我们感谢为她出色的技术援助，以顺子和田。我们非常感谢昭雄野本教授请提供PVR - IgG2a杆状病毒表达载体。这项研究是通过资助部分支持，援助促进根除脊髓灰质炎和新出现和重新出现的传染病，卫生，劳工和福利部的研究。

1。维持液（MM）的制备和生长介质（GM） 对于MM和通用汽车公司的准备，请看到世界卫生组织 1脊髓灰质炎实验室手册。贝科改良Eagle培养基（DMEM），辅以2％胎牛血清（FCS）和10％FCS可以用作替代品的MM和通用分别。 2。反PV抗体的制备<ol><li>重组反光伏的抗体（RIVM光伏输入抗血清），加入水（0.5％毫升小瓶），每小瓶抗PV抗体（1型，2或3）。将此重组抗体的解决方案（0.5毫升）4.5毫升的MM单独标记的试管。 注:反光伏抗体有些地段作为重组的形式，每小瓶（0.5毫升）提供。反光伏抗体等很多，除了用于重组的水是没有必要的。 </li><li>混合同等体积的重组反光伏抗体。对于反的PV1 2，2 3，或3 +1:将每个重组抗体1.8毫升（1.8 ML + 1.8毫升= 3.6毫升为每个混合）。对于反的PV1 2 3:将每个重组抗体1.2毫升（1.2毫升+ 1.2毫升+ 1.2毫升= 3.6毫升）。 注:光伏样品可能包含的不同血清型的光伏的混合物。要确定单一血清型光伏PV的混合物样品，反PV抗体（即1 +2，2 +3 3 1 1 2 3）池是必要的。 </li><li>每个反PV（1 2 2 3 3 +1或1 +2 +3）的抗体（3.6毫升）（最终18.2％FCS）FCS（0.72毫升）的1 / 5体积。 注:此外，为减少非特异性抗体引起的明胶颗粒凝集的FCS。 </li><li>保持在-20 ° C，直到在PA的检测中使用的抗体池。 </li></ol> 3。重组致敏明胶颗粒<ol><li>加入1.5 mL的重组缓冲区套件中包括一小瓶致敏明胶颗粒（冻干粉）。 </li><li>充分混匀，温柔攻</li><li>保持重组敏颗粒4 ° C。复原明胶颗粒可以活跃在4 ° C保存至少2周，但应检测前的准备。 </li></ol>重要注意事项: 不要混合使用不同致敏的明胶颗粒很多，因为非致敏明胶颗粒凝集的图像可以由很多的颗粒不同。对于每一个实验中，一个单一地段致敏明胶颗粒应该被使用。 4。 PA检测为光伏识别<ol><li>准备一个微滴定板。识别一个病毒样本，我们需要5口井+，至少有一个阴性对照组（在下面没有病毒和抗体致敏粒子，以及＃6。看不到例病毒和抗体控制）。井总数是5 ×样品+中至少有一个控制检测。 重要注意事项: 请至少有一个无病毒的检测和抗体控制。一小瓶敏颗粒约10个病毒样本的鉴定。 </li><li>加入12μL/孔反PV抗体或GM微滴定板。该板块的安排如下（见下面的例子）。无抗体（以及＃1和6）的样品，加入12μL的GM，而不是反光伏抗体。 重要注意事项: 如果许多光伏样品应在本实验测试，多道移液器或分配器将有助于这一步。 <table border="1"><tbody><tr><td>那么＃1 </td><td>好＃2 </td><td>那么＃3 </td><td>那么＃4 </td><td>好＃5 </td><td>那么＃6 </td></tr><tr><td>通用汽车公司</td><td>抗- P1 2 3 </td><td>抗- P1的2 </td><td>抗- P2的3 </td><td> ANI - P1的3 </td><td>通用汽车公司</td></tr></tbody></table></li><li>添加12μL/孔的病毒样本。在一个没有病毒样本（以及＃6），加12μL转基因，而不是病毒解决方案。 