Biology subjects

Seshagiri, S.

Publications and source records attributed to Seshagiri, S..

11 recordsLinked to original sources

bioRxiv2026

A FZD4/LRP5 agonist restores pericyte coverage and vascular integrity by increasing PDGFB signaling

Pericytes, specialized mural cells of capillaries, fulfill crucial physiological functions including promoting endothelial barrier function and regulating angiogenesis. Pericyte loss or dysfunction represents a central pathological feature in diabetic retinopathy (DR) and is increasingly recognized in neurodegenerative diseases as well as in poor stroke outcomes, underscoring an urgent need for therapies that restore pericyte function or promote their regeneration. Here, we utilized a Frizzled4 (FZD4) and Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5) agonist antibody (F4L5.13) to investigate the functional consequences of mimicking {beta}-catenin-dependent signaling in CNS endothelial cells (ECs), which is physiologically induced by Norrin or WNT7A/B. In platelet-derived growth factor subunit B (Pdgfb) EC-specific knockout (ECKO) mice, a model of severe developmental pericyte deficiency with secondary blood-retina barrier (BRB) defects and hemorrhages, F4L5.13 significantly promoted retinal pericyte/mural cell proliferation and coverage, improved BRB function, reduced hemorrhages, and normalized vascular morphology. F4L5.13 restored Pdgfb mRNA expression levels from non-recombined cells in Pdgfb ECKO retinas. These findings highlight interactions of {beta}-catenin-dependent signaling and PDGFB production, identify a key pharmacodynamic action of F4L5.13 distinct from anti-VEGF therapies, and suggest that FZD4/LRP5 agonists may have uses as a regenerative pharmacology approach that promotes pericyte coverage in the neurovascular unit.

developmental biology↗

bioRxiv2025

Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8+T cell exhaustion, persistence in vivo, and CAR-T function under tumor-associated chronic antigen stimulation

Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty (SB) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8 T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB-mutagenized CD8 T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2, known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1 and CD62L phenotypes, whereas Bach2high restricted differentiation predominantly to the CD62L subset. Finally, in human CART19-28{zeta} cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

immunology↗

bioRxiv2025

C1q limits cystoid edema by maintaining basal beta-catenin-dependent signaling and blood-retina barrier function

Macular edema (ME) causes significant vision impairment and occurs in several prevalent retinal diseases including diabetic retinopathy (DR), choroidal neovascularization (CNV), retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterize Tspan12 KODBM (Disrupted Barrier Maintenance) mice, a new mouse model of cystoid edema based on genetically and pharmacologically manipulating beta-catenin-dependent norrin/frizzled4 (FZD4) signaling. We assess BRB function, cystoid edema, ERG, and microglia activation outcomes in an aging study with WT, C1qa KO, Tspan12 KODBM, and Tspan12 KODBM; C1qa KO compound mutant mice. Phenotypic analyses and cell-based experiments indicate that C1QA contributes to maintaining basal beta-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12 KODBM; C1qa compound mutant mice. Activation of beta-catenin-dependent signaling by a FZD4/LRP5 agonist antibody modality achieves complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/frizzled4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides novel insights into the contribution of C1QA to BRB maintenance.

neuroscience↗

bioRxiv2024

Platelet PI3Kβ regulates breast cancer metastasis

Platelets promote tumor metastasis by several mechanisms. Platelet-tumor cell interactions induce the release of platelet cytokines, chemokines, and other factors that promote tumor cell epithelial-mesenchymal transition and invasion, granulocyte recruitment to circulating tumor cells (CTCs), and adhesion of CTCs to the endothelium, assisting in their extravasation at metastatic sites. Previous studies have shown that platelet activation in the context of thrombus formation requires the Class IA PI 3-kinase PI3K{beta}. We now define a role for platelet PI3K{beta} in breast cancer metastasis. Platelet PI3K{beta} is essential for platelet-stimulated tumor cell invasion through Matrigel. Consistent with this finding, in vitro platelet-tumor cell binding and tumor cell-stimulated platelet activation are reduced in platelets isolated from PI3K{beta} mutant mice. RNAseq and proteomic analysis of human breast epithelial cells co-cultured with platelets revealed that platelet PI3K{beta} regulates the expression of EMT and metastasis-associated genes in these cells. The EMT and metastasis-associated proteins PAI-1 and IL-8 were specifically downregulated in co-cultures with PI3K{beta} mutant platelets. PI3K{beta} mutant platelets are impaired in their ability to stimulate YAP and Smad2 signaling in tumor cells, two pathways regulating PAI-1 expression. Finally, we show that mice expressing mutant PI3K{beta} show reduced spontaneous metastasis, and platelets isolated from these mice are less able to stimulate experimental metastasis in WT mice. Taken together, these data support a role for platelet PI3K{beta} in promoting breast cancer metastasis and highlight platelet PI3K{beta} as a potential therapeutic target. SignificanceWe demonstrate that platelet PI3K{beta} regulates metastasis, broadening the potential use of PI3K{beta}-selective inhibitors as novel agents to treat metastasis.

