Biology subjects

Ghosh, P.

Publications and source records attributed to Ghosh, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

COMPASS: A Web-Based COMPosite Activity Scoring System to Navigate Health and Disease Through Deterministic Digital Biomarkers

Reproducible and clinically transferable quantification of pathway activity remains a major barrier in precision medicine, where biomarker performance often depends on cohort composition and normalization strategies. Here, we introduce COMPASS (COMPosite Activity Scoring System), a deterministic threshold-based framework that converts gene expression into quantitative pathway activity scores without reliance on reference cohorts. COMPASS derives gene-specific activation thresholds directly from data, standardizes deviations from thresholds, and integrates directionally opposing genes into a single composite score. This enabled transparent activity scoring, statistical comparisons, and survival analyses without coding. Across diverse biological and clinical datasets, COMPASS robustly quantified cellular states, benchmarked the humanness and disease relevance of new approach methodologies, and stratified outcomes. Compared to GSVA and ssGSEA, COMPASS demonstrated greater consistency across datasets and improved robustness in bootstrap analyses, particularly for bidirectional programs, including regulatory-approved sepsis gene signatures. COMPASS therefore addresses a critical unmet need for exact, interpretable, and clinically transferable biomarker discovery and outcome modeling across diverse biological and clinical settings.

bioinformatics↗

bioRxiv2025

Efficient elimination of microbial infections in wounds using plasma activated water hydrogel

Surgical site infections are among the most common postoperative complications and remain a major global health concern, further intensified by increasing antimicrobial resistance. Plasma-activated water (PAW) has emerged as a novel antimicrobial strategy, though earlier studies suggested its activity depends on an acidic pH. To overcome this limitation, we previously developed a high-strength, neutral pH-buffered formulation (hs-PAbW) optimized for human clinical use, which preserved strong antimicrobial properties. In the present work, we created a hydrogel incorporating hs-PAbW at a bactericidal yet non-cytotoxic concentration (hs-PAbW-20) and evaluated its therapeutic performance in mouse wound models. In Balb/c mice, hs-PAbW-20 gel markedly reduced Pseudomonas aeruginosa and Staphylococcus aureus loads, demonstrating efficacy comparable to neomycin and soframycin. The gel also enhanced wound closure by regulating key genes involved in tissue repair. In a diabetic C57BL/6 model, hs-PAbW-20 gel similarly lowered P. aeruginosa burden and accelerated healing. Collectively, these results highlight hs-PAbW-20 gel as a promising next-generation wound therapy capable of addressing the shortcomings of traditional antibiotic-based treatments.

microbiology↗

bioRxiv2025

Acidic transcription factors position the genome at nuclear speckles through transcription dependent and independent mechanisms

A small fraction of the genome positions reproducibly near nuclear speckles (NS), increasing expression and/or splicing efficiency of NS-associated genes. How specific genomic regions target to NS remains unclear. Here, we demonstrate that establishment of genome-wide NS-association occurs independent of active transcription. We show that DNA sequences derived from NS-associated regions integrated as transgenes autonomously target to NS. By systematically dissecting one such genomic locus, the COL1A1-SGCA locus, we identified redundant NS-targeting cis regulatory elements, including a [~]600 bp fragment with 17 binding motifs for 8 transcription factors (TFs). Four NS-targeting TFs within this fragment contain acidic activation domains (AADs) that provide both chromatin-context and transcription-dependent NS-targeting, a property that appears common among several other tested AADs. A subset of acidic activator TFs contain an additional, transcription-independent NS-targeting activity. Our findings establish diverse and partially redundant NS-targeting activities, which may facilitate dynamic gene positioning at NS periphery for context-specific transcriptional responses.

