作者FCO，DF，JN，DJH和GDS的工作上面提到的由由英国癌症研究中心实验癌症医学中心的首席科学家办公室，项目编号：ETM37和支持。 AL的工作是由皇家爱丁堡外科的护理和治疗癌症和医学研究理事会罗伯逊信托，梅尔维尔信托。 IO的支持，玛丽·居里行动和英国医学研究理事会共同资助的英国皇家学会的爱丁堡苏格兰政府奖学金。作者想感谢SCOTRRCC共同申请人及其合作者提出了有用的讨论本文讨论的主题。

1。形态学和分子肿瘤的异质性鉴定<ol><li>肿瘤从-80℃的冰箱中取出并保持在干冰上。 </li><li>划分成部分的约1厘米3的肿瘤。映射每个肿瘤的部分彼此相对，并与一个唯一的名称标签的原始位置。将样品存放在个别的离心管中，在-80℃，直到准备使用。 </li><li>涂层样品在OCT和减少在低温恒温器在-22°C。 </li><li>样品染色，苏木精和曙红counterstaining方法。 </li><li>显微镜分析冰冻切片，以确保他们是CCRCC性质，可行肿瘤和分级（低年级，高年级或混合低/高档次，具有挑战性的，以区分富尔曼等级1到4个冰冻切片）。 图1一个与B̶（H＆E染色高，低和混合级）。 </li><li>最多选择4个样本的从各形态不同的区域内每个肿瘤蛋白提取。 </li></ol> 2。从肿瘤样本中提取蛋白质<ol><li>切肿瘤样本华侨城。 </li><li>将50-75毫克组织放入2 ml离心管，用990μl的裂解缓冲液[50 mM Tris-盐酸（pH值7.5），5mM的EGTA（pH为8.5），150mM的NaCl]补充有抑肽酶（Sigma公司A6279）（10毫克/毫升），磷酸酶抑制剂鸡尾酒2（西格玛，P5716），磷酸酶抑制剂鸡尾酒3（Sigma公司，P0044）和蛋白酶抑制剂鸡尾酒（罗氏，11836153001）。 </li><li>添加一个5毫米的钢球，每个试管中，在50Hz 5分两次使用TissueLyser的水平的同质化后，每5分钟的时间内，检查和同质化。 </li><li>匀浆后的样品转移到新的2 mL离心管，用吸管离开钢球落后。 </li><li>在离心之前在4℃下以13,000 xg离心30分钟，向每个样品中加入10μl的Triton X-100的</li><li>转移上清到新鲜的离心管中。 </li><li>确定蛋白质浓度用BCA方法（ 图2）。 </li><li>标准化蛋白浓度为1毫克/毫升。 </li></ol> 3。抗体验证<ol><li>制备蛋白质样品（从合适的细胞系或组织中提取的），Western blot和10％的SDS-PAGE凝胶上运行。 </li><li>到硝酸纤维素膜过夜转移样品在4℃下</li><li>座中的膜锂肺心病奥德赛阻断缓冲液（在PBS中稀释，50:50），在室温下1小时。 </li><li>李-COR Odyssey封闭的缓冲稀释一抗（50:50 PBS稀释）在制造商推荐的稀释通常为1千。 </li><li>在4℃下孵育膜在初级抗体过夜</li><li>上涨0.1％PBS-吐温20（PBS-T 1毫升吐温20 / 1L PBS）。 </li><li>在室温下搅拌5分钟（×3），用PBS-T洗膜。 </li><li>稀释荧光标记的二抗奥德赛封闭缓冲液（50:50 PBS稀释）0.01％SDS于1:10,000稀释液（1.5μl/15毫升）的。 </li><li>在二次孵育膜抗体在室温下轻轻摇动45分钟 - ，重要的是从这样的时间的光，直到保护膜，因为它已被最终扫描。 </li><li>在室温下用PBS-T洗膜5分钟（×3），在黑暗中保持膜。 </li><li>在PBS中洗膜，在室温下搅拌5分钟（×3），以除去残留的Tween20的，再次保持在黑暗中的膜。 </li><li>躺在膜平放在一块滤纸在黑暗中，在空气中干燥 - 干的膜增强信号，降低背景，但它是无用的剥离和重新探测。 </li><li>扫描膜上的LICOR的奥德赛扫描仪。保持膜，然后在黑暗的时间，因为它已被扫描。 </li><li>仅选择的抗体，产生一个单一的主要带在正确的分子量。 图3示出了用于与RPPA可接受和不可接受的抗体的例子。 </li></ol> 4。 RPPA小学nting <ol><li>蛋白裂解液点在硝酸纤维素镀膜玻璃幻灯片（Fastslides Whatman公司）使用微网II机器人的检举。所使用的幻灯片中包含2片上的样品被发现。每个焊盘被察觉与相同的样品，在这种情况下，100个样品。其他可用的格式包括：1，第8和16个垫幻灯片。越高的焊盘数量的小的样本数，可以察觉到每个。 </li><li> 5 2 - 倍稀释液A系列作了从每个样品中，然后发现在每个一​​式三份（导致每个样品中的总共15个斑点）。 </li></ol> 5。 