Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection. (C) 2007 Elsevier B.V. All rights reserved.

基于RTI的车辆定位仿真系统数据传输研究

目前分布式体系结构的研究重点是提高系统的可扩展性、互操作性和可重用性，而对于实时性要求高的分布式仿真系统，还需要在HLA体系结构基础上，考虑如何提高系统的数据传输效率，以满足实时性要求。本文从应用层角度出发，从以下两个方面研究了改进策略： 一，从数据交互方面考虑，为了提高分布式仿真系统内部有效数据传输效率，满足系统实时性要求，以车辆定位仿真系统为问题原型，提出一种基于运行时间支撑系统数据分发管理（RTI-DDM）和套接字(Socket)的双层数据传输模型：一方面利用RTI-DDM来限定传输数据的范围，依据待交互的数据值域对数据的发送和接收进行过滤，有效降低系统内部冗余数据的传输；另一方面，利用Socket技术在仿真实体之间建立点对点的直接传输，从而提高系统的运行效率。对比实验结果表明，在相同的仿真交互数据量下，该模型相对于传统单层RTI数据传输模型，数据传输延时平均缩短70%。 二，从时间管理方面考虑，本文通过实验验证的方法，分析不同时间管理策略对仿真系统的数据传输和运行控制的影响，并针对车辆定位仿真系统中联邦成员之间的数据交互和逻辑控制关系特点，选择合适的时间管理策略，保证数据因果关系的正确性，进一步提高了系统的数据传输性能。 实验和应用结果表明，本文提出的改进策略简单有效，提高了系统数据传输效率，较好的解决了基于HLA/RTI的定位仿真系统的实时性问题。

卫星通信网络用户接入与路由选择技术研究

卫星移动通信作为下一代移动通信中一个必不可少的组成部分，用户接入切换和路由选择策略已经成为卫星移动通信领域的研究重点。同时对卫星通信网络建设方案及其关键技术进行充分的仿真建模论证也已经成为卫星通信系统建设前的一项必不可少的工作。本文围绕用户接入切换策略和路由选择策略开展了如下工作： 在卫星通信网络用户接入切换策略中，本文针对低轨卫星通信网络研究多星覆盖接入切换技术。首先在引入非均匀多业务模型的基础上，结合星上信道分配策略设计一种多业务组合加权接入策略。接着在基于HLA/RTI的卫星网络仿真系统之上进行用户接入仿真实现，并对最近卫星、最长覆盖时间和组合加权接入策略进行了仿真分析与比较，验证了组合加权策略下的系统性能。最后进一步仿真讨论加权系数取值不同对系统新呼叫阻塞概率、切换呼叫阻塞概率和切换请求到达概率的影响。 针对卫星通信网络路由策略，本文重点研究多层卫星网络，在建立星间链路预测模型的基础上，借鉴延时可容忍网络路由设计思想，设计一种链路中断容忍路由策略，利用非均匀时间段内卫星网络拓扑结构的可预测性进行路由表计算，同时提供动态的拥塞控制机制和基于洪范思想的故障中断容忍策略，解决由卫星运动、通信设备故障等引发链路中断情况下的路由问题。通过仿真，验证该路由策略的时空特性和链路利用率。

基于刚体运动学仿真的坦克训练模拟系统研究

根据坦克部队作战特点，以计算机技术、网络技术、系统仿真与模型方法为基础，将HLA与坦克分队战术训练相结合，把分散在不同地点的人与设备“连接”到同一模拟训练环境中；将虚拟现实技术与半实物仿真模拟器相结合，建立吴有时空一致性的系统合成的虚拟“战场环境”；将刚体运动学与解析几何相结合，解决坦克实体模型视景仿真和坦克直线运动与转向运动中的六自由度刚体运动学模拟技术。遵循这一技术路线，完成了以计算机仿真技术与军事训练专业的复合应用为目的的分布式坦克训练模拟与分析系统。

Chemical modifications of carboxylated chitosan

C-6-carboxylated chitosan obtained by oxidation of chitosan was selectively modified in order to obtain derivatives similar to bacterial antigens. Selective O-acetylation of 6-carboxyl chitosan afforded a modified polysaccharide with the 2-amino group available for further modifications to create carbonyl groups. A deaminative degradation reaction allowed the formation of oligosaccharides with terminal aldehyde groups. Reductive alkylation with lactose introduced lactityl branches which were oxidized with galactose oxidase to give aldehyde groups in its -galactose residues.