cell biology↗

bioRxiv2025

Structural mechanisms for the recruitment of factor H by Streptococcus pyogenes

The major bacterial pathogen Streptococcus pyogenes (Group A Streptococcus, or Strep A) recruits the negative regulator of the alternative complement pathway factor H (FH) to its surface. Antigenically sequence variable regions of several Strep A M proteins, including M5 and M6, bind FH but have no obvious sequence homology. A second Strep A surface-localized protein, FbaA, binds FH through a purported coiled-coil region, suggesting mimicry of the well-known coiled coil of M proteins. We determined the structures of fragments of M5 protein, M6 protein, and FbaA complexed with FH domains 6 and 7 (FH(6-7)). M5 and M6 proteins formed dimeric -helical coiled coils, as expected, while FbaA instead consisted of a monomeric three-helix bundle preceded by a loop. FH(6-7) accommodated different binding modes in these three proteins, with very few common interacting amino acids. Based on contributions to binding, distinct FH-binding sequence patterns were constructed for M5 and M6 proteins, enabling identification of FH-binding sequences in M or M-like Enn proteins in 32 strains of differing M types. While FbaA was allelically sequence variable, its critical FH-binding amino acids were absolutely conserved in 95 strains of differing M types. Together, FH-binding sequences were identified in about half of the known 250 Strep A strains, with the majority due to FbaA. Our structural and functional elucidation of the mechanism of FH recruitment is applicable to precise investigation of its role in Strep A virulence.

biochemistry↗

bioRxiv2025

Intracellular formate is a critical mediator of the antimicrobial fate of Salmonella Typhimurium Against meropenem and ciprofloxacin

Salmonella enterica serovar Typhimurium (STM) employs various strategies to endure antibiotic stress, but the contribution of central metabolites to resistance remains insufficiently characterized. In this work, we identify intracellular formate, produced by pyruvate-formate lyase (PflB), as a key determinant of STM susceptibility to meropenem and ciprofloxacin. Deletion of pflB disrupts cellular pH homeostasis, impairing efflux pump function, increasing reactive oxygen species, and causing membrane depolarization, collectively heightening sensitivity to antibiotics. Supplementing extracellular formate reverses these phenotypes. Mechanistically, pH imbalance activates the RpoE-csrB regulatory pathway, leading to reduced expression of efflux pump genes acrB and tolC, while extracellular formate engages the BarA/SirA two-component system to further influence the CsrA/csrB network. Disrupting the formate pool interferes with efflux-dependent resistance, revealing a previously unrecognized signalling function for intracellular metabolites. Overall, our results position formate as a metabolic driver of antibiotic outcomes in STM and highlight it as a potential target for mitigating resistance.

microbiology↗

bioRxiv2025

RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

Mislocalization and cytoplasmic condensation of nuclear proteins, such as FUS and TDP43, are key features of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, mechanisms governing the disassembly of these pathological condensates remain elusive. Here, we identify a disordered RGG peptide as a disassembly factor for mutant FUS and TDP43 condensates. Using yeast, mammalian cells, and in vitro assays, we observe that an RGG motif reduces condensate burden and toxicity while restoring nuclear localization and RNA regulatory defects associated with the FUS-P525L mutant. The RGG motif interacts with FUS, competitively inhibiting FUS self-association. Overall, our findings uncover a direct role for a disordered RGG motif in the disassembly of ALS-associated condensates, highlighting the possibility of a new therapeutic for post-symptomatic ALS.

cell biology↗

bioRxiv2025

Distinct Colitis-Associated Macrophages Drive NOD2-DependentBacterial Sensing and Gut Homeostasis

Single-cell studies have revealed that intestinal macrophages maintain gut homeostasis through the balanced actions of reactive (inflammatory) and tolerant (non-inflammatory) subpopulations. How such balance is impaired in inflammatory bowel diseases (IBD), including Crohns disease (CD) and ulcerative colitis (UC), remains unresolved. Here, we define colon-specific macrophage states and reveal the critical role of non-inflammatory colon-associated macrophages (niColAMs) in IBD recovery. Through trans-scale analyses--integrating computational transcriptomics, proteomics, and in vivo interventional studies--we identified GIV (CCDC88A) as a key regulator of niColAMs. GIV emerged as the top-ranked gene in niColAMs that physically and functionally interacts with NOD2, an innate immune sensor implicated in CD and UC. Myeloid-specific GIV depletion exacerbates infectious colitis, prolongs disease, and abolishes the protective effects of the NOD2 ligand, muramyl dipeptide, in colitis and sepsis models. Mechanistically, GIVs C-terminus binds the terminal leucine-rich repeat (LRR#10) of NOD2 and is required for NOD2 to dampen inflammation and clear microbes. The CD-associated 1007fs NOD2-variant, which lacks LRR#10, cannot bind GIV--providing critical insights into how this clinically relevant variant impairs microbial sensing and clearance. These findings illuminate a critical GIV-NOD2 axis essential for gut homeostasis and highlight its disruption as a driver of dysbiosis and inflammation in IBD.

