selective pressures acting on Eha, Ehb, and Mtr partially depend on their subunits membrane association. Integrating the evolution of all these components, we propose that the ancestor of all methane metabolizers was an autotrophic H2/CO2 methanogen that could perhaps use methanol but not oxidize alkanes. Hydrogen-dependent methylotrophic methanogenesis has since emerged multiple times independently, both alongside a vertically inherited Mcr or from a patchwork of ancient transfers. Through their methanogenesis genomic remnants, Thorarchaeota and two newly reconstructed order-level lineages in Archaeoglobi and Bathyarchaeota act as metabolically versatile players in carbon cycling of anoxic environments across the globe.


title: Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation

abstract:
One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. To suppress latent HIV-1 in a stable fashion, we depleted the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and induced DNA methylation at specific sites in the 5LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. Thus, epigenetic silencing by itself appears to be insufficient to permanently silence HIV-1 proviral transcription.

Author SummaryThe "block and lock" strategy for a functional HIV-1 cure is based on the premise that permanent inactivation of the HIV-1 can be achieved by epigenetic silencing of the proviral DNA. For cellular genes, long-term silencing is achieved during cell differentiation by the induction of specific epigenetic modifications involving histone and DNA methylation. During HIV-1 silencing, histone methylation and DNA methylation are observed, but both sets of modifications can be reversed upon activation of T-cells through the T-cell receptor or potent latency reversing agents. In an attempt to enhance silencing of HIV-1 transcription, we used an inhibitor of H3K27 demethylases to increase H3K27 methylation. This in turn led to enhanced DNA methylation of HIV-1. Unfortunately, although the treatment effectively silenced HIV-1 and prevented viral reactivation, the silencing effects were short-lived and quickly reversed after removal of the drug.


title: Nutrient limitation magnifies fitness costs of antimalarial drug resistance mutations

abstract: Drug resistance mutations tend to disrupt key physiological processes, and therefore carry a fitness cost. The size of these fitness costs is a central determinant of the rate of spread of these mutations in natural populations so are important to quantify. Head-to-head competition assays provide a standard approach to measuring differential fitness, and have been used extensively for malaria parasites. These assays typically use standardized culture media, containing RPMI 1640, which has a 1.4 to 5.5-fold (mean: 2.6-fold) higher concentration of amino acids than human blood. In this rich media we predict that fitness costs will be underestimated because resource competition is weak. We tested this prediction using an artemisinin sensitive parasite edited to contain kelch-C580Y or R561H mutations conferring resistance to artemisinin or synonymous control mutations. We examined the impact of these single amino acid mutations on fitness, using replicated head-to head competition experiments conducted in media containing (i) normal RPMI, (ii) modified RPMI with reduced amino acid concentration, (iii) RPMI containing only isoleucine, or (iv) 3-fold diluted RPMI. We found a significant 1.3 to 1.4-fold increase in fitness costs measured in modified and isoleucine-only media relative to normal media, while fitness costs were 2.5-fold higher in diluted media. We conclude that fitness costs are strongly affected by media composition and will be significantly underestimated in normal RPMI. Elevated fitness costs in nature will limit spread of ART-resistance but will also promote evolution of compensatory mutations that restore fitness, and can be exploited to maximize selection in laboratory experiments.


title: Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

abstract: TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation, and that is involved in mitochondrial replication, transcription and translation. Two variants predicted to affect TOP1MT function (V1 - R198C and V2 - V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants had been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in knockout cells. Consistent with these TOP1MT variants contributing to the patient phenotype, comprehensive characterization suggests that both variants had impaired activity. Critically, neither variant was able to restore steady state levels of mitochondrial-encoded proteins, nor reduced oxidative phosphorylation when re-expressed in TOP1MT knockout cells. However, the two variants behaved differently in some respects. While the V1 variant was better at restoring transcript levels, the V2 variant was able to restore mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome, and how impairments in this important protein may lead to human pathology.


title: CRISPR/Cas9 screen for functional MYC binding sites reveals MYC-dependent vulnerabilities in K562 cells

abstract: The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate expression of adjacent genes in a cell type- and developmental stage-specific manner. To date, a comprehensive analysis of direct MYC targets with essential roles in different types of cancer is missing. To enable identification of functional MYC binding sites and corresponding target genes, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. As a proof of principle, using this library we identified several known and novel MYC targets critical for K562 chronic myelogenous leukemia cells and uncovered specific features of essential E-boxes. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in any cancer type.


title: Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

abstract: Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.


title: Small molecule photocatalysis enables drug target identification via energy transfer

abstract: The identification of cellular targets that can be exploited for therapeutic benefit, broadly known as target ID, remains a fundamental goal in drug discovery. In recent years, the application of new chemical and biological technologies that accelerate target ID has become commonplace within drug discovery programs, as a complete understanding of how molecules react in a cellular environment can lead to increased binding selectivity, improved safety profiles, and clinical efficacy. Established approaches using photoaffinity labelling (PAL) are often costly and time-consuming due to poor signal-to-noise coupled with extensive probe optimization. Such challenges are exacerbated when dealing with low abundance membrane proteins or multiple protein target engagement, typically rendering target ID unfeasible. Herein, we describe a general platform for photocatalytic small molecule target ID, which hinges upon the generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead from the drug, catalytic signal amplification results in multiple labelling events per drug, leading to unprecedented levels of target enrichment. Through the development of cell permeable photo-catalyst conjugates, this method has enabled the quantitative target and off target identification of several drugs including (+)-JQ1, paclitaxel, and dasatinib. Moreover, this methodology has led to the target ID of two GPCRs - ADORA2A and GPR40 - a class of drug target seldom successfully uncovered in small molecule PAL campaigns.


