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Biology subjects

Kar, S.

Publications and source records attributed to Kar, S..

At least 19 recordsLinked to original sources

Endothelial gain of PIEZO/TRPV mechanosensors drives cavernous malformations

Vascular disease often begins as a failure to read mechanical force. Cerebral cavernous malformations (CCMs) are leaky, brain-invading capillary clusters that cause stroke and seizures and arise from endothelial loss of the CCM complex. They form exclusively in low-flow vessels but existing models cannot explain this selectivity. We previously established that CCM-mutant endothelium inverts its adhesive balance, weakening cell-cell junctions while building matrix-degrading adhesions. The mechanosensors imposing this switch are unknown. Here we show that the mechanosensitive Ca2+ channels PIEZO1, TRPV2 and TRPV4 are overexpressed in the endothelium of CCM lesions in a 42-patient cohort, and that lesion volume scales with their expression level. Mechanistically, PIEZO1 partners with TRPV4 to destabilise cell-cell junctions and with TRPV2 to reinforce cell-matrix adhesions; silencing TRPV4 restores junctional integrity and abolishes KLF4 induction, whereas silencing TRPV2 or inhibition with tranilast abolishes matrix degradation and invasion. In vivo, trpv4 knockdown or inhibition with GSK2798745 restores atrioventricular canal morphogenesis in ccm2-mutant zebrafish. Gain of mechanosensory channel function is thus a causal step in CCM pathogenesis, upstream of the KLF program. Combined therapeutic targeting of TRPV2 and TRPV4 with inhibitors validated in humans offers a route to therapy.

cell biology↗

Deciphering the dynamical origin of the Adipogenic commitment during stress-induced disruption of the circadian rhythm

Obesity is a major health concern worldwide and is caused by an excessive fat accumulation in the body due to adipogenesis. Recent studies suggest that excessive stress or disruption of circadian rhythms can enhance adipogenic commitment and lead to obesity. Moreover, this adipogenic commitment occurs at a specific time of day, highlighting a correlation with the circadian rhythm. However, the dynamical nature of this kind of stress-induced adipogenic differentiation remains an unresolved puzzle. Herein, we proposed a mathematical model centred on the key transcription factor PPAR{gamma}, associated with adipogenic differentiation, and demonstrated that adipogenic commitment is a bistable, irreversible differentiation process in which slow positive feedback regulation of PPAR{gamma} primarily controls the differentiation commitment threshold. The model reproduced the experimental observations regarding the extent of adipogenesis induced by constant and pulsatile stress input signals and explained the correlation between adipogenic differentiation time and circadian phase. It further predicts how to control the adipogenic commitment even under stressful conditions. Overall, our study elucidates how stress signalling dynamically governs adipogenesis.

systems biology↗

Intricate Dynamical Cross-Talk Between p53 Protein and Cell Cycle Regulators Governs Mammalian Cell Fate

In mammalian cells, under normal circumstances, the p53 protein exhibits oscillatory dynamics in response to DNA damage and maintains the cells in a cell-cycle-arrested state. Intriguingly, some cells can escape this cell-cycle-arrested state even after prolonged DNA damage, and often undergo mitotic catastrophe. In this context, the precise role of p53 dynamics and its complex interplay with cell-cycle regulation remain poorly understood. Herein, by constructing a comprehensive network model, we have identified crucial crosstalk regulations between the p53 protein and key cell-cycle regulators that enable some cells to escape cell-cycle arrest during prolonged DNA damage. The model further illustrates a probable cellular mechanism underlying mitotic catastrophe and predicts ways to induce it in a therapeutically relevant manner.

