Biology subjects

Ravindran, R.

Publications and source records attributed to Ravindran, R..

10 recordsLinked to original sources

bioRxiv2026

SARS-CoV-2 maturation driven by a mechano-active nucleocapsid-RNA condensate

The nucleocapsid (N) protein, the core component of the SARS-CoV-2 virus, binds to the 30-kb viral genomic RNA (vgRNA) to form ribonucleoprotein assemblies that are packaged into ~100 nm membrane-enclosed virions. In addition to viral assembly, the N protein performs other functions, including roles in viral mRNA transcription, replication, and immune regulation, making it a key target for developing diagnostics and vaccines. Recent studies show that N protein and RNA undergo phase separation to form a biomolecular condensate that constitutes the viral core. However, how this condensate becomes selectively enveloped by a membrane remains unclear. Here, using a minimal reconstituted system, we demonstrate that N protein-vgRNA condensates are spontaneously enveloped by lipid bilayer membranes, whereas condensates formed by N protein alone or with genomic RNA fragments fail to undergo envelopment and instead adhere to or weakly deform membranes. We show that RNA length tunes the material properties of N protein condensates, with vgRNA imparting enhanced elasticity. We propose a physical model for SARS-CoV-2 maturation in which adhesion between the membrane and the N protein-vgRNA condensate drives membrane bending, while condensate elasticity preserves core geometry. These results identify RNA-dependent material properties as an underlying physical principle for selective SARS-CoV-2 maturation.

biophysics↗

bioRxiv2022

Peroxisome biogenesis initiated by protein phase separation

Peroxisomes are organelles that perform beta-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their disfunction1. Among disease-causing mutant genes are those required for protein transport into the peroxisome. The peroxisomal protein import machinery, also shared with chloroplasts, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes2 and current models postulate a large pore formed by transmembrane proteins3. To date, however, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes membrane proteins Pex13 and Pex14 and cargo protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions (IDR) in Pex13 and Pex5 resemble those found in nuclear pore complex (NPC) proteins. Cargo transport into peroxisomes depends on the number but not patterns of aromatic residues in these IDRs, consistent with their roles as stickers in associative polymer models of LLPS4,5. Finally, imaging Fluorescence Cross-Correlation Spectroscopy (iFCCS) shows that the transport of cargo correlates with transient focusing of GFP-Pex13/14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time-frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our results suggest a model in which LLPS of Pex5-cargo with Pex13/14 results in transient protein transport channels.

cell biology↗

bioRxiv2021

Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis.

Neurofibromatosis type 1 (NF1) is a genetic disorder characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) lack NF1 and show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. Gene expression profiling and protein expression identified P2RY14 in SCs and SC precursors (SCPs) implicating P2RY14 as a candidate upstream regulator of cAMP in EGF-dependent SCP. We found that SCP self-renewal was reduced by genetic or pharmacological inhibition of P2RY14. In NF1 deficient SCs and malignant peripheral nerve sheath tumor (MPNST) cells, P2RY14 inhibition decreased EGFR-driven phospho-Akt and increased cAMP signaling. In a neurofibroma mouse model, genetic deletion of P2RY14 increased mouse survival, delayed neurofibroma initiation and rescued cAMP signaling. Conversely, elevation of cAMP diminished SCP number in vitro and diminished SC proliferation in neurofibroma bearing mice in vivo. These studies identify the purinergic receptor P2RY14 as a critical G-protein-coupled receptor (GPCR) in NF1 mutant SCPs and SCs and suggest roles for EGFR-GPCR crosstalk in facilitating SCP self-renewal and neurofibroma initiation via cAMP and EGFR-driven phospho-Akt.

neuroscience↗

bioRxiv2021

Therapeutic reversal of prenatal pontine ID1 signaling in DIPG

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumors highly infiltrative and invasive nature. Previous reports demonstrate upregulation of the transcription factor ID1 with H3K27M and ACVR1 mutations, but this has not been confirmed in human tumors or therapeutically targeted. We developed an in utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. In human tumors, elevated ID1 expression is associated with H3K27M/ACVR1-mutation, brainstem location, and reduced survival. The ID1 promoter demonstrates a similar active epigenetic state in H3K27M tumor cells and murine prenatal hindbrain cells. In the developing human brain, ID1 is expressed highest in oligo/astrocyte-precursor cells (OAPCs). These ID1+/SPARCL1+ cells share a transcriptional program with astrocyte-like (AC-like) DIPG cells, and demonstrate upregulation of gene sets involved with regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 results in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. CBD reduces proliferation through reactive oxygen species (ROS) production at low micromolar concentrations, which we found to be achievable in the murine brainstem. Further, pediatric high-grade glioma patients treated off-trial with CBD (n=15) demonstrate tumor ID1 reduction and improved overall survival compared to historical controls. Our study identifies that ID1 is upregulated in DIPG through reactivation of a developmental OAPC transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD. One Sentence SummaryThe transcription factor ID1 is upregulated in a subset of DIPG tumor cells, and ID1-driven invasiveness is therapeutically targetable with CBD.