title: Genome-wide DNA methylation and multi-omics study of human chondrocyte ontogeny and an epigenetic clock analysis of adult chondrocytes

abstract: Articular chondrocytes undergo functional changes and their regenerative potential declines with age. Although the molecular mechanisms guiding articular cartilage aging is poorly understood, DNA methylation is known to play a mechanistic role in aging. However, our understanding of DNA methylation in chondrocyte development across human ontogeny is limited. To better understand DNA methylome changes, methylation profiling was performed in human chondrocytes. This study reveals association between methylation of specific CpG sites and chondrocyte age. We also determined the putative binding targets of STAT3, a key age-patterned transcription factor in fetal chondrocytes and genetic ablation of STAT3 induced a global genomic hypermethylation. Moreover, an epigenetic clock built for adult human chondrocytes revealed that exposure of aged adult human chondrocytes to STAT3 agonist, decreased epigenetic age. Taken together, this work will serve as a foundation to understand development and aging of chondrocytes with a new perspective for development of rejuvenation agents for synovial joints.


title: Transcriptome Profiles of Anopheles gambiae Harboring Natural Low-Level Plasmodium Infection Reveal Adaptive Advantages for the Mosquito

abstract: Anopheline mosquitoes are the sole vectors for the Plasmodium pathogens responsible for malaria, which is among the oldest and most devastating of human diseases. The continuing global impact of malaria reflects the evolutionary success of a complex vector-pathogen relationship that accordingly has been the long-term focus of both debate and study. An open question in the biology of malaria transmission is the impact of naturally occurring low-level Plasmodium infections of the vector on the mosquitos health and longevity as well as critical behaviors such as host- preference/seeking. To begin to answer this, we have completed a comparative RNAseq-based transcriptome profile study examining the effect of biologically salient, salivary gland transmission- stage Plasmodium infection on the molecular physiology of Anopheles gambiae s.s. head, sensory appendage, and salivary glands. When compared with their uninfected counterparts, Plasmodium infected mosquitoes exhibit increased transcript abundance of genes associated with olfactory acuity as well as a range of synergistic processes that align with increased fitness based on both anti-aging and reproductive advantages. Taken together, these data argue against the long-held paradigm that malaria infection is pathogenic for anophelines and, instead, suggests there are biological and evolutionary advantages for the mosquito that drive the preservation of its high vectorial capacity.


title: Dissecting the import and export pathways of the human RNA helicase UPF1

abstract: The RNA helicase UPF1 is best known for its key role in mRNA surveillance but has been implicated in additional cellular processes both in the nucleus and in the cytoplasm. In human cells, the vast majority of UPF1 resides in the cytoplasm and only small amounts can be detected in the nucleus at steady state. It was previously shown that its export from the nucleus to the cytoplasm is Crm1-dependent, yet neither the nuclear export signal (NES) nor the nuclear localization signal (NLS) has been identified. Here, we provide evidence for a noncanonical NLS in UPF1, map the NES to amino acids 89-105 and show that L103 and F105 are essential for UPF1s export to the cytoplasm. Examination of additional UPF1 mutants revealed that a functional helicase domain but not the association with RNA is crucial for the shuttling capacity of UPF1.


title: Concentration of intraflagellar transport proteins at the ciliary base is required for proper train injection

abstract: Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.


title: A distinct inner nuclear membrane proteome in Saccharomyces cerevisiae gametes

abstract: The inner nuclear membrane (INM) proteome regulates gene expression, chromatin organization, and nuclear transport, however, it is poorly understood how changes in INM protein composition contribute to developmentally regulated processes, such as gametogenesis. Using a split-GFP complementation system, we compared the distribution of all C-terminally tagged transmembrane proteins in Saccharomyces cerevisiae in gametes to that of mitotic cells. Gametes contain a distinct INM proteome needed to complete gamete formation, including expression of genes linked to cell wall biosynthesis, lipid biosynthetic and metabolic pathways, protein degradation and unknown functions. Based on the inheritance pattern, INM components are made de novo in the gametes. Whereas mitotic cells show a strong preference for proteins with small extraluminal domains, gametes do not exhibit this size preference likely due to the changes in the nuclear permeability barrier during gametogenesis.


title: Iron-loaded deferiprone can support full hemoglobinization of cultured red blood cells in the absence of transferrin

abstract:
Iron is an essential nutrient in mammalian cell cultures, conventionally supplemented as iron-loaded transferrin (holotransferrin). The high cost of human transferrin represents a challenge for the large scale production of cell therapies, such as cultured red blood cells. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3{middle dot}Fe3+) at a concentration of 52mol/L could fully replace holotransferrin during erythroblast differentiation into reticulocytes, the erythroid differentiation stage with maximal iron requirements. Reticulocytes cultured in presence of Def3{middle dot}Fe3+ or holotransferrin (1000g/mL) were similar with respect to expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Def3{middle dot}Fe3+ also supported expansion of the erythroid compartment in vitro, except for the first stage when hematopoietic stem cells committed to erythroblasts, in which a reduced erythroblasts yield was observed. This suggests that erythroblasts acquired the potential to process Def3{middle dot}Fe3+ as iron source for biosynthesis pathways. Replacement of holotransferrin by Def3{middle dot}Fe3+ was also successful in cultures of six myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). These results suggest that iron-loaded deferiprone can partially replace holotransferrin in chemically defined medium formulations for the production of cultured reticulocytes and proliferation of selected myeloid cell lines. This would lead to a significant decrease in medium cost that would improve the economic perspectives of the large scale production of red blood cells for transfusion purposes.

Key pointsO_LIHolotransferrin limitations in erythroid cultures lead to lower erythroblast yields, impaired maturation and low enucleation efficiencies.
C_LIO_LIIron-loaded deferiprone can replace holotransferrin in erythroblast expansion and differentiation cultures.
C_LI


title: High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria

abstract:
BackgroundThe kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropicas digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss.

