Biology subjects

Gupta, I.

Publications and source records attributed to Gupta, I..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Complete sequencing of ape genomes

We present haplotype-resolved reference genomes and comparative analyses of six ape species, namely: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan, and siamang. We achieve chromosome-level contiguity with unparalleled sequence accuracy (<1 error in 500,000 base pairs), completely sequencing 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, providing more in-depth evolutionary insights. Comparative analyses, including human, allow us to investigate the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference. This includes newly minted gene families within lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes, and subterminal heterochromatin. This resource should serve as a definitive baseline for all future evolutionary studies of humans and our closest living ape relatives.

genomics↗

bioRxiv2024

Microarray Integrated Spatial Transcriptomics (MIST) for Affordable, Robust, and Comprehensive Digital Pathology

10X Visium, a popular Spatial transcriptomics (ST) method, faces limited adoption due to its high cost and restricted sample usage per slide. To address these issues, we propose Microarray Integrated Spatial Transcriptomics (MIST), combining conventional tissue microarray (TMA) with Visium, using laser-cutting and 3D printing to enhance slide throughput. Our design facilitates independent replication and customization in individual labs to suit specific experimental needs. We provide a step-by-step guide from designing TMAs to the library preparation step. We demonstrate MISTs cost-effectiveness and technical benefits over Visium and GeoMx Nanostring. We also introduce AnnotateMap, a novel computational tool for efficient analysis of multiple ROIs processed through MIST.

bioinformatics↗

bioRxiv2024

Discovery of Novel Long Non-Coding RNAs That May Potentially Play a Role in Zebrafish Brain Regeneration Post Traumatic Injury

Understanding brain regeneration mechanisms is vital for treating neurological conditions. Zebrafish (Danio rerio) are an excellent model due to their genetic similarity to humans and strong regenerative abilities. In this study, we identified novel long non-coding RNAs (lncRNAs) in the regenerating zebrafish brain following traumatic brain injury (TBI). RNA sequencing data of the zebrafish telencephalon from the BioStudies database was analyzed for novel long non-coding RNA expression (lncRNA) at control, one day post-lesion (early wound healing), three days post-lesion (cell proliferation), and 14 days post-lesion (differentiation). We identified 689 potential lncRNAs using HISAT2, StringTie, FEELnc, and PhastCon analysis tools. Principal component analysis (PCA) of identified lncRNAs revealed a distinct expression profile at 1-day post-lesion, indicating their significant role in early wound healing. Weighted Gene Co-expression Network Analysis (WGCNA) identified two modules (brown and turquoise) showing unique expression patterns critical to brain regeneration. Pathway enrichment analysis linked brown module lncRNAs to peptide biosynthesis, cellular amide metabolism, and ribosome biogenesis. In contrast, turquoise module lncRNAs were associated with ion transmembrane transport and cell adhesion pathways. qPCR validation confirmed co-expression patterns of selected lncRNAs and correlated genes, emphasizing their regulatory roles. This study demonstrates that lncRNAs play crucial roles in zebrafish brain regeneration by modulating gene expression during the early wound healing stage. These insights offer potential therapeutic applications of lncRNAs in neuroregenerative medicine.

bioinformatics↗

bioRxiv2024

Triphasic Thrombosis Model: A Computational Study of Type B Aortic Dissection

Thrombosis refers to the formation of a thrombus, or a blood clot, within the body, which can occur either partially or completely. It serves as a crucial indicator of the severity of a patients medical condition, with the location and characteristics of thrombosis dictating its clinical implications. Hence, accurate diagnosis and effective management of thrombosis are paramount. In our current investigation, we incorporate the porous attributes of a thrombus using the Theory of Porous Media. This involves dividing the aggregate into solid, liquid, and nutrient phases and utilising volume fractions to capture microstructural details. Fluid flow through the porous media is modelled using a modified Darcy-Brinkman type equation, with interaction terms within balance equations facilitating the modelling of the mass exchange and other phase interactions. The shorter time scales are neglected. We present a comprehensive framework of equations and assumptions governing the behaviour of a strongly coupled multiphasic porous medium problem. Additionally, we introduce scenarios involving type B Aortic Dissection and false lumen geometries, providing a detailed outline of the problem setup. Thereafter, we present the potential of the model for thrombi growth. The simulation results are compared with velocity plots aligning with Magnetic Resonance Imaging data for three distinct cases with varying entry and exit tear sizes. Consequently, our proposed model offers a promising and reasonable approach for numerically simulating thrombosis and gaining insights into the underlying growth mechanics.

