我们要感谢Vesper的铁玛丽·拉莫斯博士的批判性阅读的手稿。这项工作是由国家研究资源中心（P20 RR0115635-6，C06 RR17417-01和G20RR024001次）和美国国家神经疾病与中风研究所（2R01 NS052609）。

1。准备设备<ol><li>使用Misonix 4000 3000或Misonix的超声波仪，（纽约法明达）连接到一个热电NESLAB EX-7水浴（纽因顿，NH），以保持恒定的温度为37°C。超声波清洗的样品在200微升薄壁的PCR管带与赛默飞世尔科技（马萨诸塞州沃尔瑟姆）获得的圆顶帽。 </li><li>一个全新的超声发生器需要一个"磨合"期，连续工作9。甲两个月的磨合期由40秒的超声处理突发和孵育10分钟的周期与振幅电平设置为10。人们应该观察的，钛微孔板角（ 图1，B组）的整个表面的侵蚀。循环水看白色混浊的钛微孔板角的侵蚀。请此水每天更换。 </li><li>放大的PrP Sc的变化整个微孔板角。最好的PrP Sc的扩增效率，可以得到内从喇叭形的微孔板的中心（参见图1，面板C）的半径为5厘米。 </li><li>一个圆形的PMCA由144个时钟周期。每个周期由5秒超声和孵育10分钟。这大约涉及到24小时，每一轮的PMCA。 </li><li>的功率输出的超声处理，超声波发生器的控制面板上显示的，与PCR管中存在的超声波发生器的机架和从水浴中的循环水的温度的数目而变化。确保在37°C的水平衡，不填写超声发生器的管架（ 图1，C组）的30％以上。传播的PCR管通过微孔板角。具有至少一个行的PCR管条带之间的空的空间，并允许不超过四个管连接在一起，每带（ 图1，C组）。 </li><li>超声处理的强度是至关重要的一个成功的朊病毒应变放大。声波处理的同时，调整振幅有一个输出功率在155〜170瓦不等。在我们的实验室中，功率电平被设置为6和7分别为Misonix 3000和Misonix 4000，。 </li><li>在水浴中使用蒸馏水，以避免积累硬水渍。更换这水每周。在孵化和超声循环，冷凝水会发生内部的的微孔板喇叭盖。为避免结露，将图1所示，面板A.超声波仪的外壳中上方的一个小鱼缸加热垫</li></ol> 2。准备样品<ol><li>所有程序，涉及动物，克赖顿大学实验动物护理和使用委员会批准，并遵守实验动物护理和使用指南。在任何组织收集之前，动物必须是麻醉，和transcardially灌注的用冰冷的磷酸盐缓冲生理盐水溶液用5mM EDTA在pH = 7.4，以除去血液从大脑和避免抑制PMCA反应的血液7。迅速收集动物组织使用无菌技术，并有专门的清扫工具，为每个朊应变。解剖后，将组织的1.5 ml离心管，闪光冻结干冰，并将其存储在-80°C至同质化。 </li><li>为了制备PMCA基板（PrP的C）的解决方案，在未感染的仓鼠脑（联合国BH）至10％重量/体积（w / v的）用冰冷的转换缓冲溶液（见第3部分），使用Tenbroeck组织研磨机均匀。在均质过程中，要注意不要让车内空气磨床的解决方案，以避免过多的泡沫。澄清溶液经离心分离，在500×g离心30秒和等分试样在1.5毫升微量离心管的上清液。储存在-80℃，直到进一步使用。 </li><li>为了制备的PMCA种子（的PrP Sc）的溶液，均质传染性物质（脑，脾，淋巴结，以及其他组织或污染土壤）的10％w / v溶液，用冰冷的娃在0.6ml离心管中，r和分装解决方案。储存在-80℃，直到进一步使用。 </li></ol> 3。准备转换缓冲区<ol><li>称量0.5093克的Triton X-100的在50毫升容量瓶中（结果最终浓度为1％的Triton X-100）（Sigma-Aldrich公司的联系; Steinheim，德国）。 </li><li>一个完整的的蛋白酶抑制剂平板（罗氏诊断公司，德国曼海姆）。 </li><li>加入的EDTA0.0931克（这将导致在5mM的最终浓度为EDTA）导管（Mallinckrodt，贝克公司;新泽西州菲利普斯堡）。 </li><li>用1N NaOH将pH调节至7.4。 </li><li>调整音量至50毫升，用Dulbecco磷酸盐缓冲液（DPBS）（敏达公司，弗吉尼亚州马纳萨斯）。 </li><li>过滤该溶液，通过0.45μm的硝酸纤维素膜，用真空过滤装置。转换缓冲器可以存储在4℃下不超过1个月。 </li></ol> 4。朊病毒菌株的扩增使用PMCA <ol><li>稀释PMCA种子进入PMCA基板到PCR管的比例为1:20（ 即混合5微升PMCA种子和95微升的PMCA基板）。取出并妥善保管15μL上清置于-80°C unsonicated控制，这unsonicated控制将被用来量化的PrP Sc的放大通过蛋白酶K（PK）消化，免疫印迹法（WB）分析。一个额外unsonicated控制可以产生的PMCA反应的持续时间，在37℃下放置一个第二个15微升等分试样。至少重复三次作统计用途。 </li><li>主题的样品（70微升）的其余部分的一个轮的PMCA，如在图1上所示，面板A.摇管上的圆顶的PCR管，以避免水凝结每5小时。 </li><li>样品的管应被剥离下来，轻轻振荡（要小心这样的解决方案不进入圆顶），然后再打开后，任何程序，以确保homogeneity和避免潜在的污染。 </li><li>对于串行PMCA（sPMCA）的短潜伏期株（ 即 HY TME，HaCWD，或263K），超声波处理的材料在1:20的比例稀释，将超声处理5微升的样品，从第一轮为95微升新鲜的未受感染的脑匀浆中（ 图2，图B）。 </li><li>对于sPMCA长潜伏期株（ 即 DY TME，139H，22CH，22AH，或ME7H）的，在以1:1的比例稀释的超声处理的材料通过从圆一个50微升的超声处理的样品转移到50微升新鲜的未感染脑匀浆中（ 图2，图B）。 </li><li>卸下两个15微升等份从预先稀释的超声处理的混合物（来自步骤4.4或4.5）中，并存储其中之一，在-80℃下和在37℃下（unsonicated PMCA第二轮的控制解决方案）。一个PMCA第二轮主题的残余物。 </li><li>此过程可无限地重复。在我们的实验室中，我们已经执行最多15 PMCA轮，与应变性能保持不变的。 </li><li> PK消化的样品。对于一个典型的WB混合5μl的PK 80μg/ ml到5μl的样品（此溶液将具有PK的最终浓度为40微克/毫升），并在37℃下孵育一小时。凝胶上样缓冲液中加入10μl。此溶液中煮沸10分钟。让该溶液冷却并加载到凝胶中的10微升。 </li><li>要计算成功扩增执行WB分析估计量放大的PrP Sc的 PK消化的样品进行比较，他们要么，在unsonicated的控制20,21 图2，图C为已知金额的PK-未消化的重组PRP。 </li></ol> 5。避免交叉污染应采取的关键步骤，以减少交叉污染和误报的发生，由于新形成的蛋白酶产品耐。 </P&gt; <ol><li>朊病毒株为每个样品收集，使用一组专用的清扫工具（ 如镊子，镊子，剪刀）。 </li><li>在均匀化作为PMCA基板使用的未受感染的大脑之前，擦拭的吸移管，用1％的漂白剂。浸泡的超声波仪，PCR管架，组织匀浆器，并用1％的漂白粉10分钟的PCR管储物架，并用蒸馏水彻底清洗。 </li><li>砂劳越干一个新的实验室的浸种每次一个新的样本是同质化覆盖工作区，一个新的PM​​CA实验做好准备，或等分之间sPMCA轮。 </li><li>使用新的手套每一次新的样品处理（特别是当传输等份之间sPMCA轮）。 </li><li>使用短超声脉冲为每个PMCA周期。周期5秒超声结合孵育10分钟是足够的放大，而不会产生从头的PrP Sc的1,20,21,23,24朊病毒。其他实验室已经证明，增加声波处理时间，增加的超声处理的振幅，和延伸PMCA周期的数目增加为从头的PrP Sc的形成2的概率。 </li></ol>

