Biology subjects

Saini, N.

Publications and source records attributed to Saini, N..

16 recordsLinked to original sources

bioRxiv2026

FOXP3-engineered regulatory T cells restore intestinal barrier integrity in Crohn's disease enteroids via PDGF-AA

Epithelial regeneration and barrier integrity are impaired in inflammatory bowel diseases, including Crohns disease (CD), yet current therapies largely target immune inflammation without directly promoting mucosal repair. While regulatory T cells are classically immunomodulatory, their capacity to directly support human intestinal stem cells (ISCs) and barrier function remains unclear. In this study, we tested the hypothesis that FOXP3-expressing regulatory T cells--engineered CD4LVFOXP3 and thymic-derived Treg (tTreg)--directly support human ISC maintenance and restore epithelial barrier function independent of their immunomodulatory function. Using CD patient ISCs-derived enteroids that display disease-associated damage, we established co-culture with FOXP3-engineered Treg cell-CD4LVFOXP3 or thymic-derived Treg (tTreg). The presence of either CD4LVFOXP3 or tTreg cells enhanced enteroid growth, improved epithelial barrier function, and restored apical-basal polarity of ISCs, indicating reparative capacity. Conversely, activated conventional CD4+ T cells reduced barrier function and abrogated apical-basal polarity. Integrating secretome profiling with ligand add-back and receptor or ligand blockade, we identify the PDGF-AA-PDGFR axis as a key regulator of Treg-mediated intestinal epithelial barrier integrity, but dispensable for Treg suppressive capacity. Collectively, our data delineate a direct, human tissue-intrinsic role of FOXP3-driven Treg in the interaction with ISCs via PDGF-AA-PDGFR, enhancing epithelial barrier function and positioning CD4LVFOXP3 as a treatment approach coupling immunoregulation with epithelial repair. One Sentence SummaryRegulatory T cells improve the intestinal epithelial barrier function, primarily, through the PDGF-AA-PDGFR axis

immunology↗

bioRxiv2026

Increased replication-associated single-stranded DNA promotes formaldehyde-induced mutagenesis.

Formaldehyde (FA) is an environmentally abundant and endogenously produced aldehyde that has been shown to cause DNA damage, mutagenesis, and carcinogenesis. Several studies have demonstrated that FA induces guanine mutations resulting in a mutation signature like SBS40. In this work, we demonstrate that replication defects generating single-stranded DNA (ssDNA) caused by the downregulation of the major replicative polymerases results in elevated FA mutagenesis. We found that loss of Mrc1 (CLASPIN) resulted in a high accumulation of ssDNA and FA mutagenesis, and that these phenotypes were not dependent on Mrc1s checkpoint activity. Loss of DNA-protein crosslink repair results in elevated FA sensitivity with no alteration to mutagenesis, likely due to the inability of the fork to bypass unprocessed protein adducts. Finally, we show that FA-induced mutagenesis is dependent on Pol {zeta}-mediated translesion synthesis, while deficiencies in the template switching pathway do not alter error-free bypass of FA adducts. Overall, our work points towards replication-associated ssDNA as a major substrate for FA-induced damage and elucidates the pathways that function to prevent FA mutagenesis at replication forks.

genetics↗

bioRxiv2025

COVID-19 induces persistent transcriptional changes in adipose tissue that are not associated with Long COVID

Long COVID is a heterogeneous condition characterized by a wide range of symptoms that persist for 90 days or more following SARS-CoV-2 infection. Now more than five years out from the onset of the SARS-CoV-2 pandemic, the mechanisms driving Long COVID are just beginning to be elucidated. Adipose tissue has been proposed as a potential reservoir for viral persistence and tissue dysfunction contributing to symptomology seen in Long COVID. To test this hypothesis, we analyzed subcutaneous adipose tissue (SAT) from two cohorts: participants with subacute COVID-19 (28-89 days post-infection) compared to pre-pandemic controls, and participants with Long COVID compared to those with those classified as "indeterminate" based on the RECOVER-Adult Long COVID Research Index (12-47 months post-infection). We found no evidence of persistent SARS-CoV-2 RNA in adipose tissue in any participant. SAT from participants with subacute COVID-19 displayed significant transcriptional remodeling, including depleted immune activation pathways and upregulated Hox genes and integrin interactions, suggesting resident immune cell exhaustion and perturbations in tissue function. However, no consistent changes in gene expression were observed between Long COVID samples and samples from indeterminant participants. Thus, SAT may contribute to inflammatory dysregulation following COVID-19, but does not appear to play a clear role in Long COVID pathophysiology. Further research is needed to clarify the role of adipose tissue in COVID-19 recovery.

