Biology subjects

Singhi, A. D.

Publications and source records attributed to Singhi, A. D..

7 recordsLinked to original sources

bioRxiv2026

MitoChontrol: Adaptive mitochondrial filtering for robust single-cell RNA sequencing quality control

SummaryMitochondrial transcript abundance is a standard quality control metric in single-cell RNA sequencing, but fixed percentage thresholds fail to account for the substantial variation in mitochondrial content across cell types and tissues, risking both retention of compromised cells and exclusion of transcriptionally active viable cell populations. We present MitoChontrol, a cell-type-aware probabilistic framework for mitochondrial quality control that models the mitochondrial transcript fraction within transcriptionally coherent clusters as a Gaussian mixture distribution. Compromised-cell components are identified from the upper tail of each cluster-specific distribution, and filtering thresholds are defined as the point at which the posterior probability of cellular compromise exceeds a user-definded confidence value. Applied to controlled perturbation experiments and a pancreatic ductal adenocarcinoma single-cell dataset, MitoChontrol selectively removes transcriptionally compromised cells while preserving biologically elevated but viable populations, outperforming fixed-threshold and outlier-based approaches. Availability and ImplementationMitoChontrol is implemented in Python and integrates directly with AnnData-based workflows. It is freely available under the GNU General Public License v3 (GPL-3.0) at: https://github.com/uttamLab/MitoChontrol (DOI: https://doi.org/10.5281/zenodo.19423054)

bioinformatics↗

bioRxiv2025

SLFN11 restricts escape from telomere crisis to prevent alternative lengthening of telomeres

The tRNA nuclease SLFN11 is epigenetically silenced in [~]50% of treatment-naive tumours and is the strongest predictor of chemoresistance but why it is frequently inactivated in cancer is unknown. To acquire immortality, cancer cells can activate alternative lengthening of telomeres (ALT), typically accompanied by ATRX loss. Here, we implicate SLFN11 in sensing telomere replication stress, triggering eradication of ATRX deficient cells prior to ALT establishment. Whereas progressive telomere shortening of cells lacking telomerase and ATRX leads to telomere crisis and cell death, SLFN11 loss confers tolerance to PML-BLM dependent ALT intermediates, permitting emergence of ALT survivors. We propose that during tumorigenesis SLFN11 inactivation is selected as means to tolerate endogenous replication stress following telomere crisis, leading to the development of therapy resistant tumours before treatment.

molecular biology↗

bioRxiv2025

Spatial analysis of IPMNs defines a paradoxical KRT17-positive, low-grade epithelial population harboring malignant features

Background & AimsIntraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that represent one of the few radiologically identifiable precursors to pancreatic ductal adenocarcinoma (PDAC). Though the IPMN-bearing patient population represents a unique opportunity for early detection and interception, current guidelines provide insufficient accuracy in determining which patients should undergo resection versus surveillance, resulting in a sizable fraction of resected IPMNs only harboring low-grade dysplasia, suggesting that there may be overtreatment of this clinical entity. MethodsTo investigate the transcriptional changes that occur during IPMN progression, we performed spatial transcriptomics using the Nanostring GeoMx on patient samples containing the entire spectrum of IPMN disease including low-grade dysplasia, high-grade dysplasia, and IPMN-derived carcinoma. Single cell RNA sequencing was performed on side branch and main duct IPMN biospecimens. ResultsWe identified a subpopulation of histologically low-grade IPMN epithelial cells that express malignant transcriptional features including KRT17, S100A10 and CEACAM5, markers that are enriched in PDAC. We validated this high-risk gene signature in both single-cell RNA sequenced samples and an external ST dataset containing a larger number of IPMN samples including non-tumor bearing IPMN (i.e. low-grade IPMN in isolation). Immunofluorescence staining of a large cohort of patient tissues confirmed the presence of KRT17-positive cells, which were found to comprise a small subset of epithelial cells within histologically low-grade IPMN in a patchy distribution. ConclusionsOur study demonstrates that KRT17 marks a distinct transcriptional signature in a subpopulation of epithelial cells within histologically low-grade IPMN. This population of cells likely represents a transitional state of histologically low-grade epithelial cells undergoing progression to a higher grade of dysplasia and thus may represent a higher risk of progression to carcinoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/643943v2_ufig1.gif" ALT="Figure 1000"> View larger version (43K): org.highwire.dtl.DTLVardef@47d380org.highwire.dtl.DTLVardef@18e7ab2org.highwire.dtl.DTLVardef@1193ffeorg.highwire.dtl.DTLVardef@b84a98_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cancer biology↗

bioRxiv2023

Western diet dampens T regulatory cell function to fuel hepatic inflammation in nonalcoholic fatty liver disease

