我们承认资助CPRIT（RP100119）和韦尔奇基金会（I-1665）。

整个协议约需4天。 1。制备的siRNA和记者转染的细胞我们将在这里介绍使用一小部分的siRNA（384不同的siRNA池阵列每个池组成的4倍siRNA靶向单个基因在96孔板格式）从全基因组siRNA文库，仅供询问的siRNA库的协议。对于这个特定的研究中，我们将利用HCT116细胞表现出离经叛道的Wnt和KRAS信号，p53的活性。合适的细胞系的研究，旨在询问其他细胞的过程，还必须以类似的方式被识别和评估。 <ol><li>洗涤5×10厘米2块板的80-90％汇合的HCT116细胞用PBS然后收获细胞，用胰蛋白酶消化，用1毫升的胰蛋白酶消化溶液/板，接着用10ml的Dulbecco氏改良的Eagle培养基（DMEM）中，含2％铁河谷牛血清（FBS）和1％青霉素/链霉素/板。 </li><li>将悬浮的细胞转移到50毫升锥形管，离心〜130×g离心5分钟，收集上清，重悬细胞沉淀在10毫升的DMEM培养基/ 2％FBS / 1％青霉素/链霉素。 </li><li>与细胞计数器计数细胞数，然后使一个细胞10 5细胞/ ml的溶液，保持150毫升细胞悬浮液中的下一个步骤。板10 4个细胞（100微升）在使用多点自动液体分配器（以下简称多点）在96孔细胞培养板的各孔中。在这种情况下，电镀12×96孔板中，应足以将细胞悬浮液。 </li><li>孵育板与细胞在37℃培养箱中，5％的CO 2，同时准备转染混合物。 </li></ol> 2。准备记者构造联合解决方案<ol><li>隔离高品质的记者使用：标准midiprep套件（NucleoBond的Xtra南部生产的构建DNAClontech）中，并稀释至终浓度为1毫克/毫升的每个记者的DNA。 </li><li>准备100μl的记者构造相结合的p53-FL，30微升30微升8X TCF-RL，30微升Gal4的ELK1，6微升UAS-CL和4微升H 2 O来实现的DNA混合储备溶液最后的记者1:1:1:0.2比例。该DNA混合原液DNA终浓度为0.3毫克/毫升。 </li></ol> 3。准备的siRNA / DNA转染混合物在这个协议的一部分，我们会做好准备，这将是足够的转染12×96孔板的siRNA / DNA转染混合物。 4×96孔板的siRNA库对于这个测试，我们将在一式三份的siRNA转染每个池。转染试剂，我们将使用被称为Effectene（Qiagen公司）。在我们手中，这是唯一的试剂适用于siRNA和DNA导入培养细胞的同时交付。 <ol><li>记者构造股票溶胶稀释80微升ution在8ml的EC缓冲液（Effectene套件）中，实现了与终浓度为3微克/毫升的DNA溶液。在一般情况下，准备足够的DNA溶液混合，可直接使用。 </li><li>板20微升该DNA溶液在96孔/板的各孔中。 96孔板的数量将取决于有多少siRNA的池将被测试。例如，在这种情况下，我们将需要4×96孔板评估384座不同的siRNA。 </li><li>要测试的siRNAs的，应该是股票的浓度为5μM。如果没有，也可以是镀副本和与每个库的使用推荐的稀释缓冲液稀释至浓度。 </li><li>将2微升每5微米的siRNA股票到相应的PCR板稀释的DNA与Biomek自动化液体处理股票。根据不同的研究规模，多通道移液器可能就足够了。 </li><li>现在，我们将增加1微升增强溶液（套件Effectene），以每口井的DNA / siRNA的解放军德。这又可以实现与自动化液体处理多通道移液器。 </li><li>允许的增强反应发生在室温下5分钟，然后加入3微升Effectene转染试剂的各孔中，并在室温下孵育10分钟。 </li><li>要停止的转染复合物的形成，PCR板的各孔中的DMEM加10微升/ 2％FBS / 1％青霉素/链霉素。 </li><li> 10微升转染混合物转移到含有细胞（从细胞培养箱中检索）的96孔板的各孔中。由于每个转染将一式三份进行，相同的组合将被应用到两个额外的96孔板中的细胞。 </li><li>孵育的细胞的转染混合物在37℃下加入在湿润的环境中，5％CO 2的36小时。 </li></ol> 4。确定细胞样本的荧光素酶的活动 36小时后，准备测量荧光素酶活性在转染细胞。端点应在每个实验的基础上确定。在我们的研究中，培养时间36小时以前产生了足够强大的信号，这意味着结果的决心。由于本研究中采用多个记者（1分泌到培养基中，另外两个细胞的细胞质中表达），我们将获得从两个培养基以及细胞裂解液中的测量。 <ol><li> CL在培养基中的活性检测： <ol><li> 20μl的培养基是副本镀在一个白色的不透明的96孔板中（无论是使用Biomek的液体处理器或者多通道移液器）。 </li><li> CL测定缓冲液中加入20μl（定位系统）加入到各孔中。 </li><li>向每孔加入10μl的海萤荧光素底物（定位系统），并使用光度计（由BMG Pherastar板读数器例如）检测CL活动。 </li></ol></li><li> FL和RL活性检测： <ol><li>裂解的c通过首先除去培养基中的细胞l。这可以通过转动板倒置和扔在一个接收器中的介质中迅速完成。剩余介质可以去除纸巾轻轻拍打倒挂板。加入30微升的1X被动裂解缓冲液（Promega公司），每口井的细胞，使用多点的地方在一个平台上摇臂（BELLCO是一个公司，使这些）设置一个中等的速度为5分钟，在RT。 </li><li>加入20微升的萤光素酶检测试剂II（部分Promega公司的双荧光素酶试剂盒）使用光度计使用多点，并立即测量FL活动。接下来，添加停止格洛试剂（Promega公司），将淬火佛罗里达州的活动，并允许测量RL活动。注意：除非你正在使用的基材混合物，使信号持续时间延长（如双GLO的萤光素酶试剂盒购自Promega公司），使用闪光灯套件除了基板时需要快速的测量（5分钟内）。 </li></ol></li></ol>

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确定和LL作者挂起的有关复用荧光素酶技术专利。

有几个传播者基于荧光素酶报告系统（如Promega公司，瞄准系统，New England Biolabs公司，赛默飞世尔）的上线提供了有用的资源，对于那