Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor

Molecular Biology Reports - Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The...     

高寒地区不同海拔梯度西北小檗生境土壤微生物群落结构及多样性分析

以青海高原2 300～4 000 m海拔范围的6处西北小檗(Berberis vernae)生境土壤为试材,采用高通量测序方法,分析不同海拔梯度西北小檗生境土壤微生物群落结构及多样性。研究结果表明:(1)在西北小檗生境土壤中,细菌群落组成主要包括10个细菌门21个细菌属,真菌群落由子囊菌门、担子菌门等8个真菌门59个真菌属组成。(2)低海拔位置的海东乐都1号样点(hdld1) 0～20 cm土层的细菌群落丰富性及多样性均最高,黄南泽库样点(hnzk) 0～20 cm土层的真菌群落丰富度最高,西宁大通样点(xndt) 0～20 cm土层的真菌群落多样性最高;随着海拔的升高,0～20 cm、40～60 cm土层的细菌群落丰富度及多样性呈现出先降低再升高再降低的趋势,20～40 cm土层的细菌群落丰富度及多样性则呈现出先升高后降低的趋势,0～20 cm、20～40 cm土层土壤微生物真菌群落丰富度呈现出先升高再降低再升高的趋势,0～20 cm、40～60 cm土层真菌群落多样性呈现先升高再降低的趋势,40～60 cm土层的真菌丰富度及20～40 cm土层的真菌多样性的变化趋势不明显。(3)硝态氮、速效磷和速效钾对土壤微生物群落的影响较明显。综上可知,高寒地区不同海拔梯度西北小檗生境土壤微生物群落结构多样性呈现出一定的海拔差异趋势,其海拔差异主要受到环境条件、土壤理化性质和植被分布的影响。     

不同家系凡纳滨对虾对低盐度的适应能力及其适应机理

为研究不同遗传背景的凡纳滨对虾(Litopenaeus vannamei)在对盐度的适应能力上具有明显的差异的机理, 比较了30个凡纳滨对虾家系在3个不同盐度水体(5‰、20‰和30‰)中饲养30d后的生长性状。研究结果证实了不同家系对虾在不同盐度条件下的生长性能和适应能力存在显著差异。研究进一步对比分析了各盐度条件下不同家系间生理代谢、ATP含量及ATP合成关键酶酶活力的差异, 并检测了不同家系凡纳滨对虾鳃Na+/K+-ATPase、Ca2+-ATPase酶活水平。结果发现盐度适应力差的对虾家系的Na+/K+-ATPase、Ca2+-ATPase酶活力较弱, 这可能是由于其机体能量供给不足所导致。此外, 研究以血浆中皮质醇浓度为指标, 对比了不同盐度下不同对虾家系的机体应激水平, 结果显示盐度适应力差的对虾家系在经30d饲养后仍处于应激状态。综合研究结果得出, 不同遗传背景的对虾对盐度的适应能力不同, 可能是由其机体代谢、离子转运及能量合成能力所决定。     

样地尺度现代表土花粉与植物群落的定量关系

建立现代植被与表土花粉的精确关系,是基于孢粉记录定量重建古植被与古气候的基础与关键。截止目前,植物群落样方记录较少参与到现代植被与花粉的统计分析中,限制了其精确关系的定量表达。本文通过中国东北样带的森林、草甸草原、典型草原和荒漠草原33个表土样品分析及植被样方调查,基于Bray-Curtis相异系数,研究了东北样带现代表土花粉与植物群落组成和数量之间的定量关系。结果表明: 由于花粉传播距离和花粉产量大小的不同,单个样点的所有科属、优势和常见科属、少见和稀有科属,在组成和数量上的关系存在差异。不同植被类型间差异显著,草甸草原在组成上差异较大,而所有科属、优势和常见科属的数量关系在森林中差异较大,少见和稀有科属的数量关系则在草甸草原中差异较大。不同科属的植被-花粉关系在组成和数量上的趋势基本一致,根据Bray-Curtis相异系数可将该地区花粉类群划分为3类: 超代表性类型、低代表性类型和适中代表性类型。该系数可以获得样方水平和物种水平上植被-花粉的物种组成和数量关系,是定量描述植被-花粉关系的有效指标之一。     