注:病毒样本是文化感染的细胞，通常L20B细胞或RD细胞上清。 重要注意事项: 如果许多光伏样品应在本实验测试，多道移液器将有助于这一步。 <table border="1"><tbody><tr><td>那么＃1 </td><td>好＃2 </td><td>那么＃3 </td><td>那么＃4 </td><td>好＃5 </td><td>那么＃6 </td></tr><tr><td>病毒样本</td><td>病毒样本</td><td>病毒样本</td><td>病毒样本</td><td>病毒样本</td><td>通用汽车公司</td></tr></tbody></table></li><li>从4℃重组敏颗粒解决方案，室温，轻轻拍打完全暂停粒子。 重要注意事项: 完成悬浮颗粒的温柔攻后，应立即添加颗粒板前的悬浮颗粒沉淀TE和形式的非均质的解决方案。 </li><li>添加25μL/孔的井敏颗粒。 重要注意事项: 此外敏颗粒后，应立即受到板到板混合开始对样品的均匀反应。因此，除了敏颗粒应使用饮水机，以尽量减少板样品之间的时间滞后。 </li><li>摆上一盘混频器的微滴定板和混合30秒板。 </li><li>置于室温白纸上的微滴定板（为了方便目视观察）表（15〜30℃）2小时。 重要注意事项: 不要板块移动，期间和之后的结果进行观察和拍照的反应。形成凝集并不稳定，很容易通过移动板不安。 </li><li>拿板的照片。 重要注意事项: 照片的部分观点不能给予足够的数据以供日后检查和解释的结果进行审查。应采取多张照片，涵盖所有板块的井，重点井的凝集形象。 </li></ol> 5。结果的解释<ol><li>正面和负面的井鉴定。比较样本中没有病毒和抗体控制（明胶颗粒，非凝集沉淀）（图1和2）的凝集。应判断为阳性凝集，显然是没有病毒，没有抗体控制（非凝集，阴性对照）杰出的图像，并凝集阴性对照，类似的图像，应判断为阴性。样本，显示清晰，但只是局部的凝集，可判断为阳性，但记录阳性（部分凝集）"。 </li><li>确定的光伏。解释与反PV抗体的正面和负面的水井，以确定光伏血清型为基础的结果。结果的解释原理是相似的细胞培养体系中和试验（如抗- P1的2抗体阳性表明，样品中含有3光伏型）的。 </li></ol> 6。代表性的成果代表PA检测光伏鉴定结果如图1所示，所有的光伏株形成敏（无抗体样品）明胶颗粒凝集。在存在相应的反光伏抗体，没有病毒类似的明胶颗粒（非凝集）的降水和没有抗体控制（阴性对照）观察（例如，PV1（萨宾）中存在抗- P1的2反P3 1抗体）。 <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="图1" /> 图1。由PA检测鉴定的PV1（萨宾）PV的病毒解决方案，PV2（萨宾）和PV3（萨宾）（1.2 × 10 8赛迪顾问50 / 12μL，8.2 × 10 7赛迪顾问50 / 12μL的病毒滴度，并3.5 × 10 7赛迪顾问50 / 12μL，分别）是用于识别由PA检测。没有病毒，没有抗体控制显示（非凝集，阴性对照）明胶颗粒沉淀。 <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="图2" /> 图2。病毒解决方案的PV1（萨宾），PV2（萨宾）和PV3（萨宾）（病毒滴度为2.4 × 10 8赛迪顾问50 / 25μL，1.7 × 10 8赛迪顾问50 / 25μL，和释义的正面和负面的井 。 7.3 × 10 7赛迪顾问50 / 25μL，分别）稀释1 / 20到1 / 320，和25μL稀释的样本，然后由PA检测没有反光伏抗体检查。