immunology↗

bioRxiv2024

CORTADO: Hill Climbing Optimization for Cell-Type SpecificMarker Gene Discovery

The advent of single-cell RNA sequencing (scRNA-seq) has greatly enhanced our ability to explore cellular heterogeneity with high resolution. Identifying subpopulations of cells and their associated molecular markers is crucial in understanding their distinct roles in tissues. To address the challenges in marker gene selection, we introduce CORTADO, a computational framework based on hill-climbing optimization for the efficient discovery of cell-type-specific markers. CORTADO optimizes three critical properties: differential expression in the clusters of interest, distinctiveness in gene expression profiles to minimize redundancy, and sparseness to ensure a concise and biologically meaningful marker set. Unlike traditional methods that rely on ranking genes by p-values, CORTADO incorporates both differential expression metrics and penalties for overlapping expression profiles, ensuring that each selected marker uniquely represents its cluster while maintaining biological relevance. Its flexibility supports both constrained and unconstrained marker selection, allowing users to specify the number of markers to identify, making it adaptable to diverse analytical needs and scalable to datasets with varying complexities. To validate its performance, we apply CORTADO to several datasets, including the DLPFC 151507 dataset, the Zeisel mouse brain dataset, and a peripheral blood mononuclear cell dataset. Through enrichment analysis and examination of spatial localization-based expression, we demonstrate the robustness of CORTADO in identifying biologically relevant and non-redundant markers in complex datasets. CORTADO provides an efficient and scalable solution for cell-type marker discovery, offering improved sensitivity and specificity compared to existing methods.

bioinformatics↗

bioRxiv2024

Spatiotemporal dynamics of EEG microstate networks over the first two years of life: A multi-cohort longitudinal study

The first two years of life are marked by rapid development of large-scale brain networks that support emerging cognition and behavior. Magnetic-resonance approaches have revealed much about largescale networks in sleep, but very little is known about functional network dynamics in awake, behaving infants during this period of substantial development. Microstates are brief instances of distinct spatial topographies of largescale neural activity measured with electroencephalography (EEG) that offer a novel approach to studying whole-brain network dynamics at sub-second scale in awake infants by capturing their temporally coherent brain activity. While emerging literature is leveraging microstate dynamics in adults to understand mature largescale network function, developmental trajectories during networks rapid construction in infancy remain uncharacterized. In this study, we leveraged longitudinal resting-state EEG data from 854 infants across two geoculturally diverse cohorts to explore largescale network development through EEG microstates over the first two years of life. We provide evidence for conserved emergence of various network configurations (microstate classes A-G) through infancy across cohorts using data-driven clustering analyses. We also demonstrate significant longitudinal changes in microstate dynamics during this period, characterized by more numerous and more rapid transitions between largescale configurations, especially over early infancy. While patterns of sensory microstate development were largely consistent between cohorts, higher-order cognitive microstates showed context-specific developmental trends. Together these results provide novel insights into how large-scale brain networks functionally develop and organize across the first two years of life.

neuroscience↗

bioRxiv2024

Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled

The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance may be evolutionarily conserved within insects.

