这项工作是由INSERM的，里尔2大学的支持，MEDIALZ，LabEx（卓越实验室，计划为未来投资）和DISTALZ（创新策略的一个跨学科的方法来老年痴呆症的发展）。 FJ.FG目前的ANR（法国国家研究署/ NeuroSplice去头PROJET ANR-2010-BLAN-1114 01）收件人奖学金，但这项工作也是下授出从JCCM（西班牙）的支持。

1，同质化，并从人类和小鼠脑组织总蛋白抽提<ol><li>脑组织的同质化。均匀化用陶工玻璃（用于人类样品），或特氟隆匀浆器（用于小鼠的样品）的脑组织中的10％（重量/体积）的8M尿素，2M硫脲和1％w / v的SDS缓冲液（UTS）。超声在60 Hz 30脉冲申请0.5秒，共组织解体13，14一个超声波发生器。 <ol><li>测定蛋白浓度与Bradford法，以BSA作为标准。这UTS缓冲器提取是用一维SDS-PAGE上完全兼容，只要溶解物没有过度加热，以避免氨甲酰15。 </li><li>添加1％（重量/体积）的Amberlite时及在室温下10分钟并轻轻搅拌下孵育。此后过滤到0.22微米的针头式过滤器，最后加SDS的尿素/硫脲溶液。此步骤会降低尿酸和异氰酸的量，这是在equilibr与脲鎓溶液中，并促进其降解。 </li></ol></li><li>氯仿/甲醇沉淀蛋白。蛋白质之间100-150微克沉淀为11厘米IPG胶条和1-1.5毫克18厘米的人（独立于选定进行IEF的pH值范围内）。 <ol><li>执行在冰上或4°C的所有步骤凉爽的氯仿和甲醇，在4℃下开始沉淀步骤之前30分钟。 </li><li>添加三卷甲醇和氯仿一个卷UTS的裂解液中的一个量。手动摇动混合物，并添加三个体积的冷水。中1分钟，并通过离心在4℃下沉淀的蛋白质以12,000×g离心30分钟涡旋</li><li>用巴斯德吸管弃上清，加入3体积的冷甲醇。旋涡再次在30分钟内于4℃下12,000×g离心最后，弃上清，干燥在N 2沉淀。 </li></ol></li><li>制备蛋白质二维分析。重悬蛋白质干燥的粒料在2D-缓冲液（8M尿素，2M硫脲添加有4％CHAPS）。用Amberlite如上文所述洗解决方案，并尽量保持在500μg蛋白的比例每200微升的2D缓冲2D-DIGE。注意:2D缓冲器可以存储在-80℃或在一周期间保持在4℃（以防止尿素降解）。 <ol><li>声处理和存储的样本在-80℃下，直到进行下列步骤。值得注意的是，该协议可用于蛋白质的甲酸溶解汇总10后。简言之，将蒸发甲酸N 2下，重悬所述沉淀。 </li></ol></li></ol> 2，2D-DIGE <ol><li>样品测​​试质量。剂量样品再次用Bradford方法。稀释15微克蛋白质在LDS样品缓冲液，加热，在37℃和15加载到4-12％的聚丙烯酰胺凝胶。 </li><li>考马斯亮蓝染色。染色聚丙烯酰胺凝胶用0.1％（重量/体积）考马斯亮蓝蓝G-250，50％乙醇和10％下至少1小时（V / V）的乙酸溶液。让背景在7％乙酸和10％（体积/体积）乙醇溶液脱色过夜。 </li><li>样品的预电泳标记。在此之前进行的花青染料标记，确认样品的pH值，这应该是基本的或什至中性，因为N-羟基取代是在碱性pH下更有效和给定是最佳pH为8.6。 <ol><li>标签微创两个样本研究用Cy3或Cy5的荧光染料与50微克protein/400皮摩尔染料的比例，并保持1小时，在4℃下在黑暗中，以促进与未质子化胺的花青的NHS酯反应性基团团体赖氨酸。 </li><li>降低样品的变化作出的横标签用Cy3和Cy5染料，避免了一个样品的优先耦合到一个特定的花青。注意，花青染料最小标记可以为少量的蛋白质可适于与保持的1微克的比例公关每8皮摩尔花菁染料otein。如果是这样，微型2DE系统可用于代替大量的2D凝胶系统。这将使用于少量脑组织的2D-DIGE分析。 </li><li>标记两个样品池与的Cy2荧光染料相同量的蛋白（50微克总数）和作为内标物。使用如前面所指出的相同的条件。 </li><li>要完成总体积350μL与2D缓冲液含1.1％Destreak试剂的IPG缓冲液1.2％和溴酚蓝总150微克标记的蛋白（50微克的的Cy2，50微克的的Cy2 Cy3标记和50微克）的痕迹。在使用四个独立的带一式四份执行此步骤。此外，准备2个非标记的条带（每个样品）与350-450微克蛋白质的制备凝胶。 </li><li>离开覆盖矿物油通宵被动补液六加载条。 </li></ol></li><li>等电聚焦。开展IEF在206，C。