Biology subjects

Shanmugam, G.

Publications and source records attributed to Shanmugam, G..

4 recordsLinked to original sources

bioRxiv2025

EPHA2-dependent Ephrin-B1 signaling supports self-renewal ability and recurrence of oral cancer

Eph-Ephrin pathway that drives a bidirectional signaling regulates a plethora of biological activities with its varied level of complexity. While the canonical trans-interaction of the ligand and receptor on neighboring cells initiates forward signaling that brings about biological activities, their cis-interaction on the same cell attenuates the forward signaling, modulating the biological effects. Yet, non-canonical cis-interactions with heterotypic surface proteins and kinases regulate certain biological effects. In cancer, the canonical signaling is believed to be tumor suppressive, while the ligand-independent non-canonical signaling drives tumor progression and poor prognosis. Self-renewal ability of cancer cells is a major underlying cause of recurrence and poor prognosis of cancer. Using SILAC-based proteomics, we identified Ephrin-B1 signaling as a crucial regulator of oral cancer self-renewal. Though Ephrin-B1 is known to regulate normal stem cells, its role in cancer remains underexplored. Our biochemical analyses show that Ephrin-B1 binds to a nonconventional receptor EPHA2, which is known to regulate cancer stem cells (CSCs), though the mechanism is less explored. Contrary to the belief that the cis-interaction of receptors and ligands is a means to block functional signaling, our immunoprecipitation, FRET facilitated photoswitching analysis, proximity ligation assay, and in vitro kinase assay provide evidence for the Ephrin-B1-EPHA2 cis-interaction leading to the phosphorylation of EphrinB1 at Y324/329 and Y317. Extreme limiting dilution assay in vitro and in vivo confirmed that this cis-interaction promotes CSC enrichment. Substantiating our in vitro results, mouse orthotopic models showed that Ephrin-B1/EPHA2 interaction regulates prognosis. The clinical relevance of the finding was validated using a TCGA data set and immunohistochemical analysis of tissue microarray using samples from oral cancer patients with recurrence in comparison to patients, who showed disease-free survival. Given that Ephrin-B interacts with EphB for normal stem cell homeostasis, this unconventional EPHA2/Ephrin-B1 cis-interaction, specifically manifested in CSC niches, might serve as an attractive target for therapy, warranting further validation.

cancer biology↗

bioRxiv2024

Molecular characterization and culture optimization of marine copepod Oithona dissimilis (Landberg, 1940) from Nagore coastal waters, Southern India

This study focused on identifying the cyclopoid copepod Oithona dissimilis found in the Nagore coastal waters of Southern India. Based on both morphological and molecular approaches to identify this species. Morphological characters were confirmed by examining the arrangement of setae and spines on the exopod of swimming legs 1-4. Molecular studies were performed using the CO 1 gene, which proved an effective marker for species identification. After identifying the Oithona dissimilis, reared them under laboratory conditions to determine the effects of various environmental parameters on their survival, nauplii production, and population density. Tested different temperatures, light intensity, pH, salinity, and diets and found that the optimum conditions for rearing Oithona dissimilis under laboratory conditions were a salinity of 25 PSU, a temperature range of 24-28{degrees} C, pH of 8, light intensity of 500 lux, and a mixed algal diet at a concentration of 30,000 cells/ml. The present study confirms the importance of accurate taxonomic identification for the Oithona group at the species level. Additionally, our findings show that rearing cyclopoid copepods under laboratory conditions that mimic their natural range of environmental parameters is crucial for the thriving culture of these organisms, the aquaculture industry frequently utilizes this as a live feed. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/580272v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@ecae13org.highwire.dtl.DTLVardef@987662org.highwire.dtl.DTLVardef@1844230org.highwire.dtl.DTLVardef@158e4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2019

Impaired Regulation of Redox Transcriptome during the Differentiation of iPSCs into Induced Cardiomyocytes (iCMs)

