Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 181 records · Page 10Linked to original sources

bioRxiv2025

A scalable organoid model of urothelial aging for metabolic interrogation, infection modeling, and reversal of age-associated changes

Aging leads to a progressive decline in overall bladder function resulting in lower urinary tract symptoms and increased susceptibility to infections. However, tissue-specific mechanisms of aging, specifically the contributions of the aged urothelium remain elusive. Here, we introduce mouse bladder epithelium-derived organoids (mBEDOs) as a scalable platform to model urothelial aging. mBEDOs from aged mice recapitulate key features of age-associated cellular reprogramming, including oxidative stress, senescence, and DNA damage. We demonstrate the utility of mBEDOs for modeling Uropathogenic Escherichia coli (UPEC) infection, generating assembloids between mBEDOs and macrophages to model epithelial-immune interactions, and genetic perturbation. Using the mBEDO platform, we also identify urothelium-specific changes in purine, amino acid, and glycerophospholipid metabolism which may contribute to age-associated cellular perturbations. Lastly, supplementation with depleted metabolites, nicotinamide (NAM) and D-mannose, reduce DNA damage and oxidative stress and restore mitochondrial integrity in aged mBEDOs. These findings establish mBEDOs as an effective platform for investigating molecular and cellular underpinnings of urothelial aging and exploring metabolism-based interventions for age-associated bladder dysfunction.

bioengineering↗

bioRxiv2025

Pan-cancer prediction of tumor immune activation and response to immune checkpoint blockade from tumor transcriptomics and histopathology

Accurately predicting which patients will respond to immune checkpoint blockade (ICB) remains a major challenge. Here, we present TIME_ACT, an unsupervised 66-gene transcriptomic signature of tumor immune activation derived from TCGA (The Cancer Genome Atlas) melanoma data. First, we demonstrate that TIME_ACT scores accurately identify tumors with activated immune microenvironments across different cancer types. Further, analysis of spatial features reveals that tumor microenvironment regions with dense lymphocyte infiltration near tumor cells have high TIME_ACT scores, successfully marking localized immune activation. Second, across 25 transcriptomic ICB cohorts encompassing nine cancer types, TIME_ACT achieves a mean AUC of 0.76 and a mean odds ratio of 5.77, significantly outperforming 30 established transcriptomic signatures and prediction methods for ICB response, including a recently developed foundation model for immunotherapy response prediction. Third, we show that TIME_ACT scores can be accurately inferred from routine tumor histopathology slides and that slide-inferred TIME_ACT scores predict ICB response across nine new independent patient cohorts spanning eight cancer types, achieving a mean AUC of 0.72 and a mean odds ratio of 4.99. These findings establish TIME_ACT as a robust, pan-cancer biomarker that enables accurate, low-cost, and clinically scalable prediction of ICB response from routine histopathology.

cancer biology↗

bioRxiv2025

Lineage memory shapes viral resistance barriers in human skin

Individual cells within a given population exhibit striking variability in viral susceptibility, but it remains unknown whether this heterogeneity reflects memories encoded into the cellular lineage or true probabilistic variability. We used multi-color lineage tracing in a human primary organotypic skin model to reveal that viral resistance is encoded within specific cellular lineages. These lineages create distinct boundaries that block viral spread. Our lineage analyses in vitro confirmed that viral susceptibility exhibits strong heritability across cell generations, with siblings and cousins displaying remarkably similar infection outcomes. ATAC and proteomics profiling of resistant and susceptible clones revealed distinct epigenomic and proteomic states, with the transcription factor AP-1 emerging as a potential central regulator of lineage-encoded viral resistance. Inducing AP-1 activity with PMA rendered cells resistant to viral infection, suggesting a causative role in mediating resistance memory. Our findings demonstrate that antiviral resistance in human skin cells is encoded within cellular lineages and preserved through cell divisions, revealing how cell memory may shape infection dynamics and viral containment in tissues.

