Biology subjects

Ghosh, D.

Publications and source records attributed to Ghosh, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Native Ion Mobility-MS Reveals the Bead Ejection Mechanism in Multi-Domain Nucleic Acids

Understanding how biomolecules acquire their charge and retain their solution conformation during electrospray ionization (ESI) is crucial for native mass spectrometry (native MS) interpretation. Here, we examine the charging and gas phase conformation of nucleic acid constructs comprising folded G-quadruplex "beads" linked by unstructured polythymine regions. Under physiological ionic strength, these oligonucleotides exhibit a multimodal charge-state and collision cross section distribution, revealing multiple conformational ensembles, in contrast to the unimodal profiles typically observed for shorter oligonucleotides. Native MS observations for intermediate charge states are compatible with ion production via the recently proposed bead ejection scenario, in addition to the charge residue scenario for low charge states and chain ejection for the highest charge states or for sequences with thymine overhangs on both ends. The preservation of the local structures in ions charged above the Rayleigh limit helps to infer the presence of folded subunits. The position of the G-quadruplex subunit and ionic strength governs the charging and retention of G-quadruplex folded regions. Our findings broaden the existing conceptual framework underpinning nucleic acid ionization. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/667413v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@42d7c3org.highwire.dtl.DTLVardef@63445dorg.highwire.dtl.DTLVardef@11d7056org.highwire.dtl.DTLVardef@1ba48bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

AAV gene therapy for GBA-PD and Gaucher Disease

Mutations in GBA1, the gene encoding glucocerebrosidase (GCase), are the most common risk factor for Parkinsons Disease (PD). GBA-PD patients are a genetic subpopulation of PD carrying heterozygous mutations in GBA1. Additionally, bi-allelic mutations in GBA1 cause Gaucher Disease (GD), a lysosomal storage disorder. Loss of GCase activity, a lysosomal enzyme leads to the accumulation of lipid substrates, disrupting lipid homeostasis and promoting cellular toxicity. Here, we report an AAV-mediated GBA1 replacement strategy to treat GD and GBA-PD by a one-time infusion via intravenous (GD Type 1) or intra-CSF (GBA-PD) route of administration. We engineered human GCase to be readily secretable to facilitate broad cross-correction. We developed CBE (conduritol {beta}-epoxide) induced lipid accumulation models to assess efficacy in mice and non-human primates (NHPs) to assess efficacy of our engineered constructs. Based on data across species, across different routes of administration, we nominated AAV.GMU01 SS3-GBA1 as our lead candidate. SS3-GBA1 is robustly secreted, cross-corrected across tissues and promotes lipid clearance. By comparing human GCase levels in AAV-treated NHP brains to healthy human donor brains, we demonstrate that AAV.GMU01 SS3-GBA1 replenishes the GCase deficit seen in GBA-PD patients, thus, restoring GCase to near-physiological levels Importantly, AAV.GMU01 SS3-GBA1 is well-tolerated with no adverse findings. Collectively, we establish a therapeutic strategy for the treatment of Gaucher Disease and GBA-PD with a single gene therapy product. One Sentence SummaryA novel gene therapy strategy for GBA1-PD and Gaucher disease with an engineered payload that robustly cross-corrects enhancing therapeutic footprint

neuroscience↗

bioRxiv2025

Cover crop root channels promote bacterial adaptation to drought in the maize rhizosphere