developmental biology↗

bioRxiv2024

mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

In metazoans, epithelia perform functions of absorption, diffusion, and secretion. The actin-based apical projections on the epithelial cells, contribute to these functions and are formed via cell-autonomous mechanisms that control cell polarity, intracellular transport, and the cytoskeleton. However, the cues that function upstream of these cell-autonomous regulators remain poorly known. Using microridges on zebrafish epithelial cells as a paradigm, we show that mTOR, a metabolic sensor, regulates the formation of apical projections. Mechanistically, mTORC1 controls the RhoA-ROCK activity via S6K1 to prevent the overactivation of Non-muscle Myosin II (NMII) to restrict microridge elongation. Furthermore, genetic, biochemical, and molecular dynamics simulation analyses reveal that mTORC2 regulates microridge pattern by modulating the activity of aPKC via its differential phosphorylation at two conserved sites. We propose that mTOR integrates the developmental and/or metabolic status of epithelial cells with cell autonomously acting RhoA and aPKC to regulate tissue wide formation of apical projections.

cell biology↗

bioRxiv2024

Identification of transcriptome-wide cobalt chloride-induced hypoxia-responsive long noncoding RNAs regulated by cytoplasmic mRNA capping enzyme

Long non-coding RNAs (lncRNAs) are key regulators of gene expression in human cancers, influencing tumor microenvironment (TME). Hypoxia, a hallmark of solid tumors, elevates hypoxia-inducible factor 1 (HIF-1), which regulates hypoxia-responsive lncRNAs (HRLs). These HRLs are essential in regulating gene expression under hypoxic conditions at both transcriptional and post-transcriptional levels. However, the mechanisms governing the post-transcriptional regulation of lncRNAs are not fully understood. It has been shown that the cytoplasmic pool of mRNA capping enzyme (cCE) can post-transcriptionally regulate its substrate uncapped mRNAs and lncRNAs in the cytoplasm, thereby preventing their degradation. Our previous study showed elevated expression of cCE under hypoxic conditions. This study aimed to determine whether cCE influences the transcriptome-wide HRLs in CoCl2-induced hypoxia in osteosarcoma U2OS cells. We identified 306 known lncRNAs with significant differential expression under hypoxic conditions, including 137 upregulated and 169 downregulated by RNA sequencing. Gene Ontology enrichment analysis revealed their distinct association with various cancers. Our investigation aimed to ascertain whether the cCE post-transcriptionally regulates these HRLs. Overexpression of a dominant negative form of cCE suggested cCE might post-transcriptionally regulate these HRLs. Future research should focus on understanding how cCE influences substrate HRLs, which play a crucial role in the bidirectional signaling circuit between TME and cancer cells under hypoxic stress as well as establishing cCE as a key factor that may alter cellular responses to hypoxia by stabilizing cCE-targeted lncRNAs.

molecular biology↗

bioRxiv2024

Reactive nitrogen species (RNS) and its reaction intermediates with reactive oxygen species (ROS) in pH-neutral high-strength plasma-activated water determines the antimicrobial activity against ESKAPE Pathogens

Plasma-activated water (PAW) is an emerging antimicrobial strategy, though its efficacy has traditionally depended on acidic pH to generate reactive species. To improve clinical and practical applicability, we developed a neutral pH formulation using buffered water--hs-PAbW--and evaluated its antimicrobial activity and mechanism of action against ESKAPE pathogens. hs-PAbW significantly reduced bacterial viability during the exponential growth phase. While reactive oxygen species (ROS) alone were insufficient for bacterial killing, reactive nitrogen species (RNS), particularly peroxynitrite, mediated strong microbicidal effects via nitrotyrosine formation. These effects occurred independently of acidic conditions, demonstrating that antimicrobial activity is retained at neutral pH. Alongside, hs-PAbW effectively reduced bacterial burden on metal surfaces and exhibited superior killing compared to high doses of standard antibiotics, including ceftazidime, ciprofloxacin, and meropenem in vitro. A non-cytotoxic dose of hs-PAbW also reduced bacterial load in infected wounds and accelerated wound closure in vivo. These findings highlight the dual functionality of hs-PAbW in surface sanitation and wound care, and support its potential as a sustainable, broad-spectrum antimicrobial agent for clinical use.

