我们感谢伊夫·亚宁博士的卓有成效的意见和建议。我们要感谢HH迦得对植/仁和C Combredet她的技术支持。巴斯德弊端Infectieuses（计划福星到POV和HM- - 这项工作是由巴斯德研究所，中心法国国家科学研究（CNRS），研究所国家德拉桑特逸德拉RECHERCHEMédicale（INSERM），研究所卡诺支持L）时，国民新电倒拉RECHERCHE（ANR-RPIB，计划STING 2.0至POV），和“行政法院区域D&#39;法兰西岛大区法国”（化学库项目，赠款N°我06-222 / R和I 09-1739 / R以HM-L）。对CHIKV /任这项工作是由项目ArbOAS支持（ANR授予2010-INTB-1601-02）。

96孔子板1。准备与化合物<ol><li>将包含化合物的DMSO中的10mM储备溶液在-20℃至室温的96孔母板上。稀释5倍的化合物在DMSO在2毫米，以获得中间96孔稀释板。稀释吹打5微升到20微升的DMSO和混合来实现。 </li><li>吸取1微升的稀释板成白色，条形码组织培养96孔板干井。这第一组子板（D1）将被用来评估化合物以20μM的终浓度的毒性。店子板在-20℃备用。 </li><li>吹打从稀释板4微升到36微升的DMSO和混合再稀释化合物1:10。吸取1μL成白色，平底，条形码组织培养的96孔板干井。这第二组子板（D2）将被用来评估MV replicatio的抑制n可在化合物的2μM的最终浓度。店子板在-20℃备用。 </li></ol> 2，细胞培养制备确定化合物的毒性<ol><li>生长的HEK-293T细胞，在37℃和5％CO 2中的Dulbecco改进的Eagle培养基（DMEM）与稳定化的L-谷氨酰胺并补充有10％胎牛血清（FCS），链霉素（100微克/毫升）和青霉素（100国际单位/毫升）。细胞被胰蛋白酶消化传代，每4-5天，和1:10稀释到一个新的烧瓶中。细胞应在对数期为实验。 </li><li>在实验的当天，通过胰蛋白酶处理回收细胞，并进行细胞计数。离心沉淀细胞，并重新悬浮于其中3×10 5个细胞/培养基毫升。认为12.5毫升细胞悬液要求每个屏蔽板。 </li><li>细胞转移到低谷，并免除100微升细胞悬液中含有尖刺化学康波D1板unds。料槽内的细胞悬液定期搅动以避免沉淀。 </li><li>秒杀对照孔A1，C1，E1，G1，A12，C12，E12和G12采用1微升的DMSO。相应孔将被用作用于活细胞的参考（无毒性）。穗对照孔B1，D1，F1，H1，B12，D12，F12和H12与1微升的DMSO和2.5微升的0.5％IGEPAL溶液杀死细胞。相应井将被用作死细胞的参考（毒性高）。 </li><li>孵育细胞24小时，在37℃和5％CO 2。 </li></ol> 3，制备rMV2/Luc感染的细胞培养的<ol><li>成长，在2.1和2.2中所述恢复细胞。再次，重悬的细胞以3×10 5个细胞/培养基毫升。认为12.5毫升细胞悬液要求每个屏蔽板。 </li><li>可选：与尿苷在20微克/毫升补充培养基。 注意：当筛查病毒replicatio化学库Ñ ​​抑制剂，似乎靶向早期步骤的嘧啶生物合成途径中的化合物经常被分离13,16,27,28。加尿苷的培养基中，用于过滤掉这样的抗病毒分子。事实上，这有效地恢复时嘧啶生物合成途径上游的酶被阻断病毒的复制。 </li><li>保存细胞悬液1:10对照孔与非感染的细胞，然后继续感染剩余量。 </li><li>解冻rMV2/Luc原液合适的音量。 MV股票的解决方案通常是在每毫升10 6 -10 8传染性颗粒。培养必须感染rMV2/Luc每个靶细胞0.1感染性颗粒，其对应于0.1感染复数（MOI）的。注：rMV2/Luc从MV疫苗（Schwarz株）得出，并操纵在BSL2环境。 </li><li>病毒加入细胞悬液，轻轻混匀。转让感染细胞的低谷，并免除100微升的感染的细胞悬浮液中含有的列尖刺化学化合物D2板的2〜11。料槽内的细胞悬液定期轻轻混合。 </li><li>在列1和12，取100μl的非感染细胞的阱上2。感染的细胞中被分配在其他井。 </li><li>孵育细胞24小时，在37℃和5％CO 2。 </li></ol> 4，荧光素酶活性措施和数据分析<ol><li>从培养箱中取出D1板，并确定细胞活力通过加入50μl荧光素酶为基础的生存能力试剂直接向培养孔。混合后，于室温下10分钟。读板用发光。积分时间设定为每孔100毫秒。 </li><li>从培养箱中取出D2板，并确定荧光素酶活性通过加入50μl萤光素酶底物的直接向培养孔。孵育6分钟，在室温下，读板用的光度计如所记述上面的床。 </li><li>计算Z&#39;-因子的毒性测定的各板D1，以保证屏幕29的质量。 Z&#39;= 1-3 *（σ+σ-）/（μ+ - μ-），其中μ+和σ+对应的发光和标准偏差的HEK-293T细胞单独用DMSO（无毒性）的手段，和μ和σ-的发光和标准差与IGEPAL处理，以杀死细胞培养孔中的手段。 Z&#39;预期为0.5以上，或相应的板将被丢弃。 </li><li>在μ+ / 2设定毒性阈值。丢弃化合物与下面这个截（ 图3A）的发光值。 </li><li>计算Z&#39;因子，抗病毒试验的每个D2板。这里，μ+和σ+对应的发光和标准偏差被感染的细胞与单独的DMSO培养的手段，和μ-和σ-是发光和标准偏差为未感染的细胞的手段。再次，板与Z＆＃39;低于0.5的值被丢弃。 </li><li>计算病毒复制的抑制作用如下：抑制=（μ+ - 对化合物X荧光素酶活性）的百分比/（μ+ - μ-）* 100。设置门槛的抑制在75％，并选择化合物既减少发光值低于这个截止（ 图3B），且没有毒性，按照4.4描述的标准。 </li></ol> 5，辅助屏幕的毒性和广谱抗病毒活性<ol><li>使1点02连续稀释为每个在DMSO击中化合物，从500微米至4微米（8稀释）开始。填充白色，条形码的组织培养板用50微升培养基。加入1μl每种化合物稀释液中培养孔中。重复两次，有3个培养板具有重复连续稀释每一击化合物。 </li><li>准备37.5毫升一个HEK-293T细胞悬浮液在6×10 5个细胞/培养基如上所述毫升（以2.1和2.2）。免除2个12.5毫升在猎鹰管和感染细胞与任何rMV2/Luc以MOI = 0.1或重组CHIKV在MOI表达海肾萤光素酶（CHIKV /仁）= 0.2。颠倒混匀。 注意：CHIKV /仁从CHIKV的野生型菌株产生的，因此应该在一个BSL3环境进行操作。板可以从BSL3被移动到BSL1一次Renilla荧光底物已被添加到培养孔（见下文）。 </li><li>免除50微升未受感染，rMV2/Luc-infected，或CHIKV /仁感染的细胞中含有的命中化合物的连续稀释培养板中。培养24小时，在37°C </li><li>加入50微升萤火虫荧光素酶底物，以确定rMV2/Luc-infected井萤火虫荧光素酶的活性。加入50微升海肾萤光素酶底物来确定CHIKV /仁感染井海肾萤光素酶的活性。最后，加入50微升荧光素酶为基础的可行性分析试剂，以未感染的细胞，以确定细胞活力。 </li><li>情节数据（ 网络连接GURE 4），并确定抑制MV和CHIKV复制了50％的命中化合物浓度。忽略表示在该试验中某些毒性化合物。 </li></ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening-based+identification+of+paramyxovirus+inhibitors.">High-throughput screening-based identification of paramyxovirus inhibitors.</a> J Biomol Screen. 13, 591-608 (2008).</li><li>Mar&#233;chal, E., Roy, S., Lafanech&#232;re, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemogenomics and Chemical Genetics: A User's Introduction for Biologists, Chemists and Informaticians. , (2011).</li><li>Gentzsch, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+complete+life+cycle+screen+for+identification+of+small+molecules+with+pro-+or+antiviral+activity.">Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.</a> Antiviral Res. 89, 136-148 (2011).</li><li>Caignard, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+V+protein+of+Tioman+virus+is+incapable+of+blocking+type+I+interferon+signaling+in+human+cells.">The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.</a> PLoS One. 8, (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里描述的筛选管线的目的是选择的化合物与合适的配置文件作为RNA病毒的广谱抑制剂。当10000的化合物库中筛选与此 ​​协议，上述过滤条件被应用， 即抑制MV复制优于75％，40个化合物（0.4％）的得分阳性。此外，约一半的人表现出一定的毒性在荧光素酶为基础的可行性分析，并无视这个原因。最后，一​​应俱全的命中打在用量较大再次接受测试。这个小套很容易被重新测试两个细胞...

