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?...

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?...

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 de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.5K 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

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

在神经退行性疾病的高内涵筛选

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

医学

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

免疫与感染

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

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 ...

神经科学

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 ...