Biology subjects

Raychaudhuri, S.

Publications and source records attributed to Raychaudhuri, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Rethinking the Benign Nature of Class II Lupus Nephritis

Introduction: Class II lupus nephritis (LN) is considered clinically benign, yet up to 50% of patients progress to more severe disease and no molecular characterization exists. We aimed to define the single-cell landscape of Class II LN and identify cellular and transcriptional features associated with kidney outcome. Methods: We performed single-cell RNA sequencing on kidney biopsies from fifteen Class II LN patients (eight de novo, seven regressed from prior proliferative or membranous disease) and six healthy controls, integrated with 155 LN and 30 healthy-control samples from the Accelerating Medicines Partnership in SLE spanning proliferative, membranous, and mixed LN. Findings were correlated with 52-week renal response and supported by urinary proteomics. Results: Despite histologically minimal changes, Class II LN exhibited marked immune cell expansion comparable to proliferative and membranous LN, including CCL3+ CCL4+ intermediate monocytes producing TNF, FOLR2+ SIGLEC1+ macrophages producing TGF{beta} and PDGF, and CD69+ CD8+ effector memory T cells producing IFN{gamma}. Fibroblasts and myofibroblasts were significantly expanded and displayed divergent transcriptional programs: pro-fibrotic and inflammatory myofibroblast modules were associated with worse 52-week outcomes, while interferon-responsive and matrix-remodeling fibroblast programs were associated with improvement. Pathogenic and protective fibroblast populations received overlapping immune-derived signals, suggesting that fibroblast transcriptional state, rather than the identity of incoming signals, shapes the stromal response. Baseline chronicity index captured this stromal heterogeneity and was the clinical parameter most associated with outcome. Urinary proteomics aligned Class II with proliferative rather than membranous disease. Conclusions: Class II LN harbors substantial molecular activity not captured by routine histology. Fibroblast transcriptional programs and baseline chronicity index are candidate correlates of kidney outcome that warrant validation in larger, prospectively followed cohorts.

genomics↗

bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗

bioRxiv2026

IKK2/β mediated phosphorylation of transcription factor Ets2 at site(s) distal to DNA binding domain negatively modulates its DNA binding activity

Transcription factor Ets2 coalesce with the NF-{kappa}B pathway to regulate gene expression in specific signaling contexts. IKK2/{beta}-mediated phosphorylation events critically regulate the NF-{kappa}B pathway. However, any link between Ets2 and IKK2 remains elusive. Here we report Ets2 as a direct substrate of IKK2. In-vitro kinase assays using deletion constructs, high resolution MS-MS and site directed mutagenesis identified S295 as a prominent phosphorylation site distal to the DNA binding domain, substitution of which to phosphor-mimetic Glutamate triggers further phosphorylation of Ets2. MD simulations clearly indicate conformational constriction of the otherwise disordered N-terminal region and inhibition of DNA binding activity upon phosphorylation, which was further confirmed by Electrophoretic mobility shift assays. Our results uncover a phosphoregulatory connection between Ets2 and IKK2.

biochemistry↗

bioRxiv2026

Efficient genome-wide mapping of reproducible, context-dependent eQTLs at single-cell resolution

Single-cell technologies enable linking disease-risk variants to gene regulatory effects in specific cell-state contexts. However, most so called "single-cell eQTL" studies use a "pseudobulking" strategy to identify expression Quantitative Trait Loci (eQTLs), obscuring subtle dynamic regulatory effects of disease alleles. Here, we propose Dynema (Dynamic eQTL mapping in single cells) for fast and accurate genome-wide mapping of context-dependent and independent eQTL effects at true single-cell resolution. To identify eQTLs, Dynema uses a Poisson model with cluster robust variance estimators (CRVEs) to account for correlation of single-cell profiles from the same individual. In contrast to other common methods, Dynema achieves statistical calibration and scales to genome-wide analysis in large single-cell datasets in realistic timeframes. We applied Dynema to two independent T cell datasets and identified reproducible cell-state-dependent eQTL effects. Some cell-state-dependent eQTLs are missed by pseudobulking approaches, and many others are conditionally independent from lead eQTL effects. We show that TSPAN32 and other autoimmune loci colocalize with cell-state-dependent eQTLs. Mapping context-dependent eQTLs at single-cell resolution enables the definition of the molecular effects of complex disease alleles.