RNA病毒负责了大量的各种人类感染，并同时拥有在公共健康和经济成本方面对全世界的人口产生巨大的影响。有效的疫苗已经开发了针对几个人RNA病毒，并且被广泛地用作预防性治疗。然而，仍然有严重缺乏的治疗药物抗RNA病毒的感染。事实上，有效的疫苗不提供对主要的人类病原体，如登革热病毒，丙型肝炎病毒或人类呼吸道合胞病毒（hRSV的）。此外，RNA病毒是负责大部分新出现的疾病，它已经在频率，因为全球性的交流和对生态系统人为的影响，增加的。针对代表RNA病毒这一威胁，我们的治疗阿森纳是极其有限的，相对低效的1-3。目前的治疗方法基本上都是基于重组I型干扰素（IFN-α/β），以刺激先天性免疫，或利巴韦林的管理。虽然这核苷类似物的作用方式是有争议的，可能依赖于各种机制，细胞IMPDH（次黄嘌呤核苷酸脱氢酶），这耗尽细胞内GTP池的抑制作用，显然是必要的4。利巴韦林，与聚乙二醇化干扰素-α结合，是对丙型肝炎病毒的主要治疗手段。然而，IFN-α/β和利巴韦林治疗的疗效是比较差在体内对大多数RNA病毒的，因为它们通过毒力表达的有效钝的IFN-α/β信号因子5和经常逃避利巴韦林3。这增加了一个事实，即利巴韦林治疗是提高重要的毒性问题，尽管它最近被批准对严重hRSV的疾病，有争议的好处6。最近，一些病毒特异性治疗方法已被销售，特别是对流感病毒与发展Õf神经氨酸酶抑制剂3。但是，大的多样性和RNA病毒的出现永久排除了对他们在一个相对紧密的未来每一个具体的治疗方法的发展。总之，这强调需要有效的战略，以确定并开发强大的抗病毒分子在不久的将来。 这是微不足道的说，一个广谱抑制剂有效对抗RNA病毒大面板会解决这个问题。虽然这样的分子仍然是一个病毒学家的梦想，我们更好地了解细胞防御机制和先天免疫系统表明，一些可能性存在7,8。几个学术和工业实验室正在寻求刺激的细胞防御机制或代谢途径钝病毒复制的特定方面的分子。虽然这种化合物可能会显示出显著的副作用，对急性病毒感染的治疗会辖编为一相对短的时间，使得即使在长期的一些潜在毒性他们可以接受的。各种策略已经被开发，以识别这样的广谱抗病毒药分子。一些研究项目旨在寻找针对特定抗辩或代谢途径的分子。这包括，例如，病原体识别受体诱发的抗病毒基因的表达和9激活抗病毒因子如引入RNaseL 10，自噬机械，以促进病毒降解11，核苷合成途径12,13，或凋亡级联反应以沉淀病毒感染细胞的死亡14。其他小组已经开发出不是目标为基础13,15-17表型的屏幕。在这种情况下，抗病毒分子通过它们的能力简单地识别，以阻止病毒复制的一个给定的蜂窝式系统中。一般的假设是，抑制2-3无关RNA病毒的化合物，将有一个合适的模式为广大-SPectrum抗病毒分子。有这样的经验方法选择的命中化合物的作用方式只是在第二次确定的，最终，可能导致新的细胞目标的识别抗病毒药物。有趣的是，1999年和2008年间获得美国食品药品监督管理局新药的回顾性分析表明，在一般情况下，这种表型筛查往往表现比目标为基础的方法更好地发现先入类小分子药物18 。 在高通量的基于细胞的测定病毒的复制通常由病毒致细胞病变效应测定。细胞被感染并培养在96 - 或384 - 孔板中测试的化合物的存在。