Biology subjects

Deshmukh, S.

Publications and source records attributed to Deshmukh, S..

14 recordsLinked to original sources

bioRxiv2026

CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity

Understanding the cellular determinants of CAR-T cell-mediated cytotoxicity at the single-cell level remains a critical challenge in developing effective cellular immunotherapies. Conventional bulk co-culture assays provide only population-level measurements of cytotoxic activity and cannot resolve the functional heterogeneity of individual effector-target interactions. Here, we demonstrate the Cellanome platform, which enables the formation of CellCage Enclosures (CCEs) through spatially controlled photopolymerization, as a tool for high-resolution, high-throughput characterization of CAR-T cell-mediated cytotoxicity. By isolating defined effector-target combinations within individual CCEs and coupling encapsulation with longitudinal time-lapse imaging and automated image analysis, we resolve cytotoxic activity across thousands of individually tracked CCEs. Using anti-CD19 CAR-T effectors and NALM6-GFP targets, we show that individual CD8+ CAR-T effectors exhibit substantial functional heterogeneity, with serial killing capacity, effector-target contact dynamics, and intrinsic motility each independently correlating with cytotoxic potency. Cytotoxic efficacy scaled positively with effector number, and CD4+ T helper cells augmented CD8+-mediated killing in a dose-dependent manner, providing direct evidence for cooperative T cell behavior during tumor cell clearance. Extending the platform to a solid tumor model using AIC100 anti-ICAM-1 CAR-T cells and HeLa-GFP targets, we show that individual AIC100 effectors are insufficient to mediate effective killing, while multi-effector CCEs exhibit robust cytotoxicity scaling with effector abundance and CD4+:CD8+ composition. These data establish the Cellanome platform as a broadly applicable system for dissecting the determinants of CAR-T cell cytotoxicity at single-cell resolution, with direct translational relevance to the optimization of cellular immunotherapy.

immunology↗

bioRxiv2026

Sustained epigenetic rejuvenation of serially engrafting human iPSC-derived HSCs

Hematopoietic stem cell (HSC) function declines with age, contributing to immunosenescence and inferior transplantation outcomes. Here, we generated iPSC-derived HSCs (iHSCs) from multiple adult donors and performed integrated epigenetic, transcriptional, telomeric, and functional analyses to see if they retain youthful identity across differentiation and serial transplantation. Longitudinal DNA methylation profiling revealed that, independent of donor age, epigenetic age was reset to near zero in iPSCs and remained under seven years across differentiation and transplantation. In contrast, hematopoietic identity was established through a two-phase process: directional remodeling during in vitro differentiation extinguished pluripotency programs and initiated hematopoietic regulatory networks, while long-term engraftment was associated with a second wave of promoter methylation differences that converged toward primary adult HSCs. Notably, methylation at age-associated sites and global entropy remained stable across both phases, and single-cell telomere analysis demonstrated restoration of telomere length in iHSCs compared to primary adult HSCs. Youthful epigenetic features were maintained through secondary transplantation. These findings demonstrate that long-term HSC identity can be achieved independently of epigenetic aging and establish a framework for evaluating rejuvenated stem cell-derived grafts in regenerative medicine.

cell biology↗

bioRxiv2026

Scalable longitudinal imaging and transcriptomics of cells in dynamic enclosures

Dynamic transitions between cell states underlie both normal physiology and disease. However, most single-cell technologies capture only static snapshots. To address this gap, we developed a platform that integrates light-guided hydrogel polymerization with computer vision to generate on-demand compartments around live cells, enabling longitudinal imaging of cellular behavior paired with whole-transcriptome profiling of the same cells at scale. These data link dynamic phenotypes with molecular programs, enabling deeper characterization of cellular states. This approach revealed an adaptive, drug-resistant state in lung cancer cells characterized by potassium channel upregulation and p53-dependent quiescence. In models of adipogenesis and microglial phagocytosis, joint analysis of imaging and transcriptomic data identified key drivers of cellular function that were missed by transcriptomic clustering alone. These results establish the value of paired functional and transcriptomic analysis to resolve molecular drivers of complex cellular behaviors.

systems biology↗

bioRxiv2026

Rational Design Reveals Structural Plasticity of the CsgA β-Solenoid Enabling Programmable Autogenic Engineered Living Materials