cancer biology↗

bioRxiv2022

Near-chromosomal de novo assembly of Bengal tiger genome reveals genetic hallmarks of apex-predation

The tiger, a poster child for conservation, remains an endangered apex predator. Continued survival and recovery will require a comprehensive understanding of their genetic diversity and the use of such information for population management. A high-quality tiger genome assembly will be an important tool for conservation genetics, especially for the Indian tiger, the most abundant subspecies in the wild. Here, we present high-quality near-chromosomal genome assemblies of a female and a male wild Indian tiger (Panthera tigris tigris). Our assemblies had a scaffold N50 of >140{square}Mb, with 19{square}scaffolds, corresponding to the 19 numbered chromosomes, containing 95% of the genome. Our assemblies also enabled detection of longer stretches of runs of homozygosity compared to previous assemblies which will improve estimates of genomic inbreeding. Comprehensive genome annotation identified 26,068 protein-coding genes, including several gene families involved in key morphological features such as the teeth, claws, vision, olfaction, taste and body stripes. We also identified 301 microRNAs, 365 small nucleolar RNAs, 632 tRNAs and other noncoding RNA elements, several of which are predicted to regulate key biological pathways that likely contribute to tigers apex predatory traits. We identify signatures of positive selection in the tiger genome that are consistent with the Panthera lineage. Our high-quality genome will enable use of non-invasive samples for comprehensive assessment of genetic diversity, thus supporting effective conservation and management of wild tiger populations.

genomics↗

bioRxiv2020

South Asian patient population genetics reveal strong founder effects and high rates of homozygosity - new resources for precision medicine

Population-scale genetic studies can identify drug targets and allow disease risk to be predicted with resulting benefit for management of individual health risks and system-wide allocation of health care delivery resources. Although population-scale projects are underway in many parts of the world, genetic variation between population groups means that additional projects are warranted. South Asia has a population whose genetics is the least characterized of any of the worlds major populations. Here we describe GenomeAsia studies that characterize population structure in South Asia and that create tools for economical and accurate genotyping at population-scale. Prior work on population structure characterized isolated population groups, the relevance of which to large-scale studies of disease genetics is unclear. For our studies we used whole genome sequence information from 4,807 individuals recruited in the health care delivery systems of Pakistan, India and Bangladesh to ensure relevance to population-scale studies of disease genetics. We combined this with WGS data from 927 individuals from isolated South Asian population groups, and developed a custom SNP array (called SARGAM) that is optimized for future human genetic studies in South Asia. We find evidence for high rates of reproductive isolation, endogamy and consanguinity that vary across the subcontinent and that lead to levels of homozygosity that approach 100 times that seen in outbred populations. We describe founder effects that increase the power to associate functional variants with disease processes and that make South Asia a uniquely powerful place for population-scale genetic studies.