之后的几天，细胞层被固定和染色的染料如结晶紫。最后，吸光度用酶标仪和化合物抑制病毒复制是由它们的能力确定为往复保存细胞层确定米病毒诱导的细胞病变效应。另外，病毒介导的细胞病变效应正在使用标准的可行性分析，如MTS减少评估。此类测定法是高度易处理的和具有成本效益的，但三个主要的限制受到影响。首先，它们需要一个病毒-细胞组合，其中病毒的复制是细胞病变中只有几天，但情况并非总是可能的，从而要求替代方法19。其次，他们都缺乏定量的，因为它们是基于对病毒复制的间接测量。最后，有毒化合物可以评价为阳性命中，并且因此必须与一个计数器屏幕测定细胞活力来消除。要克服一些障碍，重组病毒或复制子已设计通过反向遗传学表达报告蛋白，如绿色荧光蛋白或荧光素酶，由一个附加的转录单位或以帧为与病毒蛋白的基因（几个例子是20-23）。当这些病毒的复制，记者公关oteins与病毒蛋白本身产生在一起。这提供了一个非常定量测定来测量病毒的复制和评估候选分子的抑制活性。这是用于重组病毒表达荧光素酶（或生物发光能力的其他酶），因为此报告系统显示出一个很宽的动态范围内具有高灵敏度，几乎没有背景，尤其如此。此外，不存在激发光源，从而防止干扰化合物的荧光24。 在这里，我们详细地高通量协议来筛选的RNA病毒的广谱抑制剂，化学文库。化合物在感染了重组麻疹病毒（MV）表达萤火虫荧光素酶25（rMV2/Luc， 如图1所示 ，主要画面）人体细胞先进行测试。 MV属于Mononegavirales秩序，往往被视为负链RNA病毒的原型成员ES。因此，MV的基因组作为模板，通过病毒聚合酶合成mRNA分子对病毒蛋白质进行编码。在重组MV株称为rMV2/Luc，萤光素酶表达是由P和M基因（ 图2A）之间插入一个附加的转录单位表达。平行地，化合物使用的是商业荧光素酶为基础的试剂，用于评估，由ATP定量测试它们的毒性对人体细胞，代谢活性细胞中培养的数量（ 图1，主屏幕）。整个化学文库可以很容易地筛选出与这两个试验，以便选择是没有毒性的，有效地阻止MV复制的化合物。然后，点击是重新测试剂量-反应抑制的MV复制，缺乏毒性以及其对损害基孔肯雅病毒（CHIKV）复制（ 图1，二次屏）的能力。 CHIKV是披膜病毒科家族的成员，其基因组是apositive单链RNA分子。因此，它是完全无关的麻疹和化合物抑制两个MV和CHIKV站在一个很好的机会，以抑制RNA病毒大面板。 CHIKV非结构蛋白是直接从病毒基因组的转化，而结构蛋白是由转录和一个亚基因组mRNA分子的翻译编码。对CHIKV我们的体外复制的测定是基于重组菌株称为CHIKV /韧，它通过NSP3和NSP4序列26（ 图2B）之间的报告基因的插入表达的Renilla荧光素酶作为非结构性多蛋白的裂解的部分。海肾荧光素酶活性的度量允许病毒复制的监视在CHIKV生命周期的早期阶段。 这种高通量的协议被用于快速识别化合物在10000 molec商业库一个合适的模式为广谱抗病毒药物ULES丰富的化学多样性。化合物基本上按照五个平斯基的规则，分子量范围从250至600道尔顿，和日志D值低于5。大多数这些分子分别在其他商业库无法使用新的化学实体。

