Name: a high content imaging assay for identification of botulinum neurotoxin inhibitors
Uploaded: 2014-11-14T13:00:00.000+00:00
Duration: 14 min 10 s
Description: Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

板20000分化的小鼠胚胎干细胞（MES）/孔在96孔聚-D-赖氨酸包被的板并在运动神经元的终末分化培养基维持5-7天。 1.复方管理和中毒与肉毒毒素/ A 执行下列所有工作在BSL2外壳，以保证符合CDC / NIH指南。 <ol><li>在聚丙烯96孔板中制备10mM储备溶液在100％DMSO中的每个库的化合物。使用10mM储备制备中间稀释板，其是10倍高于所需的筛选浓度。制备100μM的中间板通过分配10mM储备到96孔板中并用培养基稀释至100μM的。分配在细胞上11 10微升化合物对90微升的培养基。测试该化合物在10μM的最终浓度，并确保DMSO的终比例不超过0.5％。 </li><li>执行所有螺栓IES是利用活细胞和活性的毒素在生物安全二级条件。与使用12通道移液器手动80微升新鲜终末分化培养基代替旧的介质，在96孔板中。进行抽吸和分配步骤，按行，以避免细胞暴露而不媒体到环境空气中。 </li><li>预处理细胞之前通过加入10微升从源板使用12通道移液器，随后通过抽吸混合10倍的化合物应用毒素的化合物（两次）。对于每个96孔板中，治疗的6个孔的两列用10倍的DMSO稀释，在媒体上，作为高信号和低信号的控制。未经治疗的BoNT列显示全长的SNAP-25（高信号控制）的最高水平。对待其他列与肉毒毒素单独（无任何化合物）为低信号控制。使用低对照，观察SNAP-25和其检测与BACS的抗体的BoNT裂解的效率（ 图2）。 </li><li>孵育细胞30分钟，在37℃，6％CO 2，在细胞培养箱中的化合物。 </li><li>准备肉毒杆菌神经毒素A以上的磷酸盐浓度为1mg / ml的商业来源与终端分化培养基稀释缓冲液（PBS）到最终的10倍的股票工作。 </li><li>通过加入10微升10×的BoNT / A股成指定用于使用多通道移液管（ 图2）治疗和低信号控制井引发中毒。把指定为高的控制与单独媒体的井。 </li><li>净化所有的塑料制品和其他一次性用品，在水中至少20分钟的研究，浸泡在0.825％次氯酸钠溶液中谈到与肉毒毒素/ A的接触。请在生物安全柜的所有过程和​​两个前，用5％的洗涤剂消毒清洁剂实验如的MicroChem溶液后净化工作区域。 </li><li>标签板明确表示毒素的使用和incub吃板处理与1nM / L的BoNT / A 4小时，在37℃，6％CO 2下在细胞培养孵育箱。显示相应的指示牌，告知同事说，毒素是在实验室使用。 </li><li>停止BONT / A蛋白裂解甲醇固定。从各孔中除去含有毒素及化合物媒体用多道移液器，并丢弃在10％漂白溶液中。直接添加冰冷的甲醇（100微升）到每个孔中。 </li><li>培养板在室温（RT）下15分钟，以允许固定的发生。 </li><li>固定后，安全地处理废弃试剂（被认为中和）与生物安全1级协议和相应的丢弃。 </li><li>丢弃甲醇并用100微升PBS洗涤细胞并预先水化他们的免疫染色。执行两个额外的PBS洗涤。 </li><li>在最后一次洗涤后，留下的PBS中的孔中，密封板与微板粘合膜，并储存在4℃直至分析。商店板在4℃下FOř好几天。 </li></ol> 2.免疫染色免疫染色过程是劳动密集的，多步骤的操作，其包括重复的试剂分配/吸液循环和广泛板洗涤可导致潜在的引入显著板内和板间的变异性。一种半自动化的方法被应用到保存实验室人员的时间，提高测定吞吐量，并减少免疫染色可变性。 <ol><li>使用自动化工作站配备有96孔的头部能够传送量高达250微升并集成有机器人夹具臂移动至实验室器具插入模块甲板不同的位置（见材料和​​设备）来获得本协议。 <ol><li>模块化甲板被设计来承载不同类型的实验室器皿，并且可以在任一时刻支持多达11项。所述自动微处理程序配备了双杂志微孔板堆叠以保持和操纵高达每循环50的板。 </li><li>自动化工作站位于II级生物安全柜专为实验室自动化设备，以提供无菌和安全性的内部。用于自动免疫染色，在甲板上被安排为如图3所示，使试剂访问根据需要而无需人工干预。 </li></ol></li><li>预加载板根据需要为液体处理操作包含固定的细胞进入盘盒和传递到甲板上。使用96吸管头部装有250微升提示，从油井吸出PBS下降到10微升。透化细胞膜，促进抗体结合到与透化缓冲液（0.1％的Triton-X 100）的细胞内靶。之前以及将板返回到微孔板堆叠的第二存储盒为一温育15分钟，在室温（RT）下免除透化缓冲液（100微升）到每个。 </li><li>删除通透缓冲和PErform板用PBS洗涤（两次），为在步骤1.13如前所述。 </li><li>在最后一次洗涤步骤后，除去PBS缓冲液中，并分配100μl的含有在PBS中的1％马血清和0.1％吐温20封闭缓冲液中的成所有微孔板他们在RT返回到盘盒1小时温育之前。 </li><li>使用两个初级抗体为免疫染色:兔多克隆抗小鼠BACS的抗体，用于检测全长的SNAP-25和小鼠单克隆抗III微管蛋白抗体用于检测神经元（见材料和​​设备）的。温育60分钟后，除去封闭缓冲液，并分配50微升为4ug / ml的BACS的抗体和III微管蛋白的0.5微克/毫升的在封闭缓冲液入所有的板。 </li><li>孵育1小时在室温下除去第一抗体和洗涤用100μl的PBS中含有0.05％吐温（PBST）洗涤3次。 </li><li>使用抗小鼠IgG标记的Alexa Fluor 647以可视化的III微管蛋白和抗兔IgG标记的Alexa˚Fluor 568形象化BACS抗体。在封闭缓冲液中制备两种抗体为2微克/毫升的稀释和分配50μl的该混合物在每孔中。 注意:选择用于免疫染色的二级抗体标记有不同的荧光团，以允许检测其发射的荧光的使用高含量成像仪的检测器内的不同的信道不同的波长的光。 </li><li>保温1小时，在室温。吸出的第二抗体和洗涤3次，用100μl的PBST。 </li><li>最后一次洗涤后，加入100微升PBS含染料Hoechst公司（32微米）染色的DNA进行核检测。 </li><li>封板用不透光的薄膜，以保护从荧光漂白。板可被储存在4 &#160; °下星期没有显著信号丢失。 </li></ol> 3.影像注:使用高内涵成像Sys系统进行图像采集TEM（见材料和​​设备）。 <ol><li>规格高内涵成像定义的: <ol><li>选择板型，布局，字段数，目标:格雷纳ü明确，96孔板，16，分别为20倍的水。 </li><li>选择通道:核激发EX:405纳米通道4;细胞质EX:644纳米通道2;抗原特异性抗体频道EX:561纳米通道3，每个激光器的设置励磁电源，设置实验为2曝光，曝光1与一个激励，在644纳米，2曝光的两个激励在405纳米和561纳米。选择分级为斌。 </li><li>使用高对照孔用于与最大强度为SNAP-25信道和低对照孔为最小强度的曝光最优化。 </li><li>调整曝光，使每个信道的强度为约一半的饱和电平（2,000-2,500相对单位）。 </li><li>确定使用三角校正脚本小区蒙版通道的最佳Z平面。查看网络连接gure 5染色和成像后的结果。导出数据到其中的图像分析软件是驻留在服务器上。 </li></ol></li></ol> 4.图像分析（图6） 注:以下步骤描述了应用程序的哥伦布软件算法。 <ol><li>选择一个合适的算法来分割的主要对象（核）。如果需要的话，调整背景阈值和对比度参数。哥伦布软件提供4核的检测方法。选择的方法，该方法段准确细胞核通过目视检查分段核（F中igure 6a的方法M被选中）。 </li><li>选择突起算法（CSIRO轴突分析2）段中的突起如果需要，调整背景阈值和对比度参数来去除背景。参见图6b用于检测突起的参数。 </li><li>识别轴突区域（突起部分），BAS版上使用β-III微管蛋白的通道（Alexa的面粉640）作为掩模（ 图6c）的-3像素扩展的二次对象（突起）。 </li><li>计算信号通道（SNAP-25，（Alexa的面粉568）的突起区域和β-III微管蛋白通道（ 图6d＆6E）的强度。 </li><li>选择出口所需的参数，如神经突长度，SNAP-25的平均强度，β-III微管蛋白，核数，轴突段数等 （ 图1207米 ）。 </li><li>转让板使用机器人和负载到高内涵成像仪图像采集板堆垛。 </li></ol> 5.数据分析: 评估设计测定法的鲁棒性，计算从该板为基础的实验中使用以下参数。 <ol><li>信号（S）到背景（B）中:S / B =平均值高信号控制的强度）/平均值低信号控制的强度</li> &lt;利&gt;信号与背景的变化（CV）的系数（以测量特定信号的均匀性）:CV =（标准偏差（SD）的样品/平均值）×100（％），以确定在液体处理的可变性，试剂等</li><li>信噪比:S =（平均信号强度 - 指低信号控制的强度）低控制/ SD <ol><li>计算用的二分之一的96孔板的，而另一半的一个模拟中毒中毒的Z&#39;因子。 Z&#39;= 1 - 3 *（高信号控制+低信号控制的SD SD）/（平均高信号控制 - 低信号控制的意思）。用于筛选数据的进一步分析，正常化一些在平板上的基础的主原料的参数，以允许板之间和比较实验不同天之间。 </li></ol></li><li> ％的裂解，这反映了信号的全长度的SNAP-25的特异性抗体检测（BACS）相关联的降低:％裂解= 100％*（MEA高信号控制的n个强度 - 试样的强度）/平均高信号控制的力度。 </li><li>抑制裂解的百分比:抑制％= 100％*（样品的强度 - 指低信号控制的强度）/（平均高信号控制的强度 - 指低信号控制的强度）。 </li></ol>