immunology↗

bioRxiv2025

Methylglyoxal mutagenizes single-stranded DNA via Rev1-associated slippage and mispairing

Methylglyoxal (MG) is a highly reactive aldehyde that is produced endogenously during metabolism and is derived from exogenous sources such as sugary food items and cigarette smoke. Unless detoxified by glyoxalases (Glo1 and Glo2), MG can readily react with all major biomolecules, including DNA and proteins, generating characteristic lesions and glycation-derived by- products. As a result, MG exposure has been linked to a variety of human diseases, including cancers. Prior studies show that MG can glycate DNA, preferentially on guanine residues, and cause DNA damage. However, the mutagenicity of MG is poorly understood in vivo. In the context of cancer, it is essential to comprehend the true contribution of MG to genome instability and global mutational burden. In the present study, we show that MG can robustly mutagenize induced single-stranded DNA (ssDNA) in yeast, within a guanine centered mutable motif. We demonstrate that genome-wide MG mutagenesis in ssDNA is greatly elevated throughout the genome in the absence of Glo1, and abrogated in the presence of the aldehyde quencher aminoguanidine. We uncovered strand slippage and mispairing as the predominant mechanism for generation of all MG-associated mutations, and demonstrate that the translesion polymerase Rev1 is necessary in this pathway. Finally, we find that the primary MG-associated mutation is enriched in a variety of sequenced tumor datasets. We discuss the genomic impact of methylglyoxal exposure in the context of mutagenesis, DNA damage, and carcinogenesis.

genetics↗

bioRxiv2024

Patient-derived colon epithelial organoids reveal lipid-related metabolic dysfunction in pediatric ulcerative colitis

Background & AimsUlcerative colitis (UC) is associated with epithelial metabolic derangements which exacerbate gut inflammation. Patient-derived organoids recapitulate complexities of the parent tissue in health and disease; however, whether colon organoids (colonoids) model metabolic impairments in the pediatric UC epithelium is unclear. This study determined the functional metabolic differences in the colon epithelia using epithelial colonoids from pediatric patients. MethodsWe developed biopsy-derived colonoids from pediatric patients with endoscopically active UC, inactive UC, and those without endoscopic or histologic evidence of colon inflammation (non-IBD controls). We extensively interrogated metabolic dysregulation through extracellular flux analyses and tested potential therapies that recapitulate or ameliorate such metabolic dysfunction. ResultsEpithelial colonoids from active UC patients exhibit elevated oxygen consumption and proton leak supported by enhanced glycolytic capacity and dysregulated lipid metabolism. The hypermetabolic features in active UC colonoids were associated with increased cellular stress and chemokine secretion, specifically during differentiation. Transcriptomic and pathway analyses indicated a role for PPAR- in lipid-induced hypermetabolism in active UC colonoids, which was validated by PPAR- activation in non-IBD colonoids. Accordingly, limiting neutral lipid accumulation in active UC colonoids through pharmacological inhibition of PPAR- induced a metabolic shift towards glucose consumption, suppressed hypermetabolism and chemokine secretion, and improved cellular stress markers. Control and inactive UC colonoids had similar metabolic and transcriptomic profiles. ConclusionsOur pediatric colonoids revealed significant lipid-related metabolic dysregulation in the pediatric UC epithelium that may be alleviated by PPAR- inhibition. This study supports the advancement of colonoids as a preclinical human model for testing epithelial-directed therapies against such metabolic dysfunction. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSColon mucosa healing in pediatric UC requires reinstating normal epithelial function but a lack of human preclinical models of the diseased epithelium hinders the development of epithelial-directed interventions. New FindingsUsing colon biopsy-derived epithelial organoids, samples from pediatric patients with active UC show hyperactive metabolic function largely driven by enhanced lipid metabolism. Pharmacologic inhibition of lipid metabolism alleviates metabolic dysfunction, cellular stress, and chemokine production. LimitationsThough our epithelial colon organoids from active UC patients show targetable metabolic and molecular features from non-IBD controls, they were cultured under sterile conditions, which may not fully capture any potential real-time contributions of the complex inflammatory milieu typically present in the disease. Clinical Research RelevanceCurrent therapies for pediatric UC mainly target the immune system despite the need for epithelial healing to sustain remission. We identified a pharmacologic target that regulates epithelial metabolism and can be developed for epithelial-directed therapy in UC. Basic Research RelevancePediatric UC patient tissue adult stem cell-derived colon epithelial organoids retain disease-associated metabolic pathology and can serve as preclinical human models of disease. Excess reliance on lipids as an energy source leads to oxidative and inflammatory dysfunction in pediatric UC colon organoids. Preprint: This manuscript is currently on bioRxiv. doi: https://doi.org/10.1101/2024.08.22.609271 Lay Summary: Using patient tissue-derived colon epithelial organoids, the investigators identified epithelial metabolic dysfunction and inflammation in pediatric ulcerative colitis that can be alleviated by PPAR-a inhibition.