Methodology/Principal FindingsThe genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units.

Conclusion/SignificanceThese large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.

Author SummaryCutaneous leishmaniasis is mainly caused by Leishmania tropica in the Middle East, where it is known for treatment failure and a need for prolonged and/or multiple treatments. Several factors affect the clinical presentation and treatment outcome, such as host genetic variability and specific immune response, as well as environmental factors and the vector species. Little is known about the parasite genome and its influence on treatment response. By analysing the genome of 22 isolates of L. tropica, we have revealed extensive genomic variation and a complex population structure with evidence of genetic exchange within and among the isolates, indicating a possible presence of sexual or parasexual mechanisms. Understanding the Leishmania genome better may improve future treatment and better understanding of treatment failure and relapse.


title: DropletQC: improved identification of empty droplets and damaged cells in single-cell RNA-seq data

abstract: Advances in droplet-based single cell RNA-sequencing (scRNA-seq) have dramatically increased throughput, allowing tens of thousands of cells to be routinely sequenced in a single experiment. In addition to cells, droplets capture cell-free "ambient" RNA predominately caused by lysis of cells during sample preparation. Samples with high ambient RNA concentration can create challenges in accurately distinguishing cell-containing droplets and droplets containing ambient RNA. Current methods to separate these groups often retain a significant number of droplets that do not contain cells - so called empty droplets. Additional to the challenge of identifying empty drops, there are currently no methods available to detect droplets containing damaged cells, which comprise of partially lysed cells - the original source of the ambient RNA. Here we describe DropletQC, a new method that is able to detect empty droplets, damaged, and intact cells, and accurately distinguish from one another. This approach is based on a novel quality control metric, the nuclear fraction, which quantifies for each droplet the fraction of RNA originating from unspliced, nuclear pre-mRNA. We demonstrate how DropletQC provides a powerful extension to existing computational methods for identifying empty droplets such as EmptyDrops. We have implemented DropletQC as an R package, which can be easily integrated into existing single cell analysis workflows.

category: genomics


title: Microbial community of recently discovered Auka vent field sheds light on vent biogeography and evolutionary history of thermophily

abstract: Hydrothermal vents have been key to our understanding of the limits of life, and the metabolic and phylogenetic diversity of thermophilic organisms. Here we used environmental metagenomics combined with analysis of physico-chemical data and 16S rRNA amplicons to characterize the diversity, temperature optima, and biogeographic distribution of sediment-hosted microorganisms at the recently discovered Auka vents in the Gulf of California, the deepest known hydrothermal vent field in the Pacific Ocean. We recovered 325 metagenome assembled genomes (MAGs) representing 54 phyla, over 1/3 of the currently known phylum diversity, showing the microbial community in Auka hydrothermal sediments is highly diverse. Large scale 16S rRNA amplicon screening of 227 sediment samples across the vent field indicates that the MAGs are largely representative of the microbial community. Metabolic reconstruction of a vent-specific, deeply branching clade within the Desulfobacterota (Tharpobacteria) suggests these organisms metabolize sulfur using novel octaheme cytochrome-c proteins related to hydroxylamine oxidoreductase. Community-wide comparison of the average nucleotide identity of the Auka MAGs with MAGs from the Guaymas Basin vent field, found 400 km to the Northwest, revealed a remarkable 20% species-level overlap between vent sites, suggestive of long-distance species transfer and sediment colonization. An adapted version of a recently developed model for predicting optimal growth temperature to the Auka and Guaymas MAGs indicates several of these uncultured microorganisms could grow at temperatures exceeding the currently known upper limit of life. Extending this analysis to reference data shows that thermophily is a trait that has evolved frequently among Bacteria and Archaea. Combined, our results show that Auka vent field offers new perspectives on our understanding of hydrothermal vent microbiology.


title: Colon epithelial cell-specific Bmal1 deletion impairs bone formation in mice

abstract: The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis. Disruption in central circadian rhythms has been associated with a decline in bone mineral density and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to trabecular and cortical bone loss in male mice.

category: physiology


title: Deploying photons for communication within neuronal networks

abstract: Deficiencies in neurotransmission lead to neurological disorders or misinterpretation of perceived threats. To restore defects in cellular communication, we developed a synthetic, photon-assisted synaptic transmission (PhAST) system. PhAST is based on luciferases and channelrhodopsins that enable the transmission of a neuronal state across space, using photons as neurotransmitters. We demonstrate the ability to overcome synaptic barriers and rescue the behavioral deficit of a genetically engineered glutamate mutant with conditional, Ca2+-triggered photon emission between two cognate neurons of the Caenorhabditis elegans nociceptive avoidance circuit. We also deploy these ingredients for asynaptic transmission between two unrelated cells in a sexually dimorphic neuronal network. Functional PhAST could sensitize otherwise poorly responsive males to touch and hence expand the behavioral repertoire. Our study, thus, establishes a powerful framework for complex photon-based communication between neurons in a living animal, that can readily be expanded to synthetic neuronal networks, organoids or non-invasive brain-machine interfaces.


title: Transcriptomic analysis of pathways associated with alpha(v) integrin-related non-canonical autophagy in human B cells

abstract: Autophagy proteins have been linked with development of immune-mediated diseases including lupus, but the mechanisms for this are unclear. We have previously shown that non-canonical autophagy induced by v-integrins regulates B cell activation by viral and self-antigens in mice. Here we investigated the involvement of this pathway in B cells from human tissue. Our data revealed that autophagy is specifically induced in germinal-center and memory B cell sub-populations from human tonsil and spleen. Transcriptomic analysis showed that induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of non-canonical autophagy in B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells we found that v-integrin-related non-canonical autophagy limits activation of specific pathways while promoting others. These data provide new mechanistic links for autophagy and immune dysregulation in diseases such as lupus.