Microbial functional amyloids play central roles in biofilm formation and serve as foundational building blocks for autogenic engineered living materials (ELM), yet the structural design space governing their assembly and stability remains poorly defined. In Escherichia coli, the {beta}-solenoid protein CsgA functions as a canonical extracellular matrix scaffold, but prior engineering efforts have primarily focused on terminal functionalization rather than modification of the {beta}-solenoid core itself. Here, inspired by the evolutionary diversification of CsgA-like proteins, which expands along the vertical fiber axis, we explore a second orthogonal axis of structural plasticity: the horizontal dimension of the {beta}-solenoid. We rationally designed a library of CsgA variants in which the length of individual {beta}-strands was systematically reduced or extended from the native seven residues to lengths spanning 3-21 residues, while preserving conserved gate residues and loop regions. Integration of AI-based structure prediction using AlphaFold2 with all-atom molecular dynamics simulations reveals that {beta}-solenoid stability arises from a balance among strand length, residue composition, and solvent interactions, thereby defining both lower and optimal bounds for nanofiber assembly. Experimental validation demonstrates that engineered Escherichia coli can secrete and assemble these CsgA variants into extracellular nanofibers through the native curli biogenesis machinery, while preserving the characteristic cross-{beta} architecture. Additionally, the CsgA {beta}-solenoid variant library translates molecular design into macroscopic ELM, with deletion variants showing an inverse relationship between stiffness and extensibility, from highly extensible 3aa to stiff, strong 5aa films. Insertion-based variants largely retain CsgA-like extensibility while enabling tunable stiffness and strength across strand lengths. Together, these findings uncover previously unrecognized structural plasticity in microbial {beta}-solenoid proteins and establish {beta}-strand length as a generalizable design parameter linking molecular architecture to nanofiber stability, with implications spanning microbial functional amyloids and the rational design of autogenic engineered living materials.

bioengineering↗

bioRxiv2025

De novo Autogenic Engineered Living Functional Materials

Autogenic engineered living materials (ELMs) involve in situ production and engineering of native extracellular matrix (ECM). However, the existing autogenic ELMs have limited scope and functionalities. Herein, we report a platform for de novo autogenic functional ELMs. By protein mining, we have discovered CsgA-like 33,564 homologs that can find potential utility as de novo ECM of protein nanofibers. By employing AlphaFold2 and molecular dynamics simulations, we shed insights into the CsgA-like {beta}-solenoid protein structures and stability. By hacking Escherichia coli curli machinery, we demonstrate the production of de novo autogenic ELMs from CsgA-like proteins ([≤]9-times the molecular weight and {beta}-sheet repeat units) of extremophilic non-model bacteria. Additionally, we biomanufacture macroscopic materials with tunable mechanical properties (enhancing storage modulus by 3-times) and programmable functionalities (3D printability, binding to nanoparticles/antibodies). This work showcases a versatile platform to discover, rationally design, and harness the sophisticated functionalities of natural systems for futuristic autogenic ELMs.

bioengineering↗

bioRxiv2024

MrHAMER2: high-accuracy long-read RNA sequencing to decode isoform-specific variation in viral transcripts during latency

Alternative splicing (AS) greatly expands the repertoire of proteins encoded by the human genome. Viruses have been shown to hijack AS cellular pathways to sustain replication or lead to latency. In HIV-1 infection, the virus integrates into the host genome, becoming a transcriptional unit that directly engages in AS to regulate its gene expression. Sequencing advances have enabled insights into HIV-1 gene expression dynamics during productive replication. However, viral isoform dynamics during latency remain largely uncharacterized due to the low abundance of spliced viral transcripts in associated CD4+ T cell subsets, making their accurate detection and quantification challenging. MrHAMER2 is a high-accuracy long-read RNA sequencing method that leverages dual Unique Molecular Identifier (UMI) tagging of cDNA to accurately capture and quantify full-length isoforms with high dynamic range and 99.968% single-nucleotide accuracy. We used MrHAMER2 to decode the spliced HIV-1 transcriptome in a primary CD4+ T cell model of latency and showed substantial changes in viral isoforms bearing intron retentions accompanied by changes in their potential to generate translatable protein.

genomics↗

bioRxiv2024

R-Spondin Mimetic, SZN-043, Induced Proliferation and Wnt Activity, Two Features Deficient in Human Alcohol-Associated Liver Disease

