The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.) is an important staple crop for global human. The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot, a devastating disease of wheat. Herein, we identified RcMEP1, a zinc metalloproteaseencoding gene from R. cerealis genomic sequences, and characterized its pathogenesis function. RcMEP1 expressed at markedly-high levels during R. cerealis infection process to wheat. The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH). The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death, and that the predicted signal peptide functions and is required for secretion and cell death-induction. The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation, and to inhibit expression of host chitinases when infiltrated into wheat and N. benthamiana leaves, while the M43 domain-deleting peptide and negative control lacked the capacity. Moreover, compared with the control pretreatment, the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat, whereas the M43 domain-deleting peptide failed. These results suggest that RcMEP1 acted as an important pathogenicity factor during R. cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1. This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R. cerealis infection to wheat.     

草酸处理对桂七芒果冷害及细胞壁代谢的影响

为探讨草酸处理对低温冷藏下芒果冷害及细胞壁代谢的影响,本试验以桂七芒果果实为试材,采用5 mmol·L^-1草酸溶液浸泡处理,以清水浸泡处理为对照,并于4℃贮藏,分析芒果的冷害指数、丙二醛(MDA)含量、相对电导率、硬度、细胞壁物质含量、细胞壁代谢酶的变化。结果表明,与对照组相比,草酸处理显著降低了低温贮藏14 d后桂七芒果果实冷害指数、MDA含量、相对电导率、原果胶和纤维素含量,显著降低了低温贮藏28 d后果实的硬度;显著提高了低温贮藏14 d后果实水溶性果胶含量及多聚半乳糖醛酸酶(PG)、果胶甲酯酶(PME)、纤维素酶(Cx)活性,显著提高了贮藏21 d后果实β-半乳糖苷酶(β-Gal)活性。综上所述,草酸处理能减轻桂七芒果冷害,维持采后果实细胞壁降解酶较高活性和水溶性果胶含量。本研究为揭示草酸减轻芒果果实冷害机制提供了依据,可为草酸应用于其他冷敏型果实的贮藏保鲜提供理论参考。     

龙须菜细胞壁多糖对硼的吸附性能研究

为研究龙须菜(Gracilaria lemaneiformis)细胞壁多糖对硼的吸附作用机制及官能团之间的交互作用,本试验分别采用电感耦合等离子发射光谱(ICP-OES)和傅里叶变换红外光谱(FTIR)技术对其细胞壁多糖组分在吸附试验中进行硼的测定和表征。结果表明,细胞壁去琼胶后,硼吸附量减少50.13%,半纤维素去除后,硼吸附量减少21.20%,故可得纤维素吸附量占细胞壁总硼吸附量的28.67%。同时,通过细胞壁不同组分脱硼及硼胁迫后的红外光谱表征结果可知,龙须菜细胞壁及多糖在吸附硼的过程中,羟基、羧基、多糖碳链C-C为硼离子的主要结合位点,其中,琼胶、半纤维素中起主要作用的官能团为羟基、羧基,纤维素中起主要作用的官能团为多糖碳链C-C。本研究结果为进一步探究龙须菜多糖组分与硼的内在结合机制提供了基础依据。     

CUDC-907 induces DNA damage and autophagy in glioma cells

AIM:To investigate the effect of CUDC-907, a dual histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) inhibitor, on the DNA damage, cell cycle distribution and autophagy in human glioma U251 cells. METHODS:U251 cells were treated with CUDC-907 of different concentrations, and the cell viability was detected by MTT assay. The quantitative γ-H2AX foci were determined by laser scanning confocal microscopy. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was determined by Western blot analysis. RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70s6K) in the U251 cells (P<0.05). In CUDC-907-treated cells, the number of γ-H2AX foci and protein expression of γ-H2AX were increased significantly (P<0.05). CUDC-907 also induced cell arrest in the G₂/M phase by up-regulating the expression of p21, and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein 2 (Cdc2). In addition, CUDC-907 triggered cell autophagy, and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells. CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway, induces DNA damage and arrests cell cycle in G₂/M phase. Blockage of autophagy promotes CUDC-907-induced DNA damage of U251 cells.     

Hyperbaric oxygen therapy activates CREB/BDNF signaling pathway to reduce synaptic damage in hippocampal neurons of Alzheimer disease mice

AIM To investigate the effect of hyperbaric oxygen (HBO) on synaptic damage of hippocampal neurons in APP/PS1 transgenic (TG) mice and its possible mechanism. METHODS The 6-month-old male APP/PS1 TG mice were randomly divided into TG group, HBO group and cAMP response element binding protein (CREB) inhibitor H89 group, with 10 mice in each group. Ten male wild-type (WT) C57BL/6 mice of the same age were used as negative control group (WT group). The mice in HBO and H89 groups were treated with HBO for 6 cycles, while the mice in WT group and TG group were not treated. The learning and memory abilities were observed by Morris water maze. The nesting ability of the mice was detected by nesting test. The Nissl bodies in hippocampal neurons were observed by Nissl staining. The mRNA expression of CREB and brain-derived neurotrophic factor (BDNF) in hippocampus was detected by real-time PCR. The protein levels of synapsin (SYN), postsynaptic density protein 95 (PSD95), growth-associated protein 43 (GAP43), CREB, phosphorylated CREB (p-CREB) and BDNF in the hippocampus were determined by Western blot. RESULTS Compared with WT group, the learning and memory abilities of the mice in TG group were signilficantly reduced (P<0.05). In addition, the nesting score, the number of Nissl bodies in the hippocampal neurons, the mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). Compared with TG group, the learning and memory abilities of the mice in HBO group were improved (P<0.05). Meanwhile, the nesting scores of the mice were significantly increased (P<0.05), the neurons in the hippocampus were arranged neatly, and the number of Nissl bodies, the relative mRNA expression of CREB and BDNF,and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also increased significantly (P<0.05). Compared with HBO group, the mice in H89 group had poor learning and memory abilities, lowered nesting scores and decreased number of Nissl bodies. Futhermore, the relative mRNA expression of CREB and BDNF, and the protein levels of SYN, PSD95, GAP43, p-CREB and BDNF were also decreased significantly (P<0.05). CONCLUSION HBO improves the learning and memory abilities of APP/PS1 TG mice, and its mechanism may be related to activating the CREB/BDNF signaling pathway to reduce synaptic damage of hippocampal neurons in mice.     