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Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

肉毒杆菌神经毒素，相对缓解他们的武器化的高效力，导致其分类为A类（最高优先级）生物威胁剂由美国疾病控制和预防。不幸的是，没有FDA批准的治疗剂来对抗的BoNT中毒后的毒素已被内化的运动神经元。促进从肉毒毒素中毒的神经元恢复任何成药机制可能会导致对潜在疗法的发展对这种生物的威胁，以保护双方的军种和公众。在这篇文章中，我们提出了一个详细的分析HCI协议进行筛选的致命毒素抑制剂如肉毒毒素/ A。这个实验的一个不寻常的方面是毒素和实施严格的生物安全议定书，以确保实验室的安全严格交接。至尊必须小心，同时积极毒素正在使用中。肉毒毒素/经甲醇固定和微孔板decontaminati一失在用10％漂白粉，5％的表面擦拭巾是允许微孔板从生物安全柜生物安全2级实验室环境中删除下游评估的关键步骤。 由于加上缓慢的运动神经元扩展（手机号码），化合物库的不断扩大规模，HCI测定的神经毒素拮抗剂往往运行在几个星期。这就需要在板之间并跨越时间的可变性，必须消除或最小化。在这个协议中，我们提供了自动化的方法染色，图像采集和分析，以减少在测定变异性。进一步的可靠性和一致性可以通过使用该协议，包括细胞电镀，清洗，并固定相关的例行任务的自动化而实现。我们的集团正在评估这些改进与把它们纳入到我们的协议在不久的将来的意图的影响。 在这份报告中，我们描述了一个SNAP-25采用高含量成像来测量完整的SNAP-25的运动神经元中的相对荧光性裂解分析法。该测定中使用BACS和β-III微管蛋白的抗体来检测全长的SNAP-25和β-III微管蛋白分别为11。 β-III微管蛋白作为标记物特异性地鉴定神经元（ 图4）。虽然肉毒毒素/ A裂解SNAP-25，并降低了SNAP-25的信号，小分子抑制任何部分这一过程的改进在成像检测SNAP-25的信号。作为该测定法依赖于从SNAP-25分布在许多微小突起产生的荧光信号，高品质的图像是绝对必要的。因此，能产生高分辨率的共焦图象的自动显微镜，建议（ 图4）。我们经常在使用20X水浸物镜捕获6-16字段/孔在96孔格式。场的成像的数量依赖于神经突向发电机密封所需的总数目Ë统计学显著的效果。 模块化的图像分析算法，用于提取多参数数据（ 图6）。哥伦布模块化图像分析算法，比较容易产生更简单的分析方案。对于更复杂的分析，或进行复杂的读数特定参数设计，阿卡贝拉基于脚本可以在歌剧院环境以及哥伦布的上下文内使用。除了从SNAP-25和β-III微管蛋白的通道中得到的荧光强度，我们定期收集的其他参数，如总细胞数（间接测量细胞毒性），神经突长度，轴突分枝和其它端点中量化神经元的健康化验。原始端点数据被输出到统计分析软件进行进一步的数据分析和命中的选择（ 图7）。此处呈现的数据证明了的HCl测定的是effici的能力ently用于在寻求利用生理有关运动神经元模型肉毒毒素/ A抑制剂的表型筛选。