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没有利益冲突的声明。

放大传染性朊病毒蛋白质的挑战是在体内实验中的潜伏期长和费用。 PMCA技术是一种经济有效的手段，放大传染性朊病毒剂。几个实验室已证实的能力PMCA准确放大朊病毒株在体外 7，第9，第12，14，19,24。 朊病毒疾病可以物种之间发送。贝森和湿地，有效地接种仓鼠传染性貂脑病，这就产生了两个不同的地鼠源性朊病毒株5。在一个优雅的学...

<table border="1"><tbody><tr><td>试剂/设备</td><td>生产厂家</td><td>猫。数</td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck组织研磨机</td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> NESLAB EX-7水浴</td><td> Thermo Electron公司</td><td> NESLAB EX-7 </td></tr><tr><td> 0.2毫升PCR管条</td><td> Thermo Scientific的</td><td> AB-0451 </td></tr><tr><td>的Triton X-100的</td><td> Sigma Aldrich公司</td><td> T9284-100ML </td></tr><tr><td>完整的蛋白酶抑制剂</td><td>罗氏公司</td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT贝克</td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt公司贝克敏达</td><td> 21-031-CV </td></tr><tr><td>砂劳越干实验室Soakers </td><td> Fisher Scientific则</td><td> 14 206 28 </td></tr><tr><td> Repti千卡加热器</td><td>动物园地中海Laboratories公司</td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

朊病毒是传染性病原体的动物和人类9,18导致的必然致命的传染性海绵状脑病（TSE）。的朊蛋白有两个不同的异构体，非传染性的主机的编码蛋 ​​白（PrP的C）和传染性蛋白（的PrP Sc）的，异常折叠的PrP〜C 8异构体。 朊病毒代理工作所面临的挑战之一是潜伏期长，临床症状主机接种后13前的发展。传统的授权和昂贵的动物生物测定研究。此外，生物化学和生物物理性能差的特点，其特殊的构象和聚集状态的PrP Sc的 。 的PrP sc可以转换的PrPÇ的PrP Sc的种子在体外 14。 PMCA是一种体外技术，需要阿德瓦ntage这种能力，利用超声和孵化周期产生大量的PrP Sc的 ，以更快的速度，从系统中含有过量的PrP C和微量的PrP Sc的种子19。这种技术已被证明，有效地重述物种和应变特异性的PrP Sc的转换，从蛋白Ç，干扰模拟朊病毒应变，并扩增的PrP Sc的非常低的水平，从受感染的组织，体液，及环境样品6,7,16， 23。 本文详细介绍了的PMCA协议，包括减少污染，产生一致的结果，这些结果和量化的建议。我们也讨论了几个的PMCA应用，包括株传染性朊病毒，朊病毒应变干扰，检测朊病毒的环境中产生和表征。

蛋白质错误折叠循环放大（PMCA）是用来放大的PrP Sc 在体外培养 7，12，14，19，24。一个成功的PrP Sc的扩增谱带强度的增加，如在图3中所示的PK抗朊病毒蛋白（19和30之间的迁移的kDa的仓鼠派生朊病毒株）的Western印迹上所示。后其谱带强度的增加PMCA表示扩增的PK-抗性的PrP Sc的材料。成功扩增仓鼠派生朊病毒蛋白，HaCWD和DY TME，在WB分析...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

蛋白质错误折叠循环放大（PMCA）的的朊病毒转化和应变和物种障碍的研究是在体外试验。它也可以被用作朊病毒检测法。

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

protein-misfolding-cyclic-amplification-of-prions

Research

JoVE Journal

Immunology and Infection

19.4K 次观看.  University of Nebraska at Lincoln. 蛋白质错误折叠循环放大（PMCA）的的朊病毒转化和应变和物种障碍的研究是在体外试验。它也可以被用作朊病毒检测法。

视频: Protein Misfolding Cyclic Amplification of Prions

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

科研

免疫与感染

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

建立高通量筛选平台评估乳腺癌 HER2 基因扩增的异质性

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.