Biology subjects

Bandyopadhyay, D.

Publications and source records attributed to Bandyopadhyay, D..

12 recordsLinked to original sources

bioRxiv2026

A new set of DNA methylation variants in the human genome show predominant tissue specificity and sensitivity to reprogramming with a potential for disease susceptibility.

Analyses of 3,370 normal human tissues of ectodermal, endodermal and mesodermal origins identified 12,587 regions averaging [~]585 bp with significant differences in DNA methylation levels within identical tissues. These methylation variants (MeVars) occurred in 8,037 genes enriched in neurological disorders and cancers of which, majority were tissue-specific rather than being systemic. This somatic variation was reduced by reprogramming in vitro into iPSCs and in vivo during spermatogenesis. Analysis of prefrontal cortices showed a higher incidence of MeVars in the candidate genes in controls than schizophrenia patients wherein a subset showed significantly altered transcript levels. Similar effects were observed for oral tissues and skin fibroblast cells. MeVars showed significant association with SINE1, simple and low complexity repeats, H3K27me3, H3k9me3 and H3K4me1 modifications and EZH2, SUZ12 and REST binding sites. Collectively, MeVars have postzygotic origins with an ability to reset during reprogramming, adding a new dimension in the form of epigenetic diversity and its relevance to disease susceptibility in humans.

bioinformatics↗

bioRxiv2026

Reprogramming mRNA localization by targeted RNA-protein interference

RNA binding proteins (RBPs) associate with RNAs in intricate ribonucleoprotein complexes and regulate various aspects of RNA life cycle and, by extension, cell functions. Despite their significance, elucidating the functional contributions of specific RNA-RBP binding events, particularly in long-term phenotypic assays, remains challenging. Here, we harness the specificity of CRISPR/dCas13 to interfere with specific RNA-RBP interactions. We apply this methodology to GA-rich mRNA localization elements which recruit the RNA-binding protein CNBP and serve as platforms for the assembly of mRNA trabicking complexes. We show that dCas13/gRNA binds to target transcripts in a highly specific manner and interferes with CNBP recruitment leading to altered target mRNA localization and cell motility, consistent with the function of the targeted mRNAs. We describe optimizations and considerations for the stable implementation of this system. We further use it to show that interfering with the localization of the GA-containing NET1 mRNA reduces cancer cell invasiveness and metastatic lung colonization. These data reveal a potential role for mRNA localization in oncogenic progression and illustrate how this CRISPR-based system can be used for longterm functional investigations of RNA-RBP interactions in vivo.

cell biology↗

bioRxiv2026

miRNA Sensor HuR Compartmentalizes Ago2-Uncoupled miRNAs to Lipid Droplets to Buffer miRNA Activity in Mammalian Cells

miRNA activity must be optimized to ensure target gene expression in mammalian cells in response to specific needs. How and when miRNA activity is regulated remains an open question in gene expression regulation, with limited information on how cellular machinery senses miRNA levels to regulate miRNA expression and activity. Because miRNAs are stable molecules that can be reversibly used, the existence of a miRNA-sensing mechanism in mammalian cells has been anticipated. With ectopic expression of miRNAs in mammalian cells, we found a dose-dependent miRNA buffering mechanism in which miRNA export and its storage on lipid droplets are essential for miRNA activity optimization in mammalian cells. We found that the miRNA-binding protein HuR, known for its role in miRNA export, has a dual function in mammalian cells. HuR uncouples miRNAs from Ago2 to facilitate association of lipid droplets with nonfunctional miRNAs, a process that gets augmented in cells with high lipid droplet content and restricted extracellular export. Thus, HuR acts as a miRNA sensor, and the optimal activity and abundance of HuR regulate miRNA storage on lipid droplets or its export to maintain cellular miRNA homeostasis, thereby preventing detrimental effects of an excess miRNA pool in mammalian cells. Thus, targeting miRNA export or lipid droplet association is an important strategy for buffering cellular miRNA levels. ImportanceTo ensure that target gene expression in mammalian cells is appropriately regulated, miRNA activity needs to be carefully fine-tuned. Although miRNAs are stable and can be reused, the precise mechanisms by which their levels are sensed and regulated remain unclear. Our research uncovered a fascinating dose-dependent miRNA buffering process that involves export and storage of miRNAs on lipid droplets--essential steps for precise regulation. We also found that HuR, a protein that facilitates miRNA export, plays a key role by uncoupling miRNAs from Ago2 and promoting their association with lipid droplets in cells with abundant lipid droplets and limited extracellular export capacity. Overall, HuR acts as a miRNA sensor, helping to balance miRNA storage and export to maintain cellular homeostasis and prevent the harmful effects of miRNA overactivity.

cell biology↗

bioRxiv2023

A 50-gene high-risk profile predictive of COVID-19 and Idiopathic Pulmonary Fibrosis mortality originates from a molecular imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease

