Electrochemical detection of single-nucleotide mismatches: application of M-DNA

The detection of a single-nucleotide mismatch in unlabeled duplex DNA by electrochemical methods is presented. Impedance spectroscopy is used to characterize a perfect duplex monolayer and three DNA monolayers differing in the position of the mismatch. The monolayers were studied as B-DNA (normal duplex DNA) and after conversion to M-DNA (a metalated duplex). Modeling of the impedance data to an equivalent circuit provides parameters that are useful in discriminating the four monolayer configurations. The resistance to charge transfer, R(CT), was lower for all duplexes after conversion to M-DNA. Contrary to expectations, R(CT) was also found to decrease for duplexes containing a mismatch. However, R(CT) was found to be diagnostic for mismatch detection. In particular, the difference in R(CT) between B- and M-DNA (deltaR(CT)) decreased from 190(22) omega.cm(2) for a perfectly matched duplex to 95(20), 30(20), and 85(20) omega.cm(2) for a mismatch at the top (distal), middle, and bottom (proximal) positions of the monolayer with respect to the gold surface. Further, a method to form loosely packed single-stranded (ss)-DNA monolayers by duplex dehybridization that is able to rehybridize to target strands is presented. Rehybridization efficiencies were in the range of 40-70%. Under incomplete hybridization conditions, the R(CT) was the same for matched and mismatched duplexes under B-DNA conditions. However, deltaR(CT) between B- and M-DNA, under incomplete hybridization, still provided a distinction. The deltaR(CT) for a perfect duplex was 76(12) omega.cm(2), whereas a mismatch in the middle of the sequence yielded a deltaR(CT) value of 30(15) omega.cm(2). The detection limit was measured and the impedance methodology reliably detected single DNA base pair mismatches at concentrations as low as 100 pM.     

Bead-based electrochemical immunoassay for bacteriophage MS2

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.     

Absorption coefficient imaging by near-field scanning optical microscopy in bacteria

We present a method for obtaining a position-dependent absorption coefficient from near-field scanning optical transmission microscopy. We show that the optical transmission intensity can be combined with the topography, resulting into an absorption coefficient that simplifies the analysis of different materials within a sample. The method is tested with the dye rhodamine 6G, and we show some analysis in biological samples such as bacteria KIebsiella pneumoniae and Pseudomonas aeruginosa. The calculated absorption coefficient images show important details of the bacteria, in particular for P. aeruginosa, in which membrane vesicles are clearly seen.     

Mapping protein expression in mouse pancreatic islets by immunolabeling and electron energy loss spectrum-imaging

A combination of immuno-electron microscopy and electron energy-loss spectrum-imaging was used to map the distributions of endocrine polypeptide hormones and proteins in mouse pancreatic islet of Langerhans. Tissue was analyzed from control animals and from mice that were heterozygous for the Anx7 gene, which defines a Ca2+/GTP-dependent membrane fusion and ion channel protein. The heterozygous Anx7 (+/-) mouse displays defects in IP3 receptor mediated Ca2+ signaling and insulin secretion. Therefore, information was obtained about the distributions of the hormones insulin and glucagon, as well as the proteins ANX7 and the IP3 receptor. Insulin secretion appears to be defective in the mutants. It was found from immunolabeling experiments that expression of the IP3 receptor is reduced in mutant islets compared to control islets. Subcellular distributions of sulfur and nitrogen obtained by electron energy-loss spectrum-imaging showed that the insulin concentrations of beta granules were essentially the same in control and mutant islets. By contrast, immunogold labeling of mutant islets shows more insulin immunoreactivity in the beta granules. It follows that insulin may be packaged differently in mutant islets, making antigenic determinants more available to the labeling antibody. The increased rate of insulin secretion in the hyperplastic mutant islets can be explained by compensatory increases in islet size, rather than by an increased insulin concentration in the beta cells. The results indicate that reduced ANX7 expression leads to defects in the IP3 receptor expression in the endocrine cells of the mutant mouse. Increased size of the islet or of adrenal medulla may be a compensatory mechanism for secretion defect by individual endocrine cells. Defects in IP3 receptor expression, and documented consequences of a Ca2+ signaling defect, lead to other changes in organelles such as the mitochondrial number in islet beta-cells. The effects and consequences of reduced ANX7 expression on mitochondria are evident in ultrastructural observations.     