genetics↗

bioRxiv2026

Identifying the causal allele in the CD40 autoimmune locus enables discovery of context-specific trans-effects in B cells

Thousands of genetic variants are associated with autoimmune diseases, but causal variants, their mechanisms, and the pathogenic context in which they act are elusive. Knowledge of pathogenic contexts may enable effective targeted therapies, instead of broad immunosuppressive approaches. First, to focus on the genetics of immune response, we used surface marker CITE-seq data from 1,055,857 peripheral blood mononuclear cells from 356 individuals. We defined genetic associations to 148 surface proteins across eight cell types. We observed a signal in the CD40 locus, implicated in rheumatoid arthritis (RA) and other autoimmune conditions. RA risk variants increased CD40 protein expression by [~]20% on B cells, but with minimal mRNA effects. Second, we deployed base-resolution genome editing, with CRAFT-seq, capturing genomic DNA sequence at the edited site and multimodal phenotypes at single-cell resolution. We defined a single causal allele, rs1883832, within the Kozak motif. Third, we edited this allele, in primary B cells and conducted CRAFTseq to demonstrate trans-effects in >200 genes. These effects were only in the light zone germinal center-like state. Importantly, these trans-effects were not seen in population-scale cohorts of unstimulated B cells. This represents a framework to define disease causal alleles, their cis- and trans-effects. It demonstrates the power of defining causal genetic variation to find trans-effects through editing, which cannot easily be found in population studies.

genetics↗

bioRxiv2026

Integration of large, complex single-cell datasets with Harmony2

Integrating single cell RNA-seq profiles is posing new challenges as datasets are rapidly expanding, now with over 100 million cells in the public domain. We present the latest version of the Harmony integration software, which efficiently scales to >100M cells and >1K datasets without specialized hardware. Moreover, optimizations to the underlying algorithm help prevent overintegration in biologically heterogeneous datasets. Harmony2 enables efficient, accurate integration of large, complex single-cell atlases.

bioinformatics↗

bioRxiv2026

Cytosolic interaction with RNA-helicase DDX39A titrates viral RNA G-quadruplex mediated α-Synuclein amyloidogenesis

Amyloid aggregates of -Synuclein are hallmark of Parkinsons Disease (PD) and related neurodegenerative diseases. -Synuclein, being an non-canonical RNA-binding protein (RBP), associates with other RBPs within cytosolic RNA-protein granules to modulate mRNA-stability. Conversely, mRNA G-quadruplexes (rG4s) expedite -Synuclein amyloidogenesis. However, spatiotemporal control on -Synuclein amyloidogenesis by other RBPs remains unexplored. Here, we report that RNA-dependent cytosolic interaction with DEAD-box RNA-helicase DDX39A decelerates -Synuclein amyloidogenesis. Viral infections transiently elevate rG4s in cytoplasm. Perturbing interactions between Synuclein and DDX39A using viral rG4s from H1N1-influenza and SARS-CoV-2 genomes expedites intracellular amyloidogenesis. Conversely, DDX39A overexpression alleviates -Synuclein amyloidogenesis in mouse primary neurons triggered by SARS-CoV-2 infection. We demonstrate that while DDX39A unwinds viral rG4s to mitigate -Synuclein sol-gel transition, its reciprocal cooperative phase separation with -Synuclein enhances the helicases rG4-unwinding activity. We propose that accelerated -Synuclein amyloidogenesis represents a trade-off within this RNA-protein interaction equilibrium, contributing to the viral etiology of PD.

biochemistry↗

bioRxiv2026

cellGeometry: ultra-fast single-cell deconvolution of bulk RNA-Seq using a geometric solution