对于pH值3-11NL，18厘米剥离协议是:最大电流设置为50μA/条中8个阶段:1）150 V 1小时步，2）200 V为5小时，3）500 V梯度一步2小时，4）1,000 V梯度为2小时，5）2,000 V梯度为2小时，6）4,000 V梯度为2小时，7）8000 V梯度为2小时和8），总共24000 V·小时步。 IEF之后，用色谱纸调度过量的矿物油并于-20℃下储存，​​直至条带的第二维。 </li><li> IPG胶条平衡。平衡条在6M尿素，2％SDS，30％甘油，50mM的Tris-盐酸的pH 8.6缓冲液中的DTT在15分钟内1％和之后用碘乙酰胺，在相同缓冲液中的4.7％，但没有DTT。 </li><li>第二个维度或SDS-PAGE电泳。使6 12％SDS-PAGE凝胶（1.5M的Tris-HCl pH 8.6的12％丙烯酰胺/双丙烯酰胺，0.1％（重量/体积）SDS，0.072％（重量/体积）的APS和0.1％（V / V）TEMED ）在使用大型2D系统，有四个低荧光玻璃板的STRI同时PS荧光样品和其他两个玻璃板与以切除蛋白质点厚度为1.5mm的制备凝胶。 <ol><li>加载（重量/体积）琼脂糖的6 IPG胶条在凝胶的顶部，并通过层叠具有0.5％。运行缓冲液是由25毫摩尔Tris，192 mM的甘氨酸和0.1％（重量/体积）SDS。执行迁移在2.5 W /凝胶过夜用的制冷系统设定为4℃下16,17。 </li></ol></li><li>荧光图像采集和分析。使用一个台风9400扫描仪获取的荧光凝胶图像（12图像的一个实验）。扫描的Cy2，在五百二分之四百八十八纳米，五百八十分之五百三十二nm和六百七十○分之六百三十○nm的激发/发射波长，分别Cy3和Cy5图像的每个凝胶。用信息学软件分析图像。数据代表的斑点体积的与信号的整个4凝胶研究了两种样品之间的分配相关联的变化。 </li><li>考马斯亮蓝染色大的凝胶。修复制备凝胶中50％（V / V）乙醇和3％为至少24小时（体积/体积）的正磷酸溶液。工作后，用超纯水清洗两次，每次20分钟。在34％的执行着色（体积/体积）甲醇，17％（重量/体积）的硫酸铵和添加0.1％（重量/体积）考马斯亮蓝G-250前的2％的正磷酸1小时。保持凝胶的颜色至少48小时，并在脱色的超纯水。 <ol><li>斑点的质谱鉴定。一旦荧光图像进行分析，该软件提供的斑点，其表达在统计上是不同的列表。手动从制备凝胶切除这些斑点。此过程需要实践和一些手巧，以避免角蛋白污染。然后样品可以保持在水中，在-20℃，直到发送到MS。点的识别是用串联质谱（MS / MS）和蛋白质身份的相关完全相容是根据概率基于MOWSE得分为0.05（P &lt;0.05），18 P-值计算来判断。 </li> &lt;/ OL&gt; </li></ol> 3，定性小型二维超声心动图测定<ol><li>重悬最大100微克的二维缓冲蛋白质样品，直到200微升的最终体积为11厘米IPG胶条。万一过量表达系统或纯化的蛋白质，起始量可以降低到20微克。 </li><li>添加1.1％（V / V）Destreak试剂，0.55％（体积/体积）的IPG缓冲液和溴酚蓝的痕迹，以200微升的最终体积。然后，涡流，使快速自旋并加载到IPG胶条进行被动补液（使带至其原始厚度）过夜覆盖矿物油。注:IPG缓冲液的比例是相同的pH值所需的（pH值3-11，4-7，7-11，等等）的所有的间隔，但其组合物中所选择的pH范围内的函数而变化。 </li><li> Isoelectrofocalisation。在20°C下进行的IEF该节目的持续时间取决于所选择的pH值的时间间隔。值得注意的是，参数如电渗可能会干扰IEF更新和在这些情况下，在electrofocalisation时间需要优化19。注意:它不一定是一个较长的IEF，可提供更好的结果，但缩短IEF的时候也可以提高分辨率。 IEF之后，IPG胶条可以储存在-20℃下几个月。 </li><li> IPG胶条平衡。平衡的条带中含有25mM的Tris-HCl pH值为6.8，20mM的DTT，10％甘油，5％SDS，0.05％溴酚蓝的缓冲器。做15分钟3平衡沐浴不断变化的每一个沐浴间的缓冲溶液。 </li><li> SDS-PAGE电泳。用聚丙烯酰胺根据目的蛋白的分子量选择的百分比层IPG胶条到预制凝胶（IPG +1很好，11厘米IPG胶条）。然后，吸干凝胶到硝酸纤维素/ PVDF膜在100毫伏时33分钟。 </li><li>与所需的抗体孵育过夜，用过氧化物酶活性可视化。使用由二次抗体透露无关的多肽进行免疫印迹的路线。达到半quantit由量和强度的痕迹/斑点与ImageJ的软件，密度ative分析。 </li></ol>