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

搜集Masujima是新产品设计部，Fujirebio公司雇员

这个PA检测的独特之处是利用反光伏抗体，而不是2光伏检测PVR的分子。这允许特定的相互作用的一个统一的亲和力，所有三个血清型光伏3-6致敏的明胶颗粒与光伏。我们用于致敏的明胶颗粒immunoadhesin形式的PVR（PVR - IgG2a），因为单体形式的PVR只有适度的亲和力，以单一的结合位点上的病毒粒子（解离常数10 -7〜-8中号） 7， 8，成功地观察到具体的光伏株致敏明胶颗粒凝集。随着光伏鉴定的高特异性和高敏颗粒均匀亲和力，在PA检测的关键因素是:1）病毒滴度（有效的光伏检测法由PA）和2）凝集的解释（光伏正确识别） 。 一般而言，病毒滴度的光伏株在细胞培养系统是在10 6日至8赛迪顾问50 / 50μL的范围。 PA检测1型，2，3光伏（形成完整的凝集）的检出限分别为1.5x10 6赛迪顾问50 5.3x10 5赛迪顾问50和9.1x10 5赛迪顾问50，分别为2。正确识别大部分的光伏分离PA的检测，即使在其他非光伏肠道病毒的存在。然而，对于一些样品中的不同血清型的光伏的混合物，未成年人的血清型失败，被检测到。应清除凝集形成足够的病毒滴度的检测限以上。情况下，在没有抗体样本凝集不清或部分菌株应重新在RD细胞扩增增加滴度9。 部分凝集观察时，病毒滴度低，只是在检测限以下。凝集阴性对照明显的差异，可判断在PA检测阳性。然而，相当微妙的凝集差异来判断，作为积极的结果可能会误导解释。因此，结果阳性（局部凝集），应使用可能造成轻微的血清型的菌株的支持信息。然而，这些信息将帮助时，下面的分析（光伏分离或实时RT - PCR基因组测序）提出了一个可能性，样品中的不同血清型的光伏的混合物，或只有部分凝集没有抗体样本观察低的病毒滴度，将不清楚结果的重要原因。 PA光伏鉴定检测的优点是简单的程序和结果的解释。这将允许对每一个实验室进行细胞培养系统的光电隔离的检测的简单介绍，不提供特殊的设备，材料和培训。利用特定类型的单克隆抗体，可能允许分化的PV（野生型或疫苗株），将提供重要的光伏财产的信息的新方法的发展以及通过RT - PCR和基因组与遗传分化隔离排序10，11。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>敏 - 明胶颗粒</td><td> Fujirebio公司</td><td></td><td>检测试剂盒</td></tr><tr><td>重整缓冲区</td><td> Fujirebio公司</td><td></td><td>检测试剂盒</td></tr><tr><td> Microtitration板</td><td> Fujirebio公司</td><td></td><td>检测试剂盒</td></tr><tr><td>反光伏抗体</td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

在全球根除脊髓灰质炎行动，从急性弛缓性麻痹（AFP）病例粪便标本中分离鉴定光伏实验室诊断起着至关重要的作用。在世界卫生组织（WHO）的全球脊髓灰质炎实验室网络，光电隔离和鉴定，目前正在通过细胞培养系统和实时RT - PCR检测，分别进行。在光伏根除后时期，简单和快速识别程序，将有利于快速确认在国家实验室的脊髓灰质炎病例。在本研究中，我们将展示的新型PA检测程序开发光伏鉴定。该PA检测采用光伏受体（PVR）的分子和病​​毒，是具体的和统一的光伏所有血清型的亲和力的互动。程序很简单（一个反应板的步骤程序）和反应2小时内可以得到快速的（结果），结果是直观地观察（观察明胶颗粒凝集）。

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

最近开发的新颖的颗粒凝集法（PA）的测定利用病毒的受体分子，允许快速和容易识别的脊髓灰质炎病毒（PV）。在这篇文章中，我们将显示为光伏识别PA检测的过程。

脊髓灰质炎病毒识别粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.2K 次观看.  National Institute of Infectious Diseases. 最近开发的新颖的颗粒凝集法（PA）的测定利用病毒的受体分子，允许快速和容易识别的脊髓灰质炎病毒（PV）。在这篇文章中，我们将显示为光伏识别PA检测的过程。

视频: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

科研

免疫与感染

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

肽为基础的识别功能图案和具有约束力的合作伙伴

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

为检验新型抗病毒药物的鉴定对蓝舌病病毒

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...