Accurate measurement of microsatellite length by disrupting its tandem repeat structure

Tandem repeats of simple sequence motifs, also known as microsatellites, are abundant in the genome. Because their repeat structure makes replication error-prone, variant microsatellite lengths are often generated during germline and other somatic expansions. As such, microsatellite length variations can serve as markers for cancer. However, accurate error-free measurement of microsatellite lengths is difficult with current methods precisely because of this high error rate during amplification. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure of initial templates so that their sequence lengths replicate faithfully. In this work, we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring agreement from two independent first copies of an initial template, we reach error rates below one in a million. We apply this method to a thousand microsatellite loci from the human genome, revealing microsatellite length distributions not observable without mutagenesis.     

易分解有机碳输入量对武夷山不同林型土壤激发效应的影响

土壤有机碳库是陆地生态系统中最大的碳储量库,其微小的变化也能使大气中CO₂浓度发生巨大的改变,植物来源碳的输入能通过激发效应促进或抑制土壤有机碳(SOC)的分解,对SOC的动态平衡产生影响。以武夷山三个林型(阔叶林、马尾松林、针阔混交林)土壤为研究对象,通过向土壤中添加不同量的¹³C标记葡萄糖(0、100、200、400 mg C/kg)研究易分解有机碳输入量对不同林型土壤激发效应的影响,并在此基础上探讨易分解有机碳输入量对土壤激发效应影响的作用机理。结果表明,葡萄糖输入对土壤激发效应的影响与葡萄糖输入量和林型有关。葡萄糖的输入均抑制了三个林型SOC的分解(即,呈现负的激发效应)。阔叶林土壤和针阔混交林土壤激效应强度随着葡萄糖输入量的增加而增加,而马尾松林土壤的激发效应强度对葡萄糖输入量的响应并不明显。然而在马尾松林土壤中由葡萄糖所引起的激发效应强度显著高于其他两种林型土壤。研究结果表明,易分解有机碳的输入可以抑制SOC的矿化,形成负激发效应,阔叶林土壤的激发效应强度与土壤可利用氮、葡萄糖添加量与微生物碳量比值有关,而针阔混交林与马尾松林土壤...     

人乳铁蛋白(hLF)转基因山羊的遗传背景分析

以随机整合方式获得的转基因动物外源基因的拷贝数、整合位点及染色体核型等遗传背景并不清楚,可能会存在外源基因的沉默整合、无效整合、毒性整合以及其表达水平不可预测等问题。文中选取了6只原代(F0)及其相对应的子一代(F1)的人乳铁蛋白(hLF)转基因山羊作为研究对象,分别颈静脉采血、提取DNA,通过染色体核型分析、实时荧光定量PCR(qPCR)、ELISA和Westernblotting等检测技术,研究其外源基因的遗传背景与表达水平。结果显示,6只F0代转基因山羊的染色体没有明显的形态变异、数量改变等异常情况。相对拷贝数高低不同(2–16),且能够稳定地遗传给下一代,F0和F1代hLF基因拷贝数一致。F1代转基因山羊表达hLF水平最高可达1.12 g/L(L3-1,拷贝数8)。结果表明,整合的外源基因能够稳定地遗传下一代,也没有对转基因山羊个体的生长发育造成障碍,而且拷贝数高低与hLF表达水平无明显的相关性,这为转基因山羊及其他转基因动物的新品种培育奠定了基础,解析了遗传背景。     

小绒泡菌生活史及其形态建成期显微结构的观察

在完成小绒泡菌Physcaum pusillum生活史的同时,观察了小绒泡菌P.pusillum生活史不同阶段显微结构的形成.生活史研究结果表明:小绒泡菌P.pusillum的孢子萌发方式为裂式,黏变形体具两根鞭毛,较短一根不易观察到,原质团类型为显型原质团,子实体形态建成过程约需12-13h.显微结构观察结果表明:石...     

干旱胁迫对银杏叶片光合系统Ⅱ荧光特性的影响

为了探讨银杏叶片在长期干旱胁迫下光合性能的变化,采用盆栽控水法对银杏幼苗进行干旱处理,并分别在处理后第0、20、30、40、50天,对荧光动力学曲线和荧光参数进行了测定和JIP-test分析.结果表明:随着干旱的加剧,叶片叶绿素含量逐渐递减.由于干旱导致银杏叶片的光合系统Ⅱ(PSⅡ)结构破坏和稳固性减弱,其荧光动力学曲线出现了K相和L相的升高,在干旱处理40 d后升高加剧.PSⅡ结构的变化导致了一系列荧光参数的变化,初始荧光F0逐渐升高,最大荧光Fm逐渐降低;代表单位反应中心活性的参数ABS/RC、ET0/RC、TR0/RC逐渐升高;单位面积捕获的光能TR0/CS0高于对照,单位面积用于电子传递的光能ET0/ CS0后期下降;表示受体侧电子传递活性的参数Sm、Ψ0、(φ)E0逐渐下降,M0、VJ、VI逐渐升高;表示热耗散的参数DI0/RC、DI0/CS0随干旱时间延长而急剧升高;活性反应中心数目RC/CS0减少;代表PSⅡ光合效率的参数Fv/Fm、PIabs逐渐下降;代表PSⅡ供体侧活性的参数Wk随干旱时间延长而逐渐升高,OEC逐渐减少.干旱胁迫导致银杏叶片PSⅡ反应中心失活,能流分配失衡,电子传递受阻,PSⅡ放氧复合体失活和PSⅡ稳固性减弱,从而破坏PSⅡ光合功能.而反应中心失活和受体侧电子QA-的累积是造成PSⅡ电子传递活性减弱的主要原因.干旱条件下,银杏PSⅡ的PIabs比Fv/Fm对干旱的反应更灵敏,可作为银杏叶片受到伤害的指标.     

