Evaluating the affect of dimethyl sulfoxide on the viablity of H9C2 cardiomyocytes

Objective: The study was conducted to evaluate the affect of Dimethyl Sulfoxide (DMSO) concentration on the viability of H9C2 cells under the different cultural conditions. Methods: H9C2 cells were cultured under normal conditions and subjected to hypoxia/reoxygenation model. DMSOat the doses of 0,001÷10% (v/v) was added to the cultural medium during normal culture period and reoxygenation period. Cellular viability of the experimental groups was assessed by using CCK-8 kit. Results: The results indicated that the viability of H9C2 cardiomyocytes was stable in the different culture media supplied with DMSO at the doses of0,001÷0,5% (v/v). Meanwhile, supplementation of DMSO at the doses of1% and 2% significantly decreased the survival rate of H9C2 cells (v/v,p<0,05). Conclusion: The affect of DMSO on H9C2 cardiomyocytes is dose-dependant maner.

OS6.4 The hypoxia-induced glioma derived exosome miRNA-199a-3p promotes glioma cells proliferation and increased ischemic injury of the para-tumor neurons by inhibiting mTOR pathway— A pivotal ischemic mechanism in the proliferation and growth of glioma

BACKGROUND Growth of glioma cells can be promoted by hypoxia, but its underlying molecular mechanisms are not clear. Exosomes and miRNAs were reported to play crucial roles in tumor progression. Effects of exosomes and exosomal miRNAs, induced by hypoxia, on glioma cells were still unclear. MATERIAL AND METHODS Glioma samples were analyzed by HE and HIF-1α staining. Image data of these patients were also retrospectively analyzed. HT22 and C6 cell lines were co-cultured in both direct and indirect system. Hypoxia (1% oxygen) and oxygen and glucose deprivation (OGD) were applied to evaluate hypoxia effects on the growth and proliferation of cell lines, and such effects were assessed by C6/HT22 ratio, MTT and LDH assay. Hypoxia-induced glioma derived exosomes (HIGDE) and non-HIGDE (NHIGDE) were isolated and were administrated to normal culture medium to evaluate their effects on cell growing. The target miRNA was selected by performing miRNA microarray analysis. MicroRNA mimics and shRNA were constructed to overexpress or inhibit the microRNA expression. MTOR signal pathway was activated by utilizing phosphatidic acid. The RNAs expression were detected by RT-qPCR and the proteins expression was evaluated by western blotting. RESULTS Para-tumor hypoxia area shared a same region with cytotoxic edema around the glioma lesion and can be easily detected by PET/CT. The density of positive HIF-1αstaining was higher in tumor area than that in para-tumor and normal parenchyma area. In hypoxia direct co-culture system, the cell number ratio of C6/HT22 was significantly higher than that without hypoxia pretreatment; while in hypoxia mono-culture and indirect co-culture systems, the proliferation ability of HT22 was statistical lower than C6. After applying OGD, neuron cells cultured with HIGDE showed a statistical higher LDH release level than with NHIGDE or normal culture medium. The miRNA microarray analysis revealed that miRNA-199a-3p was the highest expressed in HIGDE than in NHIGDE (p < 0.05; Fold Change > 2). Transfected with mimics or shRNA, it was indicated that upregulation of miR-199a-3p aggravated HIGDE-induced OGD injury in HT22 cells. Moreover, we interfered mTOR signal pathway and the expression of HIF-1αin C6 cells. We found that miRNA-199a-3p aggravated HIGDE-neuron cell injury via suppressing mTOR signal pathway, and hypoxia related upregulation of miRNA-199a-3p in HIGDE was induced by the activation of HIF-1α in C6 cells. CONCLUSION The Hypoxia-Induced Glioma Derived Exosome miRNA-199a-3p can be upregulated by the activation of HIF-1α, and is able to promote glioma cells proliferation and increase ischemic injury of the para-tumor neurons via inhibiting mTOR pathway.

Culture and sporulation of Paenibacillus larvae larvae (White) in not specific culture media

In this experimental study the culture possibility and the sporulation ability of Paenibacillus larvae larvae (White), in simple and compound not specific culture media, in aerobic and anaerobic conditions and in an atmosphere of 5-10% C02 in air, were investigated. The results indicated that P.l.larvae could be cultured in normal culture media, in aerobic or anaerobic conditions. However its growth is significantly delayed and only after 48-72 h incubation, its demonstration becomes evident. In specific culture media like blood agar and Brucella agar and atmosphere of 5-10% C02 in air, it can be developed faster, sporulated more and demonstrated easier. Development and sporulation of P.l.larvae was better in Brucella agar than in blood agar.

Zinc Protects Oxidative Stress-Induced RPE Death by Reducing Mitochondrial Damage and Preventing Lysosome Rupture

Zinc deficiency is known to increase the risk of the development of age-related macular degeneration (AMD), although the underlying mechanism remains poorly defined. In this study, we investigated the effect of zinc on retinal pigment epithelium (RPE) survival and function under oxidative conditions. Zinc level was 5.4 μM in normal culture conditions (DMEM/F12 with 10% FCS) and 1.5 μM in serum-free medium (DMEM/F12). Under serum-free culture conditions, the treatment of RPE cells with oxidized photoreceptor outer segment (oxPOS) significantly increased intracellular ROS production, reduced ATP production, and promoted RPE death compared to oxPOS-treated RPE under normal culture condition. Serum deprivation also reduced RPE phagocytosis of oxPOS and exacerbated oxidative insult-induced cathepsin B release from lysosome, an indicator of lysosome rupture. The addition of zinc in the serum-free culture system dose dependently reduced ROS production, recovered ATP production, and reduced oxidative stress- (oxPOS- or 4-HNE) induced cell death. Zinc supplementation also reduced oxidative stress-mediated cathepsin B release in RPE cells. Our results suggest that zinc deficiency sensitizes RPE cells to oxidative damage, and zinc supplementation protects RPE cells from oxidative stress-induced death by improving mitochondrial function and preventing lysosome rupture.