bioengineering↗

bioRxiv2024

Engineered Nanobodies Bind Theranostic Main Group Metals

Targeted theranostics heavily rely on metal isotopes conjugated to antibodies. Single-domain antibodies, known as nanobodies, are much smaller in size without compromising specificity and affinity. The conventional way of conjugating metals to nanobodies involves non-specific modification of amino acid residues with bifunctional chelating agents. We demonstrate that mutagenesis of a single residue in a nanobody creates a triple cysteine motif that selectively binds bismuth which is, for example, used in targeted alpha therapy. Two mutations create a quadruple cysteine mutant specific for gallium and indium used in positron emission tomography and single-photon emission computed tomography, respectively. Labelling is quantitative within a few minutes. The metal nanobodies maintain structural integrity and stability over weeks, resist competition from endogenous metal binders like glutathione, and retain functionality.

biochemistry↗

bioRxiv2024

Logical Network Perturbations Guide Precise Target Prioritization for Drug Development

Despite advances in artificial intelligence (AI), target-based drug development remains costly, complex, and imprecise. We introduce F.O.R.W.A.R.D [Framework for Outcome-based Research and Drug Development], a network-based target prioritization platform, and demonstrate its utility in the challenging landscape of Inflammatory Bowel Diseases (IBD), a chronic, multifactorial condition. F.O.R.W.A.R.D uses real-world clinical outcomes, and a machine-learning classifier trained on transcriptomic data from seven prospective randomized trials across four drugs. It defines remission at the molecular level and calculates, using network connectivity, the likelihood that targeting a given molecule will induce remission-associated gene expression. Benchmarking against 210 completed trials across 52 targets, F.O.R.W.A.R.D achieved 100% predictive accuracy--despite variability in drug mechanisms and trial designs. Single-cell RNA-seq and a prospective biobank of patient-derived organoids confirmed that the remission signature is epithelium-specific and tracks with poor outcomes. F.O.R.W.A.R.D enables in-silico phase zero trials to inform trial design, revive shelved drugs, and guide early termination decisions. Broadly applicable and iteratively refined by emerging trial data, F.O.R.W.A.R.D has the potential to reshape drug discovery--bringing foresight to hindsight, and empowering both R&D and human-in-the-loop clinical decision-making.

bioinformatics↗

bioRxiv2024

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria

AbstractHflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX- mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic- bound inactive 50S subunits.

biochemistry↗

bioRxiv2024

Molecular Signatures for Microbe-Associated Colorectal Cancers

BackgroundGenetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. MethodsA Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum (Fn), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice (CPC-APCMin+/-) and patients (FAP, Lynch Syndrome, PJS, and JPS). ResultsThe MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APCMin+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. ConclusionsComputational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

microbiology↗

bioRxiv2024

Cell surface RNA expression modulates alveolar epithelial function

Glycosylated RNA (glycoRNA) has recently emerged as a novel constituent of the glycocalyx on cell surfaces, yet its biological functions remain largely unexplored. In this report, we present the first analysis of glycoRNA expression and functionality in alveolar epithelial cells. To this end, we optimized new techniques for the detection of glycoRNA on living cell surfaces and in cell membrane-associated RNA samples through in-gel imaging after labeling with fluorescent dye conjugates. Specifically, we used conjugation of Cy5-hydrazide following mild oxidation with sodium periodate for detection of the entire cell surface sialoglycoRNA pool. Conjugation of dibenzocyclooctyne-sulfo-Cy5 (DBCO-Sulfo-Cy5) in cells fed with tetraacetylated N-azidoacetyl-mannosamine (Ac4ManNAz) or 6-azido-L-fucose (FucAz) detected de novo formed sialoglycoRNA or fucoglycoRNA. Finally, biotinylated lectins in combination with infrared dye-conjugated streptavidin was used to differentiate between specific glycosidic linkages. Comparisons across primary alveolar epithelial cells and different alveolar-epithelial like cell lines revealed a cell-type specific variation in glycoRNA abundance. Treatment of primary alveolar epithelial cells with an RNAse cocktail reduced epithelial surface glycoRNA and was associated with a reduction in trans-epithelial electrical resistance and influenza A viral particle abundance. As such, the present work identifies glycoRNA as a novel component of the alveolar epithelial glycocalyx, suggesting its potential relevance in epithelial barrier regulation and viral infection.

physiology↗