Biology subjects

Reddy, V.

Publications and source records attributed to Reddy, V..

9 recordsLinked to original sources

bioRxiv2026

Membrane-coupled conformational switching of VISTA regulates immune checkpoint signaling via CC' loop accessibility

V-domain Ig Suppressor of T cell activation (VISTA) has emerged as a critical target for anti-cancer immunotherapy. VISTA inhibits T cell activity, suppressing the anti-cancer immune responses. Here, we use atomistic molecular dynamics simulations to investigate the conformational behavior of membrane-bound human VISTA using both a glycan-free truncated model and a glycosylated, full-length model. Our simulations show that the extracellular and transmembrane domains remain structurally stable; notably, the unusually long CC' loop is stabilized by extensive hydrogen-bonding interactions. Principal component analysis and conformational clustering reveal a dominant rotational motion of the extracellular domain relative to the membrane, giving rise to two recurrent conformational states: an "Up" state in which the CC' loop is solvent-exposed and accessible for ligand engagement, and a "Down" state in which the loop transiently associates with the lipid bilayer. These transitions are allosterically coupled to proline-mediated bending of the transmembrane helix. We propose that this membrane-coupled Up/Down conformational switching regulates CC' loop accessibility, favoring cis interactions with cognate protein partners on the same cell surface, consistent with recent experimental observations, while permitting context-dependent trans interactions. Together, our findings reveal a membrane-coupled conformational mechanism regulating VISTA immune checkpoint function.

cancer biology↗

bioRxiv2025

Inflamed Microglia like Macrophages in the Central Nervous System of Prodromal Parkinson's Disease

We hypothesized that prodromal Parkinsons Disease (PD), characterized by REM Sleep Behavior Disorder and hyposmia, is an inflammatory disorder initiated in the gut that evolves into a neurodegenerative process manifest as classical PD. To test this hypothesis, we performed single-cell RNAseq analysis of cerebrospinal fluid (CSF) and blood from 118 individuals, comparing healthy subjects with prodromal PD and manifest PD to patients with the central nervous system (CNS) autoimmune disease, multiple sclerosis (MS). Surprisingly, we identified increased numbers of immune cells in the CSF of patients with prodromal PD. Single-cell RNA sequencing revealed increases in CSF-specific microglia-like macrophages with enhanced JAK-STAT and TNF signaling signatures in prodromal PD. CSF macrophages exhibited similar transcriptional profiles to dural macrophages and gut muscularis macrophages from -synuclein-expressing PD model mice. Finally, shared memory T cell clones were found in humans between gut and CSF T cells. These findings uncover a myeloid-mediated TNF inflammatory process in the CNS of patients with prodromal PD, with an immunological linkage between the gut and the CNS.