systems biology↗

ViralMap: Predicting Features in Viral Proteins from Primary Sequence

Modern viral vaccines are designed to elicit an immune response against viral proteins that mediate infection, making those proteins important targets for characterization and engineering. To improve vaccine efficacy, the proteins often require changes to specific residues or domains to improve immunogenicity and induce a protective response. These engineering strategies vary significantly across viruses, and comprehensive and accurate protein sequence annotation is crucial for guiding vaccine design. The growing risk of novel pathogen emergence and initiatives such as the CEPI 100 Days Mission to rapidly counter "Disease X" threats heighten the need for tools that can convert viral protein sequences from newly characterized genomes or emerging variants into the annotation profiles required for antigen engineering. To address this, we developed ViralMap, a multi-label annotation model tailored for eukaryotic viral proteins. By leveraging ESM-2 language model representations, ViralMap simultaneously predicts ten distinct annotation classes spanning domain topology and localization, post-translational modifications, and structural features directly from primary sequences. The model achieves a residue-level precision-recall area under the curve (PR-AUC) of 0.75 or greater for seven of the ten classes and realizes predictive performance competitive with established tools across the eight benchmarked classes. Case studies on complex glycoproteins, including the SARS-CoV-2 spike and HIV-1 Env, illustrated the models ability to generalize across viral strains and to novel viral families not seen during training. By providing a unified, sequence-based framework for multi-label annotation, ViralMap offers a practical and scalable bridge from raw viral protein sequences to the annotation profiles required for antigen engineering.

bioinformatics↗

Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

Cisplatin (CDDP), a widely used chemotherapy drug is imported in mammalian cells by copper transporter CTR1. We observed that CDDP cytotoxicity is significantly reduced in CTR1 deficient yeast and in copper loaded mammalian cells indicating its shared uptake pathway with copper. CTR1 resides on the plasma membrane undergoes endocytosis during CDDP import as in copper, involving its amino-terminal His-Met-motifs and the conserved 150MXXM154 pore motif. Unlike Copper, CDDP directs endocytosed CTR1 toward lysosomes rather than being sorted through VPS35-positive endosome. CDDP enhances lysosomal acidity and cathepsin activity. Furthermore, CDDP selectively downregulate cuproproteins, CCS and SOD1, disrupting copper homeostasis. This study highlights the unique aspects of copper versus CDDP uptake by CTR1 and its differential downstream ramification on lysosomes and cuproproteins.

cell biology↗

Mechanisms of resistance to active state selective tri-complex RAS inhibitors

Tri-complex inhibitors (TCIs) act as molecular glues to recruit cyclophilin A (CYPA) to the active (GTP-bound or ON) conformation of RAS, which in turn prevents the activation of downstream effector proteins like RAF and PI3K. Emerging data demonstrate clinical activity, including tumor regressions, in patients with RAS driven cancers. Despite being promising therapeutic interventions, the mechanisms of resistance in patients treated with these inhibitors remain unknown. Here we studied matched baseline and post-progression specimens from patients treated with the RAS(ON) multi-selective inhibitor daraxonrasib (RMC-6236). Tissue or cell-free DNA specimens were collected from 40 patients with RAS-mutant non-small cell lung, colorectal, or other cancers. Eighteen patients (45%) were found to have acquired alterations in RAS signaling intermediates, including recurrent alterations in KRAS, BRAF, RAF1, MAP2K1/2 and PIK3CA. Preclinical resistance models mirrored the alterations observed in patients. We found that secondary KRAS Y64X mutations caused resistance by disrupting an important pi-pi interaction between KRAS and the indole ring of daraxonrasib, which lowers the affinity of the daraxonrasib:CYPA binary complex for active KRAS. We also identified kinase-dead and low-activity BRAF mutations in samples with acquired resistance. This is puzzling, because TCIs are expected to prevent the interaction between RAS and BRAF, which is needed for hypoactive mutants to dimerize and signal. We now show that RAF dimers are harder to displace from active RAS, as compared to their monomeric forms. Indeed, enhanced RAF dimerization attenuated the ability of TCIs to recruit CYPA to active RAS, resulting in diminished inhibition of oncogenic signaling and tumor growth. Thus, several clinical resistance alterations converge at attenuating the formation of the RAS:daraxonrasib:CYPA tri-complex, either by preventing daraxonrasib binding or by inducing RAF dimers.

cancer biology↗

Non-canonical regulation of the plasma membrane copper transporter CTR1 through modulation of membrane mechanical properties

We describe a non-canonical, membrane receptor-like regulation of the human copper transporter CTR1 in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on plasma membrane physical properties harboring the clusters that favors endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain MD simulation and coupled Interference Reflection Microscopy-Total Internal Reflection Fluorescence Microscopy we demonstrated that CTR1 clusters induce positive membrane curvature, increase in local membrane tension and decrease in local membrane fluctuation; alterations that favors formation of endocytic pits. Clustering is facilitated by copper-sequestering Methionine rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adapter protein AP-2. To summarize, we report a hitherto unknown pre-endocytic receptor-like phenomenon of ligand-induced clustering of a metal channel, that in-turn regulates self-endocytosis by modulating membrane properties.