BackgroundWe aim to study the source of circulating immune cells expressing a 50-gene signature predictive of COVID-19 and IPF mortality. MethodsWhole blood and Peripheral Blood Mononuclear cells (PBMC) were obtained from 231 subjects with COVID-19, post-COVID-19-ILD, IPF and controls. We measured the 50-gene signature (nCounter, Nanostring), interleukin 6 (IL6), interferon {gamma}-induced protein (IP10), secreted phosphoprotein 1 (SPP1) and transforming growth factor beta (TGF-{beta}) by Luminex. PCR was used to validate COVID-19 endotypes. For single-cell RNA sequencing (scRNA-seq) we used Chromium Controller (10X Genomics). For analysis we used the Scoring Algorithm of Molecular Subphenotypes (SAMS), Cell Ranger, Seurat, Propeller, Kaplan-Meier curves, CoxPH models, Two-way ANOVA, T-test, and Fishers exact. ResultsWe identified three genomic risk profiles based on the 50-gene signature, and a subset of seven genes, associated with low, intermediate, or high-risk of mortality in COVID-19 with significant differences in IL6, IP10, SPP1 and TGF{beta}-1. scRNA-seq identified Monocytic-Myeloid-Derived Suppressive cells (M-MDSCs) expressing CD14+HLA DRlowCD163+ and high levels of the 7-gene signature (7Gene-M-MDSC) in COVID-19. These cells were not observed in post-COVID-19-ILD or IPF. The 43-gene signature was mostly expressed in CD4 T and CD8 T cell subsets. Increased expression of the 43 gene signature was seen in T cell subsets from survivors with post-COVID-19-ILD. The expression of these genes remained low in IPF. ConclusionA 50-gene, high-risk profile in COVID-19 is characterized by a genomic imbalance in monocyte and T-cell subsets that reverses in survivors with post-COVID-19 Interstitial Lung Disease

genomics↗

bioRxiv2023

Mast-Cell Expressed Membrane Protein-1 (MCEMP1) is expressed in classical monocytes and alveolar macrophages in Idiopathic Pulmonary Fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFβ dependent manner.

BackgroundMast-Cell Expressed Membrane Protein-1 (MCEMP1) is higher in Idiopathic Pulmonary Fibrosis (IPF) patients with increased risk of death and poor outcomes. Here we seek to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. MethodsMCEMP1 expression was analyzed by single-cell RNA sequencing, immunofluorescence in Peripheral Blood Mononuclear Cells (PBMC) as well as in lung tissues from IPF patients and controls. Chromatin Immunoprecipitation (ChiP) and Proximity Ligation Assay (PLA) were used to study the transcriptional regulation of MCEMP1. Transient RNA interference and lentivirus transduction were used to knockdown and knock-in MCEMP1 in THP-1 cells to study chemotaxis, adhesion, and migration. Bulk RNA sequencing was used to identify the mechanisms by which MCEMP1 participates in monocyte function. Active RHO pull-down assay was used to validate bulk RNA sequencing results. ResultsWe identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared to controls. MCEMP1 was upregulated by TGF{beta} at the mRNA and protein levels in THP-1. TGF{beta}-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis-regulatory elements within the MCEMP1 promoter. In terms of its function, we found that MCEMP1 regulates TGF{beta}-mediated monocyte chemotaxis, adhesion, and migration. 400 differentially expressed genes were found to increase after TGF{beta} stimulation of THP-1, further increased in MCEMP1 knock-in cells treated with TGF{beta} and decreased in MCEMP1 knockdown cells treated with TGF{beta}. GO annotation analysis of these genes showed enrichment for positive regulation of RHO GTPase activity and signal transduction. While TGF{beta} enhanced RHO GTPase activity in THP-1 cells, this effect was attenuated following MCEMP1 knockdown. ConclusionMCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF. MCEMP1 is regulated by TGF{beta} and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGF{beta} in RHO activity. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.

immunology↗

bioRxiv2022

Structural analysis unravels the functional promiscuity of Quinolone synthase-mediated polyketide biosynthesis in Aegle marmelos Correa

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Based on the high-resolution protein structure of AmQNS, this study provided a mechanistic explanation of the structure to synthetic selectivity. Additionally, it displays the comparatively wide active site entry that allows the catalytic pocket to accommodate bulky substrates, which affects the enzyme catalysis. We also develop a model framework for comprehending the structural constraints on ketide insertion, and postulate that AmQNS synthetic diversity is owing to its steric and electrostatic selectivity, which allows it to bind to a variety of core substrates. We further establish that AmQNS is structurally biased toward quinolone synthesis and only synthesizes acridone when malonyl-CoA concentrations are significantly high. In a nutshell, we anticipate that addressing the structural and molecular underpinnings of AmQNS-substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. Besides, the approaches can also be expanded to other potential enzymes, which will help the pharmaceutical sector by expanding the pool of potential medication leads.