immunology↗

bioRxiv2025

Bacterial Vaginosis Toxins Impair Sperm Capacitation and Fertilization

Study questionWhat effect do toxins produced by bacterial vaginosis (BV) bacteria have on sperm function? Summary answerBacterial vaginosis toxins dysregulate sperm capacitation and intracellular calcium homeostasis and impair the ability of sperm to fertilize oocytes. What is known alreadyIn bacterial vaginosis, which is linked to infertility, overgrowth of Prevotella and Gardnerella in the vagina is accompanied by elevated concentrations of the toxins lipopolysaccharide (LPS) and vaginolysin (VLY). Study design, size, durationThis was a laboratory study in which human semen samples were collected from consenting healthy donors with normal semen parameters. Mouse sperm samples were obtained from the caudal epididymis. Participants/materials, setting, methodsMotile mouse and human sperm were isolated via swim-up and treated under non-capacitating or capacitating conditions. LPS from Escherichia coli was commercially available. VLY was produced by cloning the Gardnerella VLY protein in the ClearColi expression system. Mouse sperm were pre-incubated in in vitro fertilization medium with LPS or VLY and then co-cultured with ovulated cumulus-oocyte complexes. The effects of LPS and VLY on sperm motility and hyperactivation were assessed with computer-assisted sperm analysis. Effects on viability were assessed by Hoechst staining. Acrosomal exocytosis was assessed in sperm from transgenic Acr-eGFP mice and in human sperm stained with Pisum sativum agglutinin FITC. Intracellular calcium dynamics were assessed by staining sperm with the calcium-sensitive dye Fluo-4 AM and fluorescent imaging several sperm at the single-cell level. The effects of LPS on sperm from CatSper knock-out mice were assessed. Additionally, sperm were treated with a toll-like receptor 4 antagonist and further exposed to LPS. Main results and the role of chanceExposure of mouse sperm to LPS or VLY significantly decreased in vitro fertilization (P < 0.05). Under capacitating conditions, both toxins initially increased mouse and human sperm hyperactivation, then significantly decreased sperm motility (P < 0.05), hyperactivation (P < 0.05), and acrosomal exocytosis (P < 0.01). These changes were accompanied by a rapid and irreversible increase in intracellular calcium concentration. Effects of LPS, but not VLY, were prevented by polymyxin-B, which aggregates LPS. The LPS-induced intracellular calcium increase required external calcium but not the calcium channel CatSper and was inhibited by the Toll-like receptor 4 antagonist. Limitations, reasons for cautionFirst, the commercially available LPS we used was isolated from Escherichia coli, rather than from the BV-associated bacteria Prevotella bivia. Second, we did not quantify the absolute sperm intracellular calcium concentration before or after LPS or VLY treatment. Third, all of our experiments were in vitro. Wider implications of the findingsThese studies suggest that BV-associated toxins contribute to infertility by, in part, impairing sperm capacitation and reducing their fertilizing ability. Study funding/competing interest(s)This work was supported by the National Institutes of Health (grant #R01 HD069631). The authors declare that they have no conflict of interest.

physiology↗

bioRxiv2024

Twinfilin is a non-processive depolymerase which synergizes with formin to dramatically accelerate actin filament uncapping by 300-fold

Cellular actin networks assemble by actin filament elongation at barbed ends and are thought to disassemble primarily by depolymerization at filament pointed ends. Contrary to this conventional understanding of actin dynamics, twinfilin was recently shown to promote barbed-end depolymerization. Twinfilin has additionally been suggested to sequester monomers and cap as well as uncap filament barbed ends. As a result, the exact mechanisms by which twinfilin affects barbed-end dynamics remain controversial. Using multicolor single-molecule microscopy, we show that both mouse and yeast twinfilin are non-processive depolymerases that interact only transiently with barbed ends ([~]0.2-0.5 s). Each twinfilin binding event, on average, results in the removal of one or two actin subunits. At CP-capped barbed ends, twinfilin synergizes with formin to accelerate uncapping by up to [~]320-fold. We find that uncapping by twinfilin, alone and together with formin, depends on the nucleotide state of the filament, with the two proteins causing a much more modest enhancement of uncapping of newly assembled filaments. Our study thus establishes twinfilin as a multifunctional barbed-end binding protein capable of non-processively depolymerizing, transiently capping, and synergizing with formin to rapidly uncap actin filament barbed ends.

biochemistry↗

bioRxiv2023

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.

biochemistry↗

bioRxiv2023

Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinixtcal oncology research. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scid IL2Rgammanull (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. In the lung TRACERx PDX pipeline, lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest the presence of B cell clones with lymphoproliferative potential within primary NSCLC tumours that are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).

cancer biology↗

bioRxiv2021

Differential Vulnerability of Anterior Cingulate Cortex Cell-Types to Diseases and Drugs