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发现蛋白质的表达病理变化，寻找生物标记物和药物靶潜在途径的调节是其中neuroproteomics的目标接近30。在一片新兴的工具，在二维超声心动图场增加了一个有前途的预想。然而，一个共识应以尽量减少可变性和提高重现性在实验中达到。在这种思想的行标准化协议来执行2D-DIGE（ 图2）和随后的2DE免疫印迹（ 图3），对人类和小鼠的脑组织与一维免疫印迹完全兼容本文中详细描述。 其中最大的难题在蛋白质组学是溶解缓冲液的选择。为了增加不同的方法之间的再现性，UTS被选择，因为它是用IEF完全兼容。此外，UTS提取缓冲液产生任何PELLET或离心12,000×g离心后真的稍微一（不与SDS沉淀在4℃混淆）。这是一个关键点要考虑到许多神经退行性疾病存在的蛋白质聚集，包括AD。事实上，没有沉淀简化了性能和数据解释，因为没有独立的研究应该为可溶和不可溶级分来完成。 无论2D-DIGE或mini-2D的方法将要进行的，也有一些限制，应当说明。首先在UTS缓冲器的化学性质，这种解决方案不能被过热，或氨甲酰化发生在伯胺，因此可以用两个花青染料耦合和2D轮廓干涉，通过削弱IEF 15恶化的点分辨率。其次，现有的pH值范围，时下最宽的pH范围是其中3至11，这个因素加入到，并非所有的蛋白质进入到IPG胶条可勘探的概率泉层叠蛋白条带在染色后的凝胶的两侧。这种限制可能会阐述为什么不是整个蛋白质可以通过二维进行研究。另一个重要的限制是争议使用的沉淀。一方面，这是非常可取的做沉淀步骤，用氯仿/甲醇，使有效地降低脂质含量27，它们的盐，核酸和带电清洁剂可能干扰IEF参数和耐冲击的分辨率和/或再现性2D。此外，另一方面也不能排除有的蛋白质牢固地附着于膜可能会丢失。然而，在其他萃取缓冲器是用来代替UTS，具有或不加入蛋白酶或磷酸酶抑制剂的情况下，利用沉淀步骤的高度IEF期间被推到任何缺陷。最后，值得注意的是，2D-DIGE图像分析介绍了荧光重叠的不便斑点可能会导致信息丢失，因为软件的分析并不认为这就像一个显著的变化。 这种组合方法的新颖之处在于它们的重现性，多功能性和蛋白质表征。只有一个提取缓冲液允许一个执行两个互补的技术。 2D-DIGE提供有关蛋白质表达的变化及其随后的鉴定MS / MS定量信息。然而，它是可能的异构体，isovariants，酶原，翻译后修饰和分解代谢产物不能自重叠的荧光与其他蛋白质被识别。为了克服这个限制所以建议在并行使用微型二维超声心动图。事实上，就我们所知，可以承诺的最重要的缺陷之一是采取单维免疫印迹作为2D-DIGE结果的一个完整的验证，甚至认为任何蛋白质组学修改是没有必要的验证。 