title: The CHK-2 antagonizing phosphatase PPM-1.D regulates meiotic entry via catalytic and non-catalytic activities

abstract: The transition from the stem cell/progenitor fate to meiosis is mediated by several redundant post-transcriptional regulatory pathways in C. elegans. Interfering with all three branches causes tumorous germlines. SCFPROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in timely initiation of events of meiotic prophase I, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation e.g., DNA double strand breaks, chromosome pairing and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 by both catalytic and non-catalytic activities, where strikingly the non-catalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery and programmed SCFPROM-1 mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.


title: Lipid droplets regulate actin remodeling via prostaglandin signaling during Drosophila oogenesis

abstract: A key factor controlling oocyte quality and fertility is lipids. Even though lipid droplets (LDs) are crucial regulators of lipid metabolism, their roles in fertility are poorly understood. During Drosophila oogenesis, LD accumulation in nurse cells coincides with dynamic actin remodeling necessary for late-stage follicle morphogenesis and fertility. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when Pxt, the enzyme responsible for prostaglandin (PG) synthesis, is missing. Dominant genetic interactions and PG treatment of follicles in vitro reveal that ATGL and Pxt act in the same pathway to regulate actin remodeling, with ATGL upstream of Pxt. Further, lipidomic analysis detects arachidonic acid (AA) containing triglycerides in ovaries. Because AA is the substrate for Pxt, we propose that ATGL releases AA from LDs to drive PG synthesis necessary for follicle development. We also find that exogenous AA is toxic to follicles in vitro, and LDs modulate this toxicity. This leads to the model that LDs both sequester AA to limit toxicity, and release AA via ATGL to drive PG production. We speculate that the same pathways are conserved across organisms to regulate oocyte development and promote fertility.


title: Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets


title: Multi-Task Brain Network Reconfiguration is Inversely Associated with Human Intelligence

abstract: Intelligence describes the general cognitive ability level of a person. It is one of the most fundamental concepts in psychological science and is crucial for effective adaption of behavior to varying environmental demands. Changing external task demands have been shown to induce reconfiguration of functional brain networks. However, whether neural reconfiguration between different tasks is associated with intelligence has not yet been investigated. We used fMRI data from 812 subjects to show that higher scores of general intelligence are related to less brain network reconfiguration between resting state and seven different task states as well as to network reconfiguration between tasks. This association holds for all functional brain networks except the motor system, and replicates in two independent samples (N = 138, N = 184). Our findings suggest that the intrinsic network architecture of individuals with higher intelligence scores is closer to the network architecture as required by various cognitive demands. Multi-task brain network reconfiguration may, therefore, represent a neural reflection of the behavioral positive manifold - the essence of the concept of general intelligence. Finally, our results support neural efficiency theories of cognitive ability and reveal insights into human intelligence as an emergent property from a distributed multi-task brain network.

category: neuroscience


title: Genome-wide RNA structure changes during human neurogenesis drive gene regulatory networks

abstract: The distribution, dynamics and function of RNA structures in human development is under- explored. Here, we systematically assayed RNA structural dynamics and its relationship with gene expression, translation and decay during human neurogenesis. We observed that the human ESC transcriptome is globally more structurally accessible than that of differentiated cells; and undergo extensive RNA structure changes, particularly in the 3UTR. Additionally, RNA structure changes during differentiation is associated with translation and decay. We also identified stage-specific regulation as RBP and miRNA binding, as well as splicing is associated with structure changes during early and late differentiation, respectively. Further, RBPs serve as a major factor in structure remodelling and co-regulates additional RBPs and miRNAs through structure. We demonstrated an example of this by showing that PUM2-induced structure changes on LIN28A enable miR-30 binding. This study deepens our understanding of the wide-spread and complex role of RNA-based gene regulation during human development.


title: Rapid Directed Molecular Evolution of Fluorescent Proteins in Mammalian Cells

abstract: In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2{middle dot}107 independent random genes of fluorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of [~]8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.


title: Completion of neural crest cell production and emigration is regulated by retinoic acid-dependent inhibition of BMP signaling

abstract: Production and emigration of neural crest cells is a transient process followed by the emergence of the definitive roof plate. The mechanisms regulating the end of neural crest ontogeny are poorly understood. Whereas early crest development is stimulated by mesoderm-derived retinoic acid, we report that the end of the neural crest period is regulated by retinoic acid synthesized in the dorsal neural tube. Inhibition of retinoic acid signaling in the neural tube prevents the normal upregulation of BMP inhibitors in the nascent roof plate and prolongs the period of BMP responsiveness which otherwise ceases close to roof plate establishment. Consequently, neural crest production and emigration are extended well into the roof plate stage. In turn, extending the activity of neural crest-specific genes inhibits the onset of retinoic acid synthesis in roof plate suggesting a mutual repressive interaction between neural crest and roof plate traits. Although several roof plate-specific genes are normally expressed in the absence of retinoic acid signaling, roof plate and crest markers are co-expressed in single cells and this domain also contains dorsal interneurons. Hence, the cellular and molecular architecture of the roof plate is compromised. Collectively, our results demonstrate that neural tube-derived retinoic acid, via inhibition of BMP signaling, is an essential factor responsible for the end of neural crest generation and the proper segregation of dorsal neural lineages.

category: developmental biology


title: Macaques preferentially attend to intermediately surprising information

abstract: Normative learning theories dictate that we should preferentially attend to informative sources, but only up to the point that our limited learning systems can process their content. Humans, including infants, show this predicted strategic deployment of attention. Here we demonstrate that rhesus monkeys, much like humans, attend to events of moderate surprisingness over both more and less surprising events. They do this in the absence of any specific goal or contingent reward, indicating that the behavioral pattern is spontaneous. We suggest this U-shaped attentional preference represents an evolutionarily preserved strategy for guiding intelligent organisms toward material that is maximally useful for learning.

