南部亚利桑那州退伍军人事务部的卫生保健系统弗吉尼亚优异审查授予0142和亚利桑那州的生物医学研究委员会授予0803提供的资金。

准备组织观看幻灯片<ol><li>一个结肠镜可以通过进入结肠，直肠。一个结肠镜是一个长管，照明和视频摄像头，能够传递到结肠空气吹起来就像一条长长的气球，以便更好地可视化结肠，并有活检钳，可以通过扩展超出了视频摄像头，掐了一个3-5毫米的面积内表面的"皮肤"或结肠的粘膜层，给我们一个活检标本。活检钳也可以用来移除可能在结肠中发现的癌前息肉。 </li><li>我们获得的正常结肠黏膜组织活检，从病人的知情同意。这些患者接受胃肠诊所的一个结肠镜筛查。福尔马林罐被安置在我们的活组织切片检查。 4小时后，福尔马林取代70％的酒精。 </li><li>类似的组织样本取自在手术切除，结肠切除术的领域，即1厘米和10厘米的距离先进瘤结肠癌或腺瘤。这些组织样品也被放置在福尔马林，如果组织样本大，有些福尔马林保存在一个较长时期之前被放置在70％的酒精的。 </li><li>采取组织样本是一个组织学实验室正在处理和放置在蜡块。 </li><li>冷藏在冰箱中，使他们更容易被削减一个切片蜡块。 </li><li>从4微米厚的组织切片切组织块，用一个切片，在水中漂浮。 </li><li> ERCC1和PMS2例如，比较两种蛋白质的表达，可以拿起备用组织切片到两张幻灯片，直到每两个幻灯片上有三个组织切片。例如，第1，3，5，可放置标记ERCC1和第2，4和6可以放在一张幻灯片标记PMS2上一张幻灯片。三个组织切片一张幻灯片蛋白免疫染色可以让你看到，如果地穴一个不寻常的染色模式是神器，或者是真实的，因为文物不会在一个以上的组织切片，在幻灯片上重复。结肠隐窝直径约60微米，约15个组织切片，以便可以减少通过一个地穴，和相同的地穴可以在多个组织切片，每张幻灯片。 </li></ol>免疫组织节<ol><li>幻灯片，然后通过免疫组织化学标记过程。这是8个小时的过程，是相当标准。的过程，我们使用的部分，即不规范，使用染色Sequenza容器，并使用一个解决方案，以减少被称为"狙击手"的背景染色。 "狙击手"的解决方案是出售作为保科医疗，协和CA的专利配方。 </li><li>这里显示了一些我们的程序步骤。 </li><li>作为一个例子，PMS​​2免疫组织化学，幻灯片变化对二甲苯（3分钟），在100％的乙醇变化（2分钟），其次是2的变化在95％的乙醇（2分钟，放置第一deparaffinized每件），其次是2蒸馏水的变化（2分钟）。这些程序下进行化学与负压罩，使实验者是不暴露的蒸气。 </li><li>当福尔马林最初是用来修复组织，部分组织样本中的蛋白质交联。现在，我们需要打破交叉连接，使一种抗体可以识别蛋白。幻灯片放置在缓冲溶液中，这是在微波炉煮沸。这是由介质上设置的10分钟，随后在冰上20分钟冷却。这一步需要不同的微波炉进行测试和调整，直到达到良好的效果。请注意，免疫CcOI或Ku86我们用了9毫升柠檬酸+ + 450ML H 2 O（包括钠离子缓冲液），ERCC1我们使用的柠檬酸缓冲液，pH值约6.0 41mL柠檬酸钠柠檬酸钠缓冲液1公升2.1克柠檬酸+ H 2 O +〜5毫升氢氧化钠使pH值至6.1（最小的钠离子的缓冲），和PMS2我们用5ML的抗原的揭露解决方案（矢量）+ 533mL提出了解决方案H 2 O。不同的缓冲区，需要增加与特定的蛋白质抗原决定簇的抗体相互作用。 </li><li>一个在蒸馏水冲洗3分钟，洗净磷酸盐缓冲液（PBS）的5分钟20分钟的幻灯片，然后放入3％的双氧水（甲醇稀释）。 </li><li>幻灯片，然后放置到平面的狭窄染色架，称为Sequenza（Shandon Sequenza Thermo Scientific的免疫染色系统），并用PBS漂洗。 </li><li> ERCC1或PMS2免疫染色的幻灯片，然后暴露于10分钟犹特人接触到3滴Biocare公司获得一个专有的解决方案，被称为"狙击手"的行为，以减少非特异性染色背景蛋白的补充治疗。 </li><li>幻灯片冲洗缓冲区"TBST"，这是1毫升吐温100毫升10倍三+ 900毫升蒸馏水[10X三是Trizma + 80克氯化钠+ 1,000毫升蒸馏水和去离子水+浓盐酸（24.2克约15毫升），使pH值7.6]的解决方案。 </li><li>幻灯片，然后用缓冲液冲洗三次，和100微升DAKO公司二抗生物素在1:100稀释（2％牛血清白蛋白在TBST缓冲）添加到幻灯片和30分钟，在室温下孵育。 </li><li>此时，Vectastain ABC（亲和素生物素复合体）（矢量实验室）试剂准备，用2.5毫升PBS，1滴溶液，1滴溶液B，以及解决的办法是允许站30分钟。 </li><li>幻灯片用TBST缓冲液漂洗3次。 </li><li>然后Vectastain农行试剂3滴添加幻灯片室温孵育30分钟，用TBST漂洗2次。 </li><li>幻灯片，然后沉浸在DAB（二氨基苯甲酰）（2.9毫升民建联+ 400微升PBS + 250微升双氧水）约5分钟。 </li><li>幻灯片，然后用蒸馏水，去离子水冲洗2次。 </li><li> Counterstaining稀溶液中10秒的苏木。幻灯片在去离子水的自来水彻底冲洗。 </li><li>幻灯片，然后脱水2次，2分钟，95％的乙醇2分钟，100％的乙醇和二甲苯2分钟的2倍，2倍。 </li><li>滴Cytoseal XYL（理查德 - 阿兰科学）夫妇将被添加到幻灯片和应用盖玻片。 </li><li>免疫组化评价的ERCC1，Ku86和CcOI PMS2使用相同的协议，但与抗体取代ERCC1的，Ku86和CcOI PMS2抗体表所述。 </li></ol>隐窝缺乏ERCC1表达，PMS2，Ku86和CcOI评估组织切片<ol><li>我们将着眼于组织切片下一个麦克奥迪（DM - BA300）photomicroscope PMS2和结肠黏膜隐窝ERCC1的表达。 </li><li> PMS2和ERCC1都是DNA修复酶，所以他们的DNA是位于隐窝细胞的细胞核中，。这是表示在图1，其中棕色的冒号为ERCC1的染色，在隐窝细胞的细胞核中发生。我们首先在显微镜显示正常隐窝染色为ERCC1的，棕色的污渍显示ERCC1的位置。我们指出的隐窝，"杯状细胞"的形状有点像一个酒杯，在小区域基地外缘隐窝细胞的细胞核，出第一个热气球的细胞类型，充满了白色的粘蛋白颗粒隐窝内细胞的一部分。其他两个类型的细胞看起来有点都在我们的染色切片，和他们的肠细胞和肠内分泌细胞，其原子核也外缘隐窝细胞的基础，。在从未有过的结肠瘤的患者，或在远离任何网站结肠癌的活组织切片活检，几乎所有的所有隐窝细胞核正常显示ERCC1表达的高层次，如下所示。 </li><li>引导我们的观察，从没有结肠瘤患者的活组织切片获得的组织切片观察为表达水平PMS2，ERCC1，CcOI或在正常结肠黏膜Ku86。我们可以观察到所有的地窖，在一个正常的组织切片，用40X物镜，因为我们慢慢地经过一个完整的组织切片，点出的隐窝，间质细胞，在幻灯片上的任何淋巴小结，肌层（如果存在的话在活检）。 </li><li>我们还指出，有只是几个在干细胞的地穴基础细胞。这些干细胞产生的所有完整的隐窝细胞约2000。一个干细胞与基因突变或epimutation可以接管整个干细胞小生境。然后整个隐窝可能已经改变，因为我们在这个新的幻灯片显示，我们看到CcOI未来CcOI高表达的隐窝缺乏整个隐窝的利益酶（地穴转换）的染色。 </li><li>接下来，我们从病人与结肠癌或先进腺瘤腺瘤的幻灯片。在这张幻灯片，有隐窝为ERCC1的正常的高表达水平，一些较低水平的表达。所有的组织切片内的隐窝被拍到与10倍物镜的分辨率低，图像，使整个组织切片平铺在一个图像中看到。隐窝，然后编号。 </li><li>酶的高表达与隐窝，隐窝远来回活检的典型米癌症，被称为水平"4"染色。其他级别的"0"，如果没有表达检测，"1"如果刚刚检测到表达是显而易见的，"2"，如果有表达目前，但比4级的水平要低得多，而"3"如果表达式是目前低于4的水平，但相当强的。我们点4每个幻灯片上的低分辨率图像的蓝色，绿色，3，2黄，橙1和红色为0。在这里，我们将展示通过使用40X物镜部分从活检。我们点上的低分辨率图像的隐窝。 </li></ol><img src="/files/ftp_upload/1931/1931fig1.jpg" alt="图1" /> 图1。一个结肠隐窝，隐窝细胞的细胞核中的ERCC1高表达。在此图像的所有颜色也同样增强对比度和饱和度，使用Paint Shop Pro的5。 代表性的成果<ol><li>在单独的幻灯片的替代组织切片，让我们看看在同一结肠隐窝采用免疫组织化学显示出两个不同的蛋白质表达。 PMS2隐窝和相同的crypt染染ERCC1的形象是相邻显微镜与相邻的电脑显示器上显示。这使我们能够确定是否有联合缺乏两种蛋白质，或是否在每个蛋白质的不足之处，而每个频繁，发生独立。我们有三个组织切片，每张幻灯片，使任何一种蛋白质的表达明显缺乏可作为缺乏多个节，而不是由于对工件验证。在组织周围的结肠癌，那里有一个视野缺损引起的癌症，有高频率的缺乏隐窝ERCC1和PMS2。 </li><li>我们还采用免疫组化发现Ku86和CcOI隐窝缺陷的频率。在这张幻灯片中，我们经过一个完整的组织切片免疫染色为Ku86，使用40X物镜，和我们可以看到，几个隐窝Ku86在这个组织的第中的不足。 </li><li>同样，在这张幻灯片中，我们通过一个完整的组织切片免疫染色CcOI，使用40X物镜，我们可以看到，只有中等数量的隐窝CcOI缺乏。 </li></ol>

