Biology subjects

Bhardwaj, V.

Publications and source records attributed to Bhardwaj, V..

18 recordsLinked to original sources

bioRxiv2026

First detected incursions of avian influenza H5N1 clade 2.3.4.4b into mainland Australia from the Southern Ocean

High pathogenicity avian influenza H5N1 clade 2.3.4.4b has caused a panzootic of devastating impact to poultry and wildlife globally. The Australian continent and broader Oceania until recently remained the last major region without confirmed detections. Here we report the first H5N1 clade 2.3.4.4b detections from two live seabirds - a brown skua and a southern giant petrel - found on the south coast of Western Australia in June 2026. Virus genome sequencing showed that both viruses were most closely related to H5N1 viruses detected recently on sub-Antarctic islands in the Southern Indian Ocean. In time-calibrated phylogeographic analyses, both viruses sampled in Western Australia clustered with viruses from Heard Island, a sub-Antarctic external territory of Australia. Ancestral location reconstruction also identified Heard Island as the most probable source location, although unsampled intermediate locations cannot be excluded. The two Western Australian detections were estimated to be independent incursions from Heard Island, rather than local transmission on mainland Australia. There was no evidence of reassortment with endemic avian influenza viruses in Australia, and both virus sequences retained key avian-like genetic markers and lacked known substitutions for reduced antiviral susceptibility. These detections revealed a Southern Ocean pathway of recurrent H5N1 incursions into Australia, highlighting the risk of potential establishment on the mainland and the need for heightened surveillance and rapid, nationally-coordinated, virus genomic characterisation.

microbiology↗

bioRxiv2025

Cotranslational assembly confers specificity for in vivo target heterodimerization of paralogous H2B-like TAF12 proteins in the human fungal pathogen Candida albicans

The fidelity of assembly of multiprotein complexes is essential for the formation of stable and functional protein complexes that are critical for cell growth and survival. In this context, TBP-associated factor (TAF) subunits maintain tight specificity for their integration into TFIID and SAGA complexes. In this work, using affinity purification-coupled mass spectrometry of epitope-tagged TFIID subunits TBP and TAF11, and the SAGA subunit TAF12L we identified components of the C. albicans TFIID and SAGA complexes. Whereas TAF12 is a subunit of TFIID, the paralogous TAF12L is a subunit of the SAGA complex, and we further identified each of the TFIID and SAGA complex subunits with high confidence. We found that the steady-state levels of the H2B-H2A-like histone fold domain containing pairs, TAF12-TAF4 and TAF12L-Ada1 proteins, are mutually dependent on the stable expression of each other. Using RNA coimmunoprecipitation from polysome-containing extracts, we found that nascent TAF4 and Ada1 proteins interact with TAF12 and TAF12L, respectively, by a cotranslational mechanism in an ordered, sequential mode of assembly. Thus, our results indicate that heterodimerization of the TAF12 paralogs with cognate partners occur by sequential cotranslational assembly thereby ensuring both selectivity and stability of the H2A-H2B heterodimers in fungal pathogen C. albicans.

genetics↗

bioRxiv2025

Single-cell Analysis of Intracellular Transport and Expression of Cell Surface Proteins

Intracellular protein transport (ICT) is a tightly regulated process that orchestrates protein localization and expression, ensuring proper cellular function. Dysregulated ICT can lead to aberrant expression of surface proteins involved in cell-cell communication, adhesion, and immune responses, contributing to disease progression and therapeutic resistance. Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) enables the simultaneous measurement of mRNA and surface protein levels in the same cell, providing a powerful opportunity to investigate the molecular mechanisms underlying surface protein regulation. In this study, we introduce a novel computational frame for Modeling Protein Expression and Transport (MPET) that evaluates the contribution of ICT activity to differential surface protein expression using CITE-seq data. MPET comprises three modules for identification of ICT- surface protein regulatory circuits across biological scales and their contributions to phenotypic variation. We applied MPET to analyze single-cell data from COVID-19 patients with varying disease severity. Our analysis revealed context-dependent recruitment of ICT genes and pervasive rewiring of ICT pathways throughout the course of disease progression. Notably, we found that even when the transcriptional levels of key immune response proteins remained stable, their expression on cell surface were significantly altered due to dysregulated ICT. MPET provides a valuable new tool for dissecting complex regulatory networks and offers mechanistic insight into post-transcriptional regulation of cell surface proteins in diseases.

bioinformatics↗

bioRxiv2025

Vibrio harveyi plasmids as drivers of virulence in barramundi (Lates calcarifer)?