Single-cell analysis has rapidly expanded to produce cell atlases encompassing all human tissues. However, computational methods to deconvolute bulk samples using single-cell reference data have failed to keep pace with the increasing data size. Here we present cellGeometry, which uses non-negative geometric deconvolution (NGD), an intuitive vector projection method featuring non-negative matrix regularisation. Using matrix operations, cellGeometry scales to massive datasets and is ultrafast. Benchmarked using simulations from single-cell RNA-Seq datasets with >3 million cells, cellGeometry is more accurate than existing methods and more robust against noise simulating different sequencing chemistries. It identifies outlying residual genes which may unveil pathogenic changes in gene expression and the presence of cell types absent from the reference. cellGeometrys flexible architecture allows merging of single-cell reference signatures to expand the range of cell types being deconvoluted. Validated against real bulk RNA blood and tissue samples, cellGeometry produces more accurate and realistic results.

bioinformatics↗

bioRxiv2025

Deep profiling of lupus nephritis kidneys reveals dynamic changes in myeloid cells associated with disease progression

ObjectivesLupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aim to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. MethodsSingle-cell RNA-sequencing (scRNA-seq) was used to profile dissociated kidney samples from 156 LN patients and 30 healthy individuals. Spatial transcriptomics (ST), utilizing a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, was applied to kidney samples acquired from 6 LN patients and 2 healthy controls. ResultsWe generated a full catalog of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the NIH chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4+ T, GZMK+CD8+ T, B and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are indicated to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. ConclusionsWe suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

immunology↗

bioRxiv2025

Functional and dysfunctional T regulatory cell states in human tissues in RA and other autoimmune arthritic diseases

Regulatory T cells (Tregs), characterized by FOXP3 expression, are essential for maintaining immune homeostasis by controlling inflammation. However, in autoimmune diseases such as rheumatoid arthritis (RA), impaired Treg function contributes to immune dysregulation and disease pathology. While most studies of human Tregs have focused on blood, here we analyzed Tregs in synovial tissues from RA patients using single cell RNA sequencing (scRNAseq). We identified two predominant Treg states, CD25hiCXCR6pos Tregs with strong suppressive function, and CD25loAREGpos Tregs, a dysfunctional state exclusively enriched in synovial tissues but not in blood. Computational and in vitro analyses revealed that cortisol induced AREG expression, suppressed glycolysis, and impaired the suppressive function of CD25loAREGpos Tregs. In turn, AREG promoted an IL-33+ inflammatory phenotype in synovial fibroblasts. Importantly, we found that TNFR2 engagement can prevent or reverse this dysfunctional Treg state. In contrast to CD25loAREGpos Tregs, CD25hiCXCR6pos Tregs were highly suppressive, showed coordinated abundance with macrophages in synovial tissue, and functionally interacted with membrane-bound TNF expressed by macrophages, which promoted their functional suppressive state. These two Treg subsets were similarly found in the synovial tissue in Juvenile Idiopathic Arthritis (JIA), another inflammatory arthritic disorder, indicating conserved mechanisms across arthritic diseases. Together, our findings define distinct pathways driving divergent functional and dysfunctional Treg states in inflamed tissues and point to interventions that may prevent or reverse the development of the dysfunctional state.

immunology↗

bioRxiv2025

Transcytosis-mediated anterograde transport of TrkA receptors controls formation of presynaptic sites in sympathetic neurons

In neurons, many membrane proteins, synthesized in cell bodies, must be efficiently delivered to axons to influence neuronal connectivity. Transcytosis is an atypical transport mode, where membrane proteins internalized from soma surfaces are anterogradely transported to axons. Here, we define the trafficking dynamics and the transport vesicles involved in transcytosis of TrkA neurotrophin receptors and demonstrate that transcytosis controls formation of presynaptic sites in sympathetic neurons. Live imaging and electron microscopy in compartmentalized cultures revealed that soma surface-derived TrkA receptors undergo dynamic movements in axons and are housed in endosomes and multi-vesicular bodies. Soma-surface labeled TrkA appear in nerve terminals, demonstrating transcytosis occurs in vivo. Notably, transcytosed TrkA receptors are enriched at presynaptic varicosities. Disruption of transcytosis impairs the number and morphology of presynaptic sites and decreases synaptic transmission. These findings provide mechanistic insight into an atypical mode of receptor trafficking and highlights its physiological relevance in sympathetic neuron connectivity.

