Biology subjects

Ramasubramanian, A.

Publications and source records attributed to Ramasubramanian, A..

5 recordsLinked to original sources

bioRxiv2026

Topotecan Syndergizes with SP141 in the Management of Uveal Melanoma

Purpose: Uveal melanoma (UM) is a rare but aggressive intraocular malignancy originating in the uvea (choroid, iris, and ciliary body). Despite primary treatment, 50% of UM patients develop metastases to the liver, lungs, bones, and skin. Topotecan, a topoisomerase I inhibitor, and SP141, an MDM2 inhibitor, have been studied in cancers, including retinoblastoma. This study explores a combined approach with these drugs in the 92-1 UM cell line. Methods: Primary choroidal melanoma-derived 92-1 cells were treated with topotecan and SP141 individually to determine IC50 values. Cell viability and proliferation were assessed using the MTT assay and the xCELLigence real-time monitoring system. The synergistic effects were analyzed quantitatively using the Loewe Additivity and Highest Single-Agent (HSA) models. Flow cytometry was employed to analyze cell cycle distribution, while cell death and apoptosis were monitored via high-throughput, real-time quantitative analysis using the IncuCyte live-cell kinetic imaging system. Results: Both Topotecan and SP141 induced significant cell death in 92-1 cells. xCELLigence showed dose- and time-dependent inhibition of proliferation. Combining sub-IC50 concentrations of topotecan and SP141 exhibited significant synergistic effects, markedly reducing growth. Cell cycle analysis revealed that topotecan causes a dose-dependent biphasic arrest in G2 and S phases, while SP141 causes G2/M arrest, leading to cell death. Cytotoxicity assays corroborated increased cell death following combination therapy, and the Caspase 3/7 assay indicated that apoptosis is the mechanism underlying cell death. Conclusions: The multi-targeted approach combining topotecan and SP141 drugs demonstrated synergistic therapeutic effects in 92-1 cells, suggesting a potential treatment strategy for managing UM.

cancer biology↗

bioRxiv2024

Retinal microglia-derived S100A9 incite NLRP3 inflammasome in a Western diet fed Ossabaw pig retina

PurposeWe established S100A9 as a myeloid-derived damage-associated molecular pattern (DAMPs) protein associated with increasing severity of diabetic retinopathy (DR) in type 2 diabetic subjects. The present study investigates the retinal localization, expression, and mechanisms of action for S100A9 in the young obese Ossabaw pig retina. MethodsRetinae from Ossabaw pigs fed a Western diet for 10 weeks were evaluated for S100 and inflammatory mediator expression using quantitative PCR and Western blot. Double immunohistochemistry was performed to identify the cellular sources of S100A9 in the pig retina. Primary pig retinal microglial cells (pMicroglia) were examined for S100A9 production. S100A9-induced responses were also investigated, and inhibitor studies elucidated the mechanism of action via the NLRP3 inflammasome. A specific inhibitor, Paquinimod (ABR-215757), was administered in vitro to assess the rescue of S100A9-induced NLRP3 inflammasome activation in pMicroglia. ResultsThe expression of the S100 family in the obese Ossabaw pig retina showed a significant elevation of S100A9, consistent with increased levels of circulating S100A9. Moreover, the retina had elevated levels of inflammatory mediators IL-6, IL-8, MCP-1, IL-1{beta} and NLRP3. Retinal microglia in obese Ossabaw were activated and accompanied by an increased expression of intracellular S100A9. pMicroglia isolated from pig retina transformed from ramified to amoeboid state when activated with LPS and produced high S100A9 transcript and protein levels. The S100A9 protein, in turn, further activated pMicroglia by heightened production of S100A9 transcripts and secretion of pro-inflammatory IL-1{beta} protein. Inhibition of TLR4 with TAK242 and NLRP3 with MCC950 attenuated the production of IL-1{beta} during S100A9 stimulus. Finally, pre-treatment with Paquinimod successfully reduced S100A9-driven increases of glycosylated-TLR4, NLRP3, ASC, Caspase-1, and IL-1{beta} production. ConclusionWe demonstrated that microglial-derived S100A9 perpetuates pro-inflammatory responses via the NLRP3 inflammasome in the retina of young Western-diet-fed Ossabaw pigs exhibiting diabetic retinopathy.

