shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

High-density mapping and marker development for the powdery mildew resistance gene PmAS846 derived from wild emmer wheat (Triticum turgidum var. dicoccoides)

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important foliar disease of wheat worldwide. The dominant powdery mildew resistance gene PmAS846 was transferred to the hexaploid wheat lines N9134 and N9738 from wild emmer wheat (Triticum dicoccoides) in 1995, and it is still one of the most effective resistance genes in China. A high resolution genetic map for PmAS846 locus was constructed using two F₂ populations and corresponding F_2:3 families developed from the crosses of N9134/Shaanyou 225 and N9738/Huixianhong. Synteny between wheat and Brachypodium distachyon and rice was used to develop closely linked molecular markers to reduce the genetic interval around PmAS846. Twenty-six expressed sequence tag-derived markers were mapped to the PmAS846 locus. Five markers co-segregated with PmAS846 in the F₂ population of N9134/Shaanyou 225. PmAS846 was physically located to wheat chromosome 5BL bin 0.75–0.76 within a gene-rich region. The markers order is conserved between wheat and Brachypodium distachyon, but rearrangements are present in rice. Two markers, BJ261635 and CJ840011 flanked PmAS846 and narrowed PmAS846 to a region that is collinear with 197 and 112 kb genomic regions on Brachypodium chromosome 4 and rice chromosome 9, respectively. The genes located on the corresponding homologous regions in Brachypodium, rice and barley could be considered for further marker saturation and identification of potential candidate genes for PmAS846. The markers co-segregating with PmAS846 provide a potential target site for positional cloning of PmAS846, and can be used for marker-assisted selection of this gene.     

Prevalence of Sarcopenia and Its Relationship with Sites of Fragility Fractures in Elderly Chinese Men and Women

Objective

Sarcopenia might be associated with bone fragility in elderly individuals. This study aimed to investigate the prevalence of sarcopenia and its association with fragility fracture sites in elderly Chinese patients.

Methods

Patients (322 men and 435 women) aged 65–94 years and with a history of fragility fractures in the ankle, wrist, vertebrae or hip, and healthy men (n = 1263) and women (n = 1057) aged 65–92 years without a history of fractures were enrolled. Whole-body dual energy X-ray absorptiometry was used to analyze skeletal muscle mass index (SMI), fat mass and bone mineral density. Sarcopenia was defined as SMI less than two standard deviations below the mean of a young reference group.

Results

Sarcopenia occurrence varied with fracture location. Sarcopenia was more common in females with vertebral and hip fractures and in men with hip and ankle fractures than in the non-fracture group). Sarcopenia was significantly more prevalent in men with wrist, hip and ankle fractures than in women. SMI was correlated with BMD in different fracture groups. Logistic regression analyses revealed that lower SMI was associated with an increased risk of hip fracture both in men and women and ankle fracture in men.

Discussion

Sarcopenia may be an independent risk factor for hip and ankle fractures in men, and for hip fractures in women.     

A specific plasminogen activator inhibitor‐1 antagonist derived from inactivated urokinase

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re‐establish blood flow after thrombus formation. Plasminogen activator inhibitor‐1 (PAI‐1) inhibits urokinase‐type and tissue‐type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI‐1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI‐1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI‐1 and identified a potent PAI‐1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active‐site mutation (S195A) and four additional mutations (G37bR–R217L–C122A–N145Q). PAItrap inhibits human recombinant PAI‐1 with high potency (K_d = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI‐1. In addition, PAItrap inhibits both human and murine PAI‐1, allowing the evaluation in murine models. In vivo, using a laser‐induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI‐1 inhibitors using inactivated urokinase.     

水曲柳花发育过程中AG、SOC1基因表达的qRT-PCR分析

以水曲柳(Fraxinus mandshurica Rupr.)不同时期的雌雄花为材料,应用实时荧光定量PCR技术,分析了18S rRNA、GAPDH、β-actin和TU 4个常用内参基因在水曲柳花中的表达情况。经NormFinder软件分析选择出TU作为内参基因,并对水曲柳中的开花相关基因AG和SOC1在开花不同时期的表达情况进行了分析,结果表明：在水曲柳花发育期间AG、SOC1表达量在雌雄花间存在差异,AG基因在雌花初期表达量最高,之后逐渐降低,到了成熟胚囊时AG表达量又有所增加;而在雄花中AG在减数分裂时最高为1.53。SOC1基因在雌花中的表达量低于雄花。这一研究结果为深入了解水曲柳花发育过程中相关基因的表达提供理论依据。     

印度南瓜果皮和果肉颜色遗传分析及基因定位

以印度南瓜‘98-2-351’与‘06820-1’杂交构建F2群体,对亲本及各世代群体成熟果实果皮和果肉颜色进行调查、统计分析。结果表明:F2群体中果皮桔红色和灰色的分离比呈3∶1,说明果皮灰色是由单隐性基因控制;F2群体中果肉黄色和白色的分离比呈3∶1,说明果肉白色也是由单隐性基因控制。利用群体分离分析法结合隐性群体分析法,采用SSR分子标记,找到了2个与控制灰色果皮基因位点CmRc紧密连锁的SSR标记(PU078072和PU013839),其连锁遗传距离分别为5.9cM和14.5cM;同时找到了1个与控制白色果肉基因位点CmFc紧密连锁的SSR标记PU132712,其连锁遗传距离为6.7cM。本研究为进一步筛选与控制印度南瓜果皮和果肉颜色基因更加紧密连锁的分子标记及相关基因的精确定位奠定了基础。     

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.