Liver regeneration is impaired in patients suffering from alcohol-associated liver (ALD) diseases. Wnt ligands and their FZD receptors are dysregulated in diseased livers. R-spondin and their receptors are known to regulate Wnt activity via the stabilization of FZD receptors. Here, we investigated the components of the Wnt and R-Spondin-signaling pathways and their activity in patients with ALD. We found that while hepatocytes retained high levels of differentiation markers such as ASGR1 and ASGR2, the expression of two R-spondin co-receptors, LGR4 and LGR5, and of Wnt target genes, CYP1A2 and others, were strongly reduced. SZN-043, a hepatocyte-targeted R-Spondin mimetic, is a new investigational drug that stimulates the physiological Wnt repair pathway and proliferation of hepatocytes. Here, we show that SZN-043 induced hepatocyte proliferation in all models tested, including humanized mouse livers, a chronic-binge alcohol-induced liver injury, and a CCl4-induced fibrosis mouse model. Altogether, SZN-043 could be beneficial for the treatment of ALD.

pathology↗

bioRxiv2024

Reprogramming Human Inflammatory Macrophages in Symptomatic Carotid Stenosis: Potential Mechanisms for Stabilisation of Atherosclerotic Carotid Plaques

BackgroundInflammation is a precursor to atherosclerotic plaque destabilisation, leading to ischaemic events such as stroke. Macrophage phenotypes can be altered by the microenvironment, and certain anti-inflammatory agents may, therefore, stabilise plaques and reduce the risk of recurrent ischaemic events. MethodsThirteen carotid plaques were obtained from stroke/ Transient Ischaemic Attack (TIA) patients undergoing carotid endarterectomy. An immunofluorescence stain was used to identify common macrophage markers (pan macrophage: CD68, pro-inflammatory: CD86, anti-inflammatory: MRC1), and a novel analysis technique was used to measure the prevalence of macrophage phenotypes in carotid plaques in relation to other histological features of instability. An in vitro model of human blood-derived macrophages was also developed to evaluate the effect of statins and glucocorticoids on macrophage-specific markers using RT-qPCR, Western Blot and immunofluorescence stain. The physiological effect of dexamethasone was further evaluated on macrophages and human carotid plaques cultured ex vivo. ResultsThe macrophage population (CD68+) in the carotid plaques was dominated by "double-positive" (CD86+MRC1+) macrophages (67.8%), followed by "M1-like" (CD86+MRC1-) (16.5%), "M2-like" (CD86-MRC1+) (8.7%) and "double-negative" (CD86-MRC1-) (7.0%) macrophages. M1-like macrophages were more prevalent in unstable plaque sections than stable ones (p=0.0022). Exposure to dexamethasone increased macrophage MRC1 gene expression in vitro and ex vivo. Dexamethasone also reduced Oxidised Low-Density Lipoprotein Receptor 1 (OLR1) gene and protein expression, leading to a decreased ox-LDL uptake in foam cell assays. This was, in turn, associated with reduced lipid uptake in macrophages, as shown by Oil Red O staining. ConclusionsHuman macrophages may be "switched" to a less inflammatory phenotype by exposure to clinically relevant concentrations of glucocorticoid, potentially mediated by a reduction in Oxidised LDL uptake. This effect was not observed following macrophage exposure to statins. Glucocorticoids may have a future role in preventing ischaemic events in patients with advanced atherosclerosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/598440v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@bb389borg.highwire.dtl.DTLVardef@18151b8org.highwire.dtl.DTLVardef@11c28a1org.highwire.dtl.DTLVardef@1af6acd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA high prevalence (68% in this study) of carotid plaque macrophages express both pro-inflammatory (CD86) and anti-inflammatory (MRC1) markers. These may represent a novel macrophage population. C_LIO_LIHuman macrophages may be "reprogrammed" to a less inflammatory phenotype following exposure to glucocorticoids. C_LIO_LIDexamethasone increased MRC1 and decreased OLR1 expression in macrophages derived from human blood samples in vitro and in cells derived from cultured human carotid plaque tissue ex vivo. This was associated with reduced oxLDL uptake and reduced lipid accumulation in the macrophages. C_LIO_LIDexamethasone has the potential to stabilise carotid atherosclerotic plaques in humans. C_LI

cell biology↗

bioRxiv2023

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights{square} The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions. {square}The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs. {square}Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture. {square}The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

molecular biology↗

bioRxiv2023

Rapid evolution of pre-zygotic reproductive barriers in allopatric populations.

