Biology subjects

Joseph, A.

Publications and source records attributed to Joseph, A..

14 recordsLinked to original sources

bioRxiv2026

A ML-framework for the discovery of next-generation IBD targets using a harmonized single-cell atlas of patient tissue

Target discovery for IBD has traditionally relied on genetic associations, which lack the cellular resolution needed to identify novel, actionable, cell type-specific disease pathways. Here, we describe an integrated analytical and experimental framework that leverages harmonized single-cell data to systematically discover novel therapeutic strategies for IBD. We used AMICA DBTM, Immunais harmonized database of single-cell RNA datasets to construct a harmonized 1 million single-cell atlas of the human intestine. We applied a machine learning framework (Immune Patient Representation, IPR) to identify disease-associated transcriptional programs and cell type-specific gene targets. Candidate targets were prioritized using atlas-derived metrics, refined using custom criteria emphasizing translational actionability, and validated across independent clinical cohorts. Select candidates were evaluated in human primary-cell models reflecting the targets cell-type context. The IPR framework identified 85 disease-associated transcriptional programs and ranked 400 cell type-specific target genes across immune and stromal lineages. Disease-associated programs were interpreted using a structured AI-assisted reasoning framework for structured biological reasoning, linking them to IBD-relevant pathways and guiding the identification of novel, promising gene targets. Functional validation of two cell-type-specific candidates, PTGIR in myeloid cells and IL6ST in fibroblasts, confirmed the reduction of inflammatory and fibrotic pathways linked to IBD pathology. Multi-omic profiling and projection of in vitro phenotypes to patient datasets demonstrated the reversal of disease-associated programs via mechanisms distinct from those of existing biologics. Our single-cell anchored, machine-learning framework integrates in silico discovery with experimental validation, revealing new cell type-specific therapeutic opportunities and supporting a scalable approach for precision target discovery in IBD and other immune-mediated diseases.

bioinformatics↗

bioRxiv2025

p53Y220C-BET-bifunctionals (tPRIMEs) drive p53Y220C-mutant cancer cells into apoptosis

The transcription factor and tumor suppressor p53 is one of the most frequently mutated genes in cancer and has been difficult to target therapeutically due to its intrinsically disordered regions. The hotspot mutation p53Y220C, a mutation thermodynamically destabilizing p53, creates a unique extended crevice on the surface of the protein for which chemical matter has been identified over the last years. Advanced p53Y220C stabilizers reconstitute p53Y220C to its wildtype conformation, thereby restoring p53s role in target gene expression and inhibiting the growth of p53Y220C mutant cancer cell lines. We hypothesized that direct recruitment of the transcriptional elongation machinery to p53Y220C and its target genes may potentiate effects beyond p53 protein stabilization alone. We leveraged induced proximity to discover bifunctional molecules, p53Y220C-targeted PRoximity Induced Modulators of Expression (tPRIMEs), that specifically recognize the BET bromodomain proteins and induce stable ternary complexes with p53Y220C. p53Y220C-tPRIMEs potently inhibit proliferation and induce apoptosis of p53Y220C mutant cancer cell lines to a greater extent than the parental ligands alone or in combination. Gene expression analyses revealed that p53Y220C-tPRIMEs induce an increase in p53 target gene expression compared to parental binders. The superior antiproliferative activity, enhanced apoptosis, and increased p53 target gene expression are dependent on ternary complex formation. These data strongly suggest that a p53Y220C-tPRIME-mediated induced proximity approach between transcriptional regulators and p53Y220C - in contrast to p53 stabilization alone - can modulate the cell fate control from cell cycle inhibition to an apoptotic response, providing a compelling therapeutic modality for p53 mutant cancers.

