Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.     

Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.     

5种动物源食品中的氟虫腈及其3个代谢物残留量检测

利用QuEChERS技术结合超高效液相色谱-串联质谱的方法检测5种动物源食品(鸡肉、鸡蛋、猪肝、牛奶、猪肉)中氟虫腈及其3个代谢物残留量。样品用乙腈提取,经低温处理,多壁碳纳米管(MWCNTs)、弗罗里硅土(Florisil)和C18分散固相萃取(d-SPE)净化,外标法定量。在不同浓度的加标水平下,氟虫腈及其代谢物在5种动物源食品中的平均回收率为72.1%～113.8%,相对标准偏差RSD为1.9%～17.6%,检出限LOD在0.73～1.94μg/kg范围内,定量限LOQ在5～10μg/kg范围内。该方法具有简单、灵敏、准确等优点,适用于动物源食品中氟虫腈及其代谢物的快速筛查和定量检测。     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Possible involvement of PKC/MAPK pathway in the regulation of GnRH by dietary arachidonic acid in the brain of male tongue sole Cynoglossus semilaevis

Our previous studies have indicated that dietary arachidonic acid (ARA) significantly affects the gonadal steroidogenesis in the marine teleost tongue sole Cynoglossus semilaevis, and this effect was more positive in male fish than in female fish (Aquaculture, 468, 378–385). As a following up study, the present study was further aimed at investigating the possible mechanisms in the brain mediating the effects of dietary ARA on gonadal steroidogenesis. A 70‐day feed trial was repeated with two‐year‐old tongue sole, using three experimental diets with graded levels of ARA, 0.34%, 2.53%, and 9.63% of total fatty acids. Each diet was fed to triplicate groups of 23 fish (15 males and 8 females). The results confirmed the positive effect of dietary ARA on testosterone production in male fish. The concentration of gonadotrophin‐releasing hormone (GnRH) in serum responded to dietary ARA in a similar pattern with the testosterone concentration, but the concentration of gonadotrophin in serum was not affected by dietary ARA. The response of gene expression of PKCβ, ARRB1, ARRB2, ERK2 and ATF3 in the brain to dietary ARA was in good agreement with those of GnRH and testosterone, indicating the possible involvement of PKC‐ARRB‐ERK‐ATF3 pathway in signalling transduction of GnRH. However, the PI3K/Akt and TLR/NF‐κB pathways may not be directly involved in the regulation of GnRH metabolism by ARA. This is the first study reporting the possible involvement of PKC/MAPK pathways in regulation of reproductive endocrine processes by long‐chain polyunsaturated fatty acid in brain of marine fish.     

Effects of the severity and timing of basal leaf removal on the amino acids profiles of Sauvignon Blanc grapes and wines

The effects of the severity and timing of leaf removal(LR) on the amino acids of Sauvignon Blanc grapes and wines were studied during the 2017 growing season. High-performance liquid chromatography(HPLC) was used to analyze the amino acids profiles of grape berries and wines. The basal leaves were removed at three time points(40, 56 and 72 days after flowering, named LR40, LR56 and LR72, respectively) at two severity levels(one at which the first, third, and fifth basal leaves of each shoot were removed(50% level); and another at which the first six basal leaves were removed(100% level)). The results showed that leaf removal had little impact on total soluble solids(°Brix), titratable acidity, pH or berry weight. The LR72-50% treated grapes had higher berry weight, titratable acidity and °Brix than those of the other treatments. The highest concentrations of total amino acids and of total amino acids except proline were detected in LR72-50% treated grapes(2 952.58 and 2 764.36 mg L~(-1), respectively); the lowest were detected in LR72-100% treated grapes(2 172.82 and 2 038.71 mg L~(-1), respectively). LR72-50% treatment significantly promoted the synthesis of aspartic acid, serine, arginine, alanine, aminobutyric acid and proline at both severity levels for grapes, the concentrations of all of these amino acids were increased relative to the control concentrations. The LR72-50%, LR40-100% and LR72-100% treated wines had higher total amino acids concentrations and higher concentrations of some individual amino acids, such as arginine, alanine and serine, than did the control wines. Of all the amino acids studied, glycine, tyrosine, cysteine, methionine and lysine were not significantly influenced by the timing or severity basal defoliation in grapes and wines. The present study reveals the effects of the timing and severity of leaf removal on the amino acids profiles of grapes and wines.     

基于B/S的农业气象自动化观测系统国家级平台设计与应用

农业气象自动化观测系统国家级平台是依托中国气象局组织的农业气象自动化观测试点工作而设计开发,平台的搭建采用B/S架构实现多级用户、跨平台登录,采用XML及JSON等数据传输格式实现国家级、省级、台站级用户信息交互,实现农业气象观测XML数据传输状态的监控、台站端设备状态的评估以及数据内容的实时查询。     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

基于nanoUPLC-MS/MS的螺旋藻游离肽组成分析

为了研究螺旋藻中的多肽成分,以钝顶螺旋藻为原料,采用超滤法提取螺旋藻中的游离肽,利用nanoUPLC-MS/MS和PEAKS Studio分析谱图信息,结合NCBI数据库进行比对和从头测序分析,获得游离肽的结构组成和百分含量信息。结果表明,利用数据库比对法和从头测序法,分别得到可信的游离肽4 485个和20 597个,匹配到的蛋白质有1 036种。数据库比对结果中的游离肽主要为七肽到二十一肽,少量为二十一肽以上;从头测序结果中游离肽主要为二肽到十六肽,少量为十六肽以上。数据库比对结果中百分含量最高的游离肽是十肽,为15.95%;从头测序结果中百分含量最高的是五肽,达到24.09%。本研究结果为螺旋藻蛋白资源的进一步开发和利用提供了依据。