一个可能引起歧义的概念——“条件致病菌”辨析

"条件致病菌"是国内医学微生物学教材中普遍应用的一个基本概念,但此概念在建立与应用中都存在明显缺陷,其使用可能对医学学生理解"人体共生微生物群落"与"机会性感染"等重要的医学基础理念造成误解与歧义。谨就此提出一些粗浅的认识与观点,希望通过同行间切磋,就此建立更完善的概念与更合理的表述。     

Determination of cefazedone in human plasma by high performance liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study on Chinese volunteers

A rapid, sensitive and simple high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for determination of cefazedone in human plasma using metronidazole as internal standard (IS). The chromatographic separation was achieved on an Ultimate XB-CN column (2.1 mm × 150 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 20 mM ammonium acetate in 0.1% formic acid in water (15:85, v/v). Detection was performed using electrospray ionization in positive ion multiple reaction-monitoring mode (SRM), monitoring the transitions m/z 548.2 → 344.1 for cefazedone and m/z 172.2 → 128.1 for IS. Calibration curves were linear over a wide range of 0.20–401.12 μg/mL for cefazedone in plasma. The lower limit of quantification (LLOQ) was 0.20 μg/mL. The intra- and inter-day precisions were less than 7.2%. The average recovery of cefazedone was 90.8–91.0%. The validated method was successfully applied to the pharmacokinetic study of cefazedone in Chinese healthy volunteers following intravenous (IV) administration of 500, 1000 and 2000 mg cefazedone injection.     

Population dynamics of Bemisia tabaci (Homoptera: Aleyrodidae) parasitoids on cassava mosaic disease–resistant and susceptible varieties

Three field trials were conducted at Namulonge Agricultural and Animal Production Research Institute to investigate the population dynamics of Bemisia tabaci Gennadius and its associated aphelinid parasitoids in Uganda. Results showed that more whitefly occurred on the cassava mosaic disease-resistant variety, (Nase 4) compared to the susceptible variety, (Ebwanatereka). Two species of aphelinid parasitoids, Eretmocerus mundus Mercet and Encarsia sophia Girault and Dodd, were identified during the study. Overall percent parasitism did not differ significantly (P>0.05) between varieties in all the trials, but significant differences occurred at 13, 15 and 21 weeks after planting during late season (2000), 18 weeks after planting during early season (2001), and 8 weeks after planting during late season (2001). The trends in the build up in numbers of both parasitoids species and apparent parasitism were similar, differed significantly on certain dates. It was, however, noted that percent parasitism decreased with nymph number. The significance of this phenomenon on the potential use of these aphelinid parasitoids as biocontrol agents of the cassava whitefly is discussed.     

MiR‐616‐3p modulates cell proliferation and migration through targeting tissue factor pathway inhibitor 2 in preeclampsia

Objectives

Despite improvements in diagnosis and treatment, preeclampsia (PE) continues to pose a significant risk of maternal and foetal morbidity and mortality if not addressed promptly. An increasing number of studies have suggested that tissue factor pathway inhibitor 2 (TFPI2) acts as a suppressor gene, possibly inhibiting multiple serine proteases affecting cell proliferation and migration. It plays an essential role in the occurrence and development of PE, but the pathogenesis remains unclear.

Materials and methods

In our research, we performed western blotting, immunohistochemistry and qPCR assays to investigate TFPI2 and miR‐616‐3p expression in preeclamptic placental tissues. Cell assays were performed in HTR‐8/SVneo and JEG3 cell lines. Cell proliferation and migration events were investigated by MTT, EdU and transwell assays. In conjunction with bioinformatics analysis, luciferase reporter assays were performed to elucidate the mechanism by which miR‐616‐3p binds to TFPI2 mRNA.

Results

We established that TFPI2 protein levels were significantly upregulated in PE placental tissues. In addition, we found that miR‐616‐3p binds specifically to the 3′‐UTR region of TFPI2 mRNA. Furthermore, miR‐616‐3p knockdown or TFPI2 overexpression substantially impaired cell growth and migration, whereas miR‐616‐3p upregulation or TFPI2 knockdown stimulated cell proliferation and migration. This miR‐616‐3p / TFPI2 axis was also found to affect the epithelial‐mesenchymal transition process in PE.

Conclusions

Our results demonstrated that TFPI2 plays a vital role in the progression of PE and might provide a prospective therapeutic strategy to mitigate the severity of the disorder.

     

Biodegradation of poly(ε-caprolactone) (PCL) by a new Penicillium oxalicum strain DSYD05-1

In this study, fungi isolated from soil were screened for their ability to form clear zones on agar plates with emulsified poly(ε-caprolactone) (PCL). The most active strain, designated as DSYD05, was identified as Penicillium oxalicum on the basis of morphological characteristics and phylogenetic analysis. Mutant DSYD05-1, obtained by ultraviolet-light mutagenesis from strain DSYD05, was more effective in PCL degradation. In liquid cultures of the mutant strain with PCL emulsion, DSYD05-1 showed the highest PCL-degrading activity after 4?days of cultivation. The products of PCL degradation were analysed by mass spectrometry; the results indicated that 6-hydroxyhexanoic acid was produced and assimilated during cultivation. The degradation of PCL film by DSYD05-1 was observed by scanning electron microscopy, and was indicative of a three-stage degradation process. The degradation of amorphous parts of the film preceded that of the crystalline center and then the peripheral crystalline regions. In addition, DSYD05-1 showed a wide range of substrate specificity, with capability to degrade PCL, poly(β-hydroxybutyrate), and poly(butylene succinate), but not poly(lactic acid), indicating that the strain could have potential for application in the treatment or recycling of bio-plastic wastes.