RPPA蛋白检测<ol><li>湿幻灯片超过LICOR封闭液（50:50 PBS稀释）。在RT下孵育1小时上摆动平台。 </li><li>准备800微升的初级抗体LICOR阻断缓冲液（在PBS中稀释，50:50）中，在所需浓度，并保持在冰上。 </li><li>安装：（i）本框架芯片夹中的幻灯片或（ii）&#39;FastFrame&#39;四槽滑动架，以便紧密的密封之间形成的滑动和孵化室。 </li><li>清除残留的缓冲区中井和各孔加600μl的主要抗体。 </li><li>放置成一密封的湿盒和摆动平台4℃过夜孵育滑动和室℃。 </li><li>化妆0.1％PBS-Tween20的PBS（PBS-T;100μl的吐温20/100毫升PBS中）。 </li><li>删除幻灯片从寒冷的房间，小心地取出每口井的主要抗体。 </li><li>加入600微升的PBS-T洗上滑动，在RT下5分钟（X3）的摆动平台。 </li><li>准备荧光标记的第二抗体，稀释在Odyssey封闭缓冲液（在PBS中稀释，50:50）0.01％SDS在1:2,000稀释液（1微升/ 2毫升）中的第一个实例。 </li><li>删除缓冲区中井和各孔加600μl荧光标记的二抗。二次抗体，在室温下孵育45分钟，轻轻摇动 - 它重要的是，直至时间，因为它已被最终扫描的光保护膜。 </li><li>删除从以及，并简要洗涤（×3），在室温下在600μl的PBS-T中的二抗。从载体中移除滑动，转移到一个合适的容器中，并在过量的PBS-T洗15分钟，保持在黑暗中的膜。 </li><li>删除的PBS-T，并进一步在室温下用PBS洗膜15分钟，以除去残留的Tween-20的，再在黑暗中保持膜。 </li><li>在50°C的烘箱中10分钟，然后擦干Fastslide LI-COR的奥德赛扫描仪扫描。保持在黑暗中的幻灯片，直到它被扫描。 </li><li>在680 nm和/或800nm​​的根据二次抗体/抗体用于扫描的滑动。对于两色的检测一直使用高度交叉吸附的抗体，以减少交叉反应。仔细选择两色检测原发性和继发性抗体是必要的。最重要的是选择种不同的主机（ 例如，兔和小鼠）的两个主要的抗体。这允许与易于分辨的发射光谱染料标记的抗兔抗小鼠二次抗体的歧视。 </li><li>图像文件保存为TIFF文件。 图4（图像扫描的文件）。 </li></ol> 6。数据分析<ol><li>中启动MicroVigene软件（VigeneTech，卡莱尔，MA，USA）。 </li><li>打开TIFF图像文件包含扫描的RPPA幻灯片。 </li><li>选择预定义的模板文件，该文件将有一个网格，覆盖在图像上的RPPA幻灯片。 </li><li>点击定义区域的利息 （ROI）按钮，调出网格。 </li><li>网格定位在RPPA点。 图6a（网格图像的图像）。 </li><li>点击全选按钮以突出显示所有的投资回报率。 </li><li>单击“ 查找全部”。 MicroVigene将AUTOMatically的投资回报率，发现斑点，减去的背景下，清除所有灰尘和量化点。 </li><li>点击查看稀释曲线“按钮，将所有的样品上的RPPA的幻灯片的结果。 </li><li>单击“ 保存”稀释数据 。 </li><li>各一式三份，因为每个样本被印在星稀释点有15个点来分析，从而降低了发生错误的风险，提高的曲线拟合的质量。 MicroVigene产生四参数的逻辑对数模型“Supercurve”算法（ 图6b），结合点，以产生抗原抗体结合动力学的S型曲线。的假设是，相同的抗体-抗原结合动力学发生在每个采样点，即使在不同的样品，从而可以增加通过采取在阵列上的所有点，以适应一个共同的响应曲线的曲线拟合10,11的信心 Y = A +（（BA）/（1 + E（C * D-LN（X））） 其中x是dilutio的n因子和Y信号的强度。 样品可比较分析使用的Y0值，这在我们的分析到supercurve映射后的x值的中点对应的y值。 </li><li>情节Y0在Microsoft Excel中的数据导出， 如图7所示 。 </li><li>方差分析框架中未处理和处理过治疗的患者中，肿瘤内蛋白质的变异分别计算。结合从所有分析蛋白质的数据的方差分布进行了比较由Mann-Whitney检验（MWT）。瘤内个别蛋白质的差异进行了比较，通过F-检验，正态性和方差齐性的假设认为，分别评估使用Lillefours和Fligner测试;错误发现率（FDR）校正12。未处理和处理病人样本之间的差异蛋白质表达对于每个蛋白质进行了测试，使用学生的t检验，正态性和方差齐性的假设S分别为满足，否则MWT进行FDR应用在组合的t检验和MWT值12。蛋白表达的意义和方差Pearson相关的蛋白质，估计使用标准的方法[R参考和FDR应用12。 </li></ol>