Vibrio species are an emerging public and animal health risk in marine environments and the opportunistic bacterial pathogen Vibrio harveyi is a major disease risk for tropical aquaculture. Current understanding of virulence in V. harveyi is limited by strain-specific variability and complex host-pathogen dynamics. This study sought to integrate genomic investigation, phenotypic characterisation and in vivo challenge trials in barramundi (Lates calcarifer) to increase our understanding of V. harveyi virulence. We identified two hypervirulent isolates, Vh-14 and Vh-15 that caused 100% mortality in fish within 48 hours, and that were phenotypically and genotypically distinct from other V. harveyi isolates. Virulent isolates contained multiple plasmids, including a 105,412 bp conjugative plasmid with type III secretion system genes originally identified in Yersinia pestis. The emergence of this hypervirulent plasmid-mediated patho-variant poses a potential threat to the sustainable production of marine finfish in Southeast Asia, the Mediterranean and Australia. In addition, we observed an effect of temperature on phenotypic indicators of virulence with an increase in activity at 28{degrees}C and 34{degrees}C compared to 22{degrees}C. This suggests that temperature fluctuations associated with climate change may act as a stressor on bacteria, increasing virulence gene secretion and host adaptation. Our results utilising a myriad of technologies and tools, highlights the importance of a holistic view to virulence characterisation.

microbiology↗

bioRxiv2024

Single-cell multi-omic analysis reveals principles of transcription-chromatin interaction during embryogenesis

Establishing a cell-type-specific chromatin landscape is crucial for the maintenance of cell identity during embryonic development. However, our knowledge of how this landscape is set during vertebrate embryogenesis has been limited, due to the lack of methods to jointly detect chromatin modifications and gene expression in the same cell. Here we present a multimodal measurement of full-length transcriptome and histone modifications in individual cells during early embryonic development in zebrafish. We show that before the formation of germ layers, the chromatin and transcription states of cells are uncoupled, and become progressively connected during gastrulation and somitogenesis. Silencing of developmental genes is achieved by local spreading of repressive chromatin together with cell-type specific demethylation. Combining transcription factor (TF) expression and chromatin states within an interpretable machine learning model, we classify TFs as lineage-specific activators and repressors and identify a subset of TFs that are epigenetically regulated. Altogether, our data resolves the dynamic relationship between chromatin and transcription during early vertebrate development and clarifies how these two layers interact to establish cell identity.

genomics↗

bioRxiv2024

User-friendly exploration of epigenomic data in single cells using sincei

Emerging single-cell sequencing protocols allow researchers to study multiple layers of epigenetic regulation while resolving tissue heterogeneity. However, despite the rising popularity of such single-cell epigenomics assays, the lack of easy-to-use computational tools that allow flexible quality control and data exploration hinders their broad adoption. We introduce the Single-Cell Informatics (sincei) toolkit. sincei provides an easy-to-use, command-line interface for the exploration of data from a wide range of single-cell (epi)genomics protocols directly from aligned reads stored in .bam files. sincei can be installed via bioconda and the documentation is available at https://sincei.readthedocs.io.

bioinformatics↗

bioRxiv2024

T-ChIC: multi-omic detection of histone modifications and full-length transcriptomes in the same single cell

Epigenetic mechanisms, including histone modifications, are key regulators of transcription and influence cellular differentiation. To uncover the interplay between histone modifications and transcription, we developed T-ChIC (Transcriptome + Chromatin ImmunoCleavage), which detects full-length transcripts and histone modifications in the same single cell. We applied T-ChIC to gastruloids, an in vitro model containing cell types derived from all three germ layers. During gastruloid development we observed a switch in transcription start site usage from multiple regions marked by H3K4me3 to one marked start site per gene. H3K27me3 coverage transitions from global marking of non-transcribed regions towards localised marking of repressive regions. Upon lineage commitment, bivalent regions are sequentially resolved towards a germ layer-specific state, where endoderm retains a high abundance of H3K27me3 while ectoderm displays a widespread loss. Together, we propose a model for a cell-intrinsic epigenetic timer, which ensures sequential perceptiveness of pluripotent cells to transcriptional activation of germ layer-specific genes.

