Panax notoginseng saponins suppress oxygen-glucose deprivation/reoxygenation-induced pyroptosis of SH-SY5Y cells

AIM To investigate the effect of Panax notoginseng saponins (PNS) on pyroptosis of SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS The OGD/R was conducted to induce ischemia/reperfusion injury in SH-SY5Y cells. The effects of PNS on the viability (detected by CCK-8 assay) and membrane permeability [indicated by lactate dehydrogenase (LDH) leakage and propidium iodide (PI) staining positive cell proportion] of OGD/R-induced SH-SY5Y cells were observed. The protein levels of gasdermin D (GSDMD), GSDMD N-terminal fragment (GSDMD-N), caspase-1 and caspase-4, and the release of interleukin-1β (IL-1β) and IL-18 in the cells were also determined. RESULTS After exposure to OGD/R, the viability of SH-SY5Y cells dramatically decreased (P<0.01), while the LDH leakage, the PI staining positive cell proportion, the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4, and the release of IL-1β and IL-18 were significantly increased (P<0.01). However, PNS treatment enhanced the viability of SH-SY5Y cells inhibited by OGD/R (P<0.01), but reduced the leakage of LDH and the percentage of PI staining positive cells (P<0.05 or P<0.01). Moreover, PNS reversed the increases in the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4 and the release of IL-1β and IL-18 in OGD/R-induced SH-SY5Y cells (P<0.05 or P<0.01). CONCLUSION Treatment with PNS alleviates OGD/R-induced injury in SH-SY5Y cells. Its mechanism may be related to inhibition of SH-SY5Y cell pyroptosis induced by OGD/R.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability， promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

玉米粉中伏马毒素FB1基体标准物质的评价

为填补我国在伏马毒素基体标准物质的空白,本试验研制了以玉米粉为基体的伏马毒素FB1标准物质,玉米样品经过筛、加标、冷冻干燥、磨粉、混匀、密封分装后,用超高效液相色谱-串联质谱(UPLC-MS/MS)法检验样品的均匀性、稳定性,幵联合多家实验室对玉米粉中伏马毒素FB1含量定值,同时分析样品的不确定度。样品均匀性经斱差分析法表明F值为1.42,小于临界值F_(0.05),且伏马毒素FB1含量在觃定时间6个月内无明显变化。结果表明,均匀性与稳定性均符合标准物质的要求。样品定值为1475.56μg kg~(–1),不确定度为169.98μg kg~(–1)。该标准物质可替代进口标准物质,用于伏马毒素检测过程中的仪器校准、实验室质量控制和操作人员的水平考核等。     

Soybean molasses as an organic carbon source in the farming of Litopenaeus vannamei (Boone, 1931) in a biofloc system

Soybean molasses was evaluated as a partial replacement for sugarcane molasses as a carbon source for biofloc development in the superintensive culture of Pacific white shrimp (Litopenaeus vannamei). A 50‐day study was conducted with juvenile (3.2 g) shrimp stocked in 16 800 L tanks at a stocking density of 250 shrimp m^?3. Control of total ammonia concentration was performed by the addition of combined mixtures of soybean and sugarcane molasses to the culture water. Three different molasses treatments were evaluated using different soybean‐to‐sugarcane molasses ratios: 15–85%, 38–62% and 60–40% respectively. The control group was treated only with sugarcane molasses. Water quality, chlorophyll a concentration, heterotrophic bacterial load, Vibrio spp. concentration and zootechnical indexes were all evaluated. Total ammonia concentration was controlled by heterotrophic and chemotrophic pathways. Biofloc formation, as quantified by measuring the total suspended solids, was not altered. The Vibrio spp. concentration showed a significant reduction in treatments with soybean‐to‐sugarcane molasses ratios of 38–62% and 60–40%. All combined mixtures of soybean and sugarcane molasses could maintain water quality and productivity in the superintensive culture of L. vannamei using the biofloc system. Thus, the potential use of a residue from agroindustry as a carbon source in a biofloc culture is demonstrated.     

