ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性

利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2（Enhanced disease resistance 2），在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1，构建CRISPR/Cas9敲除载体，通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序，结果表明，共有8个转基因植株在靶位点处发生不同类型的双等位基因突变，编辑效率为32%；突变体植株生长势较弱，叶片较小，茎秆丛生、细弱。进一步对突变体植株进行抗病检测，结果表明，接种葡萄白粉菌（Erysiphe necator Schw.）5 d后，突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝，表皮细胞产生大量明显的H2O2，而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器，无明显H2O2产生。这些结果表明，可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2，提高葡萄白粉菌抗性。     

赤霉素不同处理浓度和浸种时间对托鲁巴姆发芽的影响

托鲁巴姆种子的休眠较深,发芽较困难。为了打破其种子的休眠期,加快种子萌发,提高种子发芽的整齐度,以托鲁巴姆为试材,研究了不同的赤霉素处理浓度和浸种时间对其发芽的影响。结果表明:生产中适宜的赤霉素处理浓度为750~1 250 mg/L,浸种时间为36 h,发芽率可达93.00%;可有效提高托鲁巴姆种子的发芽速率和发芽整齐度。     

H2S的信号分子作用及其对果蔬采后生理代谢的调控研究进展

硫化氢（H2S）作为气体信号分子，参与调控植物体的一系列生命活动，并在果蔬采后生理代谢过程中发挥着重要作用。本文综述了H2S的理化性质、发展应用历史及其在果蔬采后生理调控机制中的信号分子作用，讨论了H2S对果蔬生理代谢的调控作用，包括对果蔬采后抗逆性和品质的影响，以期为采后果蔬的贮藏保鲜提供理论参考。     

1株H1N1亚型猪流感病毒的全基因组特征及其对小鼠的致病性

旨在了解河南省猪流感病毒的流行情况及其遗传进化和基因组特征。2018年4月，从河南省某一出现疑似流感症状猪群中采集鼻拭子样品150份用于分离病毒，对分离病毒的全基因组进行序列测定和分析。同时感染6周龄BALB/c小鼠，研究其对小鼠的致病性。结果显示，获得1株H1N1亚型病毒[命名为A/swine/Henan/NY20/2018（H1N1）]。遗传进化表明，其HA和NA基因属于欧亚类禽H1N1分支，PB2、PB1、PA、NP和M基因属于2009甲型H1N1分支，NS基因属于经典H1N1分支。HA蛋白的裂解位点序列为PSIQSR↓GL，具有低致病性流感病毒的分子特征，在小鼠肺和鼻甲有效复制并能引起肺组织病理学变化。本研究分离到1株3源重排H1N1亚型病毒，对小鼠呈现一定致病力，提示应进一步加强对SIV的监测。     

Identification of an H1N1 subtype of swine influenza virus and serological analysis

To investigate the epizootic of swine influenza virus(SIV), 60 nasal swabs were collected from a clinical cases of pig farm in Tai'an City, Shandong Province of China in April 2017. SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced. An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1). Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch, seven genome segments belonged to avian-like H1N1 influenza virus lineage. The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G, which is a typical molecular biological characteristic. Five potential N-glycosylation sites(N14, N26, N277, N484 and N543) were found in the HA gene. To further investigate the epidemiology of SIV in this farm, the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens. The results showed that the total positive rate was 65.43%. The positive rates of single virus infection detected by EAH1N1, 2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35, 30.85 and 7.47%, respectively. The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm. These data provide evidence of an epizootic of SIV.     

神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路

NMB/NMBR通过调节A型流感病毒（IAV/H1N1/PR8）感染诱导的细胞因子表达而参与抗IAV的先天性免疫反应。为探究其发挥抗IAV/H1N1感染的信号通路，本文用PR8和WSN毒株分别感染MLE-12细胞和小鼠，用NF-κB抑制剂BAY11-7028单独或联合NMB处理MLE-12细胞，小鼠后腿肌内注射NMB和NMBRA，采用RT-PCR和qRT-PCR分析NMB、NMBR、IL-6、IFN-α和NP基因表达变化，采用Western blot分析NMB、NMBR、P65/p-P65、IκBα和NP蛋白表达的变化。结果显示，BAY11-7028可促使PR8和WSN感染的MLE-12细胞中NMB、NMBR、IL-6和IFN-α基因表达水平均下降和NP基因表达水平上升，并降低NMB、NMBR和p-P65蛋白表达水平和提升IκBα和NP蛋白表达水平。然而，NMB联合BAY 11-7028诱导PR8或WSN感染后的细胞中IL-6和NP表达出现极显著下降和IFN-α显著上升。此外，NMB抑制PR8和WSN感染的小鼠肺组织内p-P65和NP蛋白表达水平和促进IκBα蛋白表达水平；NMBRA联合NMB抵消NMB对PR8或WSN感染后的这些蛋白表达水平的调节作用。综上表明，NMB/NMBR通过调节PR8和WSN感染的MLE-12细胞和小鼠体内的NF-κB信号通路上P65蛋白磷酸化和IκBα的表达，进而影响下游细胞因子IL-6和IFN-α基因的表达，从而发挥抗IAV/H1N1感染的先天性免疫应答反应。     