多表征模型组合仿真的形式描述与因果序研究

多表征模型组合仿真是可组合仿真、遗留系统集成的重要研究领域，也是多表征建模与仿真的一项关键技术，无论在理论研究还是实际应用上都有重要意义。本文针对多表征模型组合仿真问题在以下几个方面展开了较深入的研究：（1）并发控制机制；（2）多表征模型组合的形式描述；（3）因果序保持；（4）所有权管理服务。 在分析并发表示法特点的基础上，针对并发表示法中对于交互控制过于集中的问题，将集中式的并发控制机制改为分布式的并发控制机制，改进了整个并发控制的体系结构模型；对于多表征实体中的集中式并发控制处理中容易造成服务瓶颈的问题，利用多表征仿真的分层交互规则，将原有的单个交互消解器和一致性执行器改成分布式的控制方式，以有效缓解单个交互消解器的负担。 在改进的采用分布式并发控制机制的体系结构基础上，对多表征模型组合的机制及过程进行了分析，然后使用并行离散事件规范PDEVS对多表征原子模型与耦合模型进行了形式化定义，证明了多表征耦合模型具有耦合封闭性，这使得可以将多表征耦合模型与其它的多表征原子模型或多表征耦合模型一起不断地组合成更大的系统，即多表征模型可以以构件耦合的方式层次式地组成整个仿真系统。并提出了一个多表征模型组合框架。通过多表征模型组合框架清晰地描述了多表征模型组合的过程以及与HLA/RTI协调的机制。 针对HLA时间管理服务中没有提供因果排序机制，而多表征模型组合仿真需要保证以因果序提交模型交互的问题，我们通过引入向量时间，设计了一个符合因果序提交的通信协议，并对其相关的正确性问题进行了证明。 分析了HLA所有权协议应用于多表征模型组合仿真时存在的不足，并以HLA协商型推模式所有权服务为例进行了改进，使得所有权协议可以支持指定某个成员为对象属性所有权的接收者、解决了RTI不存储释放了所有权的属性的更新值，以及HLA属性所有权服务本身不包含时间管理机制等问题。

可扩展的卫星网络建模与仿真研究

卫星网络仿真是对卫星网络进行优化设计、性能分析、效能评估的有效途径。本文针对卫星网络的建模与仿真开展了如下工作： 围绕卫星网络的建模问题，本文在分析卫星网络组成结构和运行特点的基础上，建立了卫星的轨道运行、平台状态、有效载荷模型；针对不同组网模式建立了结构模型；综合考虑卫星的状态参数和空间环境对链路的影响建立了链路模型；基于物理信道、网络层、传输层三个层次建立了通信模型，并讨论了各仿真模型的组成、适应性和可扩展性。 在建模基础上，设计了基于HLA的卫星组网仿真系统总体架构，给出了仿真联邦对象模型的详细设计与具体实现方式，设计了仿真运行流程和系统接口，并进行了仿真实验，验证了仿真模型的正确性和有效性，并进一步分析了HLA在进行面向服务扩展时的适用性，给出了利用XMSF体系结构对其进行扩展的必要性和研究思路。 通过对可扩展的建模与仿真框架（XMSF）特别是面向服务的体系结构（SOA）和仿真服务总线（SSB）的讨论，针对研究卫星网络上层应用的需要，设计了基于XMSF/HLA双层体系结构的原型系统，重点研究了基于web的C/S交互通讯模式和仿真服务总线运行模式。该系统结构支持对HLA的“web化”扩展，并可跨平台、跨语言共享，易于理解和实现。

Efficient synthesis of highly substituted pyrrolin-4-ones via PIFA-mediated cyclization reactions of enaminones

A convenient and efficient synthesis of highly substituted pyrrolin-4-ones is developed via the PIFA-mediated cyclization reactions of readily available enaminones, and a mechanism involving sequential cleavage of N-C bond, formation of new N-C bond, intramolecular addition reaction, and benzilic acid type rearrangement is proposed.

Simultaneous spectrophotometric determination of trace iron (III) and aluminum (III) with hybrid linear analysis

Hybrid linear analysis (HLA) was applied to resolution of overlapping spectra of Fe3+-salicylfluorone and Al3+-salicylfluorone complexes and simultaneous spectrophotometric determination of Fe3+ and Al3+. The absorbance matrix of 7 standard mixtures at 41 measuring points ranged from the wavelength of 550 nm to 630 nm was used for calibration. To avoid the effect of interaction between the two components on the determination, the column vector of K matrix obtained from the standard mixtures with least squares was used as the pure spectrum of component. The recoveries of the two elements for the analysis of the synthetic samples were 93.3% similar to 107.5% in the range of the concentration ratio of Fe3+:Al3+ = 10:1 to 1:8. Comparing with the partial least squares (PIS) model, the HLA method was simple, accuracy and precise.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析

迟缓爱德华氏菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文分析了9种具有保护潜能的迟缓爱德华氏菌蛋白，经过牙鲆免疫保护实验，筛选出EseD和Et18两种有显著性保护效应的抗原。为了提高其保护效应，论文使用基因工程技术将这两种抗原融合到一起，构建重组融合蛋白EEH。结果表明，融合蛋白EEH保护效应较EseD和Et18分别免疫时有所提高。ELISA和Western blotting 结果显示，三种蛋白都能诱导牙鲆产生特异抗体。这些研究为开发迟缓爱德华氏菌疫苗提供了理论基础。 论文克隆分析了迟缓爱德华氏菌AcrAB耐药系统，采用定点突变确定了acrAB、acrR的启动子序列和AcrR在acrAB启动子的结合位点。启动子分析显示，AcrR对acrAB启动子有300倍抑制效应, 对acrR启动子有3倍抑制效应。定点突变显示，K39和R45对AcrR功能具重要性；缺失突变表明，N端205个氨基酸残基是其功能必需。实验筛选出Acriflavine、Ethidium Bromide、Methyl Viologen、Sodium Dodecyl Sulfate等四种AcrR诱导物。分析AcrR过量表达菌株结果显示，其耐药性、生长状况和毒力水平较阴性对照组降低。这些研究加深了我们对迟缓爱德华氏菌耐药机制及其与毒力关系的了解。

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.