category: animal behavior and cognition


title: The ER transmembrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

abstract: Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): The guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, that are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2-Get1 and Emc6-Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6-Emc3 fusion was able to complement an{Delta} oxa1 mutant and restored its respiratory competence. The Emc6-Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2 as well as of nuclear encoded inner membrane proteins though was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.


title: OPT3 plays a central role in both copper and iron homeostasis and signaling due to its ability to deliver copper as well as iron to the phloem in Arabidopsis

abstract: Copper and iron are micronutrients but are toxic when they accumulate in cells in excess. Crosstalk between copper and iron homeostasis in Arabidopsis thaliana has been documented and includes iron accumulation under copper deficiency and vice versa. However, molecular components of this crosstalk are not well understood. Iron concentration in the phloem has been suggested to act systemically, negatively regulating iron uptake to the root. Consistently, systemic iron signaling is disrupted in A. thaliana mutants lacking the phloem companion cell-localized iron transporter, AtOPT3, and opt3 mutants hyperaccumulate iron. Here, we report that in addition to iron, AtOPT3 transports copper and mediates copper loading to the phloem for delivery from sources to sinks. As a result of this function, the opt3-3 mutant accumulates less copper in the phloem, roots, developing leaves and embryos compared to wild type, is sensitive to copper deficiency, and mounts transcriptional copper deficiency response. Because copper deficiency has been shown to stimulate iron accumulation, we propose that reduced copper concentration in the phloem of the opt3-3 mutant and its constitutive copper deficiency contribute to iron overaccumulation in its tissues. Our data assign new transport capabilities to AtOPT3 and increase understanding of copper - iron interactions and signaling.


title: Eelbrain: A Python toolkit for time-continuous analysis with temporal response functions

abstract: Even though human experience unfolds continuously in time, it is not strictly linear; instead, it entails cascading processes building hierarchical cognitive structures. For instance, during speech perception, humans transform a continuously varying acoustic signal into phonemes, words, and meaning, and these levels all have different, interdependent temporal structures. Deconvolution analysis has recently emerged as a promising tool for disentangling electrophysiological brain responses related to such complex models of perception. Here we introduce the Eelbrain Python toolkit, which makes this kind of analysis easy and accessible. We demonstrate its use, using continuous speech as a sample paradigm, with a freely available EEG dataset of audiobook listening. A companion GitHub repository provides the complete source code for the analysis, from raw data to group level statistics. More generally, we advocate a hypothesis-driven approach in which the experimenter specifies a hierarchy of time-continuous representations that are hypothesized to have contributed to brain responses, and uses those as predictor variables for the electrophysiological signal. This is analogous to a multiple regression problem, but with the addition of the time dimension. The deconvolution analysis decomposes the brain signal into distinct responses associated with the different predictor variables by estimating a multivariate temporal response function (mTRF), quantifying the influence of each predictor on brain responses as a function of time(-lags). This allows asking two questions about the predictor variables: 1) Is there a significant neural representation corresponding to this predictor variable? And if so, 2) what are the temporal characteristics of the neural response associated with it? Thus, different predictor variables can be systematically combined and evaluated to jointly model neural processing at multiple hierarchical levels. We discuss applications of this approach, including the potential for linking algorithmic/representational theories at different cognitive levels to brain responses through appropriate linking models.


title: Photorealistic reconstruction of visual texture from EEG signals

abstract: Recent advances in brain decoding have made it possible to classify image categories based on neural activity. Increasing numbers of studies have further attempted to reconstruct the image itself. However, because images of objects and scenes inherently involve spatial layout information, the reconstruction usually requires retinotopically organized neural data with high spatial resolution, such as fMRI signals. In contrast, spatial layout does not matter in the perception of  texture, which is known to be represented as spatially global image statistics in the visual cortex. This property of  texture enables us to reconstruct the perceived image from EEG signals, which have a low spatial resolution. Here, we propose an MVAE-based approach for reconstructing texture images from visual evoked potentials measured from observers viewing natural textures such as the textures of various surfaces and object ensembles. This approach allowed us to reconstruct images that perceptually resemble the original textures with a photographic appearance. A subsequent analysis of the dynamic development of the internal texture representation in the VGG network showed that the reproductivity of texture rapidly improves at 200 ms latency in the lower layers but improves more gradually in the higher layers. The present approach can be used as a method for decoding the highly detailed  impression of sensory stimuli from brain activity.


title: The Arabidopsis m6A-binding proteins ECT2 and ECT3 bind largely overlapping mRNA target sets and influence target mRNA abundance, not alternative polyadenylation

abstract: Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.


title: The Mus musculus papillomavirus type 1 E7 protein binds to the retinoblastoma tumor suppressor - implications for viral pathogenesis

abstract:
The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts specifically with RB1. We show that MmuPV1 E7 interacts through its C-terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 virus expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting non-canonical activities of RB1, which are independent of RB1s ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis.

ImportancePapillomavirus infections cause a variety of epithelial hyperplastic lesions, warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species-specific and do not infect heterologous hosts. Here we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.


title: Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry

abstract: Circulating large "preplatelets" undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry (ISFC) and super-resolution microscopy. Immature platelets, preplatelets and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia (ITP) or undergoing chemotherapy. Preplatelets and barbells were 1.9%+0.18/1.7%+0.48 (n=6) and 3.3%+1.6/0.5%+0.27 (n=12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of HLA-I with high thiazole orange and mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence upon the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with ITP had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent post-chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.


title: GSA: An Independent Development Algorithm for Calling Copy Number and Detecting Homologous Recombination Deficiency (HRD) from Target Capture Sequencing

abstract:
BackgroundThe gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair.