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没有利益冲突的声明。

RPPA这里介绍的方法代表一个高吞吐量的替代广泛使用，但相对较低的吞吐量的蛋白质分析的免疫印迹技术。该方法允许数百个样品是半定量的分析和比较，同时允许在很宽的选择的细胞系和组织样品中的关键蛋白的直接比较。复用与不同的抗体物种进一步增加电源的技术允许同时使用多种抗体。这里介绍的例子强调的能力RPPA在研究肿瘤的异质性和异质性靶向治疗的效果。 ?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>抑肽酶</td><td>西格玛</td><td> A6279 </td></tr><tr><td>磷酸酶抑制剂的鸡尾酒2 </td><td>西格玛</td><td> P5726 </td></tr><tr><td>磷酸酶抑制剂的鸡尾酒3 </td><td>西格玛</td><td> P0044 </td></tr><tr><td>蛋白酶抑制剂混合物</td><td>罗氏公司</td><td> 11836153001 </td></tr><tr><td>的Triton X-100的</td><td>的Triton-X </td><td> T8787 </td></tr><tr><td> LI-COR Odyssey封闭缓冲液</td><td> LI-COR </td><td> 927-40000 </td></tr><tr><td> TissueLyser </td><td> Qiagen公司</td><td> 85600 </td></tr><tr><td>微网II机器人去污剂</td><td> Biorobotics </td><td></td></tr><tr><td> FastFrame“四个海湾幻灯片夹</td><td> Whatman公司</td><td> 10486001 </td></tr><tr><td> FAST幻灯片 - 2 - 垫</td><td> Whatman公司</td><td> 10485317 </td></tr><tr><td> IRDye 680LT山羊抗鼠IgG </td><td> LICOR </td><td> 926-68020 </td></tr><tr><td> IRDye 800CW的山羊抗兔IgG </td><td> LICOR </td><td> 926-32211 </td></tr></tbody></table>

the use of reverse phase protein arrays (rppa) to explore protein expression variation within individual renal cell cancers