细菌肉毒梭菌产生的肉毒杆菌神经毒素，对人类已知1是最有效的生物毒素之一。有7个不同的血清型肉毒毒素（肉毒毒素/ AG）。肉毒毒素/ A-胆引起麻痹的神经肌肉接头处由于SNARE复合体蛋白水解2,3。 SNARE蛋白水解阻止神经递质囊泡膜融合，从而阻止神经递质胞吐4。具体的SNARE目标取决于所涉及的中毒过程中的特殊肉毒毒素血清型。肉毒毒素/ A和肉毒毒素/ E切割SNAP-25，而肉毒毒素/ C切割两种SNAP-25和突触5。剩下的血清型切割突触（也称为囊泡相关膜蛋白（VAMP）。肉毒毒素/ A被选为试验发展，因为它是负责自然发生的肉毒杆菌中毒的比例很高，并具有行动6。发展小分子的最长期限治疗对肉毒毒素/ A是主要目标我们的药物研发项目，并利用传统的基于目标的方法来确定活性位点的蛋白水解酶抑制剂7，8-10。然而，创建活性位点抑制剂具有抗多种血清型和后曝光功效广谱活性的将可能是具有挑战性的。 因此，我们已经实现了一个创新的，表型的药物发现，使用肉毒毒素SNAP-25的切割作为一个功能的端点，以确定小分子，可以阻止肉毒毒素介导的运动神经中毒的方法。 SNAP-25所需的神经递质的释放，如SNAP-25的降解是预测性麻痹和致死的体内 。例如，基于细胞的筛选可能导致发现的负责毒素失活或抑制毒素途径的靶细胞内的细胞因子的新调节剂。在表型检测发展的一个重要因素是生理上相关的生物模型的选择。 W¯¯e和其他人描述了小鼠胚胎干（ES）细胞衍生的运动神经元概括初级运动神经元的免疫学性质，包括SNAP-25 -11- 13的表达。重要的是，这些蜂窝系统到的BoNT / A中毒高度敏感和演示的SNAP-25的剂量依赖性裂解响应于增加毒素11,12的浓度。在分化的运动神经元也产生在量，足以适用于高通量板为基础的分析，并允许细胞试验的阵列的设计。 表型分析是利用两种不同的抗体，肉毒毒素/鼠标运动神经元文化的陶醉在量化内源性表达的全长SNAP-25的裂解免疫荧光法。羧基末端肉毒毒素/ A裂解敏感（BACS）抗体只识别全长SNAP-25允许SNAP-25的肉毒毒素/ A介导的蛋白水解的评价在小鼠运动神经元表达10。的盐酸法的示意图示于图1。

