A new 9-nor-atractylodin from Atractylodes lancea and the antibacterial activity of the atractylodin derivatives

A new compound, namely, 9-nor-atractylodin (1) and one known atractylodin (2) were isolated from the rhizomes of Atractylodes lancea. The structural modifications of atractylodin were carried out and a series of atractylodin derivatives (3-10) were obtained. The antibacterial activities of 1-10 were examined against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans. Compounds 4 and 8, which contained the α, β-unsaturated carbonyl fragment, were found to be active against E. coli and S. aureus.     

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。     

Protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats

AIM: To investigate the protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats.METHODS: SPF male Sprague-Dawley rats (n=40) were randomly divided into 5 groups: normal control (NC) group, high fat (HF) group, high fat+L6H4 treatment (FT) group, diabetes mellitus (DM) group and DM+L6H4 treatment (DT) group. The rats in the later 4 groups were fed with high-fat diet. After 4 weeks of high-fat diet fee-ding, the rats in DM and DT groups were intraperitoneally injected with streptozotocin to induce type 2 diabetes melliutus. The rats in FT and DT groups were given L6H4 by gavage for 8 weeks. Blood glucose and blood lipid levels were detected biochemically. Fasting serum insulin (FINS) level was measured by radioimmunoassay and insulin resistance index (HOMA-IR) was calculated. Serum adiponectin (APN) level was measured by ELISA. The morphological changes of the diaphragm were observed under light and transmission electron microscopes. Lipid deposition and the activity of succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) were observed by enzyme histochemical staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the diaphragm were measured by thiobarbituric acid method and hydroxylamine method, respectively. The protein expression of adiponectin receptor 1 (AdipoR1) in the diaphragm was determined by immunohistochemistry and Western blot. RESULTS: The levels of blood lipids, blood glucose, FINS and HOMA-IR in HF and DM groups were higher than those in NC group, but decreased after L6H4 treatment. The serum APN level in HF and DM groups was lower than that in NC group, but increased after treatment with L6H4. The muscle fibers of the diaphragm were shrunk, fat particles accumulated in the muscle fibers, and the mitochondria were slightly swollen in HF and DM groups. The diaphragmatic fibrosis was obvious in DM group. These lesions were relieved after L6H4 treatment. Compared with NC group, the level of MDA and the activity of SDH and NADH-TR in the diaphragm were increased in HF and DM groups, but decreased after treatment with L6H4. The activity of SOD and the expression of AdipoR1 in the diaphragm were lower than those in NC group, but increased after L6H4 treatment.CONCLUSION: The curcumin analogue L6H4 exerts a protective effect on diaphragm in type 2 diabetic rats. The strengthened protein expression of AdipoR1, the increased serum level of APN, and anti-lipid peroxidation may be involved in the process.     

Screening of lentiviral vectors carrying effective siRNA targeting S1P2 gene

AIM:To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2) gene expression in the primarily cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).METHODS:SHR and SD rats (n=5 each) were used for primarily culturing corpus cavernosum smooth muscle cells.The cells were randomly divided into 6 groups:SHR siRNA-1,SHR siRNA-2,SHR siRNA-3,SHR GFP,SHR control (SHR non-transfection group),and SD control (SD rat control group).Each group had 5 samples with 1.0×10⁵ cells of each sample.At 72 h after transfection (MOI=60) with lentiviral vectors carrying S1P2 siRNA into the SHR corpus cavernosum smooth muscle cells,the expression of GFP was observed under fluorescence microscope.The protein expression of S1P2,ROCK1,ROCK2 and eNOS in the corpus cavernosum smooth muscle cells,and the mRNA expression of S1P2,ROCK1 and ROCK2 were determined by by Western blot and RT-PCR.RESULTS:The transfection efficiency of the corpus cavernosum smooth muscle cells in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were>80%.Compared with SHR control group,the mRNA levels and the protein expression of S1P2,ROCK1 and ROCK2 in SHR GFP group showed no remarkable changes,while those in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SD control groups were significantly lower than those in SHR control group (P<0.05).The protein expression of eNOS in SHR siRNA-1,SHR siRNA-2,SHR siRNA-3 and SHR GFP groups were not significantly changed as compared with SHR control group,but that in SD control group was significantly higher than that in SHR control group.CONCLUSION:Three groups of siRNA lentiviral vectors targeting S1P2 inhibit the expression of S1P2 in the corpus cavernosum smooth muscle cells of SHR,and by silencing the S1P2 expression,the expression of ROCK1 and ROCK2 is inhibited.Among them,siRNA-1 has the highest inhibitory efficiency.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.     