biophysics↗

bioRxiv2024

PATHOGEN GROWTH AND VIRULENCE DYNAMICS DRIVE THE HOST EVOLUTION AGAINST COINFECTIONS

Coinfections, or the simultaneous infection of hosts by multiple pathogens, are widespread in nature with significant negative impacts on global health. Can hosts evolve against such coinfections as effectively as they would against individual pathogens? Also, what roles do individual pathogens play during such evolution? Here, we combined theoretical models and experiments with Tribolium castaneum populations evolving against two coinfecting bacterial pathogens, with contrasting growth and virulence dynamics, to reveal that fast-growing pathogens inflicting rapid mortality surges (i.e., fast-acting) restrict adaptive success against coinfections. While hosts rapidly evolved better survival against slow-growing bacteria causing long-lasting infections, evolution against coinfection was significantly delayed and resembled slow adaptation against fast-acting pathogens. Moreover, limited scopes of immunomodulation against fast-acting pathogens during coinfections can drive the observed adaptive patterns. Overall, we provide new insights into how adaptive dynamics and mechanistic bases against coinfections are critically regulated by individual pathogens growth and virulence dynamics.

evolutionary biology↗

bioRxiv2023

Mesenchymal Wnts are required for morphogenetic movements of calvarial osteoblasts during apical expansion

Apical expansion of calvarial osteoblast progenitors from the cranial mesenchyme (CM) above the eye is integral for calvarial growth and enclosure of the brain. The cellular behaviors and signals underlying the morphogenetic process of calvarial expansion are unknown. During apical expansion, we found that mouse calvarial primordia have consistent cellular proliferation, density, and survival with complex tissue scale deformations, raising the possibility that morphogenetic movements underlie expansion. Time lapse light sheet imaging of mouse embryos revealed that calvarial progenitors intercalate in 3D to converge supraorbital arch mesenchyme mediolaterally and extend it apically. In contrast, progenitors located further apically exhibited protrusive and crawling activity. CM cells express non-canonical Wnt/Planar Cell Polarity (PCP) core components and calvarial osteoblasts are bidirectionally polarized. We found non-canonical ligand, Wnt5a-/- mutants have less dynamic cell rearrangements, protrusive activity, and a flattened head shape. Loss of cranial mesenchyme-restricted Wntless (CM-Wls), a gene required for secretion of all Wnt ligands, led to diminished apical expansion of OSX+ calvarial osteoblasts in the frontal bone primordia in a non-cell autonomous manner without perturbing proliferation or survival. Calvarial osteoblast polarization, progressive cell elongation and enrichment for actin cytoskeleton protein along the baso-apical axis were dependent on CM-Wnts. Thus, CM-Wnts regulate cellular behaviors during calvarial morphogenesis and provide tissue level cues for efficient apical expansion of calvarial osteoblasts. These findings also offer potential insights into the etiologies of calvarial dysplasias.

developmental biology↗

bioRxiv2023

Msx genes delineate a novel molecular map of the developing cerebellar neuroepithelium.

In the early cerebellar primordium, there are two progenitor zones, the ventricular zone (VZ) residing atop the IVth ventricle and the rhombic lip (RL) at the lateral edges of the developing cerebellum. These zones give rise to the several cell types that form the GABAergic and glutamatergic populations of the adult cerebellum, respectively. Recently, an understanding of the molecular compartmentation of these zones has emerged. The Msx genes are a family of three transcription factors that are expressed downstream of Bone Morphogenetic Protein (BMP) signaling in these zones. Using fluorescent RNA in situ hybridization, we have characterized the Msx (Msh Homeobox) genes and demonstrated that their spatiotemporal pattern segregates specific regions in the progenitor regions. Msx1 and Msx2 are compartmentalized within the rhombic lip (RL), while Msx3 is localized within the ventricular zone (VZ). The relationship of the Msx genes with an early marker of the glutamatergic lineage, Atoh1, was examined in Atoh1-null mice and it was found that the expression of Msx genes persisted. Importantly, Msx1 and Msx3 expressions expanded in response to the elimination of Atoh1. These results point to early markers of cerebellar progenitor zones and more importantly to an updated view of the molecular parcellation of the RL with respect to the canonical marker of the RL, Atoh1.