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Freedom EVO platform</td>
			<td style="width:105px;">TECAN</td>
			<td style="width:119px;"> </td>
			<td style="width:316px;">Robotic platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Polystyrene Cell Culture Microplates, White</td>
			<td style="width:105px;">Greiner Bio One</td>
			<td style="width:119px;">655083</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTiter-Glo Luminescent Cell Viability Assay</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">G7570</td>
			<td>Luciferase-based viability assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bright-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2610</td>
			<td>Reagent containing firefly luciferase substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Renilla-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2710</td>
			<td>Reagent containing Renilla luciferase substrate</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Britelite plus Reporter Gene Assay System</td>
			<td style="width:105px;">Perkin-Elmer</td>
			<td style="width:119px;">6016761</td>
			<td style="width:316px;">Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity</td>
		</tr>
	</tbody>
</table>

high-throughput screening for broad-spectrum chemical inhibitors of rna viruses

RNA病毒是负责重大人类疾病，如感冒，支气管炎，登革热，丙型肝炎或麻疹。他们还表示，因为增加了世界各地的交流与人群渗透越来越多的自然生态系统的一个新威胁。这样的情况出现的一个很好的例子是2005-2006年在印度洋基孔肯雅病毒的流行。在我们的细胞途径控制病毒复制的了解最新进展表明，靶向宿主细胞的功能的化合物，而不是病毒本身，能抑制RNA病毒的大面板。一些广谱抗病毒化合物被确定，与主机针对性的分析。然而，测量病毒复制的细胞培养物的抑制使用减少的细胞病变效应的读出还是代表一个最重要的筛选策略。这种功能的屏幕已经由重组病毒表达记者的发展有了很大的提高酶CA帕布尔生物发光如荧光素酶。在本报告中，我们详细介绍了高通量筛选的管道，它结合了重组麻疹和基孔肯雅病毒与细胞活力检测，以确定化合物具有广谱抗病毒配置文件。

这种筛选流水线首先依赖于抑制MV复制化合物的选择和不显示任何显著的细胞毒性（ 图1，主屏幕）。它利用荧光素酶为基础的检测，以确定细胞毒性和病毒复制的抑制。所有吸移步骤和数据采集可以在高通量环境来执行与机器人平台。 化合物的细胞毒性是使用计算结果的基础上，ATP的存在，它对应于代谢活性细胞的定量在培养的活细胞的数目的荧光素酶为基础?...

Watch this Scientific Journal Video about High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses at JoVE.com

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

在体外测定法来测量病毒的复制已经由重组RNA病毒表达荧光素酶或能生物发光的其它酶的开发大大提高。这里我们详细介绍，结合麻疹和基孔肯雅病毒，从化学文库分离出广谱抗病毒药物，例如重组菌株的高通量筛选的管道。

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

high-throughput-screening-for-broad-spectrum-chemical-inhibitors-rna

Research

JoVE Journal

Immunology and Infection

13.6K 次观看.  Institut Pasteur, CNRS UMR3569.  在体外测定法来测量病毒的复制已经由重组RNA病毒表达荧光素酶或能生物发光的其它酶的开发大大提高。这里我们详细介绍，结合麻疹和基孔肯雅病毒，从化学文库分离出广谱抗病毒药物，例如重组菌株的高通量筛选的管道。 

视频: High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

高通量化学化合物筛选，以阐明其对细菌持久性的影响

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...