Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV‐inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3‐MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.     

赖氏衣属,中国地衣一新记录属

报道了文字衣科一中国新记录属——赖氏衣属(Reimnitzia Kalb),该属仅含1种桑蒂赖氏衣[Reimnitzia santensis (Tuck.)Kalb]。桑蒂赖氏衣生于树皮或苔藓上,地衣体壳状,无皮层;含有大的柱状草酸钙结晶;裂芽丰富,与地衣体颜色一致,无分枝,先呈球状,后变为蠕虫状,长度可达1.5 mm,顶部有黑色的孔区或者单个的小孔;具有Chroodiscus型子囊盘;子囊孢子棕色,亚砖壁型,孢子大小15～25μm×8～12μm;分生孢子器位于裂芽的顶端和地衣体疣中,分生孢子呈杆状;无次生代谢物。     

苦荞R2R3-MYB转录因子调控原花青素生物合成的研究

基于苦荞花期转录组数据,该研究筛选并克隆获得一个黄酮代谢相关的MYB类转录因子,并命名为FtMYB23。该基因ORF框长879bp,编码292个氨基酸;系统进化树分析显示,FtMYB23与SG5-MYB亚家族成员聚为一簇,属于典型的R2R3-MYB型转录因子。β-半乳糖苷酶滤纸分析表明,其具有转录激活活性。FtMYB23过表达转基因拟南芥株系的表型分析表明,3个阳性株系的种皮颜色均呈现出比野生型更深的褐色,其叶中原花青素含量均极显著增加(P 0.01),分别为野生型的4.68、3.5和2.8倍。qRT-PCR分析表明,转基因拟南芥中黄酮合成相关的AtCHS、AtCHI、AtF3H、AtF3′H、AtFLS、AtDFR和AtBAN等基因的表达量显著升高(P 0.05),而AtTT12的表达量极显著降低(P 0.01)。研究认为,FtMYB23作为典型的Subgroup5-MYB(SG5-MYB)激活型转录因子,通过促进黄酮合成途径早期关键酶基因的表达,从而提高原花青素的合成与积累。     

Notch信号通路参与卵巢生殖干细胞的调控及卵巢的衰老进程

卵巢生殖干细胞(ovarian germline stem cells, OGSCs)的发现,打破了生殖医学领域传统的"固定卵泡池"理论。近年来,OGSCs新的研究成果不断涌现,但关于OGSCs体内调控机制的研究仍然较少。Notch通路广泛参与多种成体干细胞不对称分裂的过程,并与细胞衰老密切相关,但其是否参与OGSCs的体内调控机制及卵巢的衰老进程尚不清楚。本研究以原代培养技术提取OGSCs,通过荧光双标染色发现,OGSCs标志基因MVH、Oct4与Notch信号通路相关分子Notch1、Hes1在OGSCs中存在共表达;抑制Notch信号通路活性后,cck-8检测发现,OGSCs的增殖活性呈下降趋势;而以免疫组化、荧光双标、Western印迹法检测性成熟期(2月龄)、不孕和衰老(20月龄)小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达变化,发现2月龄小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达量较高(P<0.05),而不孕和衰老小鼠卵巢皮层中,MVH、Oct4、Notch1和Hes1的表达量均明显下降。上述结果表明,Notch信号通路在小鼠OGSCs中高表达,并可能参与调控OGSCs的增殖机制及卵巢的衰老进程。     

蔓茎堇菜和柔毛堇菜多糖的抑菌抗氧化活性

利用乙醇沉淀法提取蔓茎堇菜Viola diffusa和柔毛堇菜V.principis多糖并分别进行抑菌及抗氧化试验。结果表明,蔓茎堇菜和柔毛堇菜多糖提取率分别为7.0%和8.3%。不同倍数体积无水乙醇沉淀提取的多糖抑菌和抗氧化能力不同。抑菌效果显示,蔓茎堇菜多糖对大肠杆菌和枯草芽孢杆菌的抑菌圈分别可达8.46mm和8.59mm,柔毛堇菜对大肠杆菌和枯草芽孢杆菌的抑菌圈均可达9.13mm,但两种堇菜多糖对黑曲霉和啤酒酵母未呈现抑制活性;抗氧化研究发现,蔓茎堇菜多糖抗氧化活性为243.64U·mL^-1,柔毛堇菜多糖抗氧化活性为411.78U·mL^-1。由此可见,无论是抑菌还是抗氧化活性方面,柔毛堇菜极显著优于蔓茎堇菜(P<0.01)。蔓茎堇菜和柔毛堇菜多糖都具有一定的抑菌抗氧化活性,均可作为食药两用植物资源进行开发利用。     

Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier

Neurotrophins, such as brain derived neurotrophic factor (BDNF), do not cross the blood-brain barrier (BBB). Certain monoclonal antibodies (MAb) to the human insulin receptor (HIR) do cross the BBB via receptor-mediated transport, and can act as a molecular Trojan horse to ferry across the BBB an attached drug. A genetically engineered fusion protein was produced whereby the amino terminus of human BDNF is fused to the carboxyl terminus of the heavy chain of a chimeric HIRMAb. The HIRMAb-BDNF fusion protein reacted equally with antibodies to human IgG and BDNF. The bi-functionality of the fusion protein was retained as the affinity of the fusion protein for the HIR was identical to that of the chimeric HIRMAb, and the affinity of the fusion protein for the trkB receptor was identical to that of BDNF. The fusion protein was equi-potent with BDNF in a neuroprotection assay in human neural cells. The pharmacokinetics (PK) of the fusion protein was examined in the adult Rhesus monkey. The mean residence time (MRT) of the fusion protein in blood was >100-fold longer than the MRT of BDNF. Therapeutic levels of BDNF were produced in primate brain following the intravenous administration of the fusion protein. A fusion protein tandem vector was engineered that allowed for isolation of a CHO cell line that produced the fusion protein at high levels in serum free medium. Neurotrophins, such as BDNF, can be re-formulated to enable these molecules to cross the human BBB, and such fusion proteins represent a new class of human neurotherapeutics.     

