Biology subjects

Bapat, S.

Publications and source records attributed to Bapat, S..

6 recordsLinked to original sources

bioRxiv2025

Identification and characterization of host-modulating effectors encoded by the Cluster F1 mycobacteriophage NormanBulbieJr

NormanBulbieJr (NBJ) is a temperate siphovirus isolated on the host Mycobacterium smegmatis mc2155 that encodes 102 gene products, 60 of which have no known function (NKF). Based on gene content, NBJ is classified as a Cluster F, Subcluster F1 phage and shares 70% of its encoded gene phamilies with Girr, another F1 mycobacteriophage that was recently analyzed in a genome-wide overexpression screen and found to encode 29 diverse gene products capable of inhibiting growth of M. smegmatis. Similar functional screens in other mycobacteriophages have uncovered a growing repertoire of diverse, phage-encoded bacterial growth inhibitors, providing prime candidates for dissecting novel bacterial-phage interactions. An arrayed overexpression library encoding all 102 genes was constructed and systematically screened using a plate-based cytotoxicity assay, identifying 29 genes that inhibit mycobacterial growth. Because mycobacteriophage genomes are also known to encode systems involved in phage-phage competition, we conducted additional phage defense assays for a subset of NBJ genes in our library. This analysis confirmed homotypic immunity by the predicted immunity repressor and identified gp45 as a second defense factor that protects against Cluster F phages and promotes NBJ lysogen stability. Additional mechanistic analyses indicate that gp45 functions in a secretion-dependent manner, acting after phage adsorption to reduce productive infection and early phage transcript accumulation. Escape mutant and bacterial two-hybrid analyses implicate Cluster F tape measure proteins as the likely target of gp45, consistent with disruption of an early step in phage genome delivery. Finally, we extended our analysis to explore the essentiality of all identified host-modulating genes in the NBJ life cycle, using CRISPR-enhanced recombineering to generate phage deletion mutants, revealing two host modulators that are critical for lytic growth. Conducted as part of the SEA-GENES undergraduate research consortium, this study adds to a growing functional genomics resource and provides new insights into the complex interactions between phage gene products and the mycobacterial host cell.

microbiology↗

bioRxiv2025

Paired primary-metastasis patient-derived organoids and mouse models identify phenotypic evolution and druggable dependencies of peritoneal metastasis from appendiceal cancer

Peritoneal carcinomatosis is a common yet deadly manifestation of gastrointestinal cancers, with few effective treatments. To identify targetable determinants of peritoneal metastasis, we focused on appendiceal adenocarcinoma (AC), a gastrointestinal cancer that metastasizes almost exclusively to the peritoneum. Current treatments are extrapolated from colorectal cancer (CRC), yet AC has distinct genomic alterations, mucinous morphology and peritoneum restricted metastatic pattern. Further, no stable preclinical models of AC exist, limiting drug discovery and representing an unmet clinical need. We establish a first-in-class stable biobank of 16 long-term cultured AC patient-derived organoids (PDOs), including 3 matched, simultaneously resected primary AC-peritoneal carcinomatosis (AC-PC) pairs. By enriching for cancer cells, AC PDOs enable accurate genomic characterization relative to paucicellular AC tissue. We establish an organoid orthotopic intraperitoneal xenograft model that recapitulates diffuse peritoneal carcinomatosis and show that PC-organoids retain increased metastatic capacity, decreased growth factor dependency and sensitivity to standard of care chemotherapy relative to matched primary AC organoids. Single cell profiling of AC-PC pairs reveals dedifferentiation from mucinous differentiated states in primary AC into intestinal stem cell and fetal progenitor states in AC-PC, with upregulation of oncogenic signaling pathways. Through hypothesis-driven drug testing, we identify KRASMULTI-ON inhibitor RMC-7977 and Wnt-targeting tyrosine kinase inhibitor WNTinib as novel, clinically actionable strategies to target AC-PC more effectively.

cancer biology↗

bioRxiv2025

Phosphatidylserine (PS)-targeting chimeric Interferon (IFN) fusion proteins for anti-tumor applications

In viable healthy cells, membrane phospholipids are asymmetrically distributed across the lipid bilayer, whereby the anionic phospholipid phosphatidylserine is virtually all distributed on the inner leaflet of the plasma membrane. During apoptosis, phospholipid asymmetry collapses and PS is externalized to the external leaflet where it serves as an "eat-me" signal for efferocytosis, the process whereby dying cells are engulfed and degraded by phagocytes. PS is also externalized on viable activated tumor endothelial cells, stromal cells and cancer cells in the tumor microenvironment reflecting a pathophysiological state of solid cancers that function to suppress host anti-tumor immunity. Several strategies have been envisioned to target dysregulated PS in the tumor microenvironment including PS binding proteins such as Annexin V and PS-targeting monoclonal antibodies (Bavituximab) with promising preclinical results. Here, in an attempt to enhance the efficacy of PS-targeting therapeutics, we have generated a series of recombinant chimeric fusion proteins that fuse type I and type III IFNs (IFN-{beta}-IFN-{lambda}) into a single polypeptide chain separated by a short linker. The IFN-{beta}-IFN-{lambda} fusion proteins retain functions of both type I and type III IFNs but show combined effects to improve biological function as well as enhance anti-tumor activities. To localize IFNs to sites of externalized PS, we next fused the IFN-{beta}-IFN-{lambda} chimeric protein to the PS-targeting gamma-carboxyglutamic acid-rich (Gla) domain of Growth Arrest Specific factor 6 (Gas-6), rendering these IFN biologics as PS targeting modalities. Gas6-IFN-{beta}-IFN-{lambda} proteins selectively bind PS as evident by solid-phase ELISA assays as well as bind PS-positive cells, including apoptotic cells and cells that express CDC50 subunit mutant of the ATP11C flippase. In vivo, Gas6-IFN-{beta}-IFN-{lambda} retain strong anti-tumor activities in a syngeneic model when expressed ectopically in a E0771 breast cancer model and B16-F10 melanoma models. Collectively, we report on the generation and utility of a series of novel in class IFN fusion proteins that target the immune stimulatory features of IFNs to the PS externalization in the tumor microenvironment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/634764v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@6fecceorg.highwire.dtl.DTLVardef@23d0d0org.highwire.dtl.DTLVardef@1621a39org.highwire.dtl.DTLVardef@1a7aa6f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Gas6-IFN--IFN-{lambda} (VitK) have tri-functional activities, acting on a diverse set of cell types to induce an anti-tumor affect. The Gas6 domain aids in homing to the PS rich tumor microenvironment, binding to apoptotic or live stressed PS positive tumor cells. The Gla domain directly binds to PS, whereas the EGF domains help in oligomerization and signal amplification resulting from intermolecular disulphide bonds. The IFN- domain acts on immune cells such as dendritic cells and macrophages, inducing an interferon response, whereas the IFN-{lambda} domain acts on the tumor epithelial cells, inducing tumor intrinsic anti-tumor activity. C_FIG