neuroscience↗

bioRxiv2024

High-throughput Image-based Clustering of CAR-T/Tumor Cocultures for Rapid and Facile Hit Identification

AO_SCPLOWBSTRACTC_SCPLOWChimeric antigen receptor T cell is important because of its potential to treat various diseases. As deep learning continues to advance, using unsupervised methods to classify medical images has become a significant focus because collecting high-quality labeled data for medical images is labor-intensive and time-consuming. Beyond the need for accurate labeling, there is a desire to explore the underlying characteristics of the data, even when labels may be ambiguous or uncertain. To address these challenges, we present a novel approach that combines image clustering with an insightful explanation of how these clusters are formed. Our method employs a U-net combined with a clustering algorithm to segment the dataset into different groups. After clustering, we use various techniques to interpret and elucidate the results. Moreover, our paper introduces a unique dataset focused on cell data, specifically highlighting the developmental patterns of cancer cells and T cells under various experimental conditions. This dataset offers a rich source of information and presents a complex challenge for image classification due to the diversity of conditions and cell behaviors involved. Our study thoroughly compares different architectural models on this new dataset, demonstrating the superior performance of our proposed architecture. Through experimental analysis and ablation studies, we provide substantial evidence of the benefits offered by our architecture, not only in terms of accuracy but also in its ability to reveal deeper insights into the data. This work advances the field of image classification and opens new possibilities for understanding complex biological processes through computer vision.

cell biology↗

bioRxiv2024

Identifying Factors Important for Conservation at Sites of Synonymous Variations

Synonymous mutations can have a deleterious effect leading to disease, even though they are not protein altering. Variations at genomic sites leading to synonymous variants are frequently highly conserved across species. Several prediction methods have been developed to assess the impact of synonymous mutations and are highly dependent on having validated sets of both deleterious and benign synonymous mutations. However, validated data available for deleterious synonymous mutations is sparse unlike for missense mutations. Rather than develop a model for predicting pathogenicity of synonymous variants, we seek to understand the relative importance of various factors that lead to conservation at sites of synonymous variants. Our study built machine learning models using various features on a large set of reported and generated synonymous variants (Zeng Z et al, 2019) to predict conservation (Genomic Evolutionary Rate Profiling - Rejected Substitution (GERP RS) base scores and Phylogenetic p-values for 100 vertebrates (PP100)) at genomic sites. We used the extreme gradient boosting classifier to classify sites as high, medium and low conservation at different cutoffs. Our experiments report an AUC between 0.74-0.79 and the sensitivity was significant. Of the features we explored, a few alternate allele independent properties were repeatedly flagged as having high impact. These findings provide information for predictors to further improve models for synonymous variant impact.

bioinformatics↗

bioRxiv2021

Fibrin Prestress Due to Platelet Aggregation and Contraction Increases Clot Stiffness

Efficient haemorrhagic control is attained through the formation of strong and stable blood clots at the site of injury. Although it is known that platelet-driven contraction can dramatically influence clot stiffness, the underlying mechanisms by which platelets assist fibrin networks in resisting external loads are not understood. In this study, we delineate the contribution of platelet-fibrin interactions to clot tensile mechanics using a combination of new mechanical measurements, image analysis, and structural mechanics simulation. Based on uniaxial tensile test data using custom-made microtensometer, and fluorescence microscopy of platelet aggregation and platelet-fibrin interactions, we show that integrin-mediated platelet aggregation and actomyosin-driven platelet contraction synergistically increase the elastic modulus of the clots. We demonstrate that the mechanical and geometric response of an active contraction model of platelet aggregates compacting vicinal fibrin is consistent with the experimental data. The model suggests that platelet contraction induces prestress in fibrin fibres, and increases the effective stiffness in both crosslinked and non-crosslinked clots. Our results provide evidence for fibrin compaction at discrete nodes as a major determinant of mechanical response to applied loads.

bioengineering↗