BackgroundIncreasing drought frequency poses a significant threat to agricultural productivity. A promising strategy to enhance crop resilience against drought is the utilisation of root channels left by winter cover crops, which can improve access to subsoil water and nutrients for subsequent cash crops like maize (Zea mays L.). The impact of drought on bacterial communities inhabiting these root channels remains largely unknown. Here, we investigated drought-induced shifts in maize rhizosphere bacterial communities and their functional adaptation in cover crop root channels across three soil types in northern Germany (Luvisol, Podzol, and Phaeozem). ResultsUsing a multi-omics approach (16S rRNA gene amplicon sequencing, qPCR, and metaproteomics), we identified significant taxonomic and functional responses to drought. A rise in the abundance of K-strategist bacterial communities indicate a shift towards stress-tolerant populations with drought. Under drought stress, the relative abundances of Acidobacteriota, Actinomycetota, Planctomycetota, and Pseudomonadota increased, while Chloroflexota, Methylomirabilota, Ca. Patescibacteria, and Verrucomicrobiota declined. Functional analyses revealed that drought-stressed aerobic taxa among the Pseudomonadota and Verrucomicrobiota upregulated the glyoxylate cycle, potentially enhancing carbon and energy conservation, and increased antioxidant defences (catalase-glutathione peroxidase and methionine cycle-transsulfuration pathway). These drought-mitigating strategies were especially pronounced in root channels formed by Brassicaceae and Poaceae cover crops in the Luvisol and Podzol soils. ConclusionsThese findings demonstrate the functional plasticity of rhizosphere bacterial communities in reused root channels in response to drought, highlighting the potential to leverage microbiome-mediated resilience for agricultural practices. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/651044v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@7c3f69org.highwire.dtl.DTLVardef@127182forg.highwire.dtl.DTLVardef@1a4bb61org.highwire.dtl.DTLVardef@9f7c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Breathe in, breathe out: Bacterial density determines collective migration in aerotaxis

Bacteria navigate their environment by biasing their swimming direction toward beneficial chemicals and away from harmful ones. Out of all the chemicals bacteria respond to, oxygen stands out due to its ubiquitous presence, distinct influence on bacterial metabolism and motility, and historical role in chemotaxis research. However, a coherent understanding of bacterial motility in oxygen gradients, known as aerotaxis, remains elusive, as evidenced by conflicting reports on the migration direction of the model organism Escherichia coli in self-generated oxygen gradients. Here, by combining experiments, simulations, and theory, we provide a unified framework elucidating the fundamental biophysical principle governing bacterial aerotaxis. We track the migration of bacteria in a capillary channel under self-generated oxygen gradients and show that the migration direction depends on the overall bacterial density. At high densities, bacteria migrate toward regions of higher oxygen concentration, whereas at low densities, they move in the opposite direction. We identify a critical bacterial density at which collective migration ceases, despite the presence of oxygen gradients. A kinetic theory, based on the assumption that bacteria seek an optimal oxygen concentration, is then developed to quantitatively explain our experimental findings. We validate this hypothesis by demonstrating the biased movement of individual bacteria in a dense suspension and proposing a signaling pathway that enables this behavior. Thus, by bridging the molecular level understanding of the signaling pathway, the motility of single bacteria in oxygen gradients, and the collective population dynamics shaped by oxygen diffusion and consumption, our study provides a comprehensive understanding of aerotaxis, addressing the long-standing controversy over how bacteria response to non-uniform oxygen distributions pervasive in microbial habitats.

biophysics↗

bioRxiv2025

AAV-mediated ARSA replacement for the treatment of Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is an autosomal recessive neurodegenerative disorder caused by mutations in the arylsulfatase A (ARSA) gene, resulting in lower sulfatase activity and the toxic accumulation of sulfatides in the central and peripheral nervous system. Children account for 70% of cases and become progressively disabled with death occurring within 10 years of disease onset. Gene therapy approaches to restore ARSA expression via adeno-associated viral vectors (AAV) have been promising but hampered by limited brain biodistribution. We report the development of a novel capsid AAV.GMU01, demonstrating superior biodistribution and transgene expression in the central nervous system of non-human primates (NHPs). Next, we show that AAV.GMU01-ARSA treated MLD mice exhibit persistent, normal levels of sulfatase activity and a concomitant reduction in toxic sulfatides. Treated mice also show a reduction in MLD-associated pathology and auditory dysfunction. Lastly, we demonstrate that treatment with AAV.GMU01-ARSA in NHPs is well-tolerated and results in potentially therapeutic ARSA expression in the brain. In summary, we propose AAV.GMU01-ARSA mediated gene replacement as a clinically viable approach to achieve broad and therapeutic levels of ARSA.

neuroscience↗

bioRxiv2025

Resolution of in situ inflammation in human lupus nephritis into principal immune cell trajectories