developmental biology↗

bioRxiv2023

PfAP2-MRP DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages

Malaria pathogenicity results from the parasites ability to invade, multiply within and then egress from the host red blood cell (RBC). Infected RBCs are remodeled, expressing antigenic variant proteins (such as PfEMP1, coded by the var gene family) for immune evasion and survival. These processes require the concerted actions of many proteins, but the molecular regulation is poorly understood. We have characterized an essential Plasmodium specific Apicomplexan AP2 (ApiAP2) transcription factor in Plasmodium falciparum (PfAP2-MRP; Master Regulator of Pathogenesis) during the intraerythrocytic developmental cycle (IDC). An inducible gene knockout approach showed that PfAP2-MRP is essential for development during the trophozoite stage, and critical for var gene regulation, merozoite development and parasite egress. ChIP-seq experiments performed at 16 hour post invasion (h.p.i.) and 40 h.p.i. matching the two peaks of PfAP2-MRP expression, demonstrate binding of PfAP2-MRP to the promoters of genes controlling trophozoite development and host cell remodeling at 16 h.p.i. and antigenic variation and pathogenicity at 40 h.p.i. Using single-cell RNA-seq and fluorescence-activated cell sorting, we show de-repression of most var genes in{Delta} pfap2-mrp parasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, the{Delta} pfap2-mrp parasites overexpress several early gametocyte marker genes at both 16 and 40 h.p.i., indicating a regulatory role in the sexual stage conversion. Using the Chromosomes Conformation Capture experiment (Hi-C), we demonstrate that deletion of PfAP2-MRP results in significant reduction of both intra-chromosomal and inter-chromosomal interactions in heterochromatin clusters. We conclude that PfAP2-MRP is a vital upstream transcriptional regulator controlling essential processes in two distinct developmental stages during the IDC that include parasite growth, chromatin structure and var gene expression.

genomics↗

bioRxiv2023

Novel Herpesvirus Transcripts with Putative Regulatory Roles in DNA Replication and Global Transcription

In the last couple of years, the rapid advances and decreasing costs of sequencing technologies have revolutionized transcriptomic research. Long-read sequencing (LRS) techniques are able to detect full-length RNA molecules in a single run without the need for additional assembly steps. LRS studies have revealed an unexpected transcriptomic complexity in a variety of organisms, including viruses. A number of transcripts with proven or putative regulatory role, mapping close to or overlapping the replication origins (Oris) and the nearby transcription activator genes, have been described in herpesviruses. In this study, we applied both newly generated and previously published LRS and short-read sequencing datasets to discover additional Ori-proximal transcripts in nine herpesviruses belonging to all of the three subfamilies (alpha, beta and gamma). We identified novel long non-coding RNAs (lncRNAs), as well as splice and length isoforms of mRNAs and lncRNAs. Furthermore, our analysis disclosed an intricate meshwork of transcriptional overlaps at the examined genomic regions. Our results suggest the existence of a super regulatory center, which controls both the replication and the global transcription through multilevel interactions between the molecular machineries.

molecular biology↗

bioRxiv2023

Migration and establishment of progenitor pool of melanocytes is governed by SEMA3E-PLXND1 signaling

Vertebrate pigmentation is an outcome of an interplay of several signaling pathways that result in immense diversity of pigment patterns observed across the animal kingdom. Transitory nature of these signaling events impedes deciphering pathways that control migration and establishment of melanocyte stem cells (McSC), necessary for pigment patterning. Using zebrafish and cultured mammalian melanocytes, we uncover a hitherto unknown role for Plexin D1 signaling. This pathway directs migration by F-actin modulation and further dictates subsequent functional states of melanocytes through a transcriptional response. In zebrafish, abrogation of PLXND1 derails melanocyte migration and reduces mid-line melanophores emerging from the regeneration competent McSC pool. In cultured melanocytes, activation of PLXND1 by the ligand semaphorin 3E reduces the velocity of migration, influences directional correlation, and promotes movement towards positive cues such as SCF. PLXND1 activation results in EGFR signaling necessary for McSC establishment, and induces GNAS, an effector of MC1R pathway involved in melanocyte maturation. Identification of this long-range secreted negative chemotactic signaling provides a missing player and enriches reaction diffusion model for pigment patterning.

developmental biology↗

bioRxiv2023

Benchmarking robust spatial transcriptomics approaches to capture the molecular landscape and pathological architecture of archived cancer tissues