<table><tbody><tr><td>Botulinum neurotoxin A&amp;nbsp;</td><td>Metabiologics</td><td>NA</td><td>No catalog number</td></tr><tr><td>Microtitre plates</td><td>Greiner&amp;nbsp;</td><td>655946</td><td>Poly-D-Lysine 96-well plates</td></tr><tr><td>BACs antibody</td><td>Lampire Biological</td><td>NA</td><td></td></tr><tr><td>Microchem</td><td>National&amp;nbsp;</td><td>0255</td><td></td></tr><tr><td>Methanol</td><td>Thermo Scientific</td><td>A412-20</td><td></td></tr><tr><td>Formaldehyde</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Horse serum</td><td>Invitrogen</td><td>16050</td><td></td></tr><tr><td>PE JANUS MDT Mini Automated Workstation</td><td>Perkin Elmer</td><td>AJMDT01</td><td></td></tr><tr><td>Opera</td><td>Perkin Elmer</td><td>OP-QEHS-01</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td></td></tr><tr><td>BIII tubulin antibody</td><td>R&amp;amp;D Systems</td><td>BAM1195</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-1L</td><td></td></tr><tr><td>Hoechst 33342dye&amp;nbsp;</td><td>Invitrogen</td><td>3570</td><td></td></tr><tr><td>Antimouse IgG</td><td>Invitrogen</td><td>A21236</td><td></td></tr><tr><td>Anti rabbit IgG</td><td>Invitrogen</td><td>A10042</td><td></td></tr><tr><td>Columbus Image analysis software</td><td>Perkin Elmer</td><td>Ver 2.4</td><td></td></tr><tr><td>Spotfire</td><td>Perkin Elmer</td><td>Ver 5.5</td><td></td></tr><tr><td>Clorox bleach</td><td>Fisher Scientific</td><td>18-861-284</td><td></td></tr><tr><td>PlateStack</td><td></td><td></td><td></td></tr></tbody></table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

从高和低控制数据创建两个不同的群体与两个位数超过3个标准差（图7A）的差。该筛选方法的目的是要找到样品群体中的化合物，其值越接近阳性对照群体，假定样品群体（ 图7B中，（i））内的正态分布。数据点，超出平均值的3个标准偏差被认为是从噪声统计上不同的，并且分类为有效的"命中"（ 图7B（ii）中 ，红色框）。被归类为"命中"化合物将受到在未来的研究进一步确认测试， 图7B（ⅱ）示出的阳性命中的样品的群体中检测从所述隔离板的代表性子集。所有4点过值低于（中位数的样本 - 3 *样品的SD）属于这是在4个不同的隔离板发现在不同位置的同一抑制剂。这种抑制剂表现出强大的SNAP-25保护的BoNT / A， 如图5。 <img alt="图1" src="/files/ftp_upload/51915/51915fig1highres.jpg" /> 图1:工作流程中的肉毒毒素/ A HCI测定化合物筛选。 <img alt="图2" src="/files/ftp_upload/51915/51915fig2highres.jpg" /> 图2:该96孔板中的地图为的盐酸测定高对照孔（粉红色）分别以0.5％DMSO处理的，因为只有控制和低对照孔（蓝色）分别与1nM的BoNT / A和0.5％DMSO处理。样品孔（绿色）均采用1nm的毒素和10种化合物的微米0.5％DMSO。外的列和行（灰色）都没有使用，由于不兼容的优化板垫和20X水浸泡的目的的图像边缘井无力。 <img alt="图3" src="/files/ftp_upload/51915/51915fig3highres.jpg" /> 图3:自动化工作站甲板的免疫荧光测定法的设计封闭缓冲液，一级抗体，二级抗体和细胞染色分装在96孔聚丙烯板中，以减少死体积的试剂的损失。 PBS分装在能容纳300毫升托盘。用于废液抽吸歧管显示的插图图像上。 <img alt="图4" src="/files/ftp_upload/51915/51915fig4highres.jpg" /> 图4:屏幕截图含量高成像实验建立的。 <img alt="图5" src="/files/ftp_upload/51915/51915fig5highres.jpg" /> <stro<html>纳克&gt;图5:的SNAP-25裂解位在小鼠ES衍生的运动神经元的高含量成像处理细胞与1nM的BoNT / A添加和未添加1μM的抑制剂（苦楝素）14或不进行治疗，而不毒素。 SNAP-25的BACS抗体检测。在另一个信道，使用β-III微管蛋白的抗体来创建轴突的掩模的SNAP-25的图像分析方法检测神经元。这是从一个给定的井拍摄的16张图像中的一个单一的代表性图像。蓝色表示细胞核用Hoechst 33342染色，红色是BACS信号，绿色为β-III微管蛋白的信号。图片所描述的与Opera获得。尺寸吧:10微米<img alt="图6" src="/files/ftp_upload/51915/51915fig6highres.jpg" /> 图6:屏幕截图显示的图像分析流水线的各个步骤。 的"&gt; <img alt="图7" src="/files/ftp_upload/51915/51915fig7highres.jpg" /> 图7:从人机交互屏幕所产生的数据的统计处理的可视化。 （A）。 （一）箱线图分析，从控制井计算的裂解％的;与它们各自的标准差（n = 16）表现为中间值。粉红色代表着高信号控制器（HSC）;蓝色代表低信号控制（LSC）。 Z&#39;= 0.97（ii）所述相同的值，作为上述（i）的直方图分布来演示两个控制种群之间的距离。中位数和3的SD计算从与相应的盒情节相关联的统计值。（B）中 。 192化合物的分布处理的样品来自相同的实验。 （ⅰ）盒式情节表明相比于对照以及统计学显著异常值的％裂解样品的统计值。 （ii）和（iii）中，在所有的数据群相同的值从屏幕的直方图分布（绿色）。点击数（在红场高亮显示），从有乳沟％值小于3SD从样本中（&lt;25.89％裂解）的中位数的异常值选择。在选择过程中的四命中突出显示（ⅲ）中的所有数据点的直方图具有相似的值，以高的信号控制和表示的相同化合物，掺入的板测试中，公知的BoNT / A抑制剂（苦楝素），该封端的SNAP -25乳沟。