Furthermore，迷你2DE的技术优点是:1）使用小的SDS-PAGE系统，2）所需的相对低的材料，3）可在广泛的pH值范围，4）的免疫印迹的感性和5）容易估计的可行性变化。迷你2DE技术的最挑剔的方面是量化，尽管微型2DE不能被认为是一个定量的方法，后斑点或重复对准它可能建立的酸性和碱性部分或反之之间的比率，家具这样的可靠估计的变化观察到9。 在文献中一些数据准确使用微型2DE来完成蛋白质的表征，如过度表达的蛋白具有和不具有翻译后修饰为路易体痴呆32,33，并在图3A中对一个描述31，低聚和降解途径AD患者。的信息的另一个例子用m提供<html> INI二维超声心动图是在非痴呆和AD的情况下接种疫苗的方法34的潜在目标进行β-淀粉样物质的截断。低聚物和其降解差异的存在也表示在图3B根据一个散发性或家族性AD的患者。此外，微型二维超声心动图的方法打开了一个广泛而实用的可能性的窗口，因为不同的治疗方法可以执行。例如在公元领域，磷酸酶抑制剂可以添加和发现的Tau蛋白质磷酸化的影响。还药理作用的药物上的蛋白水解途径，考虑到等电点和分子量鉴定的截短形式可根据抗体的抗原决定簇34提供的切割位点。有趣的是，最近的数据都使用mini-2DE生活方式和药物治疗与认知障碍的小鼠模型的关联，以及一个新的头突变35-37的生化指标阐明。 ntent"&gt;的方法使脑组织的2D-DIGE从人类或小鼠的起源，以及使用mini-2DE背后的验证取得的发展，可能会揭示出成发现新的药理指标和生物标志物神经系统疾病的研究。这些疾病有其原产地在大脑中的事实，迫使研究这个器官是一个理想的信息来源，但人类样本是有限的，所以使用的动物模型成为不可缺少实现这一目标。这里使用的方法的重要性平行于这两种类型的样品和验证结果。期望的是，在不久的将来可靠的候选人将被发现并在肉体体液如血浆和脑脊髓液进行测试，以便建立一个早期诊断的疾病的进展，评价和最终可用的似是而非的治疗方法进行评价。 在协议中的关键步骤，必须考虑到它的suitab乐应用。在2D-DIGE的情况下， 样品测试质量是一个关键点。这个测试许可证知道蛋白质概况和验证蛋白质的真实数量提取物沉淀后现有的2D缓冲区。彩色型材给我们关于样品之间的质量和均匀性的信息。只有具有相似的模式标本必须用于2D-DIGE研究，否则2D-DIGE可能导致染色差和​​蛋白质谱分辨率差。在这些样本模式的差异是人类比老鼠的脑组织比较常见的，因为人脑样本进行研究处理的诸多不便38,39。此外，蛋白质降解，由于验尸间隔的标识已经被报道了2D-DIGE 40,41。花青染料标记前的pH为验证，这一步必须削弱所有后路程序。 花青染料标记和SDS-PAGE电泳 ，应在黑暗中进行如尽可能地，为了不影响荧光标记。最后，聚丙烯酰胺凝胶中必须尽可能均匀其中，为了电泳迁移过程中，以消除相互变异性和方便的重叠部分和事后分析42,43。