cell biology↗

bioRxiv2025

An Inflammatory and Quiescent HSC Subpopulation Expands with Age in Humans

Aging of the blood system impacts systemic health and can be traced to hematopoietic stem cells (HSCs). Despite multiple reports on human HSC aging, a unified map detailing their molecular age-related changes is lacking. We developed a consensus map of gene expression in HSCs by integrating seven single-cell datasets. This map revealed previously unappreciated heterogeneity within the HSC population. It also links inflammatory pathway activation (TNF/NF{kappa}B, AP-1) and quiescence within a single gene expression program. This program dominates an inflammatory HSC subpopulation that increases with age, highlighting a potential target for further experimental studies and anti-aging interventions.

cell biology↗

bioRxiv2025

TCR germline diversity reveals evidence of natural selection on variable and joining alpha chain genes

T cell receptors (TCRs) orchestrate adaptive immunity, yet the complex, repetitive architecture of the TCR loci has impeded systematic characterization of human genetic variation in the genes encoding the TCR. Using public long-read sequencing data from 2,668 donors, we build a near-complete map of common alleles in TCR V, D, and J genes, revealing amino acid variation at almost every position within V genes. We discover pervasive evidence of natural selection on TCR genes, including balancing selection on a TRAJ gene recognizing an immunodominant influenza epitope and positive selection on a TRAV gene. We find TCR allelic polymorphism alters core functional properties of T cells, including thymic fate commitment, phenotypes in diseased tissues, and cell-surface receptor abundance. Collectively, our findings position inherited variation in TCR genes as a key axis of immunological diversity that may shape interindividual differences in immune responses.

immunology↗

bioRxiv2025

Growth of Paracoccus aminovorans is restricted by L-ascorbic acid (LA) and human gut isolate

Gut microbiota plays a crucial role in the outcome of cholera. In this context, Paracoccus aminovorans, a gut commensal, has been evidenced to positively influence the pathogenesis of cholera bacterium. In the present study, we examined the impact of various non-antibiotic strategies on the growth of Paracoccus aminovorans. We observed the growth of Paracoccus aminovarans was severely restricted in the presence of L-ascorbic acid as well as in co-culture growth assays with another gut commensal, Weissella confusa.

microbiology↗

bioRxiv2025

Defining effective strategies to integrate multi-sample single-nucleus ATAC-seq datasets via a multimodal-guided approach

BackgroundChromatin accessibility, measured via single-nucleus Assay for Transposase-Accessible Chromatin with sequencing (snATAC-seq), can reveal the underpinnings of transcriptional regulation across heterogeneous cell states. As the number and scale of snATAC-seq datasets increases, we need robust computational pipelines to integrate samples within a dataset and datasets across studies. These integration pipelines should correct cell-state-obfuscating technical effects while conserving underlying biological cell states, as has been shown for single-cell RNA-seq (scRNA-seq) pipelines. However, scRNA-seq integration methods have performed inconsistently on snATAC-seq datasets, potentially due to sparsity and genomic feature differences. ResultsUsing single-nucleus multimodal datasets profiling ATAC and RNA simultaneously, we can measure snATAC-seq integration method performance by comparison to independently integrated snRNA-seq gold standard embeddings and annotations. Here, we benchmark 58 pipelines, incorporating 7 integration methods plus 1 embedding correction method with 5 feature sets. Using our command-line tool, we assessed 5 multimodal datasets at 3 different resolutions using 2 novel metrics to determine the best practices for multi-sample snATAC-seq integration. ATAC features outperformed Gene Activity Score (GAS) features, and embedding correction with Harmony was generally useful. SnapATAC2, PeakVI, and ArchRs iterative Latent Semantic Indexing (LSI) performed well. ConclusionsWe recommend SnapATAC2 + Harmony with pre-defined ENCODE candidate cis-regulatory element (cCRE) features as a first-pass pipeline given its metric performance, generalizability of features, and method resource-efficiency. This and other high-performing pipelines will guide future comprehensive gene regulation maps.

