检测牛冠状病毒抗体间接ELISA方法的建立与应用

[背景]牛冠状病毒(Bovine coronavirus,BCoV)是引起新生犊牛死亡的主要病原之一,有效的检测手段是防治该病的前提。目前BCoV ELISA检测方法存在敏感性低、不稳定等缺陷。[目的]对原有BCoV ELISA方法进行改进,建立间接ELISA检测方法。[方法]应用我国BCoV流行毒株CD株n基因为模板,预测N蛋白抗原表位,通过原核表达制备可溶性的重组N蛋白作为抗原,建立间接ELISA方法,应用该方法对黑龙江省2010-2017年的BCoV感染进行血清流行病学调查。[结果]该ELISA方法最佳工作条件为:用50 mmol/LpH 9.6碳酸盐作为包被液,抗原包被浓度2.5μg/mL;用PBST作为样本稀释液,稀释浓度1:50,37℃孵育1.5 h;HRP-羊抗牛IgG稀释浓度1:7 500,37℃孵育1.0 h;用1%明胶37℃封闭30 min。阴阳性临界值为0.225。该方法与BRV、BRSV、BVDV、IBRV、BPIV3和E.coli阳性血清均无交叉反应。批内和批间变异系数均小于10%,与病毒中和试验的符合率高达93.5%。对黑龙江省部分地区共603份奶牛血清样品检测结果显示,BCoV抗体阳性率为98.84%。[结论]建立的ELISA方法特异性强、敏感性高、稳定性好,为进一步研发ELISA试剂盒提供了技术基础。     

群落构建研究的新进展:进化和生态相结合的群落谱系结构研究

群落如何构建足群落生态学中的重要问题.群落谱系结构研究将物种间的亲缘进化关系运用到群落生态学研究中,利用物种的系统发育状况推测历史因素对现有群落的影响,为推断影响群落组成的生态学机制提供了有效方法.群落谱系结构的研究方法是首先建立可代表群落物种库的超级系统进化树,然后计算群落内物种间的谱系距离,最后通过统计方法检测其与随机模型下的谱系距离是否有显著差异来获得谱系结构(如谱系聚集、谱系发散),从而揭示群落构建中的关键生态过程(如生境过滤、竞争作用).群落谱系结构与空间尺度、分类群尺度、时间尺度等不同研究尺度有关.在小的空间尺度下,随着分类群尺度降低、树木年龄级增大,群落谱系结构从聚集逐渐转为发散;而随群落空间尺度的增大,谱系趋向于聚集.谱系结构受到环境因素影响,因此分析集合群落下的谱系可以揭示区域生态过程的影响.另外,群落谱系结构研究还有助于探讨中性理论、密度制约假说等生态学理论,并预测干扰作用下的群落演化趋势.在利用谱系结构深入探讨群落构建成因时,需要基于生态特征和环境变量共同分析,同时考虑小尺度局域过程(群落的微环境或群落内种间相互作用等)和大尺度区域过程(地史过程和物种形成等),并可结合生态控制实验,以确认群落构建的关键因素.在研究方法和手段上,今后需要注重通过选择合适的基因片段建立系统树,然后通过生态特征来加以校正,以更准确地反映物种间的亲缘距离.另外,获得谱系树后还需要寻找更加合理的统计模型和指数,增加统计分析和解决问题的能力.     

从大肠杆菌C-8制备和纯化唾液酸

大肠杆菌产生的多聚唾液酸是一类线性的同聚合体,这由２００个左右的唾液酸通过α２,８酮苷键连接。我们在前文［１］中已就产多聚唾液酸的菌种筛选及产酸条件作了叙述。本文将对多聚唾液酸的水解、唾液酸的纯化及鉴别进行研究,为今后应用打下基础。１材料和方法１...     

