Biology subjects

Upadhyay, R.

Publications and source records attributed to Upadhyay, R..

11 recordsLinked to original sources

bioRxiv2026

A high-throughput screening dataset of small-molecule inhibitors across human DNA glycosylases

The base excision repair pathway removes small base lesions from DNA and is initiated in humans by one of eleven DNA glycosylases with overlapping substrate specificities. Despite their central role in genome maintenance and transcription, systematic datasets describing small-molecule binders or inhibitors of DNA glycosylases are lacking. Here, through a collaborative effort across Scandinavia, we establish a high-throughput biochemical assay platform for human DNA glycosylases and use it to generate a systematic small-molecule screening dataset. A chemogenomic library was screened against nine glycosylases with assays of sufficient quality for hit identification, yielding primary screening and concentration-response data and identifying multiple previously unreported inhibitors. All primary and processed data, along with detailed assay protocols and metadata, are publicly available to support reuse in chemical biology, further compound optimization, assay development and comparative studies of DNA repair enzymes.

biochemistry↗

bioRxiv2026

Dissecting Complex Interactions Between Ferroptosis and the Proteasome

The proteasome is an essential multiprotein complex whose inhibition can lead to apoptosis. Ferroptosis is a non-apoptotic cell death mechanism whose fundamental regulation continues to be elucidated. How proteasome function regulates ferroptosis sensitivity is poorly understood and difficult to study given the essential nature of the proteasome. Here, we isolated the effects of proteasome inhibition on ferroptosis by combining direct cell death imaging, cell death pathway-specific inhibitors, and mathematical modeling. We find that proteasome inhibition enhances sensitivity to ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition while simultaneously promoting resistance to ferroptosis induced by system x - inhibition. Sensitization to GPX4 inhibition requires protein synthesis but not the apoptosis execution machinery and is opposed by the activating transcription factor 4 (ATF4) stress response pathway. This work demonstrates a complex role for proteasome function in ferroptosis regulation and establishes new methods to dissect cross-talk between ferroptosis and essential cellular processes.

cell biology↗

bioRxiv2025

An Eligibility-Aware Pipeline for Robust ITS Diagnostics in Fungi: A Cacao Case Study with Generalizable Rules

Accurate fungal ITS diagnostics rely on public sequence archives, but heterogeneous record lengths, especially frequent truncation before the LSU/28S segment, cause naive in silico benchmarking to conflate primer performance with database incompleteness. To resolve this persistent "denominator error," we present an open and fully reproducible "eligibility-aware" framework. Our pipeline first establishes eligibility by confirming both primer sites are present before applying bench-realistic performance rules, including a strict penalty for 3-terminal mismatches. It further provides mechanistic insights by analyzing binding-site conservation and uses a rarefaction-based approach to guide efficient quality control as databases grow. We demonstrate the frameworks utility using the cacao pathosystem, a context where rapid differentiation of the fungal pathogen Moniliophthora from symptomatically similar oomycetes is critical. The result is a robust, field-ready diagnostic decision tree operable under a single touchdown (TD) PCR program. By providing a transparent and barcode-agnostic template, our eligibility-aware approach offers a significant methodological advance for designing and validating molecular assays in mycology and beyond.

microbiology↗

bioRxiv2025

Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

Antigen (Ag) escape is a frequent mechanism of relapse after CAR-T therapy, even though only [~]1% of leukemic and [~]0.1% of lymphoma cells are Ag- at baseline. In this study, we modeled extreme Ag heterogeneity (>20%) to define how Fas/FasL-dependent bystander killing contributes to tumor clearance. Across patient cohorts, Fas expression predicted survival after CD19 CAR-T therapy, particularly in CD19-low disease. In both murine and human systems, Fas-dependent bystander killing required Ag stimulation and cell contact, operated within a defined therapeutic window, and could eradicate large fractions of Ag- tumors in vivo. Pharmacologic potentiation with inhibitor of apoptosis protein antagonists or genetic stabilization of CAR-T membrane-bound FasL enhanced bystander killing but simultaneously induced CD4 T cell fratricide, which was rescued by CAR-T Fas knockout. Importantly, Fas sensitization also enabled bispecific antibody-redirected T cells to mediate bystander killing in resistant tumors. Finally, targeting tumor-associated macrophages triggered Fas-dependent clearance of neighboring tumor cells. These findings establish Fas-mediated bystander killing as a generalizable and therapeutically actionable axis to prevent Ag escape and broaden the scope of targeted T cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/677496v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@11bedorg.highwire.dtl.DTLVardef@d09d07org.highwire.dtl.DTLVardef@1f53c1dorg.highwire.dtl.DTLVardef@13d96f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Fas-mediated bystander killing can be potentiated by IAP inhibition and FasL stabilization, and further improved by decoupling CD4 T cell fratricide via Fas knockout. Schematic representation of CAR-T bystander killing across three mechanistic states. Left (Baseline): Antigen-positive (CD19) tumor cells activate CAR-T cells, leading to FasL-mediated cytotoxicity of neighboring antigen-negative (CD19-) tumor cells via membrane-bound FasL. Limited FasL expression and ADAM10-mediated shedding constrain bystander killing, and CD4 T cells are largely spared. Middle (Potentiated): Enhancing Fas signaling through IAP inhibition (IAPi), increasing FasL expression, and reducing FasL shedding result in improved bystander killing but also increased CD4 T cell fratricide. Right (Potentiated & Decoupled): Combining IAPi with FasL stabilization and CAR-T Fas knockout (FasKO) augments bystander killing while eliminating CD4 T cell fratricide.