猪RUVBL2真核表达载体的构建及其在CHO细胞中的表达

RUVBL2(RuvB-like 2)蛋白是进化上高度保守AAA+(ATPases Associated With Diverse Cellular Activities,AAA)家族成员之一,CHO(Chinese Hamster Ovary)细胞被广泛地用于表达重组DNA蛋白。为探究猪睾丸组织的RUVBL2基因是否能够在CHO细胞中表达,本实验以猪睾丸组织的cDNA为模板,PCR扩增RUVBL2目的基因,并将其克隆至pIRES2-EGFP(Mammalian Expression Vectors pIRES2-EGFP)载体上,进一步转化到DH5α中,再进行PCR、酶切及测序鉴定;将重组质粒转染到CHO细胞中,再进行荧光、RT-PCR、Western blot检测。结果显示:PCR、酶切及测序都证实了RUVBL2基因正确地插入到了载体质粒pIRES2-EGFP的多克隆位点;荧光、RT-PCR、Western blot也证实了RUVBL2基因在CHO细胞中的成功表达。本实验成功构建了猪的pIRES2-EGFP-RUVBL2真核表达载体,并证实了猪睾丸组织中的RUVBL2基因能够在CHO细胞中表达。     

马链球菌兽疫亚种烯醇化酶对小鼠肺泡巨噬细胞吞噬功能的影响

旨在研究马链球菌兽疫亚种（Streptococcus equi ssp.zooepidemicus，SEZ）烯醇化酶（enolase，Eno）对小鼠肺泡巨噬细胞（RAW264.7）吞噬能力的影响。通过构建原核表达质粒获得重组烯醇化酶（rEno），采用台盼蓝活细胞计数法，判定在不同处理浓度和时间下rEno蛋白对RAW264.7细胞的细胞毒性。将rEno蛋白与RAW264.7细胞共孵育后，用SEZ作用于细胞并检测细胞吞菌数量，判断RAW264.7细胞对SEZ的吞噬活性。进一步通过活细胞稳定同位素标记技术（SILAC）和蛋白质谱分析技术（LC-MS/MS），筛选到RAW264.7细胞中可能与SEZ Eno存在相互作用的候选蛋白。结果发现，10 μg·mL^-1 rEno蛋白处理对RAW264.7细胞有明显的细胞毒性，且10 μg·mL^-1 rEno蛋白处理RAW264.7细胞2和4 h可显著抑制其对SEZ的吞噬作用（P<0.01、P<0.05）。初步筛选到RAW264.7细胞中动力蛋白激活蛋白亚单位蛋白（dynactin subunit protein 2，Dctn）、整合素α-M蛋白（integrin alpha-M）等17种可能与Eno发生互作的蛋白。本研究获得了rEno重组表达蛋白，发现rEno可减少RAW264.7细胞对SEZ的吞噬，互作蛋白的初步筛选也为进一步揭示Eno在SEZ抗吞噬中的作用机制奠定了基础。     

Effects of DHRS3 in C2C12 Myoblast Differentiation and Mouse Skeletal Muscle Injury

Myoblast differentiation is an essential process during skeletal muscle development. C2 C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro. Dehydrogenase/reductase(SDR family) member 3(DHRS3) is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol. Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation. However, the effect of DHRS3 on mouse muscle cell differentiation was unclear. The objective of current study was to determine if DHRS3 affected muscle cell differentiation, and if DHRS3 was involved in muscle regeneration. Protein expression was determined by western blot and immunofluorescence analysis. The activation and inhibition of DHRS3 increased and decreased C2 C12 myoblast differentiation respectively, which indicated that DHRS3 could affect C2 C12 myoblast differentiation. DHRS3 expression was significantly changed during muscle regeneration, with the regeneration of muscle injury, the expression of DHRS3 tended to increase first and then decrease. It suggested that DHRS3 might be involved in muscle regeneration. In summary, this study confirmed the involvement of DHRS3 in C2 C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.     

安徽发现草地贪夜蛾为害早播小麦

2019年5月17日,在安徽省黄山市黄山区发现草地贪夜蛾为害春玉米,首次证实草地贪夜蛾入侵安徽,截至8月25日全省16市全部见虫。2019年10月22日,淮南市寿县窑口镇早播小麦田发现疑似该虫为害,经鉴定,确认为草地贪夜蛾。调查发现,全田均可见该虫为害状,百株被害率最高达84.21%,每平方米最高虫量43头。调查提示,入侵安徽的草地贪夜蛾种群可取食为害小麦,应加强其在麦田发生情况的监测。