Water structures in ion-exchange resin particles: solvation dynamics of Nile Blue A

The structures of water, partitioned in cation-exchange resin particles, were studied on the basis of fluorescence dynamics of Nile Blue A (NB). The fluorescence lifetime of NB in the resin was longer than that in water and increased with increasing cross-linking density of the resin (rho). The results demonstrated that the water structures in the resin were significantly different from those in water and dependent on rho. A study on solvation dynamics of NB in the resin, reflecting structured water around the ion-exchange group, revealed the roles of "bound water" molecules in the water structures, since the solvent relaxation time (tauS) in the resin was much longer than that in water and depended on p; tauS increased from 34 to 55 ps with increasing rho from 2 to 8%. The origin of the rho dependence of tauS was discussed in terms of the separation distance between the ion-exchange groups, and the effects of the counterion of the ion-exchange group on the solvation processes were also discussed.     

Genotypic variation among arcobacter isolates from a farrow-to-finish swine facility

Arcobacter spp. were isolated from nursing sows and developing pigs on three farms of a farrow-to-finish swine operation and market-age pigs at slaughter. Isolates were identified by polymerase chain reaction and genotypic fragment patterns were examined by pulsed-field gel electrophoresis (PFGE). Incidences of Arcobacter-positive samples increased progressively as the pigs aged, resulting in all of the pens at the end of the growth cycle in the finishing barn containing Arcobacter-positive feces. However, only 10 of 350 cecal samples from slaughtered pigs were positive. There was little similarity between genotypic patterns for Arcobacter collected from the three farms. The level of genotypic variation revealed by PFGE suggested that pigs in this farrow-to-finish operation were colonized by multiple Arcobacter parent genotypes that may have undergone genomic rearrangement, common to members of Campylobacteraceae, during successive passages through the animals. Additionally, the level of genotypic diversity seen among Arcobacter isolates from farms of a single farrow-to-finish swine operation suggests an important role for genotypic phenotyping as a source identification and monitoring tool during outbreaks.     

振动磨的周期运动稳定性与分岔

由于碰撞的存在,振动磨系统的响应呈现出复杂的周期运动或混沌运动。本文将振动磨的模型进行简化,建立一维、两自由度、受简谐激振力作用的振动磨碰撞振动力学模型。基于Poincare映射原理,根据映射Jacobi矩阵分析振动磨周期运动的稳定性,并通过理论分析和数值仿真,研究振动磨周期运动的稳定性与分岔,以及由倍周期分岔通向混沌的过程。     

一种基于故障定位的配电网简化模型

针对农村配电网T接节点多而馈线终端单元(FTU)配置相对城网少的特点,对刘健教授提出的变结构耗散网络模型进行了反变换,消去大量没有安装FTU的T接节点,采用邻接表存储结构,结合算例进行了分析验证,可以更方便地进行故障定位,隔离故障区域。     

自适应Web服务管理框架设计与实现

为了更好地管理Web服务和服务组合的运行状态，提出了一种自适应Web服务管理框架XManager.XManager框架基于自主计算思想，以RuleML语言为规则表达的定义方式形成规则库，并具有服务组件架构(SCA)模型支持的数据解析和业务执行服务组件.信息感知器采用外观模式，采取推模式和拉模式结合的信息感知方式，为框架提供运行环境数据.自适应管理引擎通过对RuleML规则的加载解析，动态调度SCA组件对面向服务运行环境进行业务操作并完成自主管理过程，降低了人工干预的频率.实验结果表明，XManager框架与Web服务运行环境有效结合，为服务平台提供了自适应管理机制，提高了平台的可管理性和服务质量.     

复杂地形条件下大截面导线张力架线施工方案

酒湖特高压直流输电线路工程(甘7标)甘陕交界段地处秦岭山脉群山峻岭区域,张力架线除考虑导地线连续上下山带来的上扬、下压、放线滑车包络角度大等影响,主要还存深山无人区域无施工运输道路,牵张场选择困难等问题,架线区段无法采用常规的顺线路牵引方式进行施工。针对上述问题,以N3662-N3678段为例,通过施工计算,优化施工方案,提出重点解决措施,可为山区张力架线施工工程提供参考。