Biology subjects

Mukherjee, G.

Publications and source records attributed to Mukherjee, G..

12 recordsLinked to original sources

bioRxiv2026

Raman spectral signatures reflect altered metabolic state of exhausted T cells

T cell exhaustion within the tumor microenvironment drives CD8+ T cells into a dysfunctional state characterized by progressive loss of proliferative capacity and effector functions, thereby limiting anti-tumor immunity and therapeutic efficacy. To investigate the biochemical alterations associated with exhaustion, an in vitro model of CD8+ T cell exhaustion was established through chronic antigenic stimulation of murine OT-1 CD8+ T cells. Phenotypic, functional, metabolic, and transcriptional characterization confirmed the acquisition of an exhausted state. Single-cell Raman spectroscopy was subsequently employed to generate biochemical signatures of activated, and exhausted CD8+ T cells. Principal component analysis (PCA) of the Raman spectral data revealed distinct separation of these two cell subsets, reflecting underlying biochemical differences associated with their functional states. Differential Raman spectral features corresponding to nucleic acids, carbohydrates, proteins, and lipids contributed significantly to this segregation, reflecting altered metabolic and biosynthetic states during exhaustion progression. Classification of the spectral data using machine-learning algorithms enabled accurate segregation of activated and exhausted T cells. Collectively, this study demonstrates that single-cell Raman spectroscopy can distinguish exhausted CD8+ T cells in a label-free and non-destructive manner, highlighting its potential as a platform for immune profiling and monitoring T cell dysfunction.

immunology↗

bioRxiv2026

Data-driven prioritization of mouse strains for improved preclinical modeling of rare and common disease

Choosing an appropriate mouse genetic background is a persistent challenge for successful translation of preclinical disease modeling. We present Strain Recommender, a genomic framework that prioritizes inbred mouse strains as relatively vulnerable or resilient to a disease state using disease-associated gene signatures and strain-specific transcriptome predictions. The method represents disease states as weighted gene scores, ranks 657 strains based on resemblance to the disease state, and estimates uncertainty via a permutation-derived false positive rate (FPR). In a prospective validation of connective tissue disorder predictions, vulnerable and resilient Collaborative Cross strains showed significantly different cardiovascular abnormalities. In a global retrospective validation predicting previously reported strain background effects, Strain Recommender achieved [≥] 90% sensitivity for 86.6% of diseases with 94.4% mean sensitivity (95% CI: 94.0-94.8%) across 5,890 diseases, including 92.3% (95% CI: 91.6-93.0%) for 2,598 rare diseases, demonstrating its potential to improve the validity of mouse models of human disease.

bioinformatics↗

bioRxiv2025

Synergy unleashed: Leishmania donovani GP63 paralogues cooperatively function to drive parasite infectivity at various pathogenic stages and regulate host survival mechanisms

Highly plastic genome of Leishmania is thought to underlie its remarkable ability to adapt to diverse host microenvironments, yet how this plasticity translates into dermal versus visceral persistence remains unclear. GP63, a zinc metalloprotease, is a well-established determinant of promastigote infectivity, particularly in cutaneous leishmaniasis. Despite this, its role in amastigote stage and in visceral disease caused by Leishmania donovani remains undefined. Here, using an integrated approach combining phylogenetics, CRISPR-mediated genome editing, and biochemical analyses, we demonstrate that GP63 paralogues in L. donovani have undergone independent evolutionary diversification to support visceral infection. While GP63-paralogues encoded on chromosome 10, previously implicated in cutaneous manifestation caused by L. major, were found severely truncated and dispensable in L. donovani, two distinct GP63-paralogues encoded on chromosome 28 and 31 were found essential to establish visceral infection. LdGP63_31 facilitates promastigote attachment to macrophages by promoting lipid raft engagement and complement inactivation, thereby enabling host entry and subsequent amastigote differentiation. LdGP63_28 is essential for intracellular amastigote survival by suppressing host inflammatory pyroptosis. Structural and enzymatic analyses revealed preferential host localization and substrate specificities, possibly resulting from distinct amino acid substitution within conserved motifs of these proteases, indicating independent evolutionary adaptations. Importantly, loss of the LdGP63_28 more severely impaired amastigote reinfection than loss of its counterpart LdGP63_31. Together, these findings reveal coordinated functional specialization of GP63-paralogues ensuring visceral adaptation.

microbiology↗

bioRxiv2024

Exploiting gene expression profiles of circulating extracellular vesicles for breast cancer detection