biochemistry↗

bioRxiv2022

A monotone single index model for missing-at-random longitudinal proportion data

Beta distributions are commonly used to model proportion valued response variables, commonly encountered in longitudinal studies. In this article, we develop semi-parametric Beta regression models for proportion valued responses, where the aggregate covariate effect is summarized and flexibly modeled, using a interpretable monotone time-varying single index transform of a linear combination of the potential covariates. We utilize the potential of single index models, which are effective dimension reduction tools and accommodate link function misspecification in generalized linear mixed models. Our Bayesian methodology incorporates the missing-at-random feature of the proportion response, and utilize Hamiltonian Monte Carlo sampling to conduct inference. We explore finite-sample frequentist properties of our estimates, and assess the robustness via detailed simulation studies. Finally, we illustrate our methodology via application to a motivating longitudinal dataset on obesity research recording proportion body fat.

developmental biology↗

bioRxiv2021

Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

Helicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.

biophysics↗

bioRxiv2021

Improved Metabolite Prediction Using Microbiome Data-Based Elastic Net Models

Microbiome data are becoming increasingly available in large health cohorts yet metabolomics data are still scant. While many studies generate microbiome data, they lack matched metabolomics data or have considerable missing proportions of metabolites. Since metabolomics is key to understanding microbial and general biological activities, the possibility of imputing individual metabolites or inferring metabolomics pathways from microbial taxonomy or metagenomics is intriguing. Importantly, current metabolomics profiling methods such as the HMP Unified Metabolic Analysis Network (HUMAnN) have unknown accuracy and are limited in their ability to predict individual metabolites. To address this gap, we developed a novel metabolite prediction method, and we present its application and evaluation in an oral microbiome study. We developed ENVIM based on the Elastic Net Model (ENM) to predict metabolites using micorbiome data. ENVIM introduces an extra step to ENM to consider variable importance scores and thus achieve better prediction power. We investigate the metabolite prediction performance of ENVIM using metagenomic and metatranscriptomic data in a supragingival biofilm multi-omics dataset of 297 children ages 3-5 who were participants of a community-based study of early childhood oral health (ZOE 2.0) in North Carolina, United States. We further validate ENVIM in two additional publicly available multi-omics datasets generated from studies of gut health and vagina health. We select gene-family sets based on variable importance scores and modify the existing ENM strategy used in the MelonnPan prediction software to accommodate the unique features of microbiome and metabolome data. We evaluate metagenomic and metatranscriptomic predictors and compare the prediction performance of ENVIM to the standard ENM employed in MelonnPan. The newly-developed ENVIM method showed superior metabolite predictive accuracy than MelonnPan using metatranscriptomics data only, metagenomics data only, or both of these two. Both methods perform better prediction using gut or vagina microbiome data than using oral microbiome data for the samples corresponding metabolites. The top predictable compounds have been reported in all these three datasets from three different body sites. Enrichment of prediction some contributing species has been detected.

microbiology↗

bioRxiv2021

Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

microRNA-122 (miR-122), a liver specific regulatory RNA, plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 is also a proinflammatory microRNA and when exported to tissue resident macrophage induces expression of inflammatory cytokines there. We found intercellular transfer of miR-122 in lipid exposed liver plays a role in liver inflammation. Exploring the mechanism of intercellular miR-122 transfer from hepatic cells, we detected MMP2 on the membrane of extracellular vesicles derived from hepatic cells which proved to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. Matrix metalloprotease 2 or MMP2 is a metalloproteinase that plays a key role in shaping and remodelling the extracellular matrix of human tissue by targeting degradation of matrix proteins. MMP2 was found to increase the movement of the EVs along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries. By targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/425217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@aec3b0org.highwire.dtl.DTLVardef@1b18e87org.highwire.dtl.DTLVardef@47e75org.highwire.dtl.DTLVardef@10a48e0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuman hepatocytes on exposure to high lipid export out miRNAs including proinflammatory miR-122. C_LIO_LIExtracellular miR-122 is taken up by tissue macrophages to get them activated to produce inflammatory cytokines. C_LIO_LIMMP2 present on the surface of the EVs released by hepatocyte is essential for miRNA transfer to macrophage cells C_LIO_LIInhibition of MMP2 prevents miR-122 transfer to macrophage and stops activation of recipient macrophage. C_LI

cancer biology↗

bioRxiv2020

Vital Insights into Prokaryotic Genome Compaction by Nucleoid-Associated Protein (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution

Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a Slacked-Dynamic state throughout the observation window where many of them have switched between multiple Wobbling States in the course of attainment of packaged form. A range of DNA Flexure Angles has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis.

biophysics↗

bioRxiv2020

CryoEM Structure of CtBP2 Confirms Tetrameric Architecture

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 -/- cells transfected with tetramer destabilizing_mutants. Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

biochemistry↗