AbtractsApplying spatial transcriptomics (ST) to explore a vast amount of formalin-fixed paraffin-embedded (FFPE) archival cancer tissues has been highly challenging due to several critical technical issues. In this work, we optimised ST protocols to generate unprecedented spatial gene expression data for FFPE skin cancer. Skin is among the most challenging tissue types for ST due to its fibrous structure and a high risk of RNAse contamination. We evaluated tissues collected from ten years to two years ago, spanning a range of tissue qualities and complexity. Technical replicates and multiple patient samples were assessed. Further, we integrated gene expression profiles with pathological information, revealing a new layer of molecular information. Such integration is powerful in cancer research and clinical applications. The data allowed us to detect the spatial expression of non-coding RNAs. Together, this work provides important technical perspectives to enable the applications of ST on archived cancer tissues.

pathology↗

bioRxiv2022

Spurious off-target signals from potential lncRNAs by 10X Visium probes

Spatial transcriptomics has revolutionized molecular profiling of tissues in a spatial context, especially in the study of cancer heterogeneity. 10X Genomics facilitates spatial gene expression profiling platforms to help work with fresh-frozen (FF) and formalin fixed paraffin embedded (FFPE) tissues. FF analysis is based on polyA capture of RNAs while FFPE analysis uses a pre-designed set of probes to capture transcripts of coding genes. Previously, we used FFPE spatial data as a negative control in a study to identify novel non-coding RNAs in FF data. Interestingly, we find and report that certain target probes used in FFPE show off-target signals from lncRNAs. The Space Ranger pipeline of 10X Visium counts the expression of these potential off-targets to be that of the corresponding target gene, some of which have known implications in cancer and its diagnosis. Therefore, relying on this technology is not ideal to investigate expression of the genes reported in this study. We hereby recommend excluding those genes in any downstream analysis of FFPE datasets and to design probes with better specificity, considering the sequence similarity between genes and non-coding RNAs.

bioinformatics↗

bioRxiv2022

Whole exome-sequencing of vitiligo lesions indicate lower burden of somatic variations: implications in risk for non-melanoma skin cancers

Mapping of somatic variations has enabled understanding the progression of clonal variations from healthy skin to cutaneous malignancies. Highlighting, the adaptive nature of pigmentation, germline mutations in albinism amplify skin cancer susceptibility. However, lower incidence of non-melanoma skin cancer among subjects with acquired depigmenting skin disorder vitiligo is enigmatic and a matter of longstanding debate. To address this, we performed high-coverage exome sequencing of matched non-lesional and lesional vitiligo skin along with whole blood to account for germline variations. Our analysis suggests lower burden of somatic cancer-associated variations in exposed depigmented lesional skin compared to the non-lesional skin. A detailed investigation of vitiligo skin transcriptome reveals elevation of DNA repair and cell-proliferation pathways. Validation by comet-assay for DNA damage and cell cycle analysis of epidermal cells suggest undamaged DNA in vitiligo lesions that could be attributed to higher proliferation-coupled repair. Endorsing this, UV-signature variations are not prominent, instead SBS5 associated with endogenous mutational processes is conspicuous in both the vitiligo tissues. Our systematic pilot study indicates lower somatic mutation burden in vitiligo skin and supports the earlier demographic observation on lower risk of non-melanoma skin cancer in vitiligo subjects, providing an opportunity to learn strategies for cancer prevention from vitiligo. Brief SummaryVitiligo skin harbors decreased somatic variation burden in cancer-associated genes and a concomitant augmentation in DNA repair response, explaining the lower incidence of cutaneous malignancies.

genomics↗

bioRxiv2022

Sustained pigmentation causes DNA damage and invokes translesion polymerase Pol κ for repair in melanocytes

The pigment melanin protects skin cells from ultraviolet (UV) radiation induced DNA damage. However, intermediates of eumelanin are highly reactive quinones that are potentially genotoxic. In this study, we systematically investigate the effect of sustained elevation of melanogenesis and map the consequent cellular repair response of melanocytes. Pigmentation increases DNA damage, causes cell cycle arrest, and invokes translesion polymerase Pol {kappa} for DNA repair in primary human melanocytes, as well as mouse melanoma cells. Confirming the causal link, CRISPR-based genetic ablation of tyrosinase, the key melanin synthesizing enzyme results in depigmented cells with low Pol {kappa} levels. However, silencing of Pol {kappa} in pigmenting cells results in unchecked proliferation despite the presence of damaged DNA, that could potentially lead to genome instability. Thereby, our results indicate Pol {kappa} to be a necessary evil to resolve melanin induced damage. Error-prone repair by Pol {kappa} in part explains the mutational landscape observed in human melanoma. Thus, our study illuminates a hitherto unknown dark side of melanin and identifies (eu)melanogenesis as a key missing link between tanning response and mutagenesis mediated via the Pol {kappa}-based low fidelity DNA repair response of melanocytes. Key HighlightsO_LISustained melanogenesis causes DNA damage in melanocytes C_LIO_LIMelanogenesis elicits replication stress and translesion repair by Pol {kappa} C_LIO_LIPol {kappa} resolves melanin-induced DNA damage and suppresses genome instability C_LIO_LIExpression of Pol {kappa} correlates with mutational load in human melanoma C_LI

cell biology↗

bioRxiv2022

Deciphering the tissue-specific regulatory role of intronless genes across cancers

