Hyperglycaemia cause vascular inflammation through advanced glycation end products/early growth response-1 axis in gestational diabetes mellitus.

Hyperglycaemia during pregnancy is the main reason for developing diabetes mediated vascular complications. Advanced glycation end products (AGEs) are formed due to non-enzymatic glycation of proteins, lipids and nucleic acids during hyperglycaemia. It has the potential to damage vasculature by modifying the substrate or by means of AGEs and receptor of AGE (RAGE) interaction. It has been linked with the pathogenesis of various vascular diseases including coronary heart disease, atherosclerosis, restenosis etc. This study was carried out to investigate the role of AGEs-EGR-1 pathway in gestational diabetes mellitus (GDM) vascular inflammation. Human umbilical vein endothelial cells (HuVECs) isolated from normal glucose tolerant mothers were subjected to various treatments including high glucose, silencing of early growth response (EGR)-1, blockade of protein kinase C (PKC) ß, blocking extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and treatment with AGEs and assayed for EGR-1, tissue factor (TF) and soluble intercellular adhesion molecule (sICAM)-1. Similarly, umbilical vein endothelial cells isolated from normal and GDM mothers were assayed for EGR-1, TF, and sICAM-1. There was a significant increase in EGR-1 and TF levels in HuVECs isolated form GDM mother's umbilical cord and normal HuVECs treated with high glucose condition. This was accompanied by elevated levels of sICAM-1 in high glucose treated cells. Our results revealed AGE-mediated activation of EGR-1 and its downstream genes via PKC ßII and ERK1/2 signaling pathway. The present study demonstrated a novel mechanism of AGEs/ PKC ßII/ ERK1/2/EGR-1 pathway in inducing vascular inflammation in GDM.

Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for mitigating MDR strains of S. aureus.

Topographic Cue from Electrospun Scaffolds Regulate Myelin-Related Gene Expressions in Schwann Cells.

Matured Schwann cells play a vital role in promoting regeneration and restoration of functional peripheral nervous tissue. In the present study, two dimensional film, three dimensional random and longitudinally aligned electrospun fibers of poly(lactide-co-glycolide) were used to evaluate the effect of topography on expressions of myelin related genes. The aligned nanofibrous scaffold demonstrated significant increase in Schwann cell adhesion using after 3, 6 and 12 hours of culture compared to the film and random fibers. Cell morphology, degree of orientation and elongation factor evaluated using a scanning electron microscope revealed that cells on aligned scaffold have spindle morphology, whereas cells on random and two dimensional films favor spherical morphology confirming the effect of topography. Significant increase in elongation factor was observed in aligned scaffold as compared to film and random fibers (p < 0.05). The gene expression analysis revealed that aligned scaffold significantly up-regulated the expression of early myelination markers: myelin-associated glycoprotein and myelin protein zero, cell adhesion molecule: neural cadherin, extracellular matrix molecule: neurocan, as well the down-regulation of non-myelinating Schwann cell marker: neural cell adhesion molecule when compared to random and film (p < 0.05). The gene expression patterns of aligned fibers favor myelination of Schwann cells when compared to film and random fibers. Thus, our results demonstrate that the aligned topography of the scaffold promotes maturation of Schwann cells and thereby its myelination to maintain its functionality.