Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Rapid mapping of candidate genes for cold tolerance in Oryza rufipogon Griff. by QTL-seq of seedlings

Cold stress is a major problem in rice production. To rapidly identify genes for cold tolerance in Dongxiang wild rice(DWR, Oryza rufipogon Griff.), sequencing-based bulked segregant analysis of QTL-seq method was used to resequence the extremely resistant(R) and susceptible(S) bulks of a backcross inbred lines(BILs) population(derived from Oryza sativa×O. rufipogon) and their parents. Single nucleotide polymorphisms(SNP)-index graphs and corresponding Δ(SNPindex) graphs(at 99 and 95% confidence levels) for R-and S-bulks detected a total of 2 609 candidate SNPs, including 58 candidate cold-tolerance genes. Quantitative real-time PCR analysis revealed that 5 out of the 58 candidate genes had significant differences in expression between O. sativa and O. rufipogon. Structural variation and functional annotations of the 5 candidate genes were also analyzed, and allowed us to identify 2 insertion-deletion(InDel) markers(12-7 and 12-16) that were linked with candidate genes on chromosome 12 in DWR. These results are helpful for cloning and using cold tolerance genes from common wild rice in cultivated rice.     

Construction and Partial Biological Characteristics Analysis of Listeriolysin S llsB Gene Deletion Strain

In order to analyze the effect of listeriolysin S (LLS) llsB gene deletion on the biological characteristics of Listeria monocytogenes (LM),this study used homologous recombination to construct the llsB gene deletion strain LM90-ΔllsB,and the biological characteristics of growth characteristics,median lethal dose (LD₅₀) and organ-borne bacteria were studied in healthy Kunming male mice at 8 weeks old and weighing 40 g±5 g.The llsB gene deletion strain was successfully constructed,and the deletion strain had good genetic stability through continuous passage to 20 generations in vitro.Based on its growth curve examination,we found that the growth rate of the mutant strain was slightly higher than that of the parent strain.The results of mice infection test showed that the LD₅₀ of the parent strain and the deletion strain were 10^6.17 and 10^6.50 CFU,respectively.Compared with the parent strain,the amount of bacteria load of the deletion strain in the liver and spleen of the mice was extremely significantly decreased (P<0.01),the results showed that the infection ability of the mutant strain on mice was obviously weakened.No Listeria monocytogenes was detected in the brain.The results suggested that llsB gene might have direct or indirect regulatory effect on some biological characteristics of LM90,and it would provide a theoretical basis for further understanding of the pathogenic mechanism of LLS,prevention and control of listeriosis.     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

12种杀虫剂对日本通草蛉不同虫态的毒力及安全性评价

为明确常见杀虫剂对日本通草蛉各虫态的影响,在室内分别采用浸渍法和喷雾法测定了12种(8类)杀虫剂对日本通草蛉卵、幼虫、蛹和成虫的毒性,并评价了杀虫剂对日本通草蛉的毒性风险。结果表明,高效氯氰菊酯对日本通草蛉为极高风险,毒死蜱、吡虫啉、烯啶虫胺和阿维菌素为高风险,溴氰虫酰胺、啶虫脒和氯虫苯甲酰胺为中等风险,噻嗪酮、吡蚜酮、螺虫乙酯和甲氧虫酰肼为低风险;与其他虫态相比,12种杀虫剂对日本通草蛉蛹的毒性均为最低。在害虫生物防治及综合治理中应尽量选用对日本通草蛉毒性低的杀虫剂,以起到保护天敌的作用。     

茶树新品种--梦茗

"梦茗"系从安徽省岳西县来榜镇优良地方群体种中采用单株选育法育成的适制绿茶新品种。经安徽省内品比、推广示范和全国区试,发现"梦茗"物候期与福鼎大白茶相当,属早生种。品质优异,全国区试3个点感官审评总分均高于对照;内含物含量高,氨基酸含量4.9%、茶多酚总量26.9%、水浸出物总量40%。产量高,品比试验高于福鼎大白茶26.45%,全国区试产量与对照相当。抗旱能力强,抗寒能力与对照相当。     

小贯小绿叶蝉在5个茶树品种（系）上的蜜露排泄量与茶树叶片结构比较

为明确茶树叶片组织结构与小贯小绿叶蝉抗性之间的相关性,在确定小贯小绿叶蝉不同龄期和虫态蜜露排泄量的基础上,比较研究了小贯小绿叶蝉雌成虫在不同茶树品种（系）上取食24 h后蜜露排泄量的差异,进而通过比较不同茶树品种（系）叶片的组织结构和色泽,分析了5个茶树品种（系）叶片的组织结构与茶树对小贯小绿叶蝉抗性能力强弱的关系。研究结果表明,小贯小绿叶蝉4龄若虫的蜜露排泄量显著高于1~3龄;雌、雄成虫的蜜露排泄量之间不具显著差异;取食中选10号的雌成虫蜜露排泄量显著低于取食其他品种;不同品种（系）茶树的叶面积、叶片总厚度、上表皮厚度、下表皮厚度以及色差等指标与雌成虫的蜜露排泄量之间无显著相关性,而叶片茸毛密度与雌成虫蜜露排泄量呈显著负相关,叶片茸毛长度与蜜露排泄量呈显著正相关。