cancer biology↗

bioRxiv2024

Role of the CTCF Binding Site in Human T-Cell Leukemia Virus-1 Pathogenesis

During HTLV-1 infection, the virus integrates into the host cell genome as a provirus with a single CCCTC binding protein (CTCF) binding site (vCTCF-BS), which acts as an insulator between transcriptionally active and inactive regions. Previous studies have shown that the vCTCF-BS is important for maintenance of chromatin structure, regulation of viral expression, and DNA and histone methylation. Here, we show that the vCTCF-BS also regulates viral infection and pathogenesis in vivo in a humanized (Hu) mouse model of adult T-cell leukemia/lymphoma. Three cell lines were used to initiate infection of the Hu-mice, i) HTLV-1-WT which carries an intact HTLV-1 provirus genome, ii) HTLV-1-CTCF, which contains a provirus with a mutated vCTCF-BS which abolishes CTCF binding, and a stop codon immediate upstream of the mutated vCTCF-BS which deletes the last 23 amino acids of p12, and iii) HTLV-1-p12stop that contains the intact vCTCF-BS, but retains the same stop codon in p12 as in the HTLV-1-CTCF cell line. Hu-mice were infected with mitomycin treated or irradiated HTLV-1 producing cell lines. There was a delay in pathogenicity when Hu-mice were infected with the CTCF virus compared to mice infected with either p12 stop or WT virus. Proviral load (PVL), spleen weights, and CD4 T cell counts were significantly lower in HTLV-1-CTCF infected mice compared to HTLV-1-p12stop infected mice. Furthermore, we found a direct correlation between the PVL in peripheral blood and death of HTLV-1-CTCF infected mice. In cell lines, we found that the vCTCF-BS regulates Tax expression in a time-dependent manner. The scRNAseq analysis of splenocytes from infected mice suggests that the vCTCF-BS plays an important role in activation and expansion of T lymphocytes in vivo. Overall, these findings indicate that the vCTCF-BS regulates Tax expression, proviral load, and HTLV pathogenicity in vivo. Author SummaryHuman T-cell leukemia virus type 1 (HTLV-1) is a cause of leukemia and lymphoma, and several inflammatory medical disorders. The virus integrates in the host cell DNA, and it includes a single binding site for a cellular protein designated CTCF. This protein is important in regulation of many viruses, as well as properties of normal and malignant cells. In order to define the role of CTCF in HTLV-1 pathogenesis in vivo, we analyzed a mutant virus lacking the binding site in humanized mice. We found that this mutation slowed virus spread and attenuated the development of disease. Gene expression studies demonstrated a dynamic role of CTCF in regulating viral gene expression and T lymphocyte activation.

microbiology↗

bioRxiv2024

Single-Cell Transcriptomic Analysis of Kaposi Sarcoma

Kaposi Sarcoma (KS) is a complex tumor caused by KS-associated herpesvirus 8 (KSHV). Histological analysis reveals a mixture of "spindle cells", vascular-like spaces, extravasated erythrocytes, and immune cells. In order to elucidate the infected and uninfected cell types in KS tumors, we examined skin and blood samples from twelve subjects by single cell RNA sequence analyses. Two populations of KSHV-infected cells were identified, one of which represented a proliferative fraction of lymphatic endothelial cells, and the second represented an angiogenic population of vascular endothelial tip cells. Both infected clusters contained cells expressing lytic and latent KSHV genes. Novel cellular biomarkers were identified in the KSHV infected cells, including the sodium channel SCN9A. The number of KSHV positive tumor cells was found to be in the 6% range in HIV-associated KS, correlated inversely with tumor-infiltrating immune cells, and was reduced in biopsies from HIV-negative individuals. T-cell receptor clones were expanded in KS tumors and blood, although in differing magnitudes. Changes in cellular composition in KS tumors were identified in subjects treated with antiretroviral therapy alone, or immunotherapy. These studies demonstrate the feasibility of single cell analyses to identify prognostic and predictive biomarkers. Author SummaryKaposi sarcoma (KS) is a malignancy caused by the KS-associated herpesvirus (KSHV) that causes skin lesions, and may also be found in lymph nodes, lungs, gastrointestinal tract, and other organs in immunosuppressed individuals more commonly than immunocompetent subjects. The current study examined gene expression in single cells from the tumor and blood of these subjects, and identified the characteristics of the complex mixtures of cells in the tumor. This method also identified differences in KSHV gene expression in different cell types and associated cellular genes expressed in KSHV infected cells. In addition, changes in the cellular composition could be elucidated with therapeutic interventions.

cancer biology↗

bioRxiv2024

HTLV-1 infected T cells cause bone loss via small extracellular vesicles

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine RANKL, suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV injected over mouse calvaria in the presence of low dose RANKL caused more osteolysis than RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukemia.