In human lupus nephritis (LuN), tubulointerstitial inflammation (TII) is prognostically more important than glomerular inflammation. However, a comprehensive understanding of both TII complexity and heterogeneity is lacking. Herein, we used high-dimensional confocal microscopy and specialized computer vision techniques to quantify immune cell populations and localize these within normal and diseased renal cortex structures. With these tools, we compared LuN to renal allograft rejection (RAR) and normal kidney. In both LuN and RAR, the 33 characterized immune cell populations formed discrete subgroups whose constituents co-varied in prevalence across biopsies. In both diseases, these co-variant immune cell subgroups organized into the same unique niches. Therefore, inflammation could be resolved into trajectories representing the relative prevalence and density of cardinal immune cell members of each co-variant subgroup. Indeed, in any one biopsy, the inflammatory state could be characterized by quantifying constituent immune cell trajectories. Remarkably, LuN heterogeneity could be captured by quantifying a few myeloid immune cell trajectories while RAR was more complex with additional T cell trajectories. Our studies identify rules governing renal inflammation and thus provide an approach for resolving LuN into discrete mechanistic categories. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/638655v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@109dd33org.highwire.dtl.DTLVardef@995037org.highwire.dtl.DTLVardef@161525dorg.highwire.dtl.DTLVardef@6692d8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

immunology↗

bioRxiv2024

Urinary biochemical ecology reveals microbiome-metabolite interactions and metabolic markers of recurrent urinary tract infection

Recurrent urinary tract infections (rUTIs) are a major clinical challenge and their increasing prevalence underscores the need to define host-microbiome interactions underlying susceptibility. How the urinary microbiota engages with the biochemical environment of the urogenital tract is yet to be fully defined. Here, we leverage paired metagenomic and quantitative metabolomic data to establish a microbe-metabolite association network of the female urinary microbiome and define metabolic signatures of rUTI. We observe unique metabolic networks of uropathogens and uroprotective species, highlighting potential metabolite-driven ecological shifts influencing rUTI susceptibility. We find distinct metabolites are associated with urinary microbiome diversity and identify a lipid signature of active rUTI that accurately distinguishes cases from controls. Finally, we identify deoxycholic acid as a prognostic indicator for UTI recurrence. Together these findings provide insight into microbiome-metabolite interactions within the female urinary tract and highlight new biomarkers for the development of new diagnostic tools to improve patient outcomes.

microbiology↗

bioRxiv2024

cytoKernel: Robust kernel embeddings for assessing differential expression of single cell data

High-throughput sequencing of single-cell data can be used to rigorously evlauate cell specification and enable intricate variations between groups or conditions. Many popular existing methods for differential expression target differences in aggregate measurements (mean, median, sum) and limit their approaches to detect only global differential changes. We present a robust method for differential expression of single-cell data using a kernel-based score test, cytoKernel. cytoKernel is specifically designed to assess the differential expression of single cell RNA sequencing and high-dimensional flow or mass cytometry data using the full probability distribution pattern. cytoKernel is based on kernel embeddings which employs the probability distributions of the single cell data, by calculating the pairwise divergence/distance between distributions of subjects. It can detect both patterns involving aggregate changes, as well as more elusive variations that are often overlooked due to the multimodal characteristics of single cell data. We performed extensive benchmarks across both simulated and real data sets from mass cytometry data and single-cell RNA sequencing. The cytoKernel procedure effectively controls the False Discovery Rate (FDR) and shows favourable performance compared to existing methods. The method is able to identify more differential patterns than existing approaches. We apply cytoKernel to assess gene expression and protein marker expression differences from cell subpopulations in various publicly available single-cell RNAseq and mass cytometry data sets. The methods described in this paper are implemented in the open-source R package cytoKernel, which is freely available from Bioconductor at http://bioconductor.org/packages/cytoKernel.

bioinformatics↗

bioRxiv2023

Electrostatic-driven Interactions Enhance Intratumoral Retention and Antitumor Efficacy of Immune Checkpoint Blockade Antibodies