cell biology↗

Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P2

The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters, whose mutations are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here, we show that two related accessory {beta}-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, -4 and -5. Cryo-EM structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl- ion pathway. Unexpectedly, we find that PI(3,5)P2 stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PI(3,5)P2 collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.

cell biology↗

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

T7 RNA polymerase has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5 methyl guanosine caps on T7 RNAP derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

synthetic biology↗

Subtle alteration in transcriptional memory governs the lineage-level cell cycle duration heterogeneities of mammalian cells

Cell cycle duration heterogeneities within a malignant tumor significantly reduce the therapeutic efficacy of cancer treatment. However, identifying factors governing such heterogeneities remains challenging. Here, we demonstrate that correlated fluctuations of the inherited transcription rates during cell cycle progression in combination with the random resetting of them during mitosis are sufficient to account for the experimentally observed cell cycle duration correlation patterns for daughter-daughter, mother-daughter, and cousin-cousin cell lineage pairs. The model elucidates that the variations in the transcriptional inheritance pattern dictate the extent of the cousin-mother inequality phenomenon. It further predicts that the longer the cell cycle duration, the lower will be the lineage pair correlations. However, higher S-G2-M phase duration will only increase daughter-daughter and mother-daughter correlations, hence, reducing the extent of cousin-mother inequality. Overall, the model shows how alteration of the inherited transcriptional fluctuations across lineages finetunes the lineage-level heterogeneities.

systems biology↗

Cis- and trans-eQTL TWAS of breast and ovarian cancer identify more than 100 risk associated genes in the BCAC and OCAC consortia

Transcriptome-wide association studies (TWAS) have investigated the role of genetically regulated transcriptional activity in the etiologies of breast and ovarian cancer. However, methods performed to date have only considered regulatory effects of risk associated SNPs thought to act in cis on a nearby target gene. With growing evidence for distal (trans) regulatory effects of variants on gene expression, we performed TWAS of breast and ovarian cancer using a Bayesian genome-wide TWAS method (BGW-TWAS) that considers effects of both cis- and trans-expression quantitative trait loci (eQTLs). We applied BGW-TWAS to whole genome and RNA sequencing data in breast and ovarian tissues from the Genotype-Tissue Expression project to train expression imputation models. We applied these models to large-scale GWAS summary statistic data from the Breast Cancer and Ovarian Cancer Association Consortia to identify genes associated with risk of overall breast cancer, non-mucinous epithelial ovarian cancer, and 10 cancer subtypes. We identified 101 genes significantly associated with risk with breast cancer phenotypes and 8 with ovarian phenotypes. These loci include established risk genes and several novel candidate risk loci, such as ACAP3, whose associations are predominantly driven by trans-eQTLs. We replicated several associations using summary statistics from an independent GWAS of these cancer phenotypes. We further used genotype and expression data in normal and tumor breast tissue from the Cancer Genome Atlas to examine the performance of our trained expression imputation models. This work represents a first look into the role of trans-eQTLs in the complex molecular mechanisms underlying these diseases.

genetics↗

Understanding the dynamics of complex in-situ enzyme-catalyzed reactions undergoing mechanical stress

Understanding the diversity in the enzymatic mechanism have utmost importance as it can temporally control the catalytic efficiency. Recent literature suggests that by influencing mechanical properties of hybrid materials, the catalytic efficiency of the enzymatic reactions can be altered significantly. Here, taking a computational and experimental approach, we have dig out the fate of the kinetics of enzyme reaction systems exhibiting relatively complex mechanism than usual Michaelis Menten kinetics involving multiple substrate/enzyme/enzyme-substrate complex. We have developed a numerical recipe improvising stochastic reaction-diffusion approach to explore the role of mechanical stress like compression-decompression cycle (C-D) on modulating the output of enzymatic reaction. The proposed methodology can be used as a theoretical tool to understand how to enhance the catalytic activity and setup appropriate reaction conditions by efficiently using the catalytic cycles. Hence, our methodology will be crucial to identify the most effective strategy to efficiently convert substrate into product in this type of mechanoresponsive materials, which will enable future development of cost-effective biomaterials. In future, the insights gained from this work may find enormous application in drug delivery, tissue engineering, bio-sensing and bio-catalysis.

systems biology↗

Interlinked Bi-stable Switches Govern the Cell Fate Commitment of Embryonic Stem Cells