赤点石斑鱼Toll样受体(TLRs)基因家族的鉴定、进化与表达

为研究赤点石斑鱼Toll样受体(TLRs)的进化、分布及表达模式,本实验基于已公开的赤点石斑鱼基因组和转录组数据,利用Blast、系统进化与共线性等生物信息学方法,分析赤点石斑鱼TLRs基因家族的系统进化、染色体分布及在各组织中的表达模式。结果显示,在赤点石斑鱼中共鉴定出17个TLR基因,这些基因分属于5个亚家族(TLR1、TLR3、TLR5、TLR7和TLR11),分别分布在24条染色体中的11条染色体上;17个TLR基因在赤点石斑鱼不同组织中具有不同的表达模式,然而,位于相同染色体上的TLR基因的不同拷贝则存在相似的表达模式。其中EaTLR18-1与EaTLR18-2均位于9号染色体上,二者的表达模式高度相似,均在心脏中高表达;同位于20号染色体上的EaTLR2-1a与EaTLR2-1b的表达模式也高度相似,均在脑中高表达;EaTLR5M与EaTLR5S同位于14号染色体上,二者不仅具有高度相似的LRR结构域,且在不同组织中的表达水平也高度相似;而位于18号染色体的EaTLR7与EaTLR8和位于4号染色体的EaTLR2-2与EaTLR3,其表达模式却存在明显差异。研究结果可为鱼类...     

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

猪生长抑素Ⅱ型受体基因(SSTR2)shRNA的构建与筛选

生长激素(GH)是调控动物生长的重要激素,GH分泌水平的高低主要受正性调控因子生长激素释放因子(GRF)和负性调控因子生长抑素(SS)的双向调节。SS通过生长抑素受体(somatostatin receptor,SSTR)发挥作用,减少GH的分泌水平。本研究设计了3条SSTR2的短发夹RNA(shRNA),构建猪(Sus scrofa)SSTR2真核表达质粒(pcDNA3.1(-)-SSTR2)和SSTR2慢病毒RNA干扰质粒(pshRNA-copGFP lentivector,LV-shRNA),并共转染中国仓鼠(Cricetulus griseus)卵巢细胞系(CHO),通过对转染细胞的荧光观察、Real-time PCR和酶联免疫法(ELISA)检测,结果表明,CHO转染细胞中猪SSTR2 mRNA表达水平不同程度的抑制,分别为90.4%、28.3%和86.3%,(P<0.05)。其中,LV-shRNA1表现出最高的沉默效率:转染效率在80%左右时,猪SSTR2mRNA水平沉默效率为90.4%,蛋白质水平降低了33.3%。本研究成功地筛选到了降低猪SSTR2基因表达的shRNA,为利用shRNA技术下调动物基因表达,构建转基因模型提供思路。     

水稻不育突变株沈农05-9的形态和细胞学特征鉴定

以辽粳5号与丰锦杂交的高代系的突变株沈农05-9和其原始株系为材料,采用醋酸洋红染色压片技术及石蜡切片技术.进行了形态学特征、小孢子发生的细胞学特征观察.结果表明:该不育突变株花粉表现典败和染败,多数花粉母细胞减数分裂不形成二分体,而形成多核的花粉母细胞;花药表现干瘪且纤维化严重,绒毡层细胞在小孢子发育过程中提前解体.该不育突变株的减数分裂方式类似于萍乡型显性不育系及早稻昆植不育系.导致败育的直接原因为绒毡层细胞提前解体.