Tumor extracellular matrix (ECM) forms a net negative charged network that interacts with and hinders the transport of molecules partly based on electrostatic interactions. The focus on drug delivery in solid tumors has traditionally been on developing neutral charge coatings to minimize interactions with the ECM for improved transport. In contrast to this prior work, we recently found a cationic peptide that interacted electrostatically with the negatively charged components of the ECM, resulting in enhanced uptake and retention of nanoparticles in tumor ECM and tumor tissue. Based on this previous study, here, we hypothesize that the electrostatically driven interactions of the cationic peptide will improve the binding and retention of immune checkpoint blockade antibodies (ICBs), ultimately enhancing their antitumor immunogenic responses. We prepared peptide antibody (Ab) conjugates by conjugating the cationic peptide to ICBs, anti-cytotoxic T lymphocyte antigen 4 ({propto}-CTLA4) and anti-programmed cell death ligand-1 ({propto}-PD-L1) Abs, using copper-free click chemistry. We confirmed an average of 1 - 2 peptides per Ab. The cationic peptide electrostatically interacted with the net negatively charged tumor ECM and improved the binding of the Abs to the tumor ECM without affecting their antigen recognition capacities. Modifying the Abs due to cationic peptide conjugation reduced the systemic exposure of the Abs and did not induce treatment-related toxicities. We quantified a significantly higher population of tumor-infiltrating CD8+ T cells and a significant depletion of regulatory T cells in the tumor and tumor-draining lymph nodes upon peptide conjugation, which resulted in a better therapeutic outcome of the ICBs. ONE SENTENCE SUMMARYElectrostatic interaction-based intratumoral retention enhances antitumor responses of immune checkpoint blockade antibodies upon local administration.

bioengineering↗

bioRxiv2023

An approach to characterize mechanisms of action of anti-amyloidogenic compounds in vitro and in situ

Aggregation of amyloidogenic proteins is associated with neurodegenerative disease and its modulation is a focus of drug development efforts. However, the physicochemical properties and structural heterogeneity of amyloidogenic proteins hinder the mechanistic understanding of anti- amyloidogenic compounds. Further, modes of interaction with amyloidogenic proteins are often probed in vitro using purified protein samples, even though these models may not capture in vivo protein structures and do not enable identification of off-target effects. We have developed a modular structural proteomic pipeline based on limited proteolysis coupled to mass spectrometry (LiP-MS) with improved, amino acid level-resolution, to probe the mechanism of action of anti- amyloidogenic compounds. We demonstrate our approach by analysing the interactions of six known or putative anti-amyloidogenic compounds and the amyloid binder Thioflavin T (ThT) with different structural forms of the amyloidogenic Parkinsons disease (PD) protein -Synuclein. Our approach enables determination of putative interaction sites, identification of whether interactions are covalent or non-covalent, and crucially, can probe for interactions of compounds with physiological structures of -Synuclein in complex cell and tissue extracts and identify off-targets. In vitro analyses with our pipeline showed that the green tea polyphenol EGCG induces an N- and C-terminus- dependent compaction of the unstructured -Synuclein monomer, detected preferential interactions of ThT with the N-terminus of -Synuclein fibrils compared to the amyloid core, and showed that the most potent inhibitors of aggregation in our study (EGCG, baicalein and AC Immune compound #2) induced similar non-fibrillar end structures despite different interactions with -Synuclein monomers. Importantly, in mammalian cell lysates, -Synuclein was either a low-affinity target (for EGCG and Baicalein) or did not show evidence of compound interaction (for ThT and doxycycline) in our experimental conditions, despite both monomeric and fibrillar forms interacting with these compounds in vitro. For EGCG, we validated this result in postmortem brain homogenates from PD patients. These in situ analyses identified many additional putative cellular targets of Doxycycline, EGCG, Baicalein and ThT, suggesting that their effects in cellular or animal models of neurodegeneration are likely due to interactions with proteins other than -Synuclein and showing that anti-amyloidogenic compounds should be analyzed in situ as well as in vitro. Our modular pipeline will enable in situ screening of drugs and PET tracers for amyloid aggregates of interest as well as detailed mechanistic studies of compound action in vitro.

systems biology↗

bioRxiv2023

Applying machine learning to identify ionizable lipids for nanoparticle-mediated delivery of mRNA