精神和神经紊乱代表全球疾病负担的13％，如病理生理机制，危险因素和前驱的生物标志物的新挑战，必须探索1。为配合这一目标，人类大脑的蛋白质组学研究成为不可或缺揭开参与像记忆，行为，情绪和神经元的可塑性，例如，不仅对生理也为病理状态过程的分子途径。因此，在使用动物模型的更具体的转基因小鼠，带来了广泛的可能性，以模仿人类神经退行性疾病2的病因。 蛋白质组学方法是时下为了实现这些新的观点在神经科学领域。二维凝胶电泳（2DE）是一种有效的和可能的简单的方法，使比较大范围的样品的蛋白质组。此外，它也是一个强大的方法，以便确定从复杂混合物中分离出的蛋白质和通过质谱法进一步分析。这种技术基本上是由在两个连续的步骤:根据其等电点（pI）1）蛋白的分离通过等电聚焦（IEF）。更精确地说，将电势穿过固丙烯酰胺条施加之间的pH梯度，然后将蛋白质转移和集中在其全球净电荷的函数所确定的等电点。 2）等电聚焦蛋白质变性并通过加入十二烷基硫酸钠（SDS带负电荷），从而在其第一结构的蛋白质根据它们的表观分子量（MW）用SDS-PAGE 3分离。这两个不同的特性使我们能够解决一个double值在蛋白质组研究中走得更远。一方面这种方法提供了可能用最小的染料2D-DIGE法进行定量分析，并在另一方面一个qualitativE解析由微型二维超声耦合到西方墨点法。 通过2D-DIGE定量分析赐蛋白表达的变化遍布样品蛋白质组。简言之，将样品标记有3花青（CY 2，Cy3和Cy5）的发光在三个不同的波长（蓝色，绿色和红色）。含有N-羟基琥珀酰亚胺酯基团，这些氟最小的染料发生反应以产生共价酰胺键4蛋白的赖氨酸残基的ε-氨基。赖氨酸残基被标记之间只有1-3％，从而防止多个标签除了每蛋白质和主要净电荷修改5，6。 Cy3和Cy5经常使用的Cy2，而标签的样本来比较相同比例的组合来标记两个独立的样本。两个主要的优点是，所有标记的​​样品混合并IEF和SDS-PAGE均在1凝胶在一次为每个步骤，避免了相互之间的变异性是由于实验来胶凝比较。此外，它提出的大约1毫微微摩尔蛋白7的高检测阈值。凝胶进行扫描和2D软件比较2D凝胶的荧光图像，其中的Cy2作为内标，允许鉴定的斑点为他们的后路鉴定通过质谱法之间的统计学差异。使用内部标准的2D分析软件实现了快速检测的超过95％的统计置信8的采样之间的差值小于10％。 由微型二维超声心动图的定性分析是蛋白质鉴定的关键一步。其原理是如前对等电点和分子量分离所描述的相同，但在这种情况下，蛋白质是从一个小的聚丙烯酰胺凝胶转移到膜和免疫印迹之后被执行。而一维凝胶电泳提供改变蛋白质表达为一个或多个表位的蛋白质在功能上的抗体，该资料N迷你 - 二维超声心动图赋予了两个额外的参数。首先，蛋白质isovariants在pI值的函数改变，表明翻译后修饰可能发生。其次，质谱鉴定可能指示合理的酶原和蛋白质的分解代谢产物。因此，通过2D-DIGE观察到的修改很可能表明相关变化的全球蛋白质组配置文件的机制。几个样品为同一蛋白表位/秒由微型双向电泳免疫印迹之间的走线可能反映酸度变化脱落光成翻译后的变化观察到轻微的，甚至不是由单维免疫印迹9,10。此外，这种分析通知关于由于代谢残基11,12的pI和分子量的知识的潜在切割位点。 这两种技术的结合，提供了一个互补的蛋白质组学分析。一方面2D-DIGE提供了一次典型电子商务的差异使多肽的表达是不同的精确分离。这些差别主要由成斑或给定的一个用荧光凝胶的软件分析的强度的增加/减少的出现或消失。然而，这些观察结果本身不太可能解释所观察到的变形的性质。由于这些原因，一旦该多肽分离和质谱鉴定，采用微型2DE使得能够精确地确认1）中分离的蛋白的同一性和差异的2）的性质:变化中的isovariant /亚型水平表达，翻译后修饰和裂解过程为实例。然而，有必要开发一种开始裂解缓冲液都用2D-DIGE和与微型2DE兼容，以便限制从使用提取的协议是非常不相似而产生的潜在的分散体。 在本文章中，我们取消刻划一个适于协议用于2D-DIGE和微型2DE技术脑蛋白从人类和小鼠组织中来制备，萃取和性能。