bioengineering↗

bioRxiv2025

Mammalian mitochondrial polyphosphate is regulated by the 5-InsP7 synthase IP6K1

Inorganic polyphosphate (polyP) is a linear polymer of varying length composed of phosphate residues linked by phosphoanhydride bonds. PolyP remains poorly understood in mammals due to its low abundance and lack of information on its synthesis and regulation. Using synthetic polyP8 as a bait, we identified polyP interacting proteins involved in RNA splicing, transcription, and mitochondrial function. We developed a DAPI fluorescence-based assay to quantify the low levels of polyP present in mammalian cell lines and tissues, and detected a higher concentration of polyP in the mitochondria compared with the nucleus and post-mitochondrial fraction. We observed that mitochondrial polyP synthesis relies on active FoF1 ATP synthase and an intact proton gradient across the inner mitochondrial membrane. Our data shows that orthophosphate (Pi) is required for the generation of mitochondrial polyP and that the presence of ATP enhances Pi-driven polyP synthesis in isolated mitochondria. We discovered that the inositol pyrophosphate 5-InsP7, synthesized by IP6K1, regulates mitochondrial polyP levels. Mice and cells deficient in IP6K1 showed a significant reduction in mitochondrial polyP synthesis compared with wild type controls. Cells lacking IP6K1 also showed impaired mitochondrial respiration and membrane potential. The expression of active IP6K1, but not its catalytically inactive form, restored mitochondrial polyP synthesis and membrane potential in IP6K1 deficient cells, but mitochondrial respiration was rescued by expression of either active or inactive IP6K1. These data show that IP6K1 regulates mitochondrial function and polyP production through both the synthesis of 5-InsP7 and via a catalytic activity-independent mechanism. Our findings uncover a link between 5-InsP7, an energy sensor, and polyP, an energy store, in the regulation of mammalian mitochondrial homeostasis.

biochemistry↗

bioRxiv2025

AI-Guided Design and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

The structural design of chimeric antigen receptors (CARs) is critical for achieving robust and durable anti-tumor responses, particularly when targeting multiple antigens to prevent tumor antigen escape. However, increasing CAR complexity can introduce structural vulnerabilities, leading to antigen-independent T cell activation, activation-induced cell death, and reduced CAR-T cell persistence. To overcome these challenges, we designed 10,824 CAR molecules across diverse formats and screened 1,452 constructs in-vitro to develop an artificial intelligence model, termed CAR-Mediated Self-Destruction (CARMSeD), which predicts CAR designs susceptible to self-activation and dysfunction. Guided by CARMSeD and structural CAR-CAR interaction modeling, we identified optimized CAR architectures incorporating ICOS and 4-1BB co-stimulatory domains. Humanized bispecific CARs targeting CD20/CD19 and CD22/CD19 demonstrated superior anti-tumor efficacy and persistence both in-vitro and in various xenograft mice models. To further extend CAR-T cell persistence, we engineered bispecific CARs integrated with an AKT3-targeted PROTAC strategy. Targeted degradation of AKT3 enhanced anti-tumor potency, promoted memory T cell formation, and enabled sustained responses even under tumor rechallenge and CD19 antigen-loss conditions. Mechanistically, these effects were mediated by metabolic reprogramming involving FOXO4; notably, FOXO4-deficient CAR-T cells exhibited impaired long-term persistence. Leveraging these mechanistic insights, we developed a trispecific CAR-T cell platform incorporating a bispecific T cell engager (BiTE) targeting CD22/CD3, combined with AKT3 PROTACs. These trispecific CAR-T cells achieved potent tumor eradication, even against malignancies lacking both CD19 and CD20 expression. Collectively, this study presents a comprehensive strategy combining structure-based design, AI-guided screening, and targeted protein degradation to engineer next-generation bi and trispecific CAR-T cells with enhanced persistence, broad antigen coverage, and superior therapeutic durability.