Adaptive divergence leading to speciation is the major evolutionary process generating diversity in life forms. The most commonly observed form of speciation is allopatric speciation which requires that gene flow be prevented between populations by physical or temporal barriers, as they adapt to their respective environments. Eventually, these adaptive responses drive the populations far apart in the genotypic space such that individuals from the two populations become reproductively isolated. A widely accepted theory is that speciation simply occurs as a by-product of adaptive response of the populations1,2. Several ecological and laboratory examples of allopatric speciation exist3-6. However, we know little about the nature (pre- or post-zygotic) of barriers that arise first in this process. Understanding the first barriers that arise between populations is key, as populations diverge towards becoming distinct species. In recent years, fungi been used as model organisms to answer questions related to evolution of reproductive isolation3,7-9. Here we show rapid evolution of pre-zygotic barriers between allopatric yeast populations. We further demonstrate that these pre-zygotic barriers arise due to altered mating kinetics of the evolved population. Moreover, our non-adaptive evolution experiments with yeast under limited selection pressure also show rapid emergence of reproductive isolation. Overall, our results show that evolution of pre-zygotic reproductive barriers can occur as result of natural selection or drift. These barriers result because of altered mating kinetics or mate preference. One sentence summaryPre-zygotic barriers to gene flow can arise due to adaptation or drift.

evolutionary biology↗

bioRxiv2020

H3 K27M and EZHIP impede H3K27-methylation spreading by inhibiting allosterically stimulated PRC2

Diffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M- and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.

cancer biology↗

bioRxiv2020

Genomic balancing selection is key to the invasive success of the fall armyworm

A successful biological invasion involves survival in a newly occupied environment. If a population bottleneck occurs during an invasion, the resulting depletion of genetic variants could cause increased inbreeding depression and decreased adaptive potential, which may result in a fitness reduction. How invasive populations survive in the newly occupied environment despite reduced heterozygosity and how, in many cases, they maintain moderate levels of heterozygosity are still contentious issues1. The Fall armyworm (FAW; Lepidoptera: Spodoptera frugiperda), a polyphagous pest, is native to the Western hemisphere. Its invasion in the Old World was first reported from West Africa in early 2016, and in less than four years, it swept sub-Saharan Africa and Asia, finally reaching Australia. We used population genomics approaches to investigate the factors that may explain the invasive success of the FAW. Here we show that genomic balancing selection played a key role in invasive success by restoring heterozygosity before the global invasion. We observe a drastic loss of mitochondrial polymorphism in invasive populations, whereas nuclear heterozygosity exhibits a mild reduction. The population from Benin in West Africa has the lowest length of linkage disequilibrium amongst all invasive and native populations despite its reduced population size. This result indicates that balancing selection increased heterozygosity by facilitating the admixture of invasive populations from distinct origins and that, once heterozygosity was sufficiently high, FAW started spreading globally in the Old World. As comparable heterozygosity levels between invasive and native populations are commonly observed1, we postulate that the restoration of heterozygosity through balancing selection could be widespread among successful cases of biological invasions.

evolutionary biology↗

bioRxiv2020

Genome-wide comparison of DNA methylation between life cycle stages of Drosophila melanogaster using high-throughput sequencing techniques.

Drosophila melanogaster undergoes holometabolous development, has very low levels of DNA methylation, and is known to possess a single known methyltransferase, dDNMT2. This study compares the DNA methylation patterns between the two life cycle stages of D. melanogaster using a combination of DNA immunoprecipitation and high throughput sequencing techniques. Our results indicate, a change in the chromosomal distribution of the sparse DNA methylation concerning genes and natural transposable elements between in the embryo and the adult stages of D. melanogaster. The differentially methylated regions localised on genes involved in the regulation of cell cycle processes of mitotic cell divisions and chromosomal segregation. dDNMT2 knockout flies exhibited altered patterns of DNA methylation. The observed differences in DNA methylation were in genes involved in cellular communication and cytoskeletal functions. The variation in DNA methylation between the two life cycle stages is indicative that it could have a role in regulatory processes during development and, dDNMT2 may have a role as a co-factor for the hitherto undiscovered DNA methyltransferase in D. melanogaster.

genomics↗

bioRxiv2019

PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Polycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors- the K27M oncohistone and the EZHIP oncohistone-mimic- that dysregulate gene silencing to promote tumorigenesis.

cancer biology↗