Lipidomic analysis of meibomian glands from type-1 diabetes mouse model and preliminary studies of potential mechanism.

Diabetes is a significant risk factor for meibomian gland dysfunction (MGD), but its mechanism is poorly understood. The main function of the meibomian glands (MGs) is to synthesize, store, and secrete lipids. In this study, we found that the amount of lipids in the meibomian acini in STZ-induced type 1 diabetic mice decreased, and the lipid droplets became larger and irregular. In all, 31 lipid subclasses were identified in the mouse MGs, which contained 1378 lipid species in total through lipidomics analysis based on LC-MS/MS. Diabetes caused a significant increase in the content of ceramides (Cer) in the MGs but a significant decrease in the ration of sphingomyelin to ceramides (SM/Cer). The quantity of meibocytes in diabetic mice was dramatically decreased, and the proliferation activity was alleviated, which may be associated with cell cycle arrest caused by diabetes-induced abnormal Cer metabolism in MGs. We found an increase in macrophage and neutrophils infiltration in the diabetic MGs, which may be related to the significant reduction of AcCa in diabetic MGs. Taken together, the results of the present study demonstrated that diabetes induced disruption of lipid homeostasis in MGs, which may mediate the decreased cell proliferation and increased inflammation caused by diabetes in MGs.

CYP3A4 and microRNA-122 are involved in the apoptosis of HepG2 cells induced by ILs 1-decyl-3-methylimidazolium bromide.

Ionic liquids (ILs) as green alternatives for volatile organic solvents are increasingly used in commercial applications. It is necessary to explore the cytotoxic mechanism of ILs to reduce the risk to human health. For this purpose, cell viability, apoptosis, cytochrome P450 3A4 (CYP3A4), glucose transporter type 2 (GLUT2), and microRNA-122 (miR-122) gene expression in HepG2 cells was evaluated after IL exposure. The results showed that ILs reduced the viability of HepG2 cells through apoptotic cell death. Moreover, ILs markedly upregulated the transcription and protein levels of CYP3A4, but did not affect the expression of GLUT2 in either messenger RNA level or protein level. Finally, ILs increased the expression of miR-122 and inhibition of miR-122 with miR-122 inhibitor blocked ILs-induced apoptosis in HepG2 cells. This finding may contribute to an increased understanding of the in vitro molecular toxicity mechanism of ILs to further understand IL-related human health risks.

Thalassotalea atypica sp. nov., isolated from seawater, and emended description of Thalassotalea eurytherma.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZG4-3-1T, was isolated from coastal seawater of Rizhao, China (119.625° E 35.517° N). The organism grew optimally at 24-28 °C, at pH 7.0 and in the presence of 2.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZG4-3-1T contained ubiquinone 8 (Q-8) as the major respiratory quinone and contained C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0 as the dominant fatty acids. The polar lipids of strain RZG4-3-1T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content of strain RZG4-3-1T was 40.1 mol%. Strain RZG4-3-1T exhibited the highest 16S rRNA gene sequence similarity value (96.0 %) to Thalassotalea eurytherma JCM 18482T. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZG4-3-1T belonged to the genus Thalassotalea. On the basis of polyphasic analyses, strain RZG4-3-1T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea atypica sp. nov. is proposed. The type strain is RZG4-3-1T (=JCM 31894T=KCTC 52745T=MCCC 1K03276T). An emended description of Thalassotalea eurytherma is also provided.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells.

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the ß-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080 cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080 cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080 cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080 cells.

Pitavastatin up-regulates eNOS production by suppressing miR-155 expression in lipopolysaccharide-stimulated human umbilical vein endothelial cells.

AIM: Pitavastatin (Pit) has been proved to efficiently inhibit the onset and progression of atherosclerosis. However, the mechanism by which Pit exerts nonlipid-related effects, such as antiinflammatory actions, is not quite clear. Our study aimed at investigating the effect of Pit on the expression of endothelial NO synthase (eNOS) and miR-155 in LPS-stimulated HUVECs to reveal the antiinflammatory mechanism of pitavastatin. METHODS: HUVECs were isolated from newborn umbilical cords and used in the experiments at passages 2-5. Cells were treated with LPS (0.05, 0.1, 1 µg/L) or LPS (0.1 µg/L)+Pit (0.01, 0.1, 1 µmol/L), untreated cells were used as control. For LPS+Pit induction, cells were firstly incubated with Pit for 1 hour before coincubation with LPS for 24 hours. eNOS mRNA and miR-155 were detected by RT-PCR, and Western blotting was used to detect protein expression of eNOS. RESULTS: Treatment of HUVECs with LPS enhanced the expression of miR-155 and reduced the expression of eNOS in mRNA and protein level in a dose-dependent manner as revealed by RT-PCR and Western blotting, respectively. Pitavastatin ameliorated LPS-induced endothelial dysfunction through upregulation of eNOS expression and downregulation of miR-155 expression. CONCLUSION: Pitavastatin increases eNOS expression and inhibits of LPS-induced miR-155 expression.