In psychiatric disorders, mismatches between disease-states and therapeutic strategies are highly pronounced, largely because of unanswered questions regarding specific vulnerabilities of different cell-types and therapeutic responses. Which cellular events (housekeeping or salient) are most affected? Which cell-types succumb first to challenges, and which exhibit the strongest response to drugs? Are these events coordinated between cell-types? How does the disease-state and drug affect this coordination? To address these questions, we analyzed single-nucleus-RNAseq (sn-RNAseq) data from the human anterior cingulate cortex--a region involved in many psychiatric disorders. Density index, a metric for quantifying similarities and dissimilarities across functional profiles, was employed to identify common (housekeeping) or salient functional themes across all cell-types. Cell-specific signatures were integrated with existing disease and drug-specific signatures to determine cell-type-specific vulnerabilities, druggabilities, and responsiveness. Clustering of functional profiles revealed cell-types jointly participating in these events. SST and VIP interneurons were found to be most vulnerable, whereas pyramidal neurons were least vulnerable. Overall, the disease-state is superficial layer-centric, largely influences cell-specific salient themes, strongly impacts disinhibitory neurons, and influences astrocyte interaction with a subset of deep-layer pyramidal neurons. Drug activities, on the other hand, are deep layer-centric and involve activating a distinct subset of deep-layer pyramidal neurons to circumvent the disinhibitory circuit malfunctioning in the disease-state. These findings demonstrate a novel application of sn-RNAseq data to explain drug and disease action at a systems level, suggests a targeted drug development and reevaluate various postmortem-based findings.

neuroscience↗

bioRxiv2021

Genome-wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

Current preimplantation genetic testing (PGT) enables the selection of embryos based on fetal aneuploidy or the presence a small number of preselected disease-associated variants. Here we present a new approach that takes advantage of the improved genome coverage and uniformity of primary template-directed amplification (PTA) to call most early embryo genetic variants accurately and reproducibly from a preimplantation biopsy. With this approach, we identified clonal and mosaic chromosomal aneuploidy, de novo mitochondrial variants, and variants predicted to cause mendelian and non-mendelian diseases. In addition, we utilized the genome-wide information to compute polygenic risk scores for common diseases. Although numerous computational, interpretive, and ethical challenges remain, this approach establishes the technical feasibility of screening for and preventing numerous debilitating inherited diseases.

bioengineering↗

bioRxiv2019

Castration-resistant prostate cancer: Androgen receptor inactivation induces telomere DNA damage, and damage response inhibition leads to cell death

Telomere stability is important for cell viability, as cells with telomere DNA damage that is not repaired do not survive. We reported previously that androgen receptor (AR) antagonist induces telomere DNA damage in androgen-sensitive LNCaP prostate cancer cells; this triggers a DNA damage response (DDR) at telomeres that includes activation of ATM, and blocking ATM activation prevents telomere DNA repair and leads to cell death. Remarkably, AR antagonist induces telomere DNA damage and triggers ATM activation at telomeres also in 22Rv1 castration-resistant prostate cancer (CRPC) cells that are not growth inhibited by AR antagonist. Treatment with AR antagonist enzalutamide (ENZ) or ATM inhibitor (ATMi) by itself had no effect on growth in vitro or in vivo, but combined treatment with ENZ plus ATMi significantly inhibited cell survival in vitro and tumor growth in vivo. By inducing telomere DNA damage and activating a telomere DDR, an opportunity to inhibit DNA repair and promote cell death was created, even in CRPC cells. 22Rv1 cells express both full-length AR and AR splice variant AR-V7, but full-length AR was found to be the predominant form of AR associated with telomeres and required for telomere stability. Although 22Rv1 growth of untreated 22Rv1 cells appears to be driven by AR-V7, it is, ironically, expression of full-length AR that makes them sensitive to growth inhibition by combined treatment with ENZ plus ATMi. Notably, this combined treatment approach to induce telomere DNA damage and inhibit the DDR was effective in inducing cell death also in other CRPC cell lines (LNCaP/AR and C4-2B). Thus, the use of ENZ in combination with a DDR inhibitor, such as ATMi, may be effective in prolonging disease-free survival of patients with AR-positive metastatic CRPC, even those that co-express AR splice variant.

cancer biology↗