mRNA lipid nanoparticles (LNPs) have a tremendous potential to treat, cure, or prevent many diseases. To identify promising candidates for each application, most studies screen dozens to hundreds of formulations with ionizable lipids synthesized using a single type of chemistry. However, this technique leaves the ionizable lipids synthesized through multi-step chemistries underexplored. This gap in the repertoire of structures is of particular significance because it affects the screening of analogs that are structurally similar to SM-102 and ALC-0315, the ionizable lipids used to formulate the clinically approved mRNA LNP COVID-19 vaccines. Herein, we address this by employing LightGBM, a machine learning algorithm, to reduce the burden of screening these types of ionizable lipids by learning from the breadth and diversity of lipids that have already been tested. We first evaluate the ability of LightGBM to predict LNP potency across heterogeneous chemistries from different studies to achieve an R2 of 0.94. After establishing the predictive capacity of the model, we then identify the number of outside carbons in the ionizable lipid as the most important factor contributing to transfection efficiency. From this finding, we subsequently apply the algorithm to predict the effect of formulating nanoluciferase mRNA LNPs using analogs of SM-102 and ALC-0315 with small changes in the number of outside carbons on luciferase activity in HEK293T cells and achieve an R2 of 0.83. Importantly, this correlation encompasses novel lipids not included within the database used to train the algorithm. Overall, this study demonstrates the potential of machine learning to accelerate the development of new ionizable lipids by simplifying the screening process.

bioengineering↗

bioRxiv2023

Intrinsic p53 Activation Restricts Gammaherpesvirus-Driven Germinal Center B Cell Expansion during Latency Establishment

Gammaherpesviruses (GHV) are DNA tumor viruses that establish lifelong latent infections in lymphocytes. For viruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68), this is accomplished through a viral gene-expression program that promotes cellular proliferation and differentiation, especially of germinal center (GC) B cells. Intrinsic host mechanisms that control virus-driven cellular expansion are incompletely defined. Using a small-animal model of GHV pathogenesis, we demonstrate in vivo that tumor suppressor p53 is activated specifically in B cells that are latently infected by MHV68. In the absence of p53, the early expansion of MHV68 latency was greatly increased, especially in GC B cells, a cell-type whose proliferation was conversely restricted by p53. We identify the B cell-specific latency gene M2, a viral promoter of GC B cell differentiation, as a viral protein sufficient to elicit a p53-dependent anti-proliferative response caused by Src-family kinase activation. We further demonstrate that EBV-encoded latent membrane protein 1 (LMP1) similarly triggers a p53 response in primary B cells. Our data highlight a model in which GHV latency gene-expression programs that promote B cell proliferation and differentiation to facilitate viral colonization of the host trigger aberrant cellular proliferation that is controlled by p53. IMPORTANCEGammaherpesviruses cause lifelong infections of their hosts, commonly referred to as latency, that can lead to cancer. Latency establishment benefits from the functions of viral proteins that augment and amplify B cell activation, proliferation, and differentiation signals. In uninfected cells, off-schedule cellular differentiation would typically trigger anti-proliferative responses by effector proteins known as tumor suppressors. However, tumor suppressor responses to gammaherpesvirus manipulation of cellular processes remain understudied, especially those that occur during latency establishment in a living organism. Here we identify p53, a tumor suppressor commonly mutated in cancer, as a host factor that limits virus-driven B cell proliferation and differentiation, and thus, viral colonization of a host. We demonstrate that p53 activation occurs in response to viral latency proteins that induce B cell activation. This work informs a gap in our understanding of intrinsic cellular defense mechanisms that restrict lifelong GHV infection.

microbiology↗

bioRxiv2023

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

CRISPR-Cas9 is the newest technology available for targeted genome editing. It is very efficient and cheap compared to other genome editing techniques. However, its therapeutic application is limited due to its off-target activity. To have a better understanding of this off-target effect, we concentrated our efforts on its mismatch-prone PAM distal end. Current off-target prediction algorithms use RNA-DNA complementation derived energy as a major factor in predicting off-target effect. RNA-DNA complementation derived energy drives Cas9 conformational change, which in turn drives its functional activity. In the case of lower RNA-DNA complementarity, a partial conformational change occurs resulting in a slower reaction rate and partial activity. However, extensive mismatches are often tolerated despite lower complementation derived energy available from RNA: DNA duplex formation. Thus, the off-target activity of Cas9 depends directly on the nature of mismatches which in turn result in deviation of the active site of the enzyme due to structural instability in the duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites and performed in vitro and cell line-based experiments to assess the effects of PAM distal mismatches in Cas9 activity. For further mechanistic validation, Molecular dynamics simulation was performed and it revealed that certain mismatch mutations induced pronounced conformational instability within the RNA-DNA duplex, leading to elevated root mean square deviation (RMSD) values. We found that, target sites having mismatches in the 18th to 16th position upstream of the PAM showed no to little activity.