cancer biology↗

bioRxiv2023

In field use of water samples for genomic surveillance of ISKNV infecting tilapia fish in Lake Volta, Ghana

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

genomics↗

bioRxiv2023

ERK1/2 Inhibition Alleviates Noise-Induced Hearing Loss While Tempering Down the Immune Response

Noise-induced hearing loss (NIHL) is a major cause of hearing impairment, yet no FDA-approved drugs exist to prevent it. Targeting the mitogen activated protein kinase (MAPK) cellular pathway has emerged as a promising approach to attenuate NIHL. Tizaterkib is an orally bioavailable, highly specific ERK1/2 inhibitor, currently in Phase-1 anticancer clinical trials. Here, we tested tizaterkibs efficacy against permanent NIHL in mice at doses equivalent to what humans are currently prescribed in clinical trials. The drug given orally 24 hours after noise exposure, protected an average of 20-25 dB SPL in three frequencies, in female and male mice, had a therapeutic window >50, and did not confer additional protection to KSR1 genetic knockout mice, showing the drug works through the MAPK pathway. Tizaterkib shielded from noise-induced cochlear synaptopathy, and a 3-day, twice daily, treatment with the drug was the optimal determined regimen. Importantly, tizaterkib was shown to decrease the number of CD45 and CD68 positive immune cells in the cochlea following noise exposure, which could be part of the protective mechanism of MAPK inhibition.

neuroscience↗

bioRxiv2023

Deep learning classification of ex vivo human colon tissues using spectroscopic OCT

Screening programs for colorectal cancer (CRC) have had a profound impact on the morbidity and mortality of this disease by detecting and removing early cancers and precancerous adenomas with colonoscopy. However, CRC continues to be the third leading cause of cancer-related mortality in both men and woman, partly because of limitations in colonoscopy-based screening. Thus, novel strategies to improve the efficiency and effectiveness of screening colonoscopy are urgently needed. Here, we propose to address this need using an optical biopsy technique based on spectroscopic optical coherence tomography (OCT). The depth resolved images obtained with OCT are analyzed as a function of wavelength to measure optical tissue properties. The optical properties can be used as input to machine learning algorithms as a means to classify adenomatous tissue in the colon. In this study, biopsied tissue samples from the colonic epithelium are analyzed ex vivo using spectroscopic OCT and tissue classifications are generated using a novel deep learning architecture, informed by machine learning methods including LSTM and KNN. The overall classification accuracy obtained was 88.9%, 76.0% and 97.9% in discriminating tissue type for these methods. Further, we apply an approach using false coloring of en face OCT images based on SOCT parameters and deep learning predictions to enable visual identification of tissue type. This study advances the spectroscopic OCT towards clinical utility for analyzing colonic epithelium for signs of adenoma.

pathology↗

bioRxiv2022

Requirement for specific bacterial genome maintenance pathways in repair of C8-linked pyrrolobenzodiazepine (PBD) bi-aryl monomer-mediated DNA damage

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and antibacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

microbiology↗

bioRxiv2020

Detection of NDM-1, VIM-1 and AIM-type Metallo-beta-lactamase genes in Gram-negative bacteria isolated from clinical samples in Tamil Nadu

The distribution of carbapenem-resistant Gram-negative bacteria has become an increasing public health concern in India. The aim of this study was to investigate the prevalence of carbapenem-resistant bacteria isolated from the clinical samples in Tamil Nadu, India. A total of 126 non-repetitive Gram-negative bacteria were taken for this study. The susceptibility to meropenem was determined by Minimum Inhibitory Concentration (MIC) by broth micro-dilution. The phenotypic resistance screening such as MHT (Modified Hodge test), EDTA disk synergy and CIM (carbapenem inactivation method) were performed. A multiplex PCR was used for the detection of carbapenemase-encoding genes. Among the 126 isolates studied, 82 (65.07%) meropenem-resistant isolates were identified by MIC. A total of 18 (21.9%) isolates were found to be positive for Metallo-{beta}-Lactamase production through EDTA synergy test. None of the isolates were carbapenemase producer by MHT and CIM. The isolates identified with resistance genes (8/82) were blaNDM-1 in two Klebsiella sp., two P. aeruginosa and one A. baumannii, blaVIM-1 in one P. aeruginosa and blaAIM-1 in one P. aeruginosa and one A. baumannii. The study showed the distribution and increase of carbapenem-resistant bacteria in the study region. Therefore, constant monitoring and effective elimination should be focused to reduce the spread of carbapenem-resistant isolates.