Characterization and the broad cross‐species applicability of 20 anonymous nuclear loci isolated from the Taiwan Hwamei (Garrulax taewanus)

We isolated 20 anonymous nuclear loci (8556 bp in total) from the Taiwan Hwamei (Garrulax taewanus), an endemic songbird of Taiwan. A panel of nine to 15 individuals with unknown relationship was used to characterize polymorphism of these loci. We identified 46 single nucleotide polymorphic sites (SNPs) in 15 polymorphic loci. Frequency of SNPs was one per every 186 bp in average. Nucleotide diversity, θ, ranged from 0.00054 to 0.00371 per locus. We also tested cross‐species applicability of these loci on 17 species from eight different passerine families. All 20 loci could be successfully amplified (ranged from one to 16 species, mean = 7.9 species).     

近视眼LASIK术后眼高阶像差变化

探讨近视眼LASIK术后眼波阵面像差高阶像差各项的变化。方法:采用NIDEK OPD-Scan ARK-1000型波前像差扫描系统对LASIK治疗近视的62只眼在术前术后眼的波前像差进行了评估。在术前、术后10天、术后一个月分别测量眼的波阵面像差。比较6mm瞳孔下高阶像差各项的变化。结果:在全部接受检查的病例中,总的高阶像差(RMSh)均有所增加。从Zernik各项绝对值来看,球差C12变化最大。术后10天比术前增加6．7742倍(P=0．0001)。其次是彗差C7 C8(X轴、Y轴上的彗差),分别增加了4．883倍(P=0．0001)、3．7523倍(P=0．0001)。结论:LASIK术后眼的总的高阶像差均方根值(RMSh)明显增加,尤其是球差和彗差;但在恢复过程中高阶像差有下降的趋势。用像差仪来指导和评估LASIK等屈光手术可以更全面、更准确。     

C3d3通过促进脾脏B细胞高表达CXCR4增强hCGβ DNA免疫体液免疫效应

本文旨在探讨分子佐剂C3d3与hCGβ融合在基因免疫中增强抗hCGβ体液免疫效应的机制。分别用质粒pCMV4-hCGB-C3d3、pCMV4-hCGβ和pCMV4免疫BALB/c小鼠,间接ELISA法检测免疫小鼠外周血IgG/IgA类抗hCGβ抗体水平;ELISPOT分析免疫鼠脾脏组织IgG/IgA类抗体分泌细胞水平(ASC);RT-PCR分析免疫鼠脾脏B细胞趋化因子受体表达,RT-PCR和FCM分析CXCR4表达水平;RT-PCR和ELISA检测脾脏组织CXCL12表达水平。结果显示,pCMV4- hCGβ-C3d3免疫组外周血IgG类抗hCGβ抗体水平明显高于pCMV4-hCGβ免疫组;而IgA类抗hCGβ抗体水平在两组间无明显差异。pCMV4-hCGβ-C3d3免疫组脾脏组织IgG类ASCs水平明显高于pCMV4-hCGβ组;两组间IgA类ASCs水平无明显差异。经pCMV4-hCGB、pCMV4-hCGβ- C3d3免疫鼠脾脏B细胞CXCR4表达明显高于对照组;且pCMV4-hCGβ-C3d3组明显高于pCMV4-hCGβ免疫组。CXCR4~ 细胞与ASCs呈正相关,r=0.966,(P<0.05)。pCMV4-hCGβ-C3d3和pCMV4-hCGβ组脾脏组织CXC L12表达均显著高于对照组。结果表明,分子佐剂C3d3与hCGβ基因融合,在基因免疫小鼠后能够显著升调节脾脏ASCs CXCR4表达,从而可能增强抗hcGβ基因疫苗的体液免疫效应。     

胰岛素抵抗在前列腺癌治疗前后动态变化的临床观察

目的:探讨胰岛素抵抗程度在前列腺癌治疗过程中动态变化及胰岛素抵抗程度与前列腺癌预后的关系。方法:选择前列腺癌惠者50名。平均年龄72岁。将前列腺癌患者根据治疗方法分为前列腺癌根治术组、手术去势组、药物去势组;根据有无复发转移分为复发转移组、无复发转移组。采用己糖激酶法测定空腹葡萄糖,放免法测定空腹胰岛素(Fasting serum insulin FINS)水平。运用HOMA模型中的胰岛素抵抗计算公式计算胰岛素抵抗指数(insulin resistance index IRI)。组间比较采用配对t检验。结果:前列腺癌患者在手术后的胰岛素抵抗指数明显下降,与手术前胰岛素抵抗指数存在统计学差异(P<0.05)。前列腺癌根治组的IRI值和手术去势组与药物去势组都存在统计学差异(P<0.05),但手术去势组和药物去势组两组之间不存在统计学差异(P>0.05)。前列腺癌治疗后无复发转移组的IRI值明显低于复发转移组,两组间IRI存在统计学差异(P<0.05)。结论:前列腺癌患者胰岛素抵抗程度与肿瘤治疗经过、效果有关。胰岛素抵抗指数有助于前列腺癌患者判断治疗效果,判断预后。