Background and aimsThe immunosuppressive T regulatory cells (Tregs) regulate immune responses and maintain immune homeostasis, yet their functions in nonalcoholic fatty liver disease (NAFLD) pathogenesis remains controversial. MethodsMice were fed a normal diet (ND) or a western diet (WD) for 16 weeks to induce NAFLD. Diphtheria toxin injection to deplete Tregs in Foxp3DTR mice or Treg induction therapy in WT mice to augment Treg numbers was initiated at twelve and eight weeks, respectively. Liver tissues from mice and NASH human subjects were analyzed by histology, confocal imaging, and qRT-PCR. ResultsWD triggered accumulation of adaptive immune cells, including Tregs and effector T cells, within the liver parenchyma. This pattern was also observed in NASH patients, where an increase in intrahepatic Tregs was noted. In the absence of adaptive immune cells in Rag1 KO mice, WD promoted accumulation of intrahepatic neutrophils and macrophages and exacerbated hepatic inflammation and fibrosis. Similarly, targeted Treg depletion exacerbated WD-induced hepatic inflammation and fibrosis. In Treg-depleted mice, hepatic injury was associated with increased accumulation of neutrophils, macrophages, and activated T cells within the liver. Conversely, induction of Tregs using recombinant IL2/IL2 mAb cocktail reduced hepatic steatosis, inflammation, and fibrosis in WD-fed mice. Analysis of intrahepatic Tregs from WD-fed mice revealed a phenotypic signature of impaired Treg function in NAFLD. Ex vivo functional studies showed that glucose and palmitate, but not fructose, impaired the immunosuppressive ability of Treg cells. ConclusionsOur findings indicate that the liver microenvironment in NAFLD impairs ability of Tregs to suppress effector immune cell activation, thus perpetuating chronic inflammation and driving NAFLD progression. These data suggest that targeted approaches aimed at restoring Treg function may represent a potential therapeutic strategy for treating NAFLD. Lay summaryIn this study, we elucidate the mechanisms contributing to the perpetuation of chronic hepatic inflammation in nonalcoholic fatty liver disease (NAFLD). We show that dietary sugar and fatty acids promote chronic hepatic inflammation in NAFLD by impairing immunosuppressive function of regulatory T cells. Finally, our preclinical data suggest that targeted approaches aimed at restoring T regulatory cell function have the potential to treat NAFLD.

pathology↗

bioRxiv2023

Selective targeting of α4β7/MAdCAM-1 axis suppresses fibrosis progression in chronic liver disease

Integrin 4{beta}7+ T cells perpetuate tissue injury in chronic inflammatory diseases, yet their role in hepatic fibrosis progression remains poorly understood. Here we report increased accumulation of 4{beta}7+ T cells in the liver of people with cirrhosis relative to disease controls. Similarly, hepatic fibrosis in the established mouse model of CCl4-induced liver fibrosis was associated with enrichment of intrahepatic 4{beta}7+ CD4 and CD8 T cells. Monoclonal antibody (mAb)-mediated blockade of 4{beta}7 or its ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 attenuated hepatic inflammation and prevented fibrosis progression in CCl4 treated mice. Improvement in liver fibrosis was associated with a significant decrease in the infiltration of 4{beta}7+ CD4 and CD8 T cells suggesting that 4{beta}7/MAdCAM-1 axis regulates both CD4 and CD8 T cell recruitment to the fibrotic liver, and 4{beta}7+ T cells promote hepatic fibrosis progression. Analysis of hepatic 4{beta}7+ and 4{beta}7-CD4 T cells revealed that 4{beta}7+ CD4 T cells enriched for markers of activation and proliferation demonstrating an effector phenotype. Notably, blockade of 4{beta}7 or MAdCAM-1 did not affect the recruitment of Foxp3+ regulatory T cells, demonstrating the specificity of 4{beta}7/MAdCAM-1 axis in regulating effector T cell recruitment to the liver. The findings suggest that 4{beta}7+ T cells play a critical role in promoting hepatic fibrosis progression, and mAb-mediated blockade of 4{beta}7 or MAdCAM-1 represents a promising therapeutic strategy for slowing hepatic fibrosis progression in chronic liver diseases.

immunology↗

bioRxiv2018

GnasR201C Induces Murine Pancreatic Cystic Neoplasms through Suppression of YAP1 Signaling and Transcriptional Reprogramming

Background & AimsSomatic \"hotspot\" mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown.\n\nMethodsWe generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC).\n\nResultsCo-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations.\n\nConclusionsGNASR201C functions not as a traditional oncogene, but rather as an \"oncomodulator\" of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

cancer biology↗

bioRxiv2018

Single Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression

BackgroundEarly detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically, intraductal papillary mucinous neoplasms (IPMNs) represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneity that accompany multistep progression from non-invasive IPMNs to PDAC.\n\nMethodsSingle cell RNA-sequencing was performed through droplet-based sequencing on 5,403 cells from two low-grade IPMNs (LGD-IPMN), two high-grade IPMNs (HGD-IPMN), and two PDACs (all surgically resected).\n\nResultsAnalysis of single cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of non-invasive dysplasia to invasive cancer. While HGD-IPMNs expressed many core-signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a pro-inflammatory immune component was readily seen in low-grade IPMNs, comprised of cytotoxic T-cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous, and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis.\n\nConclusionsThis study demonstrates the ability to perform high resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneity that accompany early cancer pathogenesis, and might be a useful substrate to identify targets for cancer interception.

pathology↗