immunology↗

bioRxiv2025

Multiple Wnt signaling pathways direct epithelial tubule interconnection in the regenerating zebrafish kidney

Epithelial tubule fusion is fundamental for kidney morphogenesis. Differentiating nephron tubules interconnect with collecting system epithelia to generate a lumenal pathway for fluid excretion. In the adult zebrafish kidney, nephrogenesis occurs as a regenerative response to injury and provides a model to explore cell signaling pathways required for tubule interconnection. We show that canonical Wnt signaling at the junction between two tubules induces a mesenchymal, invasive cell phenotype and is required, along with Src kinase and rac1, to generate basal cell protrusions. The Wnt ligands wnt9b and wnt4 are both required for new nephron formation after injury. Mutation in wnt4 or treatment with the canonical Wnt inhibitor IWR1 blocks formation of basal protrusions in forming nephrons. Mutation in the Wnt receptor frizzled9b reveals a fusion-associated non-canonical Wnt pathway that acts to 1) restrict canonical Wnt gene expression, 2) drive Rho kinase-dependent apical constriction of epithelial cells, and 3) position basal protrusions and generate orthogonal tubule lumenal connections. As a result, frizzled9b mutant nephrons fail to fully interconnect with target distal tubules. Our results indicate that canonical and non-canonical Wnt signaling interact in the same cells to orient and drive tubule interconnection in the regenerating zebrafish kidney.

developmental biology↗

bioRxiv2024

RORγt-dependent antigen-presenting cells direct regulatory T cell-mediated tolerance to food antigen

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microbes with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1-4. While ROR{gamma}t+ antigen-presenting cells (ROR{gamma}t-APCs) were shown to program gut microbiota-specific pTreg5-7, their definition remains incomplete, and the APC responsible for food tolerance has remained elusive. Here, we identify a distinct subset of ROR{gamma}t-APCs, designated tolerogenic dendritic cells (tDC), required for differentiation of both food- and microbiota-specific pTreg cells and for establishment of oral tolerance. tDC development and function require expression of the transcription factors Prdm16 and ROR{gamma}t, as well as a unique Rorc(t) cis-regulatory element. Gene expression, chromatin accessibility, and surface marker analysis establish tDC as myeloid in origin, distinct from ILC3, and sharing epigenetic profiles with classical DC. Upon genetic perturbation of tDC, we observe a substantial increase in food antigen-specific T helper 2 (Th2) cells in lieu of pTreg, leading to compromised tolerance in mouse models of asthma and food allergy. Single-cell analyses of freshly resected mesenteric lymph nodes from a human organ donor, as well as multiple specimens of human intestine and tonsil, reveal candidate tDC with co-expression of PRDM16 and RORC and an extensive transcriptome shared with mice, highlighting an evolutionarily conserved role across species. Our findings suggest that a better understanding of how tDC develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance.

immunology↗

bioRxiv2024

Discovery and characterization of dietary antigens in oral tolerance

Food antigens elicit immune tolerance through the action of intestinal regulatory T (Treg) cells. Unlike food allergens, the proteins that mediate tolerance are mostly undescribed. Here, we found that epitopes derived from seed storage proteins are targets of murine intestinal Treg cells, with the most frequent response targeting the C-terminus of the maize protein alpha-zein. An MHC tetramer loaded with this antigen revealed that zein-specific T cells are predominantly intestinal Treg cells, develop concurrently with weaning, and constitute up to 2% of the peripheral Treg cell pool. Zein-responsive Treg cells repressed naive T cell proliferation ex vivo and prior dietary exposure resulted in a constrained response upon multiple inflammatory challenges in vivo, supporting a specific role for gut-resident Treg cells in suppressing systemic immune responses. Together, our work reveals the development, immune suppressive characteristics, and function of naturally occurring Treg cells that recognize dietary seed-storage proteins, a previously undescribed class of antigens in oral tolerance. One-sentence summaryImmunodominant epitopes from seed storage proteins are a target of intestinal regulatory T cells that modulate immune challenge to food.