cancer biology↗

bioRxiv2021

Immunoinformatic approach to design a vaccine against SARS-COV-2 membrane glycoprotein

SARS-COV-2 is a pandemic virus causing COVID-19 disease which affects lungs and upper respiratory tract leading to progressive increase in the death rate worldwide. Currently, there are more than 123 million cases and over 2.71 million confirmed death caused by this virus. In this study, by utilizing an immunoinformatic approach, multiepitope-based vaccine is designed from the membrane protein which plays a vital role in the virion assembly of the novel-CoV. A total of 19 MHC class- I binders with HLA-A and HLA-B alleles have been selected with NetMHC pan EL 4.0 method from IEDB MHC-I prediction server. Four epitopes candidates from M-protein were selected based on the antigenicity, stability, immunogenicity, Ramachandran plot and scores with 100 % was taken for docking analysis with alleles HLA-A (PDB ID: 1B0R) and HLA-B (PDB ID: 3C9N) using ClusPro server. Among the four epitopes, the epitope FVLAAVYRI has the least binding energy and forms electrostatic, hydrogen and hydrophobic interactions with HLA-A (-932.8 Kcal/mol) and HLA-B (-860.7 Kcal/mol) which induce the T-cell response. Each HLA-A and HLA-B complex in the system environment achieves stable backbone configuration between 45-100 ns of MD simulation. This study reports a potent antigenic and immunogenic profile of FVLAAVYRI epitope from M-protein and further in vitro and in vivo validation is needed for its adaptive use as vaccine against COVID-19.

bioinformatics↗

bioRxiv2021

IL-1-driven stromal-neutrophil interaction in deep ulcers defines a pathotype of therapy non-responsive inflammatory bowel disease

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology, and in situ localisation, we describe heterogeneity of the tissular inflammatory response in IBD treatment failure. Among inflammatory pathotypes, we found high neutrophil infiltration, activation of fibroblasts, and vascular remodelling at sites of deep ulceration was a feature of non-response to several anti-inflammatory therapies. Activated fibroblasts in the ulcer bed display neutrophil chemoattractant properties that are IL-1R- but not TNF-dependent. The identification of distinct, localised, tissular pathotypes associated with treatment non-response will aid precision targeting of current therapeutics and provide a biological rationale for IL-1 signalling blockade in ulcerating disease.

immunology↗

bioRxiv2020

Endosomes facilitate mitochondrial clearance by enhancing Mfn2 degradation and subsequent Parkin recruitment

Mutations in ubiquitin ligase Parkin are associated with Parkinsons disease and defective mitophagy. Conceptually, Parkin-dependent mitophagy is classified into two phases; 1. Parkin recruits to and ubiquitinates mitochondrial proteins, 2. Formation of autophagosome membrane, sequestering mitochondria for degradation. Recently, endosomal machineries were reported to contribute to the later stage for membrane assembly. We report a role for endosomes in the events upstream of phase 1. We demonstrate that an endosomal ubiquitin ligase CARP2 associates with damaged mitochondria, and this association precedes that of Parkin. CARP2 interacts with Parkin, and stable recruitment of Parkin to damaged mitochondria was substantially reduced in CARP2 KO cells. Our study unravels a novel role of endosomes in modulating upstream pathways of Parkin-dependent mitophagy initiation.

cell biology↗

bioRxiv2019

Therapeutic efficacy of an immune stimulatory gene therapy strategy in a mouse model of high grade brainstem glioma