microbiology↗

bioRxiv2020

PRAK-03202: A triple antigen VLP vaccine candidate against SARS CoV-2

The rapid development of safe and effective vaccines against SARS CoV-2 is the need of the hour for the coronavirus outbreak. Here, we have developed PRAK-03202, the worlds first triple antigen VLP vaccine candidate in a highly characterized S. cerevisiae-based D-CryptTM platform, which induced SARS CoV-2 specific neutralizing antibodies in BALB/c mice. Immunizations using three different doses of PRAK-03202 induces antigen specific (Spike, envelope and membrane proteins) humoral response and neutralizing potential. PBMCs from convalescent patients, when exposed to PRAK-03202, showed lymphocyte proliferation and elevated IFN-{gamma} levels suggestive of conservation of epitopes and induction of T helper 1 (Th1)-biased cellular immune responses. These data support the clinical development and testing of PRAK-03202 for use in humans.

immunology↗

bioRxiv2020

Label-free imaging of immune cell dynamics in the living retina using adaptive optics

Our recent work characterized the movement of single blood cells within the retinal vasculature of healthy mice (Joseph et al. 2019) using adaptive optics ophthalmoscopy. Here we apply this technique to the context of acute inflammation and discover both infiltrating and tissue-resident immune cells to be visible without any labelling in the living retina using near - infrared light alone. Intravital imaging of immune cells can be negatively impacted by surgical manipulation, exogenous dyes, transgenic manipulation and phototoxicity. These confounds are now overcome, using phase contrast and time-lapse videography to reveal the dynamic behavior of myeloid cells as they interact, extravasate and survey the retina. Cellular motility and differential vascular responses can be measured noninvasively and in vivo across hours to months at the same retinal location, from initiation to the resolution of inflammation. As comparable systems are already available for clinical research, this approach could be readily translated to human application. Impact statementImmune cell motility and vascular response are imaged in vivo and label-free in the CNS for the first time, using high-resolution phase-contrast adaptive optics retinal imaging.

immunology↗

bioRxiv2019

Imaging single-cell blood flow in the smallest to largest vessels in the living retina

Impact StatementUsing a specialized camera that corrects for eye blur, millions of single blood cells are imaged and their speed measured, as they travel through the largest-to-smallest vessels of the retina. AbstractTissue light scatter limits the visualization of the microvascular network deep inside the living mammal. The transparency of the mammalian eye provides a noninvasive view of the microvessels of the retina, a part of the central nervous system. Despite its clarity, imperfections in the optics of the eye blur microscopic retinal capillaries, and single blood cells flowing within. This limits early evaluation of microvascular diseases that originate in capillaries. To break this barrier, we use adaptive optics to noninvasively measure single-cell blood flow, in one of the most widely used research animals: the C57BL/6J mouse. Flow ranged four orders of magnitude (0.0002-1.55 {micro}L min-1) across the full spectrum of retinal vessel diameters (3.2-45.8 {micro}m), without requiring surgery or contrast dye. Here we describe the data collection approach using adaptive optics and provide an analysis pipeline that can measure millions of blood cell speeds automatically.

bioinformatics↗

bioRxiv2016

Floral Radiometry: A Biophysical Basis to Characterize Landscapes.

This paper presents early results on studies of floral spectral reflectance of angiosperms and introduces floral radiometry as an emergent dimension of ecological research in India. Floral spectral reflectance of 121 angiosperm species was measured using hand-held spectroradiometer. The authors describe spectral reflectance of seven representative species within 350-800 nanometer region of the electromagnetic spectrum. Characteristic absorption and reflection of flowers, in ultraviolet and visible regions of the spectrum is reported. Near infrared reflectance was consistently high for all species studied. Flower color is unique to a species and importance of understanding flower color from pollinators perspective is highlighted.

ecology↗