cancer biology↗

bioRxiv2025

Cyclo-stationary distributions of mRNA and Protein counts for random cell division times

There is a long history of using experimental and computational approaches to study noise in single-cell levels of mRNA and proteins. The noise originates from a myriad of factors: intrinsic processes of gene expression, partitioning errors during division, and extrinsic effects, such as, random cell-cycle times. Although theoretical methods are well developed to analytically understand full statistics of copy numbers for fixed or Erlang distributed cell cycle times, the general problem of random division times is still open. For any random (but uncorrelated) division time distribution, we present a method to address this challenging problem and obtain exact series representations of the copy number distributions in the cyclo-stationary state. We provide explicit cell age-specific and age-averaged results, and analyze the relative contribution to noise from intrinsic and extrinsic sources. Our analytical approach will aid the analysis of single-cell expression data and help in disentangling the impact of variability in division times.

systems biology↗

bioRxiv2025

Proteomic characterization of major fish allergy responsive protein parvalbumins in Hilsa (Tenualosa ilisha): A commercially important fish in Southeast Asia

The protein parvalbumin (PRV)-beta (PRVB) is the primary cause of food allergies to bony fish. Although PRVB is a well-characterized protein in many bony fishes, little is known about the Hilsa, an anadromous fish with great economic importance and predominantly found in Southeast Asia. In this study, we characterized the Hilsa PRV utilizing various proteomic approaches in response to two major riverine habitats and developmental stages. Unique peptide sets corresponding to three PRV isoforms were identified in Hilsa muscle tissues. Label-free quantitative proteomic analysis coupled with ELISA revealed higher levels of PRVB in young fish compared to adults, irrespective of their riverine habitats. A comparative quantitative analysis of PRVB further demonstrated that Hilsa had less PRVB than other commonly consumed freshwater fish species. Multiple reaction monitoring (MRM)-based targeted proteomic approach showed the potential of PRV as a marker protein for allergen quantitation and authenticating the presence of Hilsa in a complex freshwater fish mixture. Our findings collectively offer fundamental knowledge on Hilsa PRVs for further investigation on the food safety and quality evaluation of Hilsa fish.

biochemistry↗

bioRxiv2025

Time Series GWAS for Iron Deficiency Chlorosis Tolerance in Soybean using Aerial Imagery

AbstractThe use of drones has become a commonly used tool by plant scientists to aid in plant phenotyping endeavors. Iron deficiency chlorosis (IDC) is a commonly observed abiotic stress in soybean fields with high soil pH levels. IDC severity is visually classified, and recent work has shown that digital imaging techniques using both ground and UAS-acquired imagery can be utilized for automated severity ratings. In our study, we compared the classification accuracy of two flight altitudes to determine the optimal flight parameters for IDC phenotyping. In addition to this, we investigated the ability to use image-predicted scores for genome wide association study (GWAS), as well as the effect of IDC on traits such as canopy area and canopy growth and development. We also report a tool for semi-automated plot extraction from orthomosaic images that can be easily integrated with UAS. We noted that 43 days after planting was an ideal time for IDC severity ratings as the highest number of significant SNPs were reported at this timepoint.

genetics↗

bioRxiv2025

Engineering Large-Scale and Innervated Functional Human Gut for Transplantation

A confined culture system (CCS) establishes methods to generate complex functional human gastrointestinal tissues. This approach yields large-scale innervated small intestinal, colonic and gastric organoids with an elongated tubular shape for both in vitro and in vivo studies. Transcriptomic and electrophysiological data demonstrate the co-development of a functional de novo enteric nervous system, which is absent from conventional organoids. When compared to traditional methods, CCS derived small intestinal, colonic and gastric organoids reached maturation supporting transplantation in half of the time, resulting in enhanced engraftment rates and sizes. Murine luminal content exposure within CCS organoids in vivo further augmented function, supporting the potential translational benefits required to model complex intestinal diseases. In summary, the CCS methodology simplifies current protocols while adding complexity and expediting the generation of clinically relevant functional gut tissues.