genomics↗

bioRxiv2023

Growth factor-induced desialylation for the fast control of endocytosis

It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na+/H+ antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

cell biology↗

bioRxiv2023

Discovery of peptides for targeted delivery of mRNA lipid nanoparticles to cystic fibrosis lung epithelia

For cystic fibrosis (CF) patients, a lung targeted gene therapy would significantly alleviate pulmonary complications associated with morbidity and mortality. However, mucus in the airways and cell entry pose huge delivery barriers for local gene therapy. Here, we used phage display technology to select for and identify mucus- and cell-penetrating peptides against primary human bronchial epithelial cells (pHBECs) from CF patients cultured at air-liquid interface (ALI). At ALI, pHBECs produce mucus and reflect CF disease pathology, making it a clinically relevant model. Using this model, we discovered a lead candidate peptide, and incorporated it into lipid nanoparticles (LNPs) to deliver mRNA to pHBECs and mouse lungs in vivo. Compared to LNPs without our peptide, peptide-LNPs demonstrated 7.8-fold and 4.8-fold higher mRNA expression in vitro and in vivo, respectively. Since gene delivery to pHBECs is a significant challenge, we are encouraged by these results and anticipate that our peptide could be used to successfully deliver CF gene therapies in future work.

bioengineering↗

bioRxiv2023

Differential Carbonic Anhydrase Activities Control EBV-Induced B-Cell Transformation and Lytic Cycle Reactivation

Epstein-Barr virus (EBV) contributes to [~]1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naive B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

microbiology↗

bioRxiv2023

Spatial reorganization of Escherichia coli chromosome contextualizes triclosan stress-related genetic, epigenetic and transcriptome changes

Changes in the spatial organization of bacterial chromosomes under stress conditions and its biological implications remain poorly understood. We mapped the structural landscape of wild-type and {Delta}dcm E. coli chromosomes under triclosan stress using Hi-C to identify triclosan-induced chromosomal interaction domains (CIDs). Two CIDs were common to the wild-type and {Delta}dcm E. coli, including a CID with a common boundary at fabI gene, which encodes the triclosan target. All mutations and structural variants under triclosan stress were observed within or in close proximity to triclosan-induced CIDs. Absence of Dcm methylation impacts both short- and long-range interactions in triclosan stress. Single-base resolution methylome maps reveal hypermethylation of adenines (in wild-type and {Delta}dcm) and cytosines (in wild-type) in the two common triclosan-induced CIDs. Furthermore, global gene expression profiling identified enrichment of highly expressed genes within the two common CIDs. Our findings suggest that stress-induced CIDs in E. coli are hotspots for genetic variations and are associated with enhanced transcriptional activity and hypermethylation of Dam/Dcm motifs.

microbiology↗

bioRxiv2023

SpaceANOVA: Spatial co-occurrence analysis of cell types in multiplex imaging data using point process and functional ANOVA

MotivationMultiplex imaging platforms have enabled the identification of the spatial organization of different types of cells in complex tissue or tumor microenvironment (TME). Exploring the potential variations in the spatial co-occurrence or co-localization of different cell types across distinct tissue or disease classes can provide significant pathological insights, paving the way for intervention strategies. However, the existing methods in this context either rely on stringent statistical assumptions or suffer from a lack of generalizability. ResultsWe present a highly powerful method to study differential spatial co-occurrence of cell types across multiple tissue or disease groups, based on the theories of the Poisson point process (PPP) and functional analysis of variance (FANOVA). Notably, the method accommodates multiple images per subject and addresses the problem of missing tissue regions, commonly encountered in such a context due to the complex nature of the data-collection procedure. We demonstrate the superior statistical power and robustness of the method in comparison to existing approaches through realistic simulation studies. Furthermore, we apply the method to three real datasets on different diseases collected using different imaging platforms. In particular, one of these datasets reveals novel insights into the spatial characteristics of various types of precursor lesions associated with colorectal cancer. AvailabilityThe associated R package can be found here, https://github.com/sealx017/SpaceANOVA. Contactsealso@musc.edu Supplementary informationThe supplementary material is attached.

bioinformatics↗