Cell fate decision-making events of embryonic stem (ES) cells are often governed by bi-stable switch-like steady state dynamics of two key transcription factors Oct4 and Nanog due to mutual positive feedback regulation. Intriguingly, for the differentiation of ES cells to either extraembryonic trophectoderm (TE) and primitive endoderm (PrE) lineages, Oct4 maintains either low (TE) or high PrE) expression levels relative to a moderate level in ES cells, while Nanog exhibits high expression in ES state and low expression in both the differentiated states. The dynamical origin for such kind of disparate steady-state level maintenance of Oct4 and Nanog remains elusive. Herein, we demonstrate that such complex steady-state dynamics can be hypothesized and explained by two different bistable switches interconnected either in a stepwise (Oct4) or in a mushroom-like (Nanog) manner. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feed-forward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.

systems biology↗

Immunogenicity and efficacy of a subcutaneously administered, adjuvanted vaccine containing modified S1 spike protein of SARS-CoV-2 variant C.1.2

During the COVID-19 pandemic, vaccines have produced protective immunity sufficient enough to cause a decrease in hospitalizations and deaths; however, the pandemic continues due to mutational events, predominantly occurring in the S1 sequence of the spike protein of SARS-CoV-2. We have developed a baculovirus-expressed, modified S1 SARS-CoV-2 protein based on the C.1.2 variant, which was first identified in South Africa.1 This was encapsulated in a vitamin E containing, nonphospholipid liposome, which was then used to subcutaneously immunize Syrian hamsters. This vaccine, when administered at day 1 generates IgG responses that react to the modified C.1.2 S1 protein; full-length spike proteins from Wuhan-Hu-1, Delta, Omicron BA.1; and the Omicron recombinant variant XBB.1.5 in 100% of the animals. The second dose administered subcutaneously on day 28 demonstrated anamnestic response in the quantitative IgG assay to the Wuhan-Hu-1 spike Receptor Binding Domain (RBD). In addition, antibody IgA and IgM responses in sera were demonstrated. Serum IgG antibody responses to the spike proteins of the modified C.1.2 S1 and full-length spike proteins Wuhan-Hu-1, Delta, Omicron BA.1, and Omicron recombinant XBB.1.5 variants are elevated for over 120 days. Challenge of vaccinated and unvaccinated hamsters at day 126 of the study with an Omicron BA.1 resulted in a difference in weight change and viral load based on the qRT-PCR assay seven days after challenge.

immunology↗

Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90% along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

immunology↗

Changes in coding and efficiency through modular modifications to a One Pot PURE system for in vitro transcription & translation

The incorporation of unnatural amino acids are attractive methods for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE for a robust and modular system of enzymatic single site-specific of unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two orthogonal aminoacyl tRNA synthetases and the suppression of two distinctive stop codons.

synthetic biology↗

Disentangling the cascading effects of Grapevine Red Blotch Virus infection on vine physiology

Grapevine Red Blotch Virus is a major grapevine pathogen and is associated with reduced carbon assimilation and delayed berry ripening in Vitis vinifera L. Recent work suggests that the virus alters leaf carbon metabolism prior to emergence of visible symptoms. Therefore, diurnal and seasonal measurements were conducted to quantify changes in leaf carbon balance and to elucidate the chronology of symptom progression in leaves and fruit. Healthy and infected vines were assayed in a commercial vineyard during which leaf water relations, photosynthesis, and nonstructural carbohydrates were measured. Additionally, sugar and anthocyanin accumulation in the fruit were monitored at the end of the season to characterize the impact of the virus on ripening. Virus infection significantly reduced carbon assimilation pre- and postveraison, but the impact was more pronounced postveraison and during the afternoon when vine water status was the lowest. Similarly, virus infection significantly increased leaf starch concentration pre- and postveraison, but increased leaf starch in infected vines was detected two weeks prior to veraison. Virus infection had the greatest impact on obstructing leaf carbon export postveraison, especially during the afternoon. The virus had no impact on chlorophyll fluorescence, indicating there was no sustained photosystem impairment and suggesting that changes in chlorophyll fluorescence were a transient response to reduced carbon assimilation and export. This study provides evidence that reduced carbon export constitutes a feedback inhibition response to accumulation of leaf starch prior to the appearance of visible symptoms or impacts to ripening, which may aid earlier detection of the virus.

plant biology↗