bioengineering↗

bioRxiv2025

Fluctuation Test with Phenotypic Switching: A Unified Stochastic Approximation Framework

This paper examines a structurally symmetric fluctuation test experiment in which cell populations grow from a single cell to a set size before undergoing treatment. During growth, cells may acquire tolerance to treatment through probabilistic events, which are passed to progeny. Motivated by recent research on drug tolerance in microbial and cancer cells, the model also allows tolerant cells to revert to a sensitive state, reflecting dynamic phenotypic switching. The master equation governing the probability distribution of tolerant cells is solved via the generating function method and the quasi-powers approximation. Depending on model parameters, the distribution may be approximated by a stable distribution (or its special case, the normal distribution) or through large deviations theory. In the regime of frequent switching, the large deviations approach provides better agreement with numerical solutions, particularly at distribution tails. Conversely, in the regime of infrequent switching, the general stable distributions offer improved accuracy over the Landau distribution, which represents a limiting distribution in case of unidirectional switching.

systems biology↗

bioRxiv2025

Bursty Gene Expression in Single Cells and Expanding Populations: A Discrete Approach

Bursty protein production is a key source of gene expression noise. In this study, we analyze a Markovian model of cellular protein dynamics, where proteins are produced in geometrically distributed bursts. Extending this model, we incorporate a feedback mechanism by assuming that higher protein levels reduce both cell growth and protein decay rates. We study both single-cell dynamics and an expanding cell population. Without feedback, the protein level follows a negative binomial distribution with the same parameters in both cases. With feedback, however, single-cell and population-level distributions differ, each expressible as a mixture of two negative binomial distributions with framework-dependent parameters. Using numerical integration of the master and population balance equations, we calculate the time-dependent distributions in both settings. This work extends previous continuous models and provides new insights into how population expansion influences intrinsic cellular heterogeneity.

systems biology↗

bioRxiv2025

Human genetic variation shapes the antibody repertoire across B cell development

The peripheral antibody repertoire is shaped by inherited genetic variation and selection during B cell development. However, how the repertoire changes across developmental stages--and the relative impact of genetics and selection in establishing the antibody repertoire--remain largely unknown. To dissect the individual and combined effects of these factors, we integrated antibody repertoire transcriptomics across pro-B, pre-B, immature, and naive B cells with single-molecule real-time long-read DNA sequencing of the immunoglobulin heavy (IGH), kappa (IGK), and lambda (IGL) chain loci. We find that IGH genetic variants establish gene usage biases at the pro-B stage that persist throughout development. In contrast, IGK and IGL repertoires are extensively remodeled during maturation, consistent with receptor editing. Surprisingly, IGH variants also exert trans effects on light chain gene usage, suggesting coordinated constraints on heavy-light chain pairing. Principal component and allele-specific analyses reveal that genetics primarily shapes the heavy chain repertoire, while selection more strongly influences light chain diversity. These findings define distinct dynamics of antibody repertoire development and underscore the importance of personal immunogenomics in understanding individual immune responses.

genomics↗

bioRxiv2025

Epstein-Barr virus and Helicobacter pylori Coinfection Drives Gut and Brain Barrier Dysfunction

Biological barriers, including blood-brain barrier (BBB) and gut epithelial barrier, are essential to maintain homeostasis and provide protection against pathogenic stress. Here, we investigated the impact of Helicobacter pylori-Epstein Barr Virus coinfection on gut-brain axis integrity. Our results demonstrate that coinfection induces significant disruptions in tight and adherens junction (TJ and AJ respectively) proteins in gastric epithelial cells, including downregulation of ZO-1 and E-cadherin. The secretome from these coinfected cells significantly compromises the integrity of BBB-derived endothelial cells, including cleavage of VE-cadherin, loss of TJ proteins such as Claudin-5, and enhanced permeability. Furthermore, the sole exposure of mice to coinfected secretome lead to the loss of cerebral VE-cadherin and ZO-1 and the induction of neuroinflammation, characterized by elevated pro-inflammatory cytokines, TNF-, amyloidogenic accumulation, and neurocognitive deficits. The secretome driving such forces potentially carries pathogenic metabolites, host inflammatory cytokines and metabolites that might contribute to disease pathogenesis as observed in this study. Together, our current study provides novel insights into how coinfections contribute to gut-brain axis dysregulation and neurological pathogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/655045v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@138ca23org.highwire.dtl.DTLVardef@15eccc4org.highwire.dtl.DTLVardef@d588c6org.highwire.dtl.DTLVardef@106a485_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗

bioRxiv2025

DNA cross-over motifs based, programmable supramolecular hydrogels for mechanoregulatory effects of cellular behaviour and cytoskeleton reorganization

The development of mechanically tunable DNA-based supramolecular hydrogels that recapitulate the dynamic mechanoelastic properties of native extracellular matrices (ECMs) is critical for advancing 3D tissue engineering. DNA supramolecular hydrogels with their precise programmable architecture and impeccable control at the nanometre scale assembly make them a highly lucrative biopolymer in the design of ECM-mimicking scaffolds. This study explores the fabrication of stiffness-customizable rigid DNA hydrogels with varying branching architectures, including Double Crossovers (DX), Paranemic Crossovers (PX), and Tensegrity motif structures with three to six arms. Our study reveals that by incorporating different numbers of crossovers, branching arms, and the type of sticky-armed linkers, it is possible to generate a range of DNA hydrogels with varying network geometry and mechanoelastic properties ranging from 50-300 kPa, without any chemical / enzymatic crosslinking aid. These DNA hydrogels mechanoregulate cellular behaviour in retinal pigment epithelial (RPE1) cells by enhancing cellular adhesion, elongation (3 - 8x area increase vs. control), viability (concentration-dependent, 1 - 8x vs. control), organellar homeostasis, including mitochondrial fragmentation and ER stress attenuation. This work establishes a framework for automating DNA hydrogel-based scaffold fabrication processes to meet the preferences of various cells/tissues with bespoke mechanical cues, advancing personalized high-throughput tissue engineering platforms. Graphical abstract

bioengineering↗

bioRxiv2025

Translation Efficiency Impacts Phage Lysis Timing and Its Precision in Single Cells

The timing of host cell lysis is a fundamental life history parameter in bacteriophages as it presents an evolutionary trade-off between maximizing intracellular phage replication and optimizing transmission to new host cells, ultimately determining viral fitness in dynamic bacterial populations. In the bacteriophage {lambda}, lysis time is dependent on the expression of a timekeeper protein, holin, which accumulates in the Escherichia coli inner membrane. Cell lysis is triggered when the membrane concentration of holin crosses a critical threshold level. In the present study, we investigated the effects of the rate of holin translation on lysis timing and its precision. We show that modulating holin translation efficiency through genetic modifications and antibiotic treatment alters bacteriophage lysis timing and its precision, providing insight into how phages optimize the evolutionary trade-off between replication and transmission. Reducing ribosomal binding affinity decreased holin expression and delayed lysis, while optimizing the Shine-Dalgarno sequence enhanced translation and accelerated lysis. Surprisingly, very low tetracycline concentrations may have improved translation efficiency and hastened lysis, whereas higher doses predictably delayed it. In all cases, longer lysis times corresponded with decreased timing variability. A model that incorporates stochastic gene expression with additional stochasticity in the initiation of holin expression was sufficient to explain the data. Thus, we demonstrated that modifying the rate of holin translation can be used as an evolutionary strategy to achieve optimum lysis timing. ImportanceOur prior work demonstrated that phage lysis timing is calibrated to optimize fitness based on environmental conditions affecting host availability and phage replication. We identified an optimal threshold--the critical holin concentration--that minimizes lysis time variability. Genetic manipulation of the holin gene altered the lysis threshold and generated phages with non-optimal early and late lysis times. The current study reveals that adjusting holin translation rates offers another mechanism for modifying lysis timing. Phages therefore have multiple means to adjust the timing of host lysis in order to maximize fitness under different selective pressures.