genomics↗

bioRxiv2024

Stem cell culture conditions affect in vitro differentiation potential and efficiency of mouse gastruloid development

By aggregating low numbers of mouse embryonic stem cells (mESCs) followed by exposure to Wnt activation, structures termed gastruloids are produced that display an anteroposterior organisation of cell types derived from all three germ layers. Since current gastruloid protocols display considerable heterogeneity between experiments in terms of morphology, elongation efficiency, and cell type composition, we investigated whether an optimised mESC pluripotency state would provide more consistent results. By growing mESCs in different intervals of ESLIF and 2i medium the pluripotency state of cells was modulated, and mESC culture as well as the resulting gastruloids were analysed. Microscopy analysis showed a pre-culture-specific effect on gastruloid formation, in terms of efficiency, elongation index and reproducibility. RNA-seq analysis of the mESC start population confirmed that short-term pulses of 2i and ESLIF modulate the pluripotency state, and result in different cellular states. With multiple epigenetic regulators among the top differentially expressed genes we further analysed genome-wide DNA methylation and H3K27me3 distributions. We observed the same divergence between conditions, most dominantly displayed in the promoter regions of developmental regulators. Lastly, when we investigated the cell type composition of gastruloids grown from these different pre-cultures, we observed that mESCs subjected to 2i-ESLIF preceding aggregation generated gastruloids more consistently, including more complex mesodermal contributions as compared to the ESLIF-only control. This indicates that optimisation of the mESCs pluripotency state allows the modulation of cell differentiation during gastruloid formation.

developmental biology↗

bioRxiv2024

Acute multi-level response to defective de novo chromatin assembly in S-phase

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered. HighlightsO_LIThe histone chaperone CAF-1 sustains DNA replication speed in single cells. C_LIO_LICAF-1 loss alters histone repertoire and delays chromatin maturation. C_LIO_LIH3K9me3 and H3K27me3 regions respond differently to acute CAF-1 depletion. C_LIO_LIImpaired S-phase chromatin assembly triggers an immediate response and a G0 arrest. C_LI

cell biology↗

bioRxiv2022

HIV-1 Vpr induces ciTRAN to prevent transcriptional silencing of the provirus

The functional relevance of circular RNA (circRNA) expression in HIV-1 infection remains unclear. By developing a customized protocol involving direct RNA nanopore sequencing here, we captured circRNAs in their native state from HIV-1 infected T cells and identified ciTRAN, a circRNA modulator of HIV-1 Transcription. We show that HIV-1 infection of monocytic, T cell lines and primary CD4+ T cells induces ciTRAN expression in a Vpr-dependent manner. ciTRAN protein interactome analysis by proximity biotinylation and mass spectrometry identified SRSF-1 as a prominent interactor of the circular RNA. SRSF-1 is known to negatively regulate HIV-1 transcription, which the virus overcomes by a yet unknown mechanism. We demonstrate that HIV-1 Vpr induced ciTRAN sequesters SRSF1 away from the viral transcriptional complex to promote efficient viral transcription. Accordingly, ciTRAN depletion by CRISPR-Cas phenocopied the effects of SRSF1 overexpression and improved SRSF1 association with HIV-1 transcriptional complex. Finally, we show that an SRSF-1-inspired competing peptide can inhibit HIV-1 transcription regardless of ciTRAN induction. The hijacking of a host circRNA thus represents a new facet of primate lentiviruses in overcoming transmission bottlenecks.

microbiology↗

bioRxiv2022

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae. Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.

microbiology↗

bioRxiv2020

Creating Clear and Informative Image-based Figures for Scientific Publications

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in three fields; plant sciences, cell biology and physiology (n=580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

bioinformatics↗

bioRxiv2020

Coevolution of retroviruses with SERINCs following whole-genome duplication divergence