BackgroundLiquid biopsy-based biomarkers offer several advantages since they are minimally invasive, can be useful in longitudinal monitoring of the disease and have higher patient compliance. We hypothesize that RNA content of circulating EVs differs in breast cancer patients and healthy women. EV RNAs may provide an opportunity to diagnose, detect subtypes and metastatic states. Experimental DesignRNA-seq analysis and qRT-PCR from matched tumor biopsy, circulating EVs from breast cancer patients (EV-C) and healthy donors (EV-H) was performed to find genes that discriminate between these groups. ResultsEV-C to EV-H comparison yielded 320 DEGs (adjusted p value [≤]0.05) enriched for cancer related pathways like Myc, Reactive oxygen species, and Angiogenesis. Allograft rejection and Interferon pathway genes were over-represented in the cancer group. Top 6 genes were validated by qRT-PCR in a validation cohort. 5 genes consistently and correctly classified cancer and healthy groups. An independent set of 374 and 640 DEGs could segregate ER positive/ER negative and non-metastatic versus metastatic samples, respectively. EVs from metastatic samples had higher variability in gene expression patterns whereas those from non-metastatic samples showed better correlation. Ability of 4 genes to classify metastases state was explored. ConclusionWe report five EV RNAs that can be used to diagnose breast cancer in a subtype independent manner. Initial analysis indicates that EV RNA content differs based on subtype specificity and metastasis status.

cancer biology↗

bioRxiv2024

Multiresolution molecular dynamics simulations reveal the interplay between conformational variability and functional interactions in membrane-bound cytochrome 2B4

Cytochrome P450 2B4 (CYP 2B4) is one of the best characterized CYPs and serves as a key model system for understanding the mechanisms of microsomal class II CYPs, which metabolize most known drugs. The highly flexible nature of CYP 2B4 is apparent from crystal structures that show the active site with either a wide open or a closed heme binding cavity. Here, we investigated the conformational ensemble of the full-length CYP 2B4 in a phospholipid bilayer, using multiresolution molecular dynamics (MD) simulations. Coarse-grained MD simulations revealed two predominant orientations of CYP 2B4s globular domain with respect to the bilayer. Their refinement by atomistic resolution MD showed adaptation of the enzymes interaction with the lipid bilayer, leading to open configurations that facilitate ligand access to the heme binding cavity. CAVER analysis of enzyme tunnels, AquaDuct analysis of water routes, and Random Acceleration Molecular Dynamics simulations of ligand dissociation support the conformation-dependent passage of molecules between the active site and the protein surroundings. Furthermore, simulation of the re-entry of the inhibitor bifonazole into the open conformation of CYP 2B4 resulted in binding at a transient hydrophobic pocket within the active site cavity that may play a role in substrate binding or allosteric regulation. Together, these results show how the open conformation of CYP 2B4 facilitates binding of substrates from and release of products to the membrane, whereas the closed conformation prolongs the residence time of substrates or inhibitors and selectively allows the passage of smaller reactants via the solvent and water channels. ImpactOur findings from multiresolution molecular dynamics simulations elucidate the structural dynamics of the membrane-bound CYP 2B4 enzyme and how these affect substrate recognition and processing, as well as inhibitor binding. As CYP 2B4 is a representative of the class of cytochrome P450 enzymes that metabolize most known drugs, the insights from this study are pertinent to the prediction of drug metabolism and the design of CYP inhibitors as therapeutics.

biochemistry↗

bioRxiv2024

O6-Alkylguanine-DNA Alkyltransferase Maintains Genomic Integrity During Peroxynitrite-Mediated DNA Damage by Forming DNA-Protein Crosslinks

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases and may be fatal. Peroxynitrite (PN) is a major reactive nitrogen species (RNS) generated during inflammation. It produces various DNA lesions including labile 8-nitroguanine which spontaneously converts into abasic sites resulting in DNA strand breakage. Here, we report the discovery of a previously unrecognized function of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate can spontaneously react with 8-nitroguanine in DNA to form a stable DNA-protein crosslink (DPC). Interestingly, the process of DPC formation provides protection from PN-mediated genome instability. The Cys145-mutant of hAGT failed to form DPC and provide protection against inflammation-associated, PN-mediated cytotoxicity. Gel shift, dot blot and UV-Vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs may seem detrimental, there are multiple systems in place in normal cells for their repair.

pharmacology and toxicology↗

bioRxiv2023

A Robust Deep Learning Approach for Joint Nuclei Detection and Cell Classification in Pan-Cancer Histology Images

Advanced image processing methods have shown promise in computational pathology, including the extraction of crucial microscopic features from histology images. Accurate detection and classification of cell nuclei from whole-slide images (WSI) play a crucial role in capturing the molecular and morphological landscape of the tissue sample. They enable widespread downstream applications, including cancer diagnosis, prognosis, and discovery of novel markers. Robust nuclei detection and classification are challenging due to the high intra-class variability and inter-class similarity of the microscopic morphological features. This is further compounded by the domain shift arising due to the variability in tissue types, staining protocols, and image acquisition. Motivated by the ability of the recent deep learning techniques to learn complex patterns in a biasfree manner, we develop a novel and robust deep learning model TransNuc, based on vision transformers, for simultaneous detection and classification of cell nuclei from H&E stained WSI. We benchmarked TransNuc on the comprehensive Open Pan-cancer Histology Dataset (PanNuke), sampled from over 20,000 WSI, comprising 19 different tissue types and five clinically important cell classes, namely, Neoplastic, Epithelial, Inflammatory, Connective, and Dead cells. TransNuc exhibited superior performance compared to the state-of-theart, including Hover-Net and Micro-Net. TransNuc was able to learn robust feature representations and thereby perform consistently better for the abundant classes such as neoplastic, and the under-represented classes such as dead cells. Similar performance gains were also obtained for epithelial and connective classes that have a significant inter-class morphological similarity.

