MicroRNA-197-3p mediates damage to human coronary artery endothelial cells via targeting TIMP3 in Kawasaki disease

Kawasaki disease (KD) causes cardiovascular system injury in children. However, the pathogenic mechanisms of KD have not been well defined. Recently, strong correlation between aberrant microRNAs and KD nosogenesis has been revealed. A role of microRNA-197-3p (miR-197-3p) in the pathogenesis of KD is identified in the present study. Cell proliferation assay showed human coronary artery endothelial cells (HCAECs) were suppressed by serum from KD patients, which was correlated with high levels of miR-197-3p in both KD serum and HCAECs cultured with KD serum. The inhibition of HCAECs by miR-197-3p was confirmed by cells expressing miR-197-3p mimic and miR-197-3p inhibitor. Comparative proteomics analysis and Ingenuity Pathway Analysis (IPA) revealed TIMP3 as a potential target of miR-197-3p, which was demonstrated by western blot and dual-luciferase reporter assays. Subsequently, by detecting the endothelium damage markers THBS1, VWF, and HSPG2, the role of miR-197-3p/TIMP3 in KD-induced damage to HCAECs was confirmed, which was further validated by a KD mouse model in vivo. The expressions of miR-197-3p and its target, TIMP3, are dramatically variational in KD serum and HCAECs cultured with KD serum. Increased miR-197-3p induces HCAECs abnormal by restraining TIMP3 expression directly. Hence, dysregulation of miR-197-3p/TIMP3 expression in HCAECs may be an important mechanism in cardiovascular endothelium injury in KD patients, which offers a feasible therapeutic target for KD treatment.

     

逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。     

Synthesis of the novel liqustrazine derivatives and Their protective effect on injured vascular endothelial cell damaged by hydrogen peroxide

A series of novel 2-acyloxymethyl-3,5,6-trimethylpyrazine derivatives was designed and synthesized. Most compounds were found to be 1.5-4.5-fold higher potency than tetramethylpyrazine (TMP) in stimulating the proliferation of normal vascular endothelial cells and in protecting against hyperoxic acute injury. The most active one is the 2-nicotinoyl ester 5a exhibiting the maximum proliferation rate (P(max)) of 88.57% at the concentration of 0.1 mmol L(-1). Structure-activity relationships of these compounds were discussed.     

Isolation and characterisation ofCandida sp. mutants enriched in S-adenosylmethionine (SAM)

The ability to accumulate S-adenosylmethionine (SAM) of 572 yeast strains isolated from environmental sources were surveyed. An S-adenosylmethionine enriching strain S42-12, identified asCandida sp., was chose to develop a SAM-accumulating mutant successfully. The final SAM-accumulating mutant strain YQ-5 was isolated by UV radiation or by NTG treatment using ethionine selection and nystatin selection method. The mutant strain YQ-5 accumulated 112.1 mg per gram biomass, was 3.14-fold higher than the original strain S42-12. When cultivated in the optimal medium with a favourable fermentation conditions, SAM content of the mutant strain reached at 1740 mg L^?1. Trend of SAM and ergosterol contents and methionine adenosyltransferase activity of SAM-accumulating mutants during fermentation were analysed. The results suggested that one of the reasons why the mutants accumulated SAM in significantly high amounts may be the lower consumption of SAM for ergosterol biosynthesis, other than improvement of methionine adenosyltransferase activity.     

Progesterone-modulated induction of apoptosis by interferon-tau in cultured epithelial cells of bovine endometrium

Interferon-tau (IFN-tau) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN-tau reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN-tau would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-tau, or 10 microg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-alpha protein expression. The results showed that IFN-tau and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN-tau to induce apoptosis (14.6%) compared with IFN-tau alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF-tau and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN-tau and CHX treatment resulted in a greater expression of the proapoptotic protein Bax-alpha compared with that in control cultures. These data demonstrate that IFN-tau can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN-tau might play a critical role in the remodeling of the endometrium around the time of implantation.     

Analysis of the status of subject recruitment in clinical trials in Shandong, China

Although subject recruitment for clinical trials in Shandong has been carried out with an awareness of international regulatory and ethical frameworks, there have been some defects in the recruitment process. The objective of this study is to analyze the current status of subject recruitment in Shandong. We conducted a survey among 198 principal investigators (PIs) and 543 subjects. The results were summarized and calculated as a percentage according to the responses to each question by PIs and subjects. This survey indicated that the ethics committee should strengthen the review of subject recruitment and enhance ethics training among board members. PIs should seek to improve the recruitment process.     

Association Analysis of Genomic Loci Important for Grain Weight Control in Elite Common Wheat Varieties Cultivated with Variable Water and Fertiliser Supply

Grain weight, an essential yield component, is under strong genetic control and markedly influenced by the environment. Here, by genome-wide association analysis with a panel of 94 elite common wheat varieties, 37 loci were found significantly associated with thousand-grain weight (TGW) in one or more environments differing in water and fertiliser levels. Five loci were stably associated with TGW under all 12 environments examined. Their elite alleles had positive effects on TGW. Four, two, three, and two loci were consistently associated with TGW in the irrigated and fertilised (IF), rainfed (RF), reduced nitrogen (RN), and reduced phosphorus (RP) environments. The elite alleles of the IF-specific loci enhanced TGW under well-resourced conditions, whereas those of the RF-, RN-, or RP-specific loci conferred tolerance to the TGW decrease when irrigation, nitrogen, or phosphorus were reduced. Moreover, the elite alleles of the environment-independent and -specific loci often acted additively to enhance TGW. Four additional loci were found associated with TGW in specific locations, one of which was shown to contribute to the TGW difference between two experimental sites. Further analysis of 14 associated loci revealed that nine affected both grain length and width, whereas the remaining loci influenced either grain length or width, indicating that these loci control grain weight by regulating kernel size. Finally, the elite allele of Xpsp3152 frequently co-segregated with the larger grain haplotype of TaGW2-6A, suggesting probable genetic and functional linkages between Xpsp3152 and GW2 that are important for grain weight control in cereal plants. Our study provides new knowledge on TGW control in elite common wheat lines, which may aid the improvement of wheat grain weight trait in further research.     

Expression of the Neisseria gonorrhoeae major outer membrane protein PI in Escherichia coli

Summary To actively express an outer membrane protein, protein I (PI), from different strains of Neisseria gonorrhoeae in E.␣coli, PI gene fragments from two reference strains and four clinical isolates of Neisseria gonorrhoeae were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. Subsequently, they were cloned into an expression vector pET-30b (+) to generate pET-PI recombinants. After inducing with isopropyl-β-d-thiogalactopyranoside (IPTG), the expressed PI proteins were analysed by SDS-PAGE, Western blotting and ELISA. The results implied that we had successfully constructed the PI gene recombinants from both reference strains and clinical isolates and obtained the recombinant proteins expressed in E. coli at relatively high levels, and the expressed proteins had the immunological activity with the corresponding antibodies. This research will be very helpful for the further study of these proteins in generating preventive vaccines on Neisseria gonorrhoeae infection and clinical diagnosis.     

逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。