genomics↗

bioRxiv2020

Human ACE2 receptor polymorphisms predict SARS-CoV-2 susceptibility

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease (COVID-19) that has resulted in a global pandemic. It is a highly contagious positive strand RNA virus and its clinical presentation includes severe to critical respiratory disease that appears to be fatal in [~]3-5% of the cases. The viral spike (S) coat protein engages the human angiotensin-converting enzyme2 (ACE2) cell surface protein to invade the host cell. The SARS-CoV-2 S-protein has acquired mutations that increase its affinity to human ACE2 by [~]10-15-fold compared to SARS-CoV S-protein, making it highly infectious. In this study, we assessed if ACE2 polymorphisms might alter host susceptibility to SARS-CoV-2 by affecting the ACE2 S-protein interaction. Our comprehensive analysis of several large genomic datasets that included over 290,000 samples representing >400 population groups identified multiple ACE2 protein-altering variants, some of which mapped to the S-protein-interacting ACE2 surface. Using recently reported structural data and a recent S-protein-interacting synthetic mutant map of ACE2, we have identified natural ACE2 variants that are predicted to alter the virus-host interaction and thereby potentially alter host susceptibility. In particular, human ACE2 variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R are predicted to increase susceptibility. The T92I variant, part of a consensus NxS/T N-glycosylation motif, confirmed the role of N90 glycosylation in immunity from non-human CoVs. Other ACE2 variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y are putative protective variants predicted to show decreased binding to SARS-CoV-2 S-protein. Overall, ACE2 variants are rare, consistent with the lack of selection pressure given the recent history of SARS-CoV epidemics, however, are likely to play an important role in altering susceptibility to CoVs.

genetics↗

bioRxiv2019

Accurate assembly of the olive baboon (Papio anubis) genome using long-read and Hi-C data

Besides macaques, baboons are the most commonly used nonhuman primate in biomedical research. Despite this importance, the genomic resources for baboons are quite limited. In particular, the current baboon reference genome Panu_3.0 is a highly fragmented, reference-guided (i.e., not fully de novo) assembly, and its poor quality inhibits our ability to conduct downstream genomic analyses. Here we present a truly de novo genome assembly of the olive baboon (Papio anubis) that uses data from several recently developed single-molecule technologies. Our assembly, Panubis1.0, has an N50 contig size of ~1.46 Mb (as opposed to 139 Kb for Panu_3.0), and has single scaffolds that span each of the 20 autosomes and the X chromosome. We highlight multiple lines of evidence (including Bionano Genomics data, pedigree linkage information, and linkage disequilibrium data) suggesting that there are several large assembly errors in Panu_3.0, which have been corrected in Panubis1.0.

genomics↗

bioRxiv2019

Massively parallel single-cell B-cell receptor sequencing enables rapid discovery of diverse antigen-reactive antibodies

Using high-throughput single-cell B-cell receptor sequencing (scBCR-seq) we obtained accurately paired full-length heavy- and light-chain variable domains from thousands of individual B cells in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells that were subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages that were not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.

immunology↗

bioRxiv2019

SCINA: Semi-Supervised Analysis of Single Cells in silico

Advances in single-cell RNA sequencing (scRNA-Seq) have allowed for comprehensive analyses of single cell data. However, current analyses of scRNA-Seq data usually start from unsupervised clustering or visualization. These methods ignore the prior knowledge of transcriptomes and of the probable structures of the data. Moreover, cell identification heavily relies on subjective and inaccurate human inspection afterwards. We reversed this paradigm and developed SCINA, a semi-supervised model, for analyses of scRNA-Seq and flow cytometry/CyTOF data, and other data of similar format, by automatically exploiting previously established gene signatures using an expectation-maximization (EM) algorithm. We applied SCINA on a wide range of datasets, and showed its accuracy, stableness and efficiency exceeded most popular unsupervised approaches. Notably, SCINA discovered an intermediate stage of oligodendrocyte from mouse brain scRNA-Seq data. SCINA also detected immune cell population shifting in Stk4 knock-out mouse cytometry data. Finally, SCINA identified a new kidney tumor clade with similarity to FH-deficient tumors from bulk tumor data. Overall, SCINA provides both methodological advances and biological insights from perspectives different from traditional analytical methods.

bioinformatics↗

bioRxiv2018

Evaluating the quality of the 1000 Genomes Project data

Data from the 1000 Genomes project is quite often used as a reference for human genomic analysis. However, its accuracy needs to be assessed to understand the quality of predictions made using this reference. We present here an assessment of the genotype, phasing, and imputation accuracy data in the 1000 Genomes project. We compare the phased haplotype calls from the 1000 Genomes project to experimentally phased haplotypes for 28 of the same individuals sequenced using the 10X Genomics platform. We observe that phasing and imputation for rare variants are unreliable, which likely reflects the limited sample size of the 1000 Genomes project data. Further, it appears that using a population specific reference panel does not improve the accuracy of imputation over using the entire 1000 Genomes data set as a reference panel. We also note that the error rates and trends depend on the choice of definition of error, and hence any error reporting needs to take these definitions into account.

genomics↗