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<ol><li>每张幻灯片中有3个组织切片，这是非常重要。气泡可以发生时，媒体携带抗体，使一些隐窝或部分隐窝可能不能充分染色只是因为泡沫阻止反应的抗体与组织切片。囊直径约60微米，和4微米厚的组织切片，所以可以通过多达15个相同的地穴组织切片。它通常是可以找到相同的地穴，在多个组织部分放在幻灯片上，使用形态为指导的地穴。 </li><li>在我们最近的研究中，对于大的组织样本，在一个Sequenza染色可能不给组织良好的抗体覆盖。在这种情况下，人们可能更喜欢使用手工染色的组织，是对每个组织样本的手放在一滴含有抗体媒体。 </li><li>要清楚地看到棕黄色染色，显示表达蛋白的免疫染色，蓝染色显示在计算机显示器上的核DNA，麦克奥迪photomicroscope软件调整如下:增益0，偏移为0，增强，伽马1.00元，0，255 0， 1锐度，颜色校正7，R，G，B增益1.00，和R，G，B亮度0。分辨率设置为1024 × 768，白平衡启用。 </li><li>我们的方法可以用于任何蛋白质被认为是重要进展结肠癌，以评估他们缺乏的频率，或可能的过度表达，在周边视野缺损先进瘤结肠癌或腺瘤。 </li><li> 视野缺损进展为癌症的一部分 术语"领域癌变"最早是由屠宰等。在1953年1来形容一个地区或上皮细胞的"场"已很大程度上是未知的进程（当时）为前提，从而易患对癌症的发展。初次使用在口腔癌中。从那时起，"领域癌变"和"视野缺损"被更广泛地使用来形容在这新的癌症更可能出现的任何癌前组织，临床肿瘤学领域癌变的概念已日益受到重视2 。我们最近检讨了在胃肠癌3的视野缺损的证据。在这次检讨，包括在结肠的视野缺损的证据提供了十几个研究的结果。 在结肠黏膜的视野缺损可能出现一个地穴等干细胞中的突变的细胞或细胞表观遗传学改变的自然选择，一个干细胞生存的利基继承4。遗传不稳定或赋值函数表型，可能是由于DNA的核苷酸切除修复或DNA错配修复的减少，会加速这一进程（以前经常在PMS2 缺陷报告结肠癌5周围地区）。如果在正常人群结肠黏膜干细胞，细胞有选择性的优势获得通过突变或epimutation，往往会牺牲邻国干细胞克隆扩大。在结肠黏膜，≤5个细胞，然后会引起所有（约2,000）隐窝细胞的干细胞小生境被占领。一个干细胞小生境的接管由突变或表观遗传学改变的细胞类型的结果，在"地穴转换"7。 结肠上皮细胞的突变克隆传播（转换隐窝）最常发生隐窝裂变8。隐窝裂变的一个例子是如图2所示。在这种方式出现的异常组织的修补程序。在这样一个补丁，第二次这样的突变可能会出现这样一个给定的地穴获得的优势，相比其他隐窝内的补丁，并克隆形成一个次要的修补程序，这隐窝内将扩大原有的补丁。在这个新的补丁，这个过程可以反复几个更多的时间，也许几十年，直到出现一种恶性干细胞克隆扩展成癌症。如果这是一般的过程，出现零星结肠腺癌，然后结肠腺癌一般应与，和前面，异常的领域。因此，转换隐窝的补丁可以预料，在该地区的先进腺瘤腺瘤周围，或周围的结肠癌的检出缺陷。 从这个模型前肿瘤的进展，希望找到采取的非肿瘤性结肠肿瘤性病变周围的地区，有可能进展为癌症的结肠黏膜组织样本中的肿瘤进展相关的基因或遗传学改变。特别是，在DNA修复酶，或凋亡所必需的蛋白质，缺陷将有助于进展，加强基因组的不稳定，以及预期可能被发现有缺陷的领域。 </li><li> 选择在PMS2细胞是D时，适用于肾阳虚eficient ERCC1和DNA损伤剂 奈良等 。同比增长25文化43 - 3B，中国仓鼠卵巢细胞与DNA修复基因ERCC1的失活突变，并处理与DNA损伤剂的文化，MNNG。幸存的细胞DNA损伤的治疗中，从每一种文化中分离单个细胞，并允许以形成一个克隆。所有的克隆分离出约10倍以上的抗MNNG比亲本菌株杀害。孤立的克隆，有22人被证明是在DNA错配修复（MMR）缺陷。这22个克隆中有5个进行了测试，经测序，三个五个PMS2缺陷。选择这PMS2缺陷可能是因为PMS2缺乏引起的抗细胞凋亡，并因此生存的能力，在细胞内的剩余ERCC1的缺席时的DNA损伤。 （通常情况下，PMS2感官DNA损伤水平，并导致细胞发生凋亡，DNA损伤是过度时。）因此，选择在PMS2缺乏ERCC1基因缺陷的细胞DNA损伤时。无性系中ERCC1和PMS2缺陷约500至1000倍的突变率，当与紫外线比亲本细胞照射。 </li><li> 胆汁酸引起氧化应激，DNA损伤剂，并在发展为结肠癌的重要 去氧胆酸，胆酸，增加通过氧化应激反应，生成活性氧（ROS）由多个进程，包括破坏线粒体 10 。最近有证据活性氧造成11后生变化，至少有15个研究表明，胆汁酸，在伴随着高脂肪的饮食浓度的增加，导致DNA 损伤 12 。伯恩斯坦等 12审查，是膳食脂肪的消耗和结肠癌的发病率之间呈正相关。在饮食中脂肪的胆汁酸被分泌到肠道，胆汁酸作为洗涤剂，以帮助脂肪的消化。因此，高脂肪的饮食与胆汁酸的分泌增加。粪便胆汁酸浓度升高与结肠癌的发病率很高的人群中，过度暴露于胆汁酸作为结肠致癌 13的主要危险因素。胆汁酸可能是在结肠的视野缺损的原因。 </li></ol><img src="/files/ftp_upload/1931/1931fig2.jpg" alt="图2" /> 图2。结肠隐窝裂变成3隐窝。 结论我们发现，在DNA修复基因ERCC1和PMS2在大视场周围结肠癌的缺陷（PMS2也需要凋亡）缺陷的隐窝高频。 ERCC1和PMS2（由于后生变化或突变）的不足之处，可在中央进展结肠癌的遗传不稳定的早期步骤。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>PMS2 antibody</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556415</td>
<td>&nbsp;</td>
<tr>
<td>ERCC1 antibody</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>MS-671-P1</td>
<td>&nbsp;</td>
<tr>
<td>Ku86 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotech.</td>
<td>SC-9034</td>
<td>&nbsp;</td>
<tr>
<td>CcO1 antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>459600</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated secondary</td>
<td>&nbsp;</td>
<td>DAKO</td>
<td>E0413</td>
<td>&nbsp;</td>
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<td>22% BSA</td>
<td>&nbsp;</td>
<td>Informagen, Inc.</td>
<td>2327</td>
<td>&nbsp;</td>
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<td>Sniper</td>
<td>&nbsp;</td>
<td>Biocare Medical</td>
<td>BS966</td>
<td>&nbsp;</td>
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<td>Vectastain ABC</td>
<td>&nbsp;</td>
<td>Vector Labs</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>DAB</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>34001</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>&nbsp;</td>
<td>USB</td>
<td>20605</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal XYL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48212-196</td>
<td>&nbsp;</td>
<tr>
<td>Trizma</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1503</td>
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deficient pms2, ercc1, ku86, ccoi in field defects during progression to colon cancer