cancer biology↗

bioRxiv2024

KAT3 mutations impair neural crest migration through EMT regulators snai1b and snai2 in Rubinstein Taybi Syndrome

Rubinstein Taybi syndrome, a rare congenital disease is caused by mutation in KAT3 genes, EP300 and CREBBP. A subset of tissues affected in RSTS have their origin in neural crest cells, prompting our exploration into the role of KAT3 in neural crest development. Our zebrafish RSTS models generated by knocking down or mutating ep300a and cbpa genes, reveal defects in neural crest migration and its derived tissues when KAT3 genes are perturbed. We also demonstrate that the effects on neural crest can be reversed by HDAC inhibition in in morphant embryos. KAT3 knockdown causes downregulation of EMT regulators, snai1b and snai2. Snai2 is known to repress cdh6b in neural crest cells facilitating their delamination from neural tube and migration. We generated RSTS patient-derived iPSC line and differentiated them into neural crest cells in vitro. We show that role of KAT3 proteins in neural crest migration is conserved in human iPSC derived neural crest cells. Our findings make a case for classifying RSTS as a neurocristopathy. HighlightsO_LIPerturbation of KAT3 gene expression in zebrafish recapitulates the Rubinstein Taybi patient defects C_LIO_LIThe zebrafish model of Rubinstein Taybi model reveals defects in neural crest cell migration C_LIO_LIKAT3 proteins regulate snai2, snai1b and cdh6, genes important for neural crest migration C_LIO_LIThe neural crest migration defects in the zebrafish model can be partially rescued by modulating the global acetylation levels C_LIO_LIStudy of RSTS patient-derived neural crest cells reveals that the role of KAT3 in neural crest migration is conserved across vertebrates C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/593474v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1761fceorg.highwire.dtl.DTLVardef@220e21org.highwire.dtl.DTLVardef@1cdd311org.highwire.dtl.DTLVardef@1dc0e53_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2021

Machine learning algorithms in big data analyses identify determinants of insulin gene transcription

Machine learning (ML)-workflows enable unprejudiced/robust evaluation of complex datasets. Here, we analyzed over 490,000,000 data points to compare 10 different ML-workflows in a large (N=11,652) training dataset of human pancreatic single-cell (sc-)transcriptomes to identify genes associated with the presence or absence of insulin transcript(s). Prediction accuracy/sensitivity of each ML-workflow were tested in a separate validation dataset (N=2,913). Ensemble ML-workflows, in particular Random Forest ML-algorithm delivered high predictive power (AUC=0.83) and sensitivity (0.98), compared to other algorithms. The transcripts identified through these analyses also demonstrated significant correlation with insulin in bulk RNA-seq data from human islets. The top-10 features, (including IAPP, ADCYAP1, LDHA and SST) common to the three Ensemble ML-workflows were significantly dysregulated in scRNA-seq datasets from Ire-1{beta}-/- mice that demonstrate dedifferentiation of pancreatic {beta}-cells in a model of type 1 diabetes (T1D) and in pancreatic single cells from individuals with type 2 Diabetes (T2D). Our findings provide direct comparison of ML-workflows in big data analyses, identify key determinants of insulin transcription and provide workflows for future analyses.

genetics↗

bioRxiv2019

Obesity Potentiates TH2 Immunopathology via Dysregulation of PPAR

How obesity affects immune function is not well understood. Clinically, obesity is strongly associated with severe TH2 immunopathology1-3, though the physiological, cellular, and molecular underpinnings of this association remain obscure. Here, we demonstrate that obese mice are susceptible to severe atopic dermatitis (AD), a major manifestation of TH2 immunopathology and disease burden in humans4,5. Mechanistically, we show that dysregulation of the nuclear hormone receptor (NHR) PPAR{gamma} (peroxisome proliferator-activated receptor gamma) in T cells is a causal link between obesity and the increased TH2 immunopathology. We find that PPAR{gamma} oversees a cellular metabolic transcriptional program that restrains nuclear gene expression of the chief TH2 priming and effector cytokine interleukin-4 (IL-4). Accordingly, thiazolidinediones (TZDs), potent PPAR{gamma} agonists, robustly protect obese mice from TH2 immunopathology. Collectively, these findings establish PPAR{gamma} as a molecular link between obesity and TH2 immune homeostasis and identify TZDs as novel therapeutic candidates for TH2 immunopathology. Fundamentally, these findings demonstrate that shifting physiologic metabolic states can shape the tone of adaptive immune responses to modulate differential disease susceptibility.

immunology↗