目前还没有治愈的疾病治疗的转移性肾透明细胞癌，最常见的变种。这种治疗性的一个关键因素被认为是分子复杂性的疾病1。如酪氨酸激酶抑制剂（TKI），舒尼替尼靶向治疗已被使用，但只有40％的病人会响应，这些患者绝大多数复发1年以内2。因此在肾细胞癌患者的内在和获得性耐药的问题是高度相关的。 为了研究抗酪氨酸激酶抑制剂，制定有效的个性化治疗的最终目标，有针对性的治疗后，在规定的顺序组织是必需的，已被证明是成功的做法，在慢性粒细胞白血病4。然而，这样的策略在肾细胞癌的应用是复杂的，高层次的b超视距间和肿瘤的异质性，这是一个功能的肾细胞癌5,6以及其他实体瘤7。即使在建立类似的介绍，肿瘤的分期，分级的患者Intertumoral异质性由于转录和遗传差异的。此外，它是明确的，也有很大的形态（肿瘤内）的异质性RCC，这很可能代表更大的分子异质性。 RCC肿瘤的综合形态分析和Fuhrman分级详细的映射和分类，用户可以通过选择有代表性的地区蛋白质组学分析。 基于蛋白质的分析的RCC 8是有吸引力的，但由于其广泛的可用性，在病理实验室，它的应用可以是有问题的，因为供应量有限，特异性抗体9。由于斑点印迹性质的反相蛋白质阵列（RPPA），抗体的特异性必须Ë预先验证的，使用的抗体，这种严格的质量控制是非常重要的。尽管这一局限斑点印迹格式不允许检测的小型化，使数百个样品用于印刷到一个单一的硝酸纤维素滑动。然后，印刷幻灯片可以用类似的方式进行分析，Western印迹分析与特定目标初级抗体和荧光标记的第二抗体的使用，允许复用。在所有的样品在幻灯片上的差异蛋白质表达，然后可以同时分析在一个更具成本效益和高通量的方式进行比较的相对水平的荧光。

一个的扫描RPPA滑动一个例子可以看出，在图4（i）与680和800 nm的通道所示。由图4（ⅱ）中的波长，使分离的图像每个焊盘上的RPPA滑动以进行分析，并确定图4（ⅲ）中的单独的蛋白表达。正如图4可以看出，在（iii）该个别蛋白质表达的整个样品与凝溶胶蛋白具有高的表达水平横跨幻灯片cMYC具有低得多的蛋白表达相比是独特的，但仍?...

Watch this Scientific Journal Video about The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers at JoVE.com

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA支持数百个样品，印在硝酸纤维素幻灯片同时进行审讯，用荧光标记的抗体蛋白的表达。这项技术已被应用在肾透明细胞癌的药物治疗的非均质性研究的效果。

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

the-use-reverse-phase-protein-arrays-rppa-to-explore-protein

Research

JoVE Journal

Medicine

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

33.5K 次观看.  University of Edinburgh. RPPA支持数百个样品，印在硝酸纤维素幻灯片同时进行审讯，用荧光标记的抗体蛋白的表达。这项技术已被应用在肾透明细胞癌的药物治疗的非均质性研究的效果。

视频: The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase 1. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% 4. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy 5. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI 6. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis 6.The occurrence of AKI is associated with dramatic increases of morbidity and mortality 1. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed 3, 7-9. 
To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required 7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle 14. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.

A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.

一个悬重系统使用隔离肾动脉闭塞

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia 1-3. A recent study of hospitalized patients ...

科研

医学

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

淀粉样前体蛋白胞内结构域的分离纯化和聚集

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

精确实施最小红斑剂量（MED）测试，以评估人体炎症反应中的个体变异

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

通过肾脏动脉直接向肾脏输送药物

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...