Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

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.     

一个潜在催化莱鲍迪D苷合成的新型糖基转移酶

尿苷二磷酸糖基转移酶(uridine diphosphate glycosyltransferases,UGTs)催化糖基转移反应,与植物次生代谢密切相关。本研究根据甜叶菊(Stevia rebaudiana)转录组数据库,克隆到一个催化莱鲍迪D苷(rebaudioside D,RD)合成的新型糖基转移酶候选基因,对其开展生物信息学分析。结果表明,该基因开放阅读框长1380 bp,编码459个氨基酸,等电点(pI)预测为5.54,理论分子量约49.66 kD,系统发育分析表明该基因与向日葵中的UGT89A2同源,故将其命名为SrUGT89A2。构建pET28a-SrUGT89A2原核表达载体,并在大肠杆菌(BL21(DE3))中诱导表达得到重组蛋白,HPLC检测表明粗酶液能催化甜叶菊提取液形成一个新的色谱峰,该峰保留时间与莱鲍迪D苷一致。经进一步纯化UGT89A2蛋白,添加不同甜菊糖苷标准品为催化底物,但未鉴定出该蛋白催化的具体糖苷。该潜在催化甜菊糖RD苷合成的新型糖基转移酶基因SrUGT89A2的发现,为RD苷的生物合成和甜菊糖苷的生物途径研究提供新的理论依据。     

NO对大鼠中脑腹侧被盖区DA神经元的调节

实验观察了微量注射 NOS抑制剂 L -NAME及微量联合注射 NO的前体 L-精氨酸( L-Arg) L-NAME于大鼠中脑腹侧被盖区( VTA)对该部位多巴胺神经元的调节。发现VTA注射 L -NAME( 1 mg/ 5μL )后 ,伏隔核( Acb)多巴胺 ( DA )代谢产物—双羟苯乙酸( DOPAC)水平升高到注射前的 1 2 2 .5% ( P <0 .0 0 1 ) ,小剂量注射 L-NAME( 0 .2 mg/ 5μL)对伏隔核 DOPAC水平无明显影响 ;同样方法联合注射 L-Arg( 3 0 0 μg/ 5μL) L-NAME( 1 mg/5μL)后 ,伏隔核 DOPAC水平无明显变化。结论 :VTA微量注射 L-NAME兴奋了该部位的DA神经元 ,而 L -Arg L -NAME联合注射 ,却不能影响 DA神经元的活动 ,说明 NO可以通过L-Arg-NOS-NO途径参与 VTA多巴胺神经元的调节     

三种PCR方法诊断猪伪狂犬病的比较研究

针对伪狂犬病毒gD基因的不同片段设计三对引物，分别进行PCR扩增用于猪伪狂犬病的诊断，扩增的三个片段的长度分别为262bp、217pb以及1203bp的全基因。通过比较发现，这三种PCB诊断方法均具有很高的特异性和敏感性，值扩增gD基因内部262bp的PCR诊断方法种具有突出优点，其退火与延伸合成一步，其操作可于1h内完成，敏感性更高，更适于猪伪狂犬病的快速诊断。     

茶多酚(TP)对家禽免疫功能的研究

选用14日龄健康的海佩科内用仔鸡48羽,随机分成试验(24羽)和对照(24羽)两组.试验组在饲粮中添加0.25％的茶多酚(TP),研究TP对鸡免疫功能的作用.试验结果表明,TP对自然感染法氏囊病鸡,有明显提高(p<0.05)鸡血液中红细胞C_(3b)受体和免疫复合物的含量及保护鸡脾脏正常的免疫功能,同时也发现TP对公母鸡法氏囊的重量有不同影响.本试验表明,TP具有提高家禽免疫功能的作用.