亚热带同质园11个树种新老叶非结构性碳水化合物含量比较

叶片中非结构性碳水化合物(NSC)不仅是植物维持代谢活动的重要物质基础, 也随凋落物归还土壤并为土壤微生物提供碳源, 对凋落物分解和土壤有机质形成具有重要意义。该研究比较了同质园中11个亚热带代表性树种新鲜叶与凋落叶NSC (可溶性糖、淀粉)含量。结果表明, 所有树种新鲜叶NSC含量均显著高于凋落叶, 新鲜叶中NSC含量为68.7-126.3 mg∙g^-1, 而凋落叶中NSC含量为31.4-79.5 mg∙g^-1。同时, 可溶性糖含量在新鲜叶和凋落叶中的变化幅度均远大于淀粉: 可溶性糖在新鲜叶中的平均含量是凋落叶的3.3倍; 而淀粉在新鲜叶中的平均含量仅为凋落叶的1.2倍。另外, 对不同功能类群的比较发现, 常绿阔叶树种与落叶阔叶树种NSC含量差异并不显著, 而针叶树种NSC含量明显低于阔叶树种。具体表现为: 在新鲜叶中, 常绿阔叶、落叶阔叶树种NSC含量平均为99.7和96.8 mg∙g^-1, 而常绿针叶树种平均为75.4 mg∙g^-1; 在凋落叶中, 常绿阔叶、落叶阔叶树种NSC含量平均为47.2和50.7 mg∙g^-1, 而常绿针叶树种平均为33.3 mg∙g^-1。这些结果表明, NSC作为林木碳代谢组分, 在叶片衰老前可能向新鲜叶转移, 反映了林木叶片碳存储策略。然而, 不管是新鲜叶还是凋落叶, 杉木(Cunninghamia lanceolata)、马尾松(Pinus massoniana)等针叶树种叶片NSC含量显著低于阔叶树种, 这可能降低这些针叶树种凋落叶初始基质质量。     

Are There Gender Differences in Coronary Artery Disease? The Malaysian National Cardiovascular Disease Database – Percutaneous Coronary Intervention (NCVD-PCI) Registry

Objectives

To assess whether gender differences exist in the clinical presentation, angiographic severity, management and outcomes in patients with coronary artery disease (CAD).

Methods

The study comprised of 1,961 women and 8,593 men who underwent percutaneous coronary intervention (PCI) and were included in the Malaysian NCVD-PCI Registry from 2007–2009. Significant stenosis was defined as ≥70% stenosis in at least one of the epicardial vessels.

Results

Women were significantly older and had significantly higher rates of diabetes mellitus, hypertension, chronic renal failure, new onset angina and prior history of heart failure whereas smokers and past history of myocardial infarction were higher in men. In the ST-elevation myocardial infarction (STEMI) cohort, more women were in Killip class III-IV, had longer door-to-balloon time (169.5 min. vs 127.3 min, p<0.052) and significantly longer transfer time (300.4 min vs 166.3 min, p<0.039). Overall, women had significantly more left main stem (LMS) disease (1.3% vs 0.6%, p<0.003) and smaller diameter vessels (<3.0 mm: 45.5% vs 34.8%, p<0.001). In-hospital mortality rates for all PCI, STEMI, Non-STEMI (NSTEMI) and unstable angina for women and men were 1.99% vs 0.98%, Odds ratio (OR): 2.06 (95% confidence interval (CI): 1.40 to 3.01), 6.19% vs 2.88%, OR: 2.23 (95% CI: 1.31 to 3.79), 2.90% vs 0.79%, OR: 3.75 (95% CI: 1.58 to 8.90) and 1.79% vs 0.29%, OR: 6.18 (95% CI: 0.56 to 68.83), respectively. Six-month adjusted OR for mortality for all PCI, STEMI and NSTEMI in women were 2.18 (95% CI: 0.97 to 4.90), 2.68 (95% CI: 0.37 to 19.61) and 2.66 (95% CI: 0.73 to 9.69), respectively.

Conclusions

Women who underwent PCI were older with more co-morbidities. In-hospital and six-month mortality for all PCI, STEMI and NSTEMI were higher due largely to significantly more LMS disease, smaller diameter vessels, longer door-to-balloon and transfer time in women.     