physiology↗

bioRxiv2024

Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to systematically delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We found that deletion of the nucleotide excision repair (NER) genes, RAD1 or RAD14, led to an increase in mutagenesis upon acetaldehyde exposure. Acetaldehyde-induced mutations were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair in{Delta} rad1 strains. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C[->]A changes coupled with an enrichment of gCn[->]A changes in the acetaldehyde-treated{Delta} rad1 isolates. The gCn[->]A mutation signature has been shown to be diagnostic of acetaldehyde exposure in yeast and in human cancers. We also demonstrated that the deletion of the two DNA-protein crosslink (DPC) repair proteases, WSS1 and DDI1, also led to increased acetaldehyde-induced mutagenesis. Defects in base excision repair (BER) led to a mild increase in mutagenesis, while defects in mismatch repair (MMR), homologous recombination repair (HR) and post replicative repair pathways did not impact mutagenesis upon acetaldehyde exposure. Our results in yeast were further corroborated upon analysis of whole exome sequenced liver cancers, wherein, tumors with defects in ERCC1 and ERCC4 (NER), FANCD2 (ICL repair) or SPRTN (DPC repair) carried a higher gCn[->]A mutation load than tumors with no deleterious mutations in these genes. Our findings demonstrate that multiple DNA repair pathways protect against acetaldehyde-induced mutagenesis.

genetics↗

bioRxiv2023

PROTEOMICS OF HYPOTHERMIC ADAPTATION REVEALS THAT RBM3 ENHANCES MITOCHONDRIAL METABOLISM AND MUSCLE STEM-CELL DIFFERENTIATION

Adaptation to hypothermic stress is important for skeletal muscle cells, but a comprehensive knowledge of molecular mediators is lacking. We show that adaptation to mild hypothermia (320C) improves the ability of skeletal muscle myoblasts to differentiate into myotubes in vitro. We performed proteomic analysis of mouse myoblasts exposed to mild hypothermia for various time points and identified dynamic changes in mitochondrial metabolism and proteostasis. This revealed that RBM3, an RNA-binding protein, increases progressively with acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation upon hypothermic adaptation. We also demonstrate that overexpression of RBM3 at physiological temperatures is sufficient to (i) enhance mitochondrial metabolism as judged by a decrease in the AMPK energy-sensing pathway, (ii) increase levels of proteins associated with translation and increase levels of 4E-BP1 phosphorylation, (iii) increase stem cell markers (MyoD1, PAX7), and improve differentiation of myoblasts from both young and aged mice.