致癌，是公认的"视野缺损"临床上，由于某些癌症的发展相同的组织类型，经常在附近的位置，其他恶性肿瘤的幸存者倾向高。这种视野缺损已表示在结肠癌。在组织周围的结肠腺癌或先进瘤（一个结肠癌的方式）腺瘤周围的正常组织，应在高频率检测，负责在结肠的视野缺损的分子异常，但在低频在无结肠腺瘤患者结肠黏膜。 采用免疫组织化学，在整个结肠腺癌或先进结肠neoplasias两侧10厘米隐窝被发现经常减少或两个DNA修复蛋白，PMS2和/或ERCC1的表达缺席。 PMS2是具有双重作用的蛋白质，DNA错配修复的积极多余的DNA损伤的细胞凋亡以及在需要。 ERCC1的是活跃于DNA的核苷酸切除修复。减少或缺的ERCC1和PMS2表达会造成细胞既增加了生存的能力（凋亡抵抗）和易变性增加。减少或缺的ERCC1和PMS2表达很可能发展为结肠癌的早期步骤。 DNA修复基因Ku86（活跃在DNA非同源末端连接）和细胞色素C氧化酶亚基我参与细胞凋亡的各据报道，在粘膜地区接近结肠癌中的表达下降。然而，免疫组化的表达水平的评价结果​​表明只有从低到适度的频率，他们在现场周围结肠癌或先进的结肠肿瘤周围的缺陷表达缺乏的隐窝。 我们表明，在这里，我们表达ERCC1的，PMS2，Ku86和CcOI隐窝的评价方法。我们表明，整个隐窝PMS2缺陷的频率和ERCC1的往往是高达70％至95％，20厘米长地区周围的结肠瘤的伟大，而缺乏在Ku86频率隐窝中值的2％和隐窝频率CcOI缺乏有在这些地区16％的中值。整个结肠是150厘米长（约5英尺），并在其粘膜层约10万隐窝。 PMS2和ERCC1周围结肠癌的缺陷，从而可能包括1万隐窝。它是从结肠癌的发生​​有缺陷的crypt。