Effects of dietary protein and lipid levels on growth performance,fatty acid composition and antioxidant‐related gene expressions in juvenile loach Misgurnus anguillicaudatus

Dojo loach (Misgurnus anguillicaudatus) is one of the most important cultured freshwater fish in several East Asian countries. However, a little information is available in its nutritional requirements. Thus, this study was conducted to evaluate the effects of feeding varying levels of dietary protein and lipid on growth, fatty acid composition and antioxidant‐related gene expressions in juvenile loach. Six practical diets at three levels of protein (30%, 40% and 50%) and two levels of lipid (6% and 12%) were fed to loach juveniles (initial weight 0.40 g) in triplicated groups (20 fish per replicated) for a period of 8 weeks. Results showed that regardless of lipid level, body weight gain of fish was significantly increased with incremental dietary protein level. Meanwhile, feed conversion ratio was significantly decreased by dietary protein levels, and the lowest value was observed in fish fed dietary protein levels of 50%, regardless of dietary lipid level. Moreover, the percentage of 22:6n‐3 in viscera was significantly increased by different protein levels. The expression level of catalase was significantly increased with incremental dietary protein level with both lipid levels. Meanwhile, the expression level of hepatic nuclear factor erythroid 2‐related factor 2 (Nrf2) was downregulated with incremental dietary protein level with 6% of lipid level, but the expression was upregulated with incremental dietary protein level with 12% of lipid level. In conclusion, these data suggested that 6% lipid and 50% protein in diet was optimal for loach during early development stage.     

基于UPLC-QTOF/MS的小麦发芽代谢组学分析方法

为探究不同提取方法对发芽小麦代谢物提取效果的影响,本研究建立了基于超高效液相色谱-四级杆飞行时间质谱(UPLC-QTOF/MS)的发芽小麦非靶向代谢组学样品前处理方法和分析方法,以发芽2 d周麦26籽粒为材料,设计提取溶剂、提取方式、提取时间3个因素在3个水平上L9(34)正交试验,并通过主成分分析和聚类分析确定提取效果最佳的组合。以80%乙腈(0.1%甲酸)震摇提取30 min可检测出1609个代谢峰,说明提取溶剂对代谢物提取效果起主要作用。共鉴定出92种小麦代谢物。     

一个CRISPR/Cas9-VQR基因编辑系统的构建

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术,但该编辑系统的使用范围受PAM (proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9 (SpCas9)编码氨基酸(1135位的天冬氨酸D突变成缬氨酸V, 1335位的精氨酸R突变为谷胱氨酸Q, 1337位的苏氨酸T突变为精氨酸R,命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的3′UTR序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点,并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%～70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%～70.5%,平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围,特别是NGA PAM位点较高的作物。     

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.     

利用CRISPR/Ca9技术靶向编辑芥蓝BoaZDS

以芥蓝(Brassicaoleraceavar.alboglabra)为材料,以ζ–胡萝卜素脱氢酶(ζ-Carotene desaturase,ZDS)基因为目标基因,建立其CRISPR/Cas9基因组编辑体系。在BoaZDS的编码区近5′端选择靶位点,构建了CRISPR/Cas9表达载体,通过农杆菌介导的遗传转化方法获得了19个芥蓝转基因阳性植株,Sanger测序分析发现其中13株成功突变,CRISPR/Cas9载体在芥蓝上的突变效率为68.42%,且所有突变植株均表现出明显的白化表型。     

基于转录组测序的半滑舌鳎长链非编码RNA的初步鉴定与分析

以抗病家系与易感家系半滑舌鳎为材料,进行哈维弧菌感染实验,并对易感家系感染前(CsSU)、易感家系感染后(CsSC)、抗病家系感染前(CsRU)、抗病家系感染后(CsRC)4组进行转录组测序,根据RNA-seq数据挖掘半滑舌鳎长链非编码RNA信息;通过生物信息学分析,筛选出与抗哈维弧菌病相关的差异长链非编码RNA(long non-coding RNA, lncRNA)。结果显示,共识别出4 584个lncRNA座位,包含5 714个转录本;其基本特征与编码基因的比较分析,lncRNA的GC含量低于编码基因,单外显子基因数多于编码基因,转录本的平均长度长于编码基因,基因表达量低于编码基因。对4组样品进行两两比较(CsRU vs CsSU、CsRC vs CsSC、CsRC vs CsRU、CsSC vs CsSU)分别筛选出818、813、261、140个差异表达lncRNA,其中CsRU与CsSU之间、CsRC与CsSC之间lncRNA数目差异最多,通过聚类分析确定了各实验组的表达模式之间的联系,CsRU与CsSU之间的表达模式最为相近。通过共表达分析,预测出lncRNA和274个编码基因可能存在14 539种相互关系,并进行了功能注释,进而筛选出7个关键lncRNA。qRT-PCR结果显示,差异表达lncRNAs的表达模式和转录组数据得到的基本一致。研究结果为揭示lncRNA在半滑舌鳎抗哈维弧菌免疫调控反应中的作用提供重要的参考数据。