利用CRISPR/Cas9技术编辑BADH2-1/BADH2-2创制香米味道玉米新种质

【目的】香味是作物的重要食味品质之一。2-乙酰-1-吡咯啉（2-acetyl-1-pyrroline,2-AP）是主要香味物。BADH2是控制水稻等作物香味性状的关键基因,敲除该基因可以产生香味稻米。利用CRISPR/Cas9基因编辑技术在北京市农林科学院自育的玉米骨干亲本京724上敲除BADH2同源基因,以期获得有香米味道的玉米新种质材料。【方法】利用Ensembl数据库在线BLAST工具,将水稻OsBADH2蛋白序列在拟南芥、水稻和玉米蛋白序列数据库中进行序列比对,获得上述3个物种的BADH基因家族成员,并利用UniProt蛋白数据库中的结构域信息进行验证。进一步使用MEGA软件进行系统进化分析,获得玉米BADH2同源基因作为候选编辑靶标。基于CRISPR/Cas9基因编辑技术的原理,在候选基因的外显子处设计特异性靶点,并构建入CRISPR/Cas9基因编辑载体。再以玉米自交系京724为受体,利用农杆菌介导的遗传转化方法,通过磷酸甘露糖异构酶基因（phosphomannose isomerase,PMI）抗性筛选获得阳性转基因植株。转基因株系经测序明确其在靶基因中产生的突变类型。利用气相色谱质谱联用仪（gas chromatography-mass spectrometry,GC-MS）检测基因编辑株系T₁籽粒中香米主要香味物质2-AP的含量,以确认京724在基因编辑前后2-AP含量的变化。【结果】系统进化分析发现,玉米中存在2个BADH2同源基因,分别命名为ZmBADH2-1和ZmBADH2-2。ZmBADH2-1位于第4染色体,ZmBADH2-2位于第1染色体。2个基因均包含15个外显子和14个内含子,第4外显子间的核苷酸序列高度同源。在2个基因的第4外显子区域设计靶点并构建入CRISPR/Cas9基因编辑载体,通过遗传转化获得28株转基因株系。PCR扩增及测序分析结果显示,其中10株材料的2个ZmBADH2s在靶点区域均发生突变,突变基因型包括双等位突变和多等位突变,突变类型为不同数量的碱基缺失和插入。质谱检测结果显示玉米ZmBADH2双基因突变体籽粒中存在与香稻同样成分的2-AP。随机选取的4个T₁代基因编辑株系籽粒中,2-AP平均含量分别为438.29、404.63、348.65和161.82 μg·kg^-1,而未经过编辑的京724中未检测到2-AP。【结论】利用CRISPR/Cas9技术对玉米ZmBADH2-1和ZmBADH2-2同时进行定点敲除,创制出籽粒中具有香米味道的玉米骨干亲本新种质材料。     

Nutritional Qualities of the Low-Value Bivalve Mollusks Paphia malabarica and Villorita cyprinoides at the Estuarine Waters of the Southwestern Coast of India

The bivalve mollusks Paphia malabarica and Villorita cyprinoides collected from the estuarine system located in the southwest coastal waters of India were evaluated for their nutritional composition. A balanced essential to nonessential amino acid ratio (> 1.0) with a greater quantity of sulfur-containing amino acids in these species demonstrated that they could provide well-balanced protein depositions. The n-3/n-6 polyunsaturated fatty acid ratio in P. malabarica was found to be greater than 2, and, therefore, can be considered as a healthy diet. The C₂₀–C₂₂ long-chain polyunsaturated n-3 fatty acids (for example, docosahexaenoic acid and eicosapentaenoic acid) accounted for more than 23% of the total fatty acid content in P. malabarica as compared to about 7% in V. cyprinoides. P. malabarica showed greater hypocholesterolemic/hypercholesterolemic ratio (1.7) and lesser atherogenicity (0.7), thrombogenicity (0.3) indices when contrasted with those in V. cyprinoides, thereby indicating the nutritional superiority of the former. A greater content of vitamin D₃ (> 150 IU), along with significant quantities of calcium and phosphorus (> 500 mg/100 g) in the clams, signified their importance in preventing osteoporosis. This study demonstrated the importance of bivalves, in particular, P. malabarica as a valued species for human consumption.