Watch this Scientific Journal Video about Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer at JoVE.com

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

减少/缺失PMS2和/或整个隐窝ERCC1的表达是在10厘米的结肠腺癌，可能与高可变性和发展为癌症的视野缺损的基础上两侧的频繁事件。 Ku86或CcOI虚寒经常在这些领域的缺陷要少得多。

缺乏PMS2，ERCC1，Ku86，视野缺损CcOI在结肠癌进展

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

deficient-pms2-ercc1-ku86-ccoi-field-defects-during-progression-to

Research

JoVE Journal

Biology

12.3K 次观看.  University of Arizona, Tucson. 减少/缺失PMS2和/或整个隐窝ERCC1的表达是在10厘米的结肠腺癌，可能与高可变性和发展为癌症的视野缺损的基础上两侧的频繁事件。 Ku86或CcOI虚寒经常在这些领域的缺陷要少得多。

视频: Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

科研

生物学

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

确定的能源系统，在运动的贡献

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction defects can be assessed by tracking methylene blue dye flow within the urinary system. Methylene blue dye is injected into the renal pelvis of perinatal embryonic kidneys and dye flow is monitored from the renal pelvis of the kidney through the ureter and into the bladder lumen after applying hydrostatic pressure. Dye accumulation will be evident in the bladder lumen of the normal perinatal urinary tract, but will be constrained between the renal pelvis and the end point of an abnormal ureter, if urinary tract obstructions occur. This method facilitates the confirmation of urinary tract junction obstructions and visualization of hydronephrosis and hydroureter. This manuscript describes a protocol for methylene blue dye injection into the renal pelvis to confirm urinary tract junction obstructions.

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

亚甲蓝染料注射液评价泌尿道结肠梗阻的缺陷

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...