microbiology↗

bioRxiv2025

Induced tolerance to UV stress drives survival heterogeneity in isogenic E. coli cell populations

The emergence of transiently tolerant bacterial subpopulations challenges our understanding of stress tolerance mechanisms. While much is known about antibiotic tolerance, it remains unclear whether similar mechanisms contribute to survival under ultraviolet (UV) stress. Here, we employed a modified Luria-Delbruck fluctuation test to investigate the presence of pre-existing UV-tolerant subpopulations in Escherichia coli. Our results showed no significant evidence of pre-stress UV tolerance. Instead, the data suggest that survival is primarily driven by inducible DNA repair responses activated after UV exposure. Furthermore, sequential low-dose UV exposures yielded higher-than-expected survival, suggesting that transient tolerance can be induced following initial UV exposure, likely through active DNA repair processes. These findings indicate that E. coli survives UV stress via an induced, rather than pre-existing, mechanism of tolerance.

microbiology↗

bioRxiv2025

Hemolytik2: An Updated Database of Hemolytic Peptides and Proteins

Hemolytik 2.0 (http://webs.iiitd.edu.in/raghava/hemolytik2/) is a comprehensive, manually curated database that provides experimentally validated information on both hemolytic and non-hemolytic peptides. Data were meticulously extracted from peer-reviewed publications and established peptide repositories, including the Antimicrobial Peptide Database (APD), UniProt, the Collection of Anti-Microbial Peptides (CAMP-R4), and the Dragon Antimicrobial Peptide Database (DAMPD). This updated version of the original Hemolytik resource encompasses 13,215 entries, representing approximately 8,700 unique peptides. Each entry in Hemolytik 2.0 offers detailed annotations, including peptide name, amino acid sequence, biological source and origin, functional characterization, terminal modifications, stereochemistry, structural classification (linear or cyclic), and experimentally determined hemolytic activity. In addition, the database provides molecular representations of peptides in SMILES (Simplified Molecular Input Line Entry System) format, alongside predicted tertiary structures and annotated secondary structural states. Additionally, a RESTful API has been integrated into the Hemolytik 2.0 repository to enable programmatic access and automated retrieval of peptide data. Hemolytik 2.0 serves as a valuable resource for the scientific community, particularly for researchers involved in the design and development of therapeutic peptides, by facilitating the identification and optimization of peptide candidates with minimal hemolytic potential and enhanced safety profiles.

bioinformatics↗

bioRxiv2025

Gene regulatory network transitions reveal the central transcription factors in lung adenocarcinoma progression

Transcription factors play a central role in cancer growth, progression, and metastasis, and contribute to intratumor phenotypic plasticity which enable drug tolerance and cancer relapse. Changes in the regulatory activities of transcription factors in cancer may not always be detected by investigating mutational signatures or differential expression of the transcription factor, as done in traditional analysis. In addition, past studies have focused on the activities of transcription factors in tumors as a whole and thus, have not fully captured the heterogeneity in gene regulation among different cell types within the tumor microenvironment. In this work, through an analysis of the transitions in regulatory network architecture and gene regulation dynamics, we identify the central transcription factors associated with lung adenocarcinoma progression. The gene NR2F1, associated with neurodevelopment and cancer dormancy, emerge as a key transcription factor in the progression of lung adenocarcinoma. We further identify transcription factors that are only active in cancer samples and uncover how changes in gene regulation dynamics influence intratumor heterogeneity. Taken together, our work elucidates the transitions in gene regulatory network during cancer progression, identifies central transcription factors in this process, and reveals the complex regulatory changes cooccurring in different cell types within the tumor microenvironment.

systems biology↗