Cortical Endogenic Neural Regeneration of Adult Rat after Traumatic Brain Injury

Focal and diffuse neuronal loss happened after traumatic brain injury (TBI). With little in the way of effective repair, recent interest has focused on endogenic neural progenitor cells (NPCs) as a potential method for regeneration. Whether endogenic neural regeneration happened in the cortex of adult rat after TBI remains to be determined. In this study, rats were divided into a sham group and a TBI group, and the rat model of medium TBI was induced by controlled cortical impact. Rats were injected with BrdU at 1 to 7 days post-injury (dpi) to allow identification of differentiated cells and sacrificed at 1, 3, 7, 14 and 28 dpi for immunofluorescence. Results showed nestin⁺/sox-2⁺ NPCs and GFAP⁺/sox-2⁺ radial glial (RG)-like cells emerged in peri-injured cortex at 1, 3, 7, 14 dpi and peaked at 3 dpi. The number of GFAP⁺/sox-2⁺ cells was less than that of nestin⁺/sox-2⁺ cells. Nestin⁺/sox-2⁺ cells from posterior periventricle (pPV) immigrated into peri-injured cortex through corpus callosum (CC) were found. DCX⁺/BrdU⁺ newborn immature neurons in peri-injured cortex were found only at 3, 7, 14 dpi. A few MAP-2⁺/BrdU⁺ newborn neurons in peri-injured cortex were found only at 7 and 14 dpi. NeuN⁺/BrdU⁺ mature neurons were not found in peri-injured cortex at 1, 3, 7, 14 and 28 dpi. While GFAP⁺/BrdU⁺ astrocytes emerged in peri-injured cortex at 1, 3, 7, 14, 28 dpi and peaked at 7 dpi then kept in a stable state. In the corresponding time point, the percentage of GFAP⁺/BrdU⁺ astrocytes in BrdU⁺ cells was more than that of NPCs or newborn neurons. No CNP⁺/BrdU⁺ oligodendrocytes were found in peri-injured cortex. These findings suggest that NPCs from pPV and reactive RG–like cells emerge in peri-injured cortex of adult rats after TBI. It can differentiate into immature neurons and astrocytes, but the former fail to grow up to mature neurons.     

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.     

棘孢曲霉固态发酵柚皮产柚苷酶及其在柑橘果汁脱苦中的应用

为了建立柚皮固态发酵产柚苷酶的工艺以及阐明其柚苷酶的应用价值,利用棘孢曲霉Aspergillus aculeatus JMUdb058对柚皮进行固态发酵,并研究其产酶特性。采用高效液相色谱法检测酶活力,通过单因素实验方法研究豆饼粉及培养基灭菌对柚苷酶发酵的影响;采用酶活力、还原糖以及生物量的变化解析发酵的动态规律;采用柚皮苷的水解活性表征柚苷酶对柑橘果汁的脱苦效果。结果表明,棘孢曲霉JMUdb058固态发酵柚皮可产生柚苷酶,额外添加豆饼粉可大幅度提高其柚苷酶活力;对培养基进行121℃20min的灭菌将导致柚苷酶活力急剧下降;当培养基组成为5g柚皮粉、0.75g豆饼粉、5.75mL蒸馏水,经30℃发酵8d时,柚苷酶和α-L-鼠李糖苷酶活力分别达到5.81IU/gds和6.08IU/gds;通过对产酶进行动力学拟合,发现柚苷酶和α-L-鼠李糖苷酶主要在棘孢曲霉的对数生长期内合成,它们的积累属于生长关联型;该柚苷酶可高效水解柚汁中的柚皮苷,柚皮苷去除率高达99.6%。因此,柚皮是固态发酵柚苷酶的良好原料,用棘孢曲霉对柚皮进行固态发酵是生产柚苷酶的有效方法。     

CXCR4慢病毒载体的构建及其在人脐带血间充质干细胞的表达

目的：构建趋化因子CXC亚族CXCR4的慢病毒表达载体并观察其转染人脐带间充质干细胞后的表达。方法：用逆转录PCR方法获取CXCR4基因编码区片段,将构建的慢病毒载体质粒pLVTHM-EGFP-CXCR4与包装质粒psPAX2和包膜质粒pMD2．G共转染293T细胞,包装生产慢病毒。用相同滴度的慢病毒转导等量间充质干细胞（Mesenchymal Stem Cell,MSCs）,后采用Real time PCR检测CXCR4 mRNA、Western Blot方法检测蛋白质的表达。结果：PCR、酶切和测序结果表明成功的构建了CXCR4基因重组慢病毒载体。同时用该慢病毒载体转染MSCs后可有效地增加MSCs中CXCR4的表达。结论：成功构建了CXCR4的慢病毒表达载体并能在MSCs中表达,为进一步研究其在干细胞移植中的应用奠定基础。’