BackgroundReprogramming of somatic cells into pluripotent stem cells (iPSC) and subsequent differentiation into iPSC-derived cardiomyocytes (iCM) seems to be a promising strategy for cardiac regenerative therapy. However, recent failure or poor outcomes in cardiac cell therapy warrants further investigation focusing on the infarction/wound environment (site of healing) to improve the cardiac regenerative medicine. Here, using next generation sequencing (NGS), we analyzed the global transcriptome to discover the unidentified genes/pathways that are crucial for cell survival, cytoprotection and mitochondrial dynamics during the differentiation of iPSC into iCM.\n\nMethodsHigh throughput NGS was performed RNA from human iPSCs and iCMs (n=3/group) and analyzed the global changes in the transcriptome during differentiation. Furthermore, Ingenuity Pathway Analysis (IPA) and Gene Ontology (GO) for biological process were performed to understand the transcriptional networks that are involved during iCM differentiation. RNA-seq data were further validated by qRT-PCR analyses.\n\nResultsGlobal transcriptome analysis revealed that ~9,290 genes (log2 FC >2) were significantly altered in human iCMs compared to the parent iPSCs, in which 4,784 transcripts were substantially upregulated and 4,506 transcripts were down-regulated during differentiation. GO enrichment and IPA analyses revealed the top 10 regulatory networks (i.e. hierarchical order) involved in differentiation of iCMs including cardiomyocyte remodeling, integrin-linked kinase signaling, Rho family of GTPases, etc. Surprisingly, none of the top 10 pathways listed the genes liable for redox signaling networks that are crucial for the basal cellular redox homeostasis, Nrf2-dependent antioxidant defense, mitochondrial functions and cell survival. Our deeper and unbiased analysis of this data revealed that the genes involved in above canonical signaling pathways are found in the middle of the inverted vertical cone. Of note, although these pathways are significantly altered during the differentiation (of iPS into cardiomyocytes), a majority of them are ranked low in the hierarchical list (>150). Validation of the randomly selected genes representing various pathways real-time qPCR confirmed the global transcriptome changes observed in NGS.\n\nConclusionWe highlight the significance of Nrf2-redox and mitochondrial transcriptome during differentiation of iPSC into iCMs. Thus, targeting the redox signaling mechanisms in iCMs may enhance their efficiency for cell therapy and improved myocardial repair.

cell biology↗

bioRxiv2018

Basal Role for Nrf2-transgne on transcriptional (mRNA/miRNA) regulation in the mouse myocardium

BackgroundNFE2L2 (nuclear factor, erythroid 2 like 2; Nrf2), is a stress-responsive transcription factor that regulates cellular redox homeostasis. The action mechanism of Nrf2 occurs through sensing oxidative stress in the cellular environment in responses to various stresses including altered metabolism, toxic insult and xenobiotic stresses. However, under the basal condition, the role of excess Nrf2 on global miRNA-mRNA interactions in the myocardium is unknown. Here, we tested a hypothesis that excess Nrf2 (transgene) will promote the transcription of antioxidants, which then might escalate the basal defense mechanisms in the heart. Furthermore, we investigated whether changes in the miRNA profile might have a strong role on the transcriptome in TG hearts.\n\nMethodsNon-transgenic (NTG), and Cardiac specific Nrf2 transgenic (Nrf2-TG) mice in the C57/BL6 background at the age of 6-8 months were used to examine transcriptional and post-transcriptional signatures (mRNA and miRNA expressions) by performing next generation sequencing (NGS) for RNA (RNAseq) and small RNA (sRNAseq) for miRNA expressions in the heart (n=3-6/group). Validation of the NGS data was performed by quantitative real time PCR (qRT-PCR) using specific primers targeting selected miRNAs or mRNAs. Finally, in-silico analyses were performed to determine the miRNA-mRNA interactome networks and the pathways that are potentially regulated by these networks.\n\nResultsNGS analysis for mRNA indicated that there were 5727 differently expressed genes (DEGs) in the Nrf2-TG vs. NTG myocardium. Of which, 3552 were upregulated and 2175 were downregulated significantly. Small RNAseq analysis revealed that 112 miRNAs were significantly altered in Nrf2-TG versus NTG hearts. Among these miRNAs, 68 were upregulated and 44 were downregulated significantly. Validation of key targets for mRNA and miRNA by qPCR confirmed the NGS results. In-silico analysis (IPA) revealed that the majority of the miRNAs and mRNAs that are significantly altered in response to transgene expression are potentially involved in Nrf2 mediated oxidative stress response, ILK Signaling, hypoxia signaling in the cardiovascular system and glutathione-mediated detoxification etc.\n\nConclusionThese high-throughput sequencing data sets from cardiac specific Nrf2-TG mice revealed the transcriptome-wide effects of Nrf2 in the myocardium. Furthermore, our comprehensive analysis indicates that Nrf2 may directly or indirectly regulates these sub-sets of cardiac miRNA-mRNA interactome networks under basal physiological setting.

molecular biology↗