ResultsIn this study, a new algorithm called Genomic Scar Analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples.

ConclusionsThis new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.


title: WGA-LP: a pipeline for Whole Genome Assembly of contaminated reads

abstract:
AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSSummaryC_ST_ABSWhole Genome Assembly (WGA) of bacterial genomes with short reads is a quite common task as DNA sequencing has become cheaper with the advances of its technology. The process of assembling a genome has no absolute golden standard (Del Angel et al. (2018)) and it requires to perform a sequence of steps each of which can involve combinations of many different tools. However, the quality of the final assembly is always strongly related to the quality of the input data. With this in mind we built WGA-LP, a package that connects state-of-art programs and novel scripts to check and improve the quality of both samples and resulting assemblies.

WGA-LP, with its conservative decontamination approach, has shown to be capable of creating high quality assemblies even in the case of contaminated reads.

Availability and ImplementationWGA-LP is available on GitHub (https://github.com/redsnic/WGA-LP) and Docker Hub (https://hub.docker.com/r/redsnic/wgalp). The web app for node visualization is hosted by shinyapps.io (https://redsnic.shinyapps.io/ContigCoverageVisualizer/).

ContactNicolo Rossi, olocin.issor@gmail.com

Supplementary informationSupplementary data are available at bioRxiv online.


title: A mechanosensing mechanism mediated by IRSp53 controls plasma membrane shape homeostasis at the nanoscale

abstract:
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by the I-BAR protein IRSp53, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.

TeaserCell stretch cycles generate PM evaginations of {approx}100 nm which are sensed by IRSp53, triggering a local event of actin polymerization that flattens and recovers PM shape.

category: biophysics


title: Cancer-associated fibroblasts exert a pro-angiogenic activity in Merkel cell carcinoma

abstract: The tumor microenvironment (TME) is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although TME is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. Here, we provide the first molecular and functional characterization of cancer- associated fibroblasts (CAFs), the major TME component, in MCC patient-derived xenografts. We show that subcutaneous co-injection of patient-derived CAFs and human MCC MKL-1 cells into SCID mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We also provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the APA/Ang II-III/AT1R axis, with the expression of aminopeptidase A (APA) in CAFs being the upstream triggering event. Altogether, our findings point to APA as a potential marker for MCC prognostic stratification and a novel candidate therapeutic target.

category: cancer biology


title: Arabidopsis Mediator subunit 17 connects transcription with DNA repair after UV-B exposure

abstract:
Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the Nucleotide Excision DNA Repair system. We here analyzed the role of MED17 in Arabidopsis plants exposed to UV-B radiation, which role has not been previously described. Comparison of med17 mutant transcriptome to that of WT plants showed that almost one third of transcripts with altered expression in med17 plants are also changed by UV-B exposure in WT plants. To validate the role of MED17 in UV-B irradiated plants, plant responses to UV-B were analyzed, including flowering time, DNA damage accumulation and programmed cell death in the meristematic cells of the root tips. Our results show that med17 and OE MED17 plants have altered responses to UV-B; and that MED17 participates in various aspects of the DNA damage response (DDR). Increased sensitivity to DDR after UV-B in med17 plants can be due to altered regulation of UV-B responsive transcripts; but additionally MED17 physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we here also show that MED17 is necessary to regulate the DDR activated by ATR, and that PDCD5 overexpression reverts the deficiencies in DDR shown in med17 mutants. Together, the data presented demonstrates that MED17 is an important regulator of the DDR after UV-B radiation in Arabidopsis plants.

One sentence summaryIn Arabidopsis, MED17 regulates the DNA damage response after UV-B exposure transcriptionally modulating the expression of genes and possibly also physically interacting with DNA repair proteins.


title: PEERS - an open science "Platform for the Exchange of Experimental Research Standards" in biomedicine

abstract:
Laboratory workflows and preclinical models have become increasingly diverse and complex. Confronted with the dilemma of assessing a multitude of information with ambiguous relevance for their specific experiments, scientists run the risk of overlooking critical factors that can influence the planning, conduct and results of studies and that should have been considered a priori. Negligence of such crucial information may result in sub-optimal study design and study execution, bringing into question the validity of generated outcomes. As a corollary, a lot of resources are wasted on biomedical research that turns out to be irreproducible and not sufficiently robust for further project development.

To address this problem, we present  PEERS (Platform for the Exchange of Experimental Research Standards), an open-access online platform that is built to aid scientists in determining which experimental factors and variables are most likely to affect the outcome of a specific test, model or assay and therefore ought to be considered during the design, execution and reporting stages.

The PEERS database is categorized into in vivo and in vitro experiments and provides lists of factors derived from scientific literature that have been deemed critical for experimentation. Most importantly, the platform is based on a structured and transparent system for rating the strength of evidence related to each identified factor and its relevance for a specific method/model. In this context, the rating procedure will not solely be limited to the PEERS working group but will also allow for a community-based grading of evidence.

To generate a proof-of-concept that the PEERS approach is feasible, we focused on a set of in vitro and in vivo methods from the neuroscience field, which are presented in this article. On the basis of the Open Field paradigm in rodents, we describe the selection of factors specific to each experimental setup and the rating system, but also discuss the identification of additional general items that transcend categories and individual tests. Moreover, we present a working format of the PEERS prototype with its structured information framework for embedding data and critical back end/front end user functionalities. Here, PEERS not only offers users the possibility to search for information to facilitate experimental rigor, but also draws on the engagement of the scientific community to actively expand the information contained within the platform through a standardized approach to data curation and knowledge engineering.

As the database grows and benefits become more apparent, we will expand the scope of PEERS to any area of applied biomedical research.