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.6K 次观看.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

视频: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

科研

化学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神经科学

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

癌症研究

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

使用 TLC-Direct 生物自闭造影测定鉴定用于生物控制的天然产物

Bioassay-Guided Identification of Natural Products for Biocontrol by Thin Layer Chromatography-Direct Bioautography

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are antagonistic against a target pathogen. It is a rapid, inexpensive, and simple option for the bioassay-guided isolation and identification of NPs that hinges on separation by TLC coupled with the direct application of a target pathogen to examine bioactivity. It is typically used for the analysis of bioactive plant extracts, detecting inhibitory activity against bacteria, fungi, and enzymes. That being said, it has great potential in bacterial NP discovery, particularly for evaluating bacterial NPs against pertinent agricultural pathogens, which is valuable for discovering and developing novel biopesticides for the agriculture industry. Furthermore, it is a tunable protocol that could be applied to other target pathogens or sources of NPs in research programs concerning the discovery and identification of bioactive compounds. Herein, we describe a model system for discovering and identifying biopesticide NPs using TLC-DB with Bacillus spp. and the agricultural pathogen Sclerotinia sclerotiorum.

作者聚焦:从生物活性土壤微生物衍生的天然产品中发现新的生物农药

We describe the use of thin-layer chromatography direct bioautography assay and liquid chromatography-mass spectrometry to identify microbial natural products that display antagonism against fungal pathogens using the pathogen Sclerotinia sclerotiorum and biopesticidal Bacillus isolates as a model system.

生物测定 - 通过薄层色谱法 - 直接生物自闭法鉴定用于生物防治的天然产物

Thin layer chromatography-direct bioautography (TLC-DB) is a well-established bioassay used to separate and identify natural products (NPs) that are ...

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

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A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

高分辨率人类中脑功能磁共振成像方法

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

听觉皮层在成年猫用高场磁共振成像功能成像

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

生物测定 - 通过薄层色谱法 - 直接生物自闭法鉴定用于生物防治的天然产物

生物测定 - 通过薄层色谱法 - 直接生物自闭法鉴定用于生物防治的天然产物