genomics↗

bioRxiv2025

In vivo efficacy of L-ascorbic acid in restricting cholera pathogenesis

In the last three centuries, our world has experienced seven cholera pandemics. Cholera is a deadly diarrheal disease caused by Vibrio cholerae, an important member of the gamma-proteobacteria. Despite conventional treatments like antibiotics, vaccines, and ORS, the 7th cholera pandemic is still a major threat to developing nations. L-ascorbic acid was recently shown to effectively kill the Vibrio cholerae cells in vitro under various growth conditions mimicking the in vivo host conditions, including growth in the presence of bile salts, growth of acid-adapted V. cholerae, and growth in the presence of various ORS components. In the current study, we extend this work and test the efficacy of L-ascorbic acid in animal models (rabbit ileal loop and the removable intestinal tie adult rabbit diarrhea). We show that L-ascorbic acid can effectively reduce the bacterial load in both the rabbit models as well as fast-track the recovery from the diarrheal symptoms. ImportanceCholera is a severe human diarrheal disease that affects millions each year. Cholera treatment is primarily oral rehydration to balance the voluminous fluid loss. Previous studies have found that L-ascorbic acid (L-AA) is effective at inhibiting growth of Vibrio cholerae, the causative agent of cholera. Thus L-AA has potential as am inexpensive cholera therapeutic. Here, L-AA is tested in rabbit models to determine whether it can inhibit the effects of V. cholerae infection. Results suggest that L-AA treatment inhibits bacterial proliferation and leads to shortened recovery from disease.

microbiology↗

bioRxiv2025

Estimating the cis-heritability of gene expression using single cell expression profiles controls false positive rate of eGene detection

For gene expression traits, cis-genetic heritability can quantify the strength of genetic regulation in particular cell types, elucidating the cell-type-specificity of disease variants and genes. To estimate gene expression heritability, standard models require a single gene expression value per individual, forcing data from single cell RNA-sequencing (scRNA-seq) experiments to be "pseudobulked". Here, we show that applying standard heritability models to pseudobulk data overestimates gene expression heritability and produces inflated false positive rates for detecting cis-heritable genes. Therefore, we introduce a new method called scGeneHE (single cell Gene expression Heritability Estimation), a Poisson mixed-effects model that quantifies the cis-genetic component of gene expression using individual cellular profiles. In simulations, scGeneHE has a consistently well-calibrated false positive rate for eGene detection and unbiasedly estimates cis-heritability at many parameter settings. We applied scGeneHE to scRNA-seq data from 969 individuals, 11 immune cell types, and 822,552 cells from the OneK1K cohort to infer cell-type-specificity of genetic regulation at risk genes for immune-mediated diseases and trace the fluctuation of cis-heritability across cellular populations of varying resolution. In summary, we developed a new statistical method that resolves the analytical challenge of estimating gene expression cis-heritability from native scRNA-seq data.

genetics↗

bioRxiv2025

Early and late RNA eQTL are driven by different genetic mechanisms

Understanding the genetic regulation of RNA abundance is essential to defining disease mechanisms. However, conventional expression quantitative loci (eQTL) studies quantify RNA molecules across the transcript lifecycle. While most eQTL likely affect transcription by altering promoter or enhancer function within the nucleus, it is also possible that they modulate any processes after transcription, including chemical modifications and RNA stability in the cytosol. To elucidate distinct eQTL mechanisms of early versus late RNA, we compared eQTL from mature cellular RNA and nascent nuclear RNA in the brain and the kidney. Across tissues, we identified different causal variants for cellular and nuclear eQTL for the same eGene. Cellular eQTL were enriched in transcribed regions (P=3.3x10-126), suggesting the importance of post-transcriptional regulation. Conversely, nuclear eQTL were enriched in distal regulatory elements (P=7.0x10-32), highlighting the role of DNA transcriptional regulation. For example, we identified stop-gain eQTL variants likely acting through nonsense-mediated decay in cellular eQTL that had no effect in nuclear eQTL. Cellular eQTL were enriched for loci with multiple causal variants in linkage disequilibrium within the transcribed regions, where they may in concert affect RNA stability. We also identified examples of nuclear eQTL variants within enhancers that had no effect in cellular eQTL. We show that such eQTL (e.g., TUBGCP4) sometimes uniquely colocalize with disease alleles (schizophrenia). This study reveals key differences in the genetic mechanisms of cellular and nuclear eQTL.

genomics↗