biochemistry↗

bioRxiv2022

Acetaldehyde makes a distinct mutation signature in single-stranded DNA

Acetaldehyde (AA), a by-product of ethanol metabolism, is acutely toxic due to its ability to react with various biological molecules including DNA and proteins, which can greatly impede key processes such as replication and transcription and lead to DNA damage. As such AA is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Previous in vitro studies have shown that AA generates bulky adducts on DNA, with signature guanine-centered (GG[->]TT) mutations. However, due to its weak mutagenicity, short chemical half-life, and the absence of powerful genetic assays, there is considerable variability in reporting the genotoxic effects of AA in vivo. Here, we used an established yeast genetic reporter system and demonstrate that AA is highly mutagenic and makes strand-biased mutations on guanines (G[->]T) at a high frequency on single stranded DNA (ssDNA). We further demonstrate that AA-derived mutations occur through lesion bypass on ssDNA by the translesion polymerase Pol{zeta}. Finally, we describe a unique mutation signature for AA, which we then identify in several whole-genome and -exome sequenced cancers, particularly those associated with alcohol consumption.

genetics↗

bioRxiv2022

Methionine uptake via SLC43A2 transporter is essential for regulatory T lymphocyte survival

It is increasingly clear that cell death, survival or growth decisions of T lymphocyte subsets depend on interplay between cytokine-dependent and metabolic processes. What the metabolic requirements of T regulatory cells (Tregs) for their survival are, and how these requirements are satisfied remain to be fully understood. In this study, we identified a necessary requirement of methionine uptake and utilization for Tregs survival upon interleukin 2 (IL-2) deprivation. Activated Tregs have high methionine uptake and consumption to S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH). This methionine uptake is essential for Tregs survival, and is regulated by Notch1 activity. Notch1 controls the expression of the solute carrier protein SLC43A2 transporter during IL-2 deprivation. SLC43A2 is necessary for sufficient methionine uptake, and determines Tregs viability upon IL-2 withdrawal. Collectively, we identify a specifically regulated mechanism of methionine import in Tregs that is necessary for the survival of these cells. This highlights the need for methionine availability and metabolism in contextually regulating cell death in an immunosuppressive population of T lymphocytes.

immunology↗

bioRxiv2021

Clonal hematopoiesis is associated with increased toxicity in large B-cell lymphoma patients treated with chimeric antigen receptor T cell therapy

To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [≥]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [≥]3 ICANS [58.9% vs. 25%, p=0.02] and grade [≥]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028). Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.

genetics↗

bioRxiv2021

Effects of substrate stiffness and actin velocity on fibronectin fibril morphometry and mechanics

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also used the model to look at two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both tissue stiffness and contractile phenotype, with intermediate contractility and high tissue stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of tissue stiffness and cell contractility on FN fibril assembly.

biophysics↗

bioRxiv2020

Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic

Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella - another positive single stranded (ss) RNA virus. Each of the rubella isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2 - likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.

genetics↗

bioRxiv2020

Integrated genomic view of SARS-CoV-2 in India

India first detected SARS-CoV-2, causal agent of COVID-19 in late January-2020, imported from Wuhan, China. March-2020 onwards; importation of cases from rest of the countries followed by seeding of local transmission triggered further outbreaks in India. We used ARTIC protocol based tiling amplicon sequencing of SARS-CoV-2 (n=104) from different states of India using a combination of MinION and MinIT from Oxford Nanopore Technology to understand introduction and local transmission. The analyses revealed multiple introductions of SARS-CoV-2 from Europe and Asia following local transmission. The most prevalent genomes with patterns of variance (confined in a cluster) remain unclassified, here, proposed as A4-clade based on its divergence within A-cluster. The viral haplotypes may link their persistence to geo-climatic conditions and host response. Despite the effectiveness of non-therapeutic interventions in India, multipronged strategies including molecular surveillance based on real-time viral genomic data is of paramount importance for a timely management of the pandemic.