immunology↗

bioRxiv2023

SignEEG v1.0 : Multimodal Electroencephalography and Signature Database for Biometric Systems

Handwritten signatures in biometric authentication leverage unique individual characteristics for identification, offering high specificity through dynamic and static properties. However, this modality faces significant challenges from sophisticated forgery attempts, underscoring the need for enhanced security measures in common applications. To address forgery in signature-based biometric systems, integrating a forgery-resistant modality, namely, noninvasive electroencephalography (EEG), which captures unique brain activity patterns, can significantly enhance system robustness by leveraging multimodalitys strengths. By combining EEG, a physiological modality, with handwritten signatures, a behavioral modality, our approach capitalizes on the strengths of both, significantly fortifying the robustness of biometric systems through this multimodal integration. In addition, EEGs resistance to replication offers a high-security level, making it a robust addition to user identification and verification. This study presents a new multimodal SignEEG v1.0 dataset based on EEG and hand-drawn signatures from 70 subjects. EEG signals and hand-drawn signatures have been collected with Emotiv Insight and Wacom One sensors, respectively. The multimodal data consists of three paradigms based on mental, & motor imagery, and physical execution: i) thinking of the signatures image, (ii) drawing the signature mentally, and (iii) drawing a signature physically. Extensive experiments have been conducted to establish a baseline with machine learning classifiers. The results demonstrate that multimodality in biometric systems significantly enhances robustness, achieving high reliability even with limited sample sizes. We release the raw, pre-processed data and easy-to-follow implementation details.

neuroscience↗

bioRxiv2023

Multimodal characterization of antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection.

The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8+ T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8+ populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.

genomics↗

bioRxiv2022

Electroencephalogram Data Collection for Student Engagement Analysis with Audio-Visual Content

Recognizing and monitoring students attention during learning is crucial to successful knowledge acquisition since it influences cognitive function. As a result, gaining a precise picture of a learners mental state may enable interactive learning systems to alter tutoring content, devise effective help tactics, and improve learning outcomes. In computer-based learning environments, keeping track of students mental states is vital. Investigating the feasibility of utilizing active learning in enhancing student engagement index when exposed to various visual stimuli is the genesis of this work. The research includes collecting EEG data from 20 participants (ten males, ten females) while resting and being subjected to various virtual infotainment/educational content. The EEG data were collected using the Allengers Neuro PLOT, a 40-channel wet electrode system. The work includes raw and pre-processed EEG data under quiescent and audio-visual continuous cues. The recorded data is accommodated by a sophisticated EEG data pre-processing pipeline and will be available to the research community for usage. Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@340852org.highwire.dtl.DTLVardef@e5c51org.highwire.dtl.DTLVardef@cefb1borg.highwire.dtl.DTLVardef@c7d208org.highwire.dtl.DTLVardef@ae4824_HPS_FORMAT_FIGEXP M_TBL C_TBL

neuroscience↗

bioRxiv2022

Characterizing Cone Spectral Classification by Optoretinography

Light propagation in photoreceptor outer segments is affected by photopigment absorption and the phototransduction amplification cascade. Photopigment absorption has been studied using retinal densitometry, while recently, optoretinography (ORG) has provided an avenue to probe changes in outer segment optical path length due to phototransduction. With adaptive optics (AO), both densitometry and ORG have been used for cone spectral classification, based on the differential bleaching signatures of the three cone types. Here, we characterize cone classification by ORG, implemented in an AO line-scan OCT and compare it against densitometry. The cone mosaics of five color normal subjects were classified using ORG showing high probability ([~]0.99), low error (<0.22%), high test-retest reliability ([~]97%) and short imaging durations (< 1 hour). Of these, the cone spectral assignments in two subjects were compared against AOSLO densitometry. High agreement (mean: 91%) was observed between the two modalities in these 2 subjects, with measurements conducted 6-7 years apart. Overall, ORG benefits from higher sensitivity and dynamic range to probe cone photopigments compared to densitometry, and thus provides greater fidelity for cone spectral classification.

neuroscience↗