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

Developmental programming: deficits in reproductive hormone dynamics and ovulatory outcomes in prenatal, testosterone-treated sheep

Prenatal testosterone excess leads to neuroendocrine, ovarian, and metabolic disruptions, culminating in reproductive phenotypes mimicking that of women with polycystic ovary syndrome (PCOS). The objective of this study was to determine the consequences of prenatal testosterone treatment on periovulatory hormonal dynamics and ovulatory outcomes. To generate prenatal testosterone-treated females, pregnant sheep were injected intramuscularly (days 30-90 of gestation, term=147 days) with 100 mg of testosterone-propionate in cottonseed oil semi-weekly. Female offspring born to untreated control females and prenatal testosterone-treated females were then studied during their first two breeding seasons. Sheep were given two injections of prostaglandin F2alpha 11 days apart, and blood samples were collected at 2-h intervals for 120 h, 10-min intervals for 8 h during the luteal phase (first breeding season only), and daily for an additional 15 days to characterize changes in reproductive hormonal dynamics. During the first breeding season, prenatal testosterone-treated females manifested disruptions in the timing and magnitude of primary gonadotropin surges, luteal defects, and reduced responsiveness to progesterone negative feedback. Disruptions in the periovulatory sequence of events during the second breeding season included: 1) delayed but increased preovulatory estradiol rise, 2) delayed and severely reduced primary gonadotropin surge in prenatal testosterone-treated females having an LH surge, 3) tendency for an amplified secondary FSH surge and a shift in the relative balance of FSH regulatory proteins, and 4) luteal responses that ranged from normal to anovulatory. These outcomes are likely to be of relevance to developmental origin of infertility disorders and suggest that differences in fetal exposure or fetal susceptibility to testosterone may account for the variability in reproductive phenotypes.     

基于热扩散技术的三倍体毛白杨单木及林分蒸腾耗水研究

定量分析单木及林分的蒸腾耗水特征,是林木水分管理的关键环节。采用热扩散式边材液流检测技术,结合自动气象站,对三倍体毛白杨树干边材液流及环境因子进行了连续2年的动态观测。结果表明:(1)单株尺度上,三倍体毛白杨边材液流速率日变化在晴天表现为"单峰型",关键影响因子为水汽压亏缺(VPD)和太阳辐射(Qs),日平均液流速率在4—10月分别为0.65×10-3、2.12×10-3、2.09×10-3、1.78×10-3、1.84×10-3、1.76×10-3、1.04×10-3cm/s;(2)林分尺度上,三倍体毛白杨在2008、2009年(栽植第4年和第5年)的蒸腾耗水量分别为339.52和410.62 mm,主要影响因素为气孔导度(Gc)、相对湿度(RH),以及VPD;(3)多元线性回归模型可以较好的模拟三倍体毛白杨边材液流速率对环境因子的响应特征(P0.01,2008年),模型预测值较实测值偏大6.39%(2009年),二者极显著线性相关(R2=0.910,Sig.=0.00054,n=1008)。     

Stabilization of IGF2BP1 by USP10 promotes breast cancer metastasis via CPT1A in an m6A-dependent manner

Metastasis leads to the vast majority of breast cancer mortality. Increasing evidence has shown that N6-methyladenosine (m6A) modification and its associated regulators play a pivotal role in breast cancer metastasis. Here, we showed that overexpression of the m6A reader IGF2BP1 was clinically correlated with metastasis in breast cancer patients. Moreover, IGF2BP1 promoted distant metastasis in vitro and in vivo. Mechanistically, we first identified USP10 as the IGF2BP1 deubiquitinase. USP10 can bind to, deubiquitinate, and stabilize IGF2BP1, resulting in its higher expression level in breast cancer. Furthermore, by MeRIP-seq and experimental verification, we found that IGF2BP1 directly recognized and bound to the m6A sites on CPT1A mRNA and enhanced its stability, which ultimately mediated IGF2BP1-induced breast cancer metastasis. In clinical samples, USP10 levels correlated with IGF2BP1 and CPT1A levels, and breast cancer patients with high levels of USP10, IGF2BP1, and CPT1A had the worst outcome. Therefore, these findings suggest that the USP10/IGF2BP1/CPT1A axis facilitates breast cancer metastasis, and this axis may be a promising prognostic biomarker and therapeutic target for breast cancer.