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consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting

二维凝胶电泳（2DE）是一个功能强大的工具来揭示蛋白质组的修改可能涉及到不同的生理或病理条件。基本上，这种技术是基于根据其等电点在第一步骤中，根据它们的分子量经SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）的蛋白质的分离，其次。在这份报告中对人类的验尸和小鼠脑组织中少量的优化样品制备协议描述。此方法使得能够同时执行二维荧光差异凝胶电泳（2D-DIGE）和迷你2DE免疫印迹。这些方法的组合，使人们不仅发现新的蛋白质和/或蛋白质修饰在它们的表达由于其与质谱检测的兼容性，又是一个新的洞察标记验证。因此，微型2DE耦合到西方墨点法证识别和验证后翻译后修饰，蛋白质分解代谢，并提供在不同的条件和/或治疗方法的定性比较。本文中，我们提供了研究中的AD和路易体痴呆，如β-淀粉样肽和α-突触核蛋白中找到的蛋白质聚集体的成分的方法。我们的方法，因此可以适用于蛋白质组的分析和不溶性蛋白质从人脑组织和小鼠模型中提取了。同时，它可提供用于所涉及的神经变性疾病以及潜在的新型生物标志物和治疗靶标分子和细胞途径的研究的有用信息。

对脑组织蛋白质组学仍然具有挑战性，因为没有理想的缓冲区存在收回100％的蛋白质，特别是膜相关或细胞骨架蛋白。在第一组实验都集中于寻找与这两种方法和使以回收蛋白质的大面板兼容合适的裂解缓冲液。因此，3裂解缓冲液进行测定，以确定最合适的一个。首先，它是用来将共同的生化和分子生物学缓冲的Tris-HCl 20在10mM的含1％（重量/体积）SDS（图1A）。此外，Tris-盐酸具有强大的应用如聚丙烯酰胺和琼脂糖凝胶电泳缓冲液中。其他两种裂解缓冲液为UTS（图1B）和2DE 1。的Tris-SDS产生一个贫穷的分辨率和斑点的数目被大大降低（图1B）在与悉尼科技大学，其中一个高点分离，无畸变和FA观察到的分布比较Ř更好回收蛋白的观察（图1B）。这一点是由悉尼科技大学萃取率几乎没有残留颗粒，而的Tris-HCl欠了厚厚一本板凳离心后的事实支持。该图案显示在图1B清楚地给出了关于其他缓冲液如的Tris-HCl在UTS较高的蛋白质溶解度的证据。 Notheworthy，即使2DE缓冲器也被测试，它的使用，因为排除了尽管沉淀既不观察，脂质不溶于它们留在上层difficulting的操纵。与这些数据一致，则建议使用UTS缓冲几个原因1）它的中性离液剂是通过破坏nonconvalent和离子键的氨基酸残基之间，并避免蛋白酶的降解细胞蛋白21 2）另外的活性蛋白质变性硫脲的脲溶液的增强显着的疏水性膜蛋白的溶解性和蛋白质容易发生聚合22,23和3）的阴离子洗涤剂的SDS的红利破坏脂质通过疏水相互作用结合的蛋白质，以及增加的蛋白酶和磷酸酶的活性抑制24,25。此外，它已决定增加一个沉淀步骤，消除impureties可能扰乱IEF。以这种方式，如沉淀法确定26和甲醇已被描述为是最合适的溶剂中，以除去脂质这是丰富的脑组织中的蛋白质的溶解度，这给我们带来了可供选择的氯仿/甲醇沉淀27。两性离子洗涤剂CHAPS（3 - [（3 -胆酰胺丙基）二甲氨基] -1 -丙烷磺酸酯）的利用，用于重悬沉淀的蛋白质在二维缓冲器是由于其适合用于促进在第一维步骤28中的蛋白质的入口。 人类和小鼠的大脑公关的个人资料oteome通过2D-DIGE是分别示于图2A和2B。在图2A合并荧光图像从人来控制和AD皮质样品可以观察到，在图2B一事不再理的小鼠样本的AD样病理头村29模型的海马。 CY2，Cy3和Cy5分别示出用于图人体样品2C-2E。两者合并的图案（ 图2A和2B）呈现高光点的分辨率反映了高品质的等电聚焦，并与在图1B中所观察到的更新协议的第二维分离。这点画质一起被以消除蛋白质的一种可能偏好某一特定花青，使这些荧光图像（ 图2C-2E）很容易在整个软件分析对齐进行交联的事实。此外，制备凝胶染成蓝色有限公司omassie证据具有丰富的斑点遍​​布利用的pH值范围，允许一个经过软件分析，并为他们鉴定后通过MS / MS切除点一个丰富的蛋白质图谱。 通过免疫印迹法对蛋白质进行定性迷你2DE分析提供了它的识别和修正高价值的信息，不仅是对翻译后的，但也为低聚物和截短（ 图3A和3B）。在小型二维超声心动图进行该技术的可行性提供了有关感兴趣的蛋白质快速和精确的数据。关于从患者受路易体痴呆（LBD）组织，α-突触核蛋白的表征能够看到一个良好定义的4-7的pH分布，其中天然蛋白是在4.67电点和18 kDa的分子量，然而与微型2DE可以进一步看到二聚体（ 图3A中，星号 ）的在同一pI值比天然形式存在，但也UBIQ的存在uitinated形式，是更基本的和具有较高的分子量时更泛素化它们（ 图3A中，箭头 ）。密切相关的广告的另一蛋白质是β-淀粉样肽，由淀粉样前体蛋白（APP）的复分解代谢产生的。在图3B它相比的散发性AD的情况下用一个家族一个阴谋。