pathology↗

bioRxiv2022

Comparative analysis of drug-salt-polymer interactions by experiment and molecular simulation improves biopharmaceutical performance

The propensity of poorly water-soluble drugs to aggregate at supersaturation impedes their bioavailability. The emergence of supersaturated amorphous drug-salt-polymer systems provides a new approach to this problem. However, the effects of polymers on drug-drug interactions in aqueous phase are largely unexplored and it is unclear how to choose an optimal salt-polymer combination for a particular drug. We describe a comparative experimental and computational characterization of amorphous solid dispersions containing the drug celecoxib, and PVP-VA or HPMCAS polymers with or without Na+/K+ salts. Classical models for drug-polymer interactions fail to identify the best drug-salt-polymer combination. In contrast, more stable drug-polymer interaction energies computed from molecular dynamics simulations correlate with prolonged stability of supersaturated amorphous drug-salt-polymer systems, along with better dissolution and pharmacokinetic profiles. The celecoxib-salt-PVP-VA formulations exhibit excellent biopharmaceutical performance, offering the prospect of less frequent administration and lower doses of this widely used anti-inflammatory, thereby increasing cost-effectiveness, and reducing side-effects.

pharmacology and toxicology↗

bioRxiv2022

mTORC2 Drives ZFX-mediated Ganglioside Biosynthesis to Promote Breast Cancer Progression.

Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside biosynthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in luminal breast cancer-specific MCF-7 and BT-474 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian luminal breast cancer tissues, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.

cancer biology↗

bioRxiv2021

Targeting "Immunogenic Hotspots" in Dengue and Zika Virus: A Novel Approach to a Common Vaccine

Diseases caused by Dengue (DENV) and Zika (ZIKV) viruses cause significant mortality and illness globally. Due to the high sequence similarity of the viral proteins and the purported cross-reactive immune responses against the viruses, we envisioned a common multi-epitope vaccine (MEV) against both viruses by adopting a novel approach of identifying "immunogenic hotspots". These stretches of the structural and non-structural proteins are enriched with MHC class I and class II supertype-restricted T cell epitopes, and B cell epitopes, in addition to being highly conserved between different DENV serotypes and ZIKV. Such an approach ensures inclusion of multiple overlapping T and B cell epitopes common to both viruses, and also warrants high population coverage. Importantly, epitopes known to cause antibody-dependent-enhancement of infection have been excluded. These immunogenic hotspots have then been stitched together with linkers in-silico along with an adjuvant, CTxB to develop the MEV candidate. Four structural models of the MEV were selected on the basis of conformational preservation of CTxB, and their biophysical parameters, which also conserved the immunogenicity of the multiple epitopes. Importantly, each of the MEV candidates were found to interact with TLR4-MD2 complex by molecular docking studies, indicative of their ability to induce TLR-mediated immune responses.

immunology↗

bioRxiv2020

Investigation of COVID-19 comorbidities reveals genes and pathways coincident with the SARS-CoV-2 viral disease

The emergence of the SARS-CoV-2 virus and subsequent COVID-19 pandemic initiated intense research into the mechanisms of action for this virus. It was quickly noted that COVID-19 presents more seriously in conjunction with other human disease conditions such as hypertension, diabetes, and lung diseases. We conducted a bioinformatics analysis of COVID-19 comorbidity-associated gene sets, identifying genes and pathways shared among the comorbidities, and evaluated current knowledge about these genes and pathways as related to current information about SARS-CoV-2 infection. We performed our analysis using GeneWeaver (GW), Reactome, and several biomedical ontologies to represent and compare common COVID-19 comorbidities. Phenotypic analysis of shared genes revealed significant enrichment for immune system phenotypes and for cardiovascular-related phenotypes, which might point to alleles and phenotypes in mouse models that could be evaluated for clues to COVID-19 severity. Through pathway analysis, we identified enriched pathways shared by comorbidity datasets and datasets associated with SARS-CoV-2 infection.

bioinformatics↗

bioRxiv2020

Electron Transfer from Cytochrome P450 Reductase to Cytochrome P450: Towards a Structural and Dynamic Understanding

Cytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP-CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP-CPR ET rates are low compared to those of soluble bacterial CYPs.

biochemistry↗