Collectively, by helping scientists to search for specific factors relevant to their experiments, and to share experimental knowledge in a standardized manner, PEERS will serve as the ultimate exchange and analysis tool to enhance data validity and robustness as well as the reproducibility of preclinical research. PEERS offers a vetted, independent tool by which to judge the quality of information available on a certain test or model, identifies knowledge gaps and provides guidance on the key methodological considerations that should be prioritized to ensure that preclinical research is conducted to the highest standards and best practice.

category: scientific communication and education


title: Membrane channel hypothesis of lysosomal permeabilization by beta-amyloid

abstract:
Alzheimers disease (AD) is the most common cause of dementia affecting millions of people. Neuronal death in AD is initiated by oligomeric amyloid-{beta} (A{beta}) peptides. Recently, we proposed the amyloid degradation toxicity hypothesis, which explains multiple major observations associated with AD - such as autophagy failure and a decreased metabolism. According to the hypothesis, the key event in the cellular toxicity of amyloid is the formation of non-selective membrane channels in lysosomal membranes by amyloid fragments that are produced by the digestion of A{beta} previously absorbed by endocytosis. Electrophysiological data suggest that amyloid-formed channels have different sizes, which can be explained by the fact that barrel-shaped amyloid aggregates which create channels can consist of different number of monomers.

To estimate the ability of channels to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in non-conductive membranes that pass spheres with a density of average globular proteins. As a basis, we used the conductance distribution taken from the previously published experimental dataset, in which single channels with a conductance reaching one nanosiemens were registered. Our calculations show that channels with a giant conductance can allow for passing macromolecules such as lysosomal cathepsins implicated in the activation of apoptosis. The formation of giant channels is disproportionally promoted in an acidic environment. Also, amyloid fragments leaking from permeabilized lysosomes can reach the internal leaflet of the plasma membrane and permeabilize it.

We conclude that while dissipation of the proton gradient by any - even the smallest amyloid channel - readily explains lysosomal failure, the relatively rare events of lysosomal permeabilization to large macromolecules can be an alternative mechanism of cellular death induced by exposure to A{beta}.


title: Investigating the Contribution of White Matter Hyperintensities and Cortical Thickness to Empathy in Neurodegenerative and Cerebrovascular Diseases

abstract:
Introduction: Change in empathy is an increasingly recognised symptom of neurodegenerative diseases and contributes to caregiver burden and patient distress. Empathy impairment has been associated with brain atrophy but its relationship to white matter hyperintensities (WMH) is unknown. We aimed to investigate the relationships amongst WMH, brain atrophy, and empathy deficits in neurodegenerative and cerebrovascular diseases.

Methods: 513 participants with Alzheimers Disease/Mild Cognitive Impairment, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia (FTD), Parkinsons Disease, or Cerebrovascular Disease (CVD) were included. Empathy was assessed using the Interpersonal Reactivity Index. WMH were measured using a semi-automatic segmentation and FreeSurfer was used to measure cortical thickness.

Results: A heterogeneous pattern of cortical thinning was found between groups, with FTD showing thinning in frontotemporal regions and CVD in left superior parietal, left insula, and left postcentral. Results from both univariate and multivariate analyses revealed that several variables were associated with empathy, particularly cortical thickness in the fronto-insulo-temporal and cingulate regions, sex(female), global cognition, and right parietal and occipital WMH.

Conclusions: Our results suggest that cortical atrophy and WMH may be associated with empathy deficits in neurodegenerative and cerebrovascular diseases. Future work should consider investigating the longitudinal effects of WMH and atrophy on empathy deficits in neurodegenerative and cerebrovascular diseases.


title: Ruminant-associated Listeria monocytogenes isolates belong preferentially to dairy-related hypervirulent clones: a longitudinal study in 19 farms

abstract:
The increasing prevalence of Listeria monocytogenes infections is a public health issue. Although studies have shown that ruminants constitute reservoirs of this foodborne pathogen, little is known about its epidemiology and genetic diversity within ruminant farms. Here we conducted a large-scale genomic and epidemiologic longitudinal study of Listeria spp. in dairy ruminants and their environments, comprising 19 farms monitored for three consecutive seasons (N=3251 samples). L. innocua was the most prevalent Listeria spp, followed by L. monocytogenes. L. monocytogenes was detected in 52.6% of farms (prevalence in feces samples 3.8%, in farm environment samples 2.5%) and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was prevalent in environmental samples. 61 different L. monocytogenes CTs (cgMLST sequence types) were found, 17 of them (27.9%) common to different animals and/or surfaces within the same farms. L. monocytogenes prevalence was not affected by farm hygiene but by season: the overall prevalence of L. monocytogenes in cattle farms was higher during winter, and in sheep farms was higher during winter and spring. Cows in their second lactation had a higher probability of L. monocytogenes fecal shedding than other lactating cows. This study highlights that dairy farms constitute a reservoir for hypervirulent L. monocytogenes and the importance of continuous animal surveillance to reduce the burden of human listeriosis.