The SERINC gene family comprises of five paralogs in humans of which SERINC3 and SERINC5 inhibit HIV-1 infectivity and are counteracted by Nef. The origin of this anti-retroviral activity, its prevalence among the remaining paralogs, and its ability to target retroviruses remain largely unknown. Here we show that despite their early divergence, the anti-retroviral activity is functionally conserved among four human SERINC paralogs with SERINC2 being an exception. The lack of activity in human SERINC2 is associated with its post-whole genome duplication (WGD) divergence, as evidenced by the ability of pre-WGD orthologs from yeast, fly, and a post-WGD-proximate SERINC2 from coelacanth to inhibit nef-defective HIV-1. Intriguingly, potent retroviral factors from HIV-1 and MLV are not able to relieve the SERINC2-mediated particle infectivity inhibition, indicating that such activity was directed towards other retroviruses that are found in coelacanth (like foamy viruses). However, foamy-derived vectors are intrinsically resistant to the action of SERINC2, and we show that a foamy virus envelope confers this resistance. Despite the presence of weak arms-race signatures, the functional reciprocal adaptation among SERINC2 and SERINC5 and, in response, the emergence of antagonizing ability in foamy virus appears to have resulted from a long-term conflict with the host.

microbiology↗

bioRxiv2018

The NSL complex mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise

Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDR and the precise positioning of the +1 nucleosome is maintained on active genes remains unclear. Here, we report that the Drosophila Non-Specific Lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. The NSL complex is not only essential for transcription but is required for accurate TSS selection for genes with multiple TSSs. Further, loss of NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

genomics↗

bioRxiv2018

snakePipes enable flexible, scalable and integrative epigenomic analysis

The scale and diversity of epigenomics data has been rapidly increasing and ever more studies now present analyses of data from multiple epigenomic techniques. Performing such integrative analysis is time-consuming, especially for exploratory research, since there are currently no pipelines available that allow fast processing of datasets from multiple epigenomic assays while also allow for flexibility in running or upgrading the workflows. Here we present a solution to this problem: snakePipes, which can process and perform downstream analysis of data from all common epigenomic techniques (ChIP-seq, RNA-seq, Bisulfite-seq, ATAC-seq, Hi-C and single-cell RNA-seq) in a single package. We demonstrate how snakePipes can simplify integrative analysis by reproducing and extending the results from a recently published large-scale epigenomics study with a few simple commands. snakePipes are available under an open-source license at https://github.com/maxplanck-ie/snakepipes.

genomics↗

bioRxiv2017

Community-driven data analysis training for biology

The primary problem with the explosion of biomedical datasets is not the data itself, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web-browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences.

bioinformatics↗

bioRxiv2017

Microbial mutualism at a distance

The exchange of diffusive metabolites is known to control the spatial patterns formed by microbial populations, as revealed by recent studies in the laboratory. However, the matrices used, such as agarose pads, lack the structured geometry of many natural microbial habitats, including in the soil or on the surfaces of plants or animals. Here we address the important question of how such geometry may control diffusive exchanges and microbial interaction. We model mathematically mutualistic interactions within a minimal unit of structure: two growing reservoirs linked by a diffusive channel through which metabolites are exchanged. The model is applied to study a synthetic mutualism, experimentally parameterised on a model algal-bacterial co-culture. Analytical and numerical solutions of the model predict conditions for the successful establishment of remote mutualisms, and how this depends, often counterintutively, on diffusion geometry. We connect our findings to understanding complex behaviour in synthetic and naturally occurring microbial communities.

biophysics↗

bioRxiv2017

High-resolution TADs reveal DNA sequences underlying genome organization in flies

Eukaryotic chromatin is partitioned into domains called TADs that are broadly conserved between species and virtually identical among cell types within the same species. Previous studies in mammals have shown that the DNA binding protein CTCF and cohesin contribute to a fraction of TAD boundaries. Apart from this, the molecular mechanisms governing this partitioning remain poorly understood. Using our new software, HiCExplorer, we annotated high-resolution (570 bp) TAD boundaries in flies and identified eight DNA motifs enriched at boundaries. Known insulator proteins bind five of these motifs while the remaining three motifs are novel. We find that boundaries are either at core promoters of active genes or at non-promoter regions of inactive chromatin and that these two groups are characterized by different sets of DNA motifs. Most boundaries are present at divergent promoters of constitutively expressed genes and the gene expression tends to be coordinated within TADs. In contrast to mammals, the CTCF motif is only present on 2% of boundaries in flies. We demonstrate that boundaries can be accurately predicted using only the motif sequences, along with open chromatin, suggesting that DNA sequence encodes the 3D genome architecture in flies. Finally, we present an interactive online database to access and explore the spatial organization of fly, mouse and human genomes, available at http://chorogeome.ie-freiburg.mpg.de.

genomics↗