Gold based nano-photonic approach for point-of-care detection of circulating long non-coding RNAs.

Development of a rapid, sensitive and easy to use point of care assay for detection of circulating long non-coding RNAs (lncRNAs) is of great importance. These biomolecules possess the ability to regulate vital cellular processes and act as biomarkers for various human non-communicable diseases. The present work aimed to develop a simplified and reliable cytometric fluorescence-based approach for precise recognition of circulating lncRNAs in a given sample using biotinylated uracil-modified oligonucleotide tethered AlexaFluor488-labeled streptavidin gold colloidal (BiO-StrAG) nano-conjugates. The fluorophores in close proximity to the gold nanoparticles result in quenching of fluorescence; however, specific recognition of target lncRNAs increases this distance which causes plasmonic enhancement of fluorescence. As per the flow cytometry and fluorometry investigations, the developed methodology provides a precise and sensitive approach for detection of the target lncRNAs (up to 5 nM in any given sample). With advantages of high selectivity and feasibility, our strategy offers great potential of being developed as a promising tool for interrogating aberrant regulation of lncRNAs functions, especially indicated in various diseased states.

Clostridium perfringens phospholipase C impairs innate immune response by inducing integrated stress response and mitochondrial-induced epigenetic modifications.

Clostridium perfringens, a rod-shaped, gram-positive, anaerobic, spore-forming bacterium is one of the most widely occurring bacterial pathogens, associated with a spectrum of diseases in humans. A major virulence factor during its infection is the enzyme phospholipase C encoded by the plc gene, known as Clostridium perfringens phospholipase C (CpPLC). The present study was designed to understand the role of CpPLC in inducing survival mechanisms and mitochondrial-induced epigenetic changes in a human lymphocyte cell culture model. Following exposure to CpPLC, a significant generation of mitochondrial reactive oxygen species was observed, which coincided with the changes in the expression of vital components of MAP/ERK/RTK signaling cascade that regulates the downstream cellular functions. These disturbances further led to alterations in the mitochondrial genome and functioning. This was supported by the observed upregulation in the expression of mitochondrial fission genes Drp1, Fis1, and Mff, and mitochondrial fusion genes MFN1, MFN2, and OPA1 following CpPLC exposure. CpPLC exposed cells showed upregulation of OMA1, DELE1, and HRI genes involved in the integrated stress response (ISR), which suggests that it may induce the ISR that provides a pro-survival mechanism to the host cell. CpPLC also initiated immune patho-physiologic mechanisms including mitochondrial-induced epigenetic modifications through a mitochondrial-ROS driven signaling pathway. Interestingly, epigenetic machinery not only play a pivotal role in lymphocyte homeostasis by contributing to cell-fate decisions but thought to be one of the mechanisms by which intracellular pathogens survive within the host cells. Importantly, the impairment of mtDNA repair among the CpPLC exposed cells, induced alterations within mtDNA methylation, and led to the deregulation of MT-CO1, MT-ND6, MT-ATPase 6, and MT-ATPase8 gene expression profiles that are important for mitochondrial bioenergetics and subsequent metabolic pathways. This was further confirmed by the changes in the activity of mitochondrial electron chain complexes (complex I, II, III, IV and V). The altered mtDNA methylation profile was also found to be closely associated with the varied expression of mitomiRs and their targets. CpPLC exposed cells showed up-regulation of miR24 expression and down-regulation of miR34a, miR150, and miR155, while the increased expression of mitomiR target genes i.e. of K-Ras, MYC, EGFR, and NF-kß was also observed in these cells. Altogether, our findings provide novel insights into the derailment of redox signaling machinery in CpPLC treated lymphocytes and its role in the induction of survival mechanisms and mitochondrial-induced epigenetic modifications.