IMPORTANCEListeria monocytogenes is a bacterial pathogen responsible for listeriosis, the foodborne disease with the highest hospitalization and case-fatality rate. Despite increasing evidence that dairy products and ruminant farms are important reservoirs of L. monocytogenes, little is known about the epidemiology and genetic diversity of Listeria spp. within dairy ruminant farms. We report the largest Listeria spp. longitudinal study in individual domestic animals, and the first using whole-genome sequencing for a deep isolate characterization. Here, we show that domestic ruminants can be asymptomatic carriers of pathogenic Listeria, that L. monocytogenes fecal shedding is often intermittent, and that hypervirulent L. monocytogenes clones are overrepresented in dairy farms. Moreover, we uncover the effect of seasons and lactation number on the prevalence of L. monocytogenes in ruminants. Our study highlights the need for Listeria spp. monitoring in farm animals to control the spread of hypervirulent L. monocytogenes and reduce the burden of human listeriosis.


title: Novel compound inhibitors of HIV-1NL4-3 Vpu

abstract: HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we developed a cell-based  gain of function assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. It was conceivable that the compounds inhibited the formation of infectious virions by targeting host cell proteins instead of Vpu directly, so we developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu.


title: Microbiome assembly predictably shapes diversity across a range of disturbance frequencies

abstract: Diversity is frequently linked to the functional stability of ecological communities. However, its association with assembly mechanisms remains largely unknown, particularly under fluctuating disturbances. Here, we subjected complex bacterial communities in bioreactor microcosms to different frequencies of organic loading shocks, tracking temporal dynamics in their assembly, structure and function. Null modelling revealed a stronger role of stochasticity at intermediate disturbance frequencies, preceding the formation of a peak in -diversity. Communities at extreme ends of the disturbance range had the lowest -diversity and highest within-treatment similarity in terms of {beta}-diversity, with stronger deterministic assembly. Stochasticity prevailed during the initial successional stages, coinciding with better specialized function (nitrogen removal). In contrast, general functions (carbon removal and microbial aggregate settleability) benefited from stronger deterministic processes. We showed that changes in assembly processes predictably precede changes in diversity under a gradient of disturbance frequencies, advancing our understanding of the mechanisms behind disturbance-diversity-function relationships.

category: ecology


title: NewWave: a scalable R/Bioconductor package for the dimensionality reduction and batch effect removal of single-cell RNA-seq data

abstract:
SummaryWe present NewWave, a scalable R/Bioconductor package for the dimensionality reduction and batch effect removal of single-cell RNA sequencing data. To achieve scalability, NewWave uses mini-batch optimization and can work with out-of-memory data, enabling users to analyze datasets with millions of cells.

Availability and implementationNewWave is implemented as an open-source R package available through the Bioconductor project at https://bioconductor.org/packages/NewWave/

Supplementary informationSupplementary data are available at Bioinformatics online.


title: AlphaMap: An open-source Python package for the visual annotation of proteomics data with sequence specific knowledge

abstract:
SummaryIntegrating experimental information across proteomic datasets with the wealth of publicly available sequence annotations is a crucial part in many proteomic studies that currently lacks an automated analysis platform. Here we present AlphaMap, a Python package that facilitates the visual exploration of peptide-level proteomics data. Identified peptides and post-translational modifications in proteomic datasets are mapped to their corresponding protein sequence and visualized together with prior knowledge from UniProt and with expected proteolytic cleavage sites. The functionality of AlphaMap can be accessed via an intuitive graphical user interface or - more flexibly - as a Python package that allows its integration into common analysis workflows for data visualization. AlphaMap produces publication-quality illustrations and can easily be customized to address a given research question.

Availability and implementationAlphaMap is implemented in Python and released under an Apache license. The source code and one-click installers are freely available at https://github.com/MannLabs/alphamap.

Supplementary informationA detailed user guide for AlphaMap is provided as supplementary data.


title: Are flexible school start times associated with higher academic grades? A 4-year longitudinal study.

abstract:
The mismatch between teenagers late sleep phase and early school start time results in acute and chronic sleep reductions. This is not only harmful for students learning in the short-term but may impact on students career prospects and widen social inequalities. Delaying school start times has been shown to improve sleep but whether this translates to better achievement is unresolved. The current evidence is limited due to a plethora of outcome measures and the many factors influencing sleep and grade/score trajectories. Here, we studied whether 0.5-1.5 years of exposure to a flexible school start system, with the daily choice of an 8AM or 8:50AM-start (intervention), allowed secondary school students (n=63-157, 14-19 years) to improve their quarterly school grades in a 4-year longitudinal pre-post design. We investigated whether sleep, changes in sleep or frequency of later starts predicted grade improvements in the flexible system. Our mixed model regressions with 5,111-16,724 official grades as outcomes did not indicate meaningful grade improvements in the flexible system per se or with previously observed sleep variables (nor their changes) - the covariates academic quarter, discipline and grade level had a greater, more systematic effect in our sample. Importantly, this finding does not preclude improvements in learning and cognition in our sample. However, at the  dose received here, intermittent sleep benefits did not obviously translate into detectable grade changes, which is in line with several other studies and highlights that grades are suboptimal to evaluate timetabling interventions despite their importance for future success.

Significance statementEarly school start times worldwide clash with teenagers delayed sleep-wake rhythms. This mismatch results in sleep restrictions below healthy amounts, which compromises health and performance and further aggravates social disparities. Since adequate sleep is important for learning and concentration, there is the strong expectation that counteracting sleep deprivation with delayed school starts results in better academic achievement. We add important high-resolution, longitudinal data to this unresolved scientific debate. When controlling for confounders, our results do not support that improved sleep leads to grade changes within 1.5 years in a flexible start system. While grades are suboptimal to measure later school start effects on performance, they nevertheless open doors to higher education worldwide and thus determine future trajectories.


title: Dynamic inventory of moths of Savitribai Phule Pune University (Pune, India) through crowdsourcing via iNaturalist

abstract:
We present here the checklist of moths (Lepidoptera: Heterocera) for the campus of Savitribai Phule Pune University, situated in the metropolis of Pune in the state Maharashra in India. We report identification of 189 unique genera along with 154 unique species. Despite the relative small size of the observation area and the location being at the heart of a busy metropolis, the moths were found to be of diverse variety, with 26 different families and 76 tribes.

The identifications of the species was crowd-sourced via iNaturalist.org. An automated program was developed to fetch the identification and generate the list. The program is also being made available open source. As a result the checklist remains relevant all the time with newer identification, correcting existing identification (if required) and taxonomic updates of the future.

category: zoology