在家族性AD的情况下，可以观察到如何β-淀粉样1-42减小相对于散发性AD，而且，该isovariantsβ-淀粉样的x 42（4 kDa的）也被下调遍布间隙，同时在8 kDa的发现的低聚物反映这一事实不是由于蛋白质的量降低，因为斑点强度是在家族性AD（ 图3B）更相关。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51339/51339fig1highres.jpg" width="500" /> 图1:二维超声公关ofile中的10mM Tris 1％SDS w / v的缓冲器提取（A）和UTS利用率（B）后所得到的图案。之后在面板乙可以看出怎样的斑点，强度和分辨率的数目比1高得多面板（A）。使用UTS允许几乎总蛋白的提取，它体现在二维超声心动图考马斯亮蓝染色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51139/51139fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51339/51339fig2highres.jpg" width="500" /> 图2:2D-DIGE访问人类（A）和小鼠（B）示出了图像（A）和（B）所代表的合并图像为三花青在为18cm条状一个3-11的pH范围内。在图片中（C），（D）和（&lt;STRONG&gt; E）的花青独立暴露（CY蓝色，Cy3标记为绿色和Cy5红色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51139/51139fig2highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51339/51339fig3highres.jpg" width="500" /> 图3:在上部面板可以观察到在LBD患者的蛋白α-突触核蛋白的微型2DE更新本机的pI（4.4）和在二聚化（星号）的函数分子量（18 kDa）的变化，其中兆瓦。是双层的，泛素化，从而改变电点，直到一个更基本的一（箭头）（A）。向下的图片揭示AD的零星和遗传情况的β-淀粉样肽抗体的轮廓。 2DE图案示出了根据这种病的病因如何低聚和降解发生在以不同的方式酶。这种免疫印迹清晰地指出了这两个条件之间的APP代谢的差异。一方面是在关于家族1（ 箭头 ）的散发性AD的增加分解代谢产物中的，而低聚物似乎不太明显（B）。这些实验已经11厘米的长条分别执行在一个12％的Bis-Tris凝胶4-7 pH值的差距，并在16.5％的Tris-甲基甘氨酸一（A，B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51139/51139fig3highres.jpg" target="_blank">点击这里查看大图。</a>

Watch this Scientific Journal Video about Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting at JoVE.c

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

用尿素/硫脲/ SDS缓冲液为人类和小鼠脑组织中一种常见的蛋白提取协议允许由蛋白质2D-DIGE并随后将其表征迷你2DE免疫印迹鉴定。这种方法使一个人从活组织切片检查和实验模型获得更多的可重复性和可靠的结果。

共识脑源性蛋白，人类和小鼠脑蛋白质组使用两个2D-DIGE和Mini双向电泳免疫印迹法研究提取协议

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

consensus-brain-derived-protein-extraction-protocol-for-study-human

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.0K 次观看.  Inserm UMR 837. 用尿素/硫脲/ SDS缓冲液为人类和小鼠脑组织中一种常见的蛋白提取协议允许由蛋白质2D-DIGE并随后将其表征迷你2DE免疫印迹鉴定。这种方法使一个人从活组织切片检查和实验模型获得更多的可重复性和可靠的结果。 

视频: Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

科研

神经科学

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

研究风味编码的新方法

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

果蝇岛试验中运动行为的高通量分析

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...