microbiology↗

bioRxiv2019

Signaling activated by nucleolar localised Notch4 Intracellular Domain underlies protection from genomic damage

The assembly of signaling hierarchies and their spatiotemporal organization together, contribute to diverse signaling outcomes. This is evident in the Notch pathway, which regulates an array of cellular processes, despite a small number of core components. Here, we describe a Notch4 activated signaling cascade, dependent on the nucleolar localization of the Notch4 Intracellular Domain (NIC4), that protects cells from genotoxic damage. Localization was assessed by immune-staining for endogenous Notch4 and visualization by confocal microscopy, in breast cancer cell lines. Live-cell, imaging-based, biophysical analysis of NIC4-GFP expressing cells, indicated unhindered mobility between the nucleolus and nucleoplasm and a stable nucleolar pool of NIC4-GFP. RNAi-mediated ablations, coupled with analysis of recombinant forms of NIC4 with modifications of its nucleolar localization sequence, confirmed nucleolar localization and identified the nucleolar proteins, Nucleolin and Fibrillarin, as key intermediates in the NIC4-activated signaling cascade. The transcriptional control of ribosome biogenesis (47s and 45s pre-rRNA transcription), emerged as another unexpected consequence of the subcellular distribution of NIC4. Taken together, this study describes intrinsic features of NIC4 that confer spatial flexibility and expand the repertoire of Notch4 signaling.

cancer biology↗

bioRxiv2018

MiR-195 regulates mitochondrial function by targeting mitofusin-2 in breast cancer cells

Mitochondrial dynamics is a highly dysregulated process in cancer. Apoptosis and mitochondrial fission are two concurrent events wherein increased mitochondrial fragmentation serves as a hallmark of apoptosis. We have shown earlier that miR-195 exerts pro-apoptotic effects in breast cancer cells. Herein, we have demonstrated miR-195 as a modulator of mitochondrial dynamics and function. Imaging experiments upon miR-195 treatment have shown that mitochondria undergo extensive fission. We validated mitofusin2 as a potential target of miR-195. Which may provide a molecular explanation for the respiratory defects induced by miR-195 over-expression in breast cancer cells? Active, but not total, mitochondrial mass, was reduced with increasing levels of miR-195. We have further shown that miR-195 enhances mitochondrial SOD-2 expression but does not affect PINK1 levels in breast cancer cells. Collectively, we have revealed that miR-195 is a modulator of mitochondrial dynamics by targeting MFN2 thereby impairing mitochondrial function. Concomitantly, it enhances the scavenger of reactive oxygen species (SOD-2) to maintain moderate levels of oxidative stress. Our findings suggest a therapeutic potential of miR-195 in both ER-positive as well as ER-negative breast cancer cells.

cancer biology↗

bioRxiv2017

Germline determinants of the somatic mutation landscape in 2,642 cancer genomes

Cancers develop through somatic mutagenesis, however germline genetic variation can markedly contribute to tumorigenesis via diverse mechanisms. We discovered and phased 88 million germline single nucleotide variants, short insertions/deletions, and large structural variants in whole genomes from 2,642 cancer patients, and employed this genomic resource to study genetic determinants of somatic mutagenesis across 39 cancer types. Our analyses implicate damaging germline variants in a variety of cancer predisposition and DNA damage response genes with specific somatic mutation patterns. Mutations in the MBD4 DNA glycosylase gene showed association with elevated C>T mutagenesis at CpG dinucleotides, a ubiquitous mutational process acting across tissues. Analysis of somatic structural variation exposed complex rearrangement patterns, involving cycles of templated insertions and tandem duplications, in BRCA1-deficient tumours. Genome-wide association analysis implicated common genetic variation at the APOBEC3 gene cluster with reduced basal levels of somatic mutagenesis attributable to APOBEC cytidine deaminases across cancer types. We further inferred over a hundred polymorphic L1/LINE elements with somatic retrotransposition activity in cancer. Our study highlights the major impact of rare and common germline variants on mutational landscapes in cancer.

genomics↗