Intronless genes (IGs) or single-exon genes lacking an intron are found across most Eukaryotes. Notably, IGs display a higher transcriptional fidelity as they are not regulated through alternative splicing, suggesting better predictability biomarkers and easier regulation as targets for therapy. Cancer is a complex disease that relies on progressive uncontrolled cell division linked with multiple dysfunctional biological processes. Tumor heterogeneity remains the most challenging feature in cancer diagnosis and treatment. Given the clinical relevance of IGs, we aim to identify their unique expression profiles and interactome, that may act as functional signatures across eight different cancers. We identified 940 protein-coding IGs in the human genome, of which about 35% were differentially expressed across the analyzed cancer datasets. Specifically, [~]78% of differentially expressed IGs were undergoing transcriptional reprogramming with elevated expression in tumor cells. Remarkably, in all the studied tumors, a highly conserved induction of a group of deacetylase-histones located in a region of chromosome 6 enriched in nucleosome and chromatin condensation processes. This study highlights that differentially expressed human intronless genes across cancer types are prevalent in epigenetic regulatory roles participating in specific PPI networks for ESCA, GBM, and LUAD tumors. We determine that IGs play a key role in the tumor phenotype at transcriptional and post-transcriptional levels, with important mechanisms such as interactomics rewiring.

genomics↗

bioRxiv2021

Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

Telomeres are crucial for cancer progression. Immune signalling in the tumour microenvironment has been shown to be very important in cancer prognosis. However, the mechanisms by which telomeres might affect tumour immune response remain poorly understood. Here, we observed that interleukin-1 signalling is telomere-length dependent in cancer cells. Mechanistically, non-telomeric TRF2 (Telomeric Repeat binding Factor 2) binding at the IL-1-receptor type-1 (IL1R1) promoter was found to be affected by telomere length. Enhanced TRF2 binding at the IL1R1 promoter in cells with short telomeres directly recruited the histone-acetyl-transferase (HAT) p300, and consequent H3K27 acetylation activated IL1R1. This altered NF-kappa B signalling and affected downstream cytokines like IL6, IL8 and TNF. Further, IL1R1 expression was telomere-sensitive in triple-negative breast cancer (TNBC) clinical samples. Infiltration of tumour-associated macrophages (TAM) was also sensitive to the length of tumour cell telomeres and highly correlated with IL1R1 expression. The use of both IL1 Receptor antagonist (IL1RA) and IL1R1 targeting ligands could abrogate M2 macrophage infiltration in TNBC tumour organoids. In summary, using TNBC cancer tissue (>90 patients), tumour-derived organoids, cancer cells and xenograft tumours with either long or short telomeres, we uncovered a heretofore undeciphered function of telomeres in modulating IL1 signalling and tumour immunity.

cancer biology↗

bioRxiv2021

Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality

The precise molecular mechanisms behind life-threatening lung abnormalities during severe SARS-CoV-2 infections are still unclear. To address this challenge, we performed whole transcriptome sequencing of lung autopsies from 31 patients suffering from severe COVID-19 related complications and 10 uninfected controls. Using a metatranscriptome analysis of lung tissue samples we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant "classical" signature (n=23) showed upregulation of unfolded protein response, steroid biosynthesis and complement activation supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) potentially representing "Cytokine Release Syndrome" (CRS) showed upregulation of cytokines such IL1 and CCL19 but absence of complement activation and muted inflammation. Further, dissecting expression of individual genes within enriched pathways for patient signature suggests heterogeneity in host response to the primary infection. We found that the majority of patients cleared the SARS-CoV-2 infection, but all suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Staphylococcus cohnii in "classical" patients and Pasteurella multocida in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients that can be identified through the status of the complement activation, presence of specific cytokines and characteristic microbiome. This information can be used to design personalized therapy to treat COVID-19 related complications corresponding to patient signature such as using the identified drug molecules or mitigating specific secondary infections.

molecular biology↗

bioRxiv2020

Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast

Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

molecular biology↗