PurposeDiffuse intrinsic pontine glioma (DIPG) bears a dismal prognosis. A genetically engineered brainstem glioma model harboring the recurrent DIPG mutation, ACVR1-G328V (mACVR1), was developed for testing an immune-stimulatory gene therapy. Experimental DesignWe utilized the Sleeping Beauty transposase system to generate an endogenous mouse model of mACVR1 brainstem glioma. Histology was used to characterize and validate the model. We performed RNAseq analysis on neurospheres (NS) harboring mACVR1. mACVR1 NS were implanted into the pons of immune competent mice to test the therapeutic efficacy and toxicity of immune stimulatory gene therapy using adenoviruses expressing thymidine kinase (TK) and fms-like tyrosine kinase 3 ligand (Flt3L). mACVR1 NS expressing the surrogate tumor antigen ovalbumin were generated to investigate if TK/Flt3L treatment induces the recruitment of tumor-antigen specific T cells. ResultsHistological analysis of mACVR1 tumors indicates that they are localized in the brainstem and have increased downstream signaling of bone morphogenetic pathway as demonstrated by increased phospho-smad1/5 and Id1 levels. Transcriptome analysis of mACVR1 NS identified an increase in the transforming growth factor beta (TGF-{beta}) signaling pathway and the regulation of cell differentiation. Adenoviral delivery of TK/Flt3L in mice bearing brainstem gliomas resulted in anti-tumor immunity, recruitment of anti-tumor specific T cells and increased median survival. ConclusionsThis study provides insights into the phenotype and function of the tumor immune microenvironment in a mouse model of brainstem glioma harboring mACVR1. Immune stimulatory gene therapy targeting the hosts anti-tumor immune response inhibits tumor progression and increases median survival of mice bearing mACVR1 tumors. Translational RelevanceThe therapeutic efficacy of anti-DIPG immune responses is limited due to a low number of immune cells in the tumor microenvironment. We have uncovered a novel treatment strategy that utilizes adenoviral delivery of therapeutic genes, thymidine kinase (TK) and fms tyrosine kinase 3 ligand (Flt3L) into the tumor, eliciting a reprograming of the hosts own immune system to recognize and kill tumor cells. We demonstrate that TK/Flt3L therapy generates an effective anti-tumor response and can be safely delivered into the brainstem. This treatment approach could provide a novel translational approach towards potentiating an anti-DIPG immune response to overcome the current limitations in the treatment of patients with DIPG.

cancer biology↗

bioRxiv2019

Paradigm of Vanadium pentoxide nanoparticle-induced autophagy and apoptosis in triple-negative breast cancer cells

Chemo-resistance remains the main hurdle to cancer therapy, challenging the improvement of clinical outcomes in cancer patients. Therefore, exploratory studies to address chemo-resistance through various approaches are highly rewarding. Nanomedicine is a promising recent advancement in this direction. Comprehensive studies to understand the precise molecular interactions of nanomaterials is necessary to validate their specific "nano induced" effects. Here, we illustrate in detail the specific biological interactions of vanadium pentoxide nanoparticles (VnNp) on triple-negative breast cancer cells and provide initial insights towards its potential in breast cancer management at the cellular level. VnNp shows a time-dependent anti-oxidant and pro-oxidant property in vitro. These nanoparticles specifically accumulate in the lysosomes and mitochondria, modulate various cellular processes including impaired lysosomal function, mitochondrial damage, and induce autophagy. At more extended periods, VnNp influences cell cycle arrest and inhibits cell migration potentiating the onset of apoptosis. Preliminary in vivo studies, on exposing healthy Swiss albino mice to VnNp demonstrated normal blood parameters, organ distribution, and tissue redox balance which further indicated the absence of any adverse organ toxicity. Hence, we foresee tumor-targeting VnNp as a potential drug molecule for future cancer management.

scientific communication and education↗

bioRxiv2018

Patterns of recurrence after curative-intent surgery for pancreas cancer reinforce the importance of locoregional control and adjuvant chemotherapy.

IntroductionThe pattern of recurrence after surgical excision of pancreas cancer may guide alternative pre-operative strategies to either detect occult disease or need for chemotherapy. This study investigated patterns of recurrence after pancreatic surgery.\n\nMethodsRecurrence patterns were described in a series of resected pancreas cancers over a 2-year period and recurrence risk expressed as odds ratio (OR) with 95% confidence interval (C.I.). Survival was displayed by Kaplan-Meier curves.\n\nResultsOf 107 pancreas resections, 69 (65%) had pancreatic cancer. R0 resection was achieved in 21 of 69 (30.4%). Analysis was based on 66 patients who survived 30 days after surgery with median follow up 21 months. Recurrence developed in 41 (62.1%) patients with median time to first recurrence of 13.3 months (interquartile range 6.9, 20.8 months). Recurrence site was most frequently locoregional (n=28, 42%), followed by liver (n=23, 35%), lymph nodes (n=21, 32%), and lungs (n=13, 19%). In patients with recurrence, 9 of 41 had single site recurrence; the remaining 32 patients had more than one site of recurrence. Locoregional recurrence was associated with R+ resection (53% vs 25% for R+ vs R0, respectively; OR 3.5, 95% C.I. 1.1-11.2; P=0.034). Venous invasion was associated with overall recurrence risk (OR 3.3, 95% C.I. 1.1-9.4; P=0.025). In multivariable analysis, R-stage and adjuvant chemotherapy predicted longer survival.\n\nDiscussionThe predominant locoregional recurrence pattern, multiple sites of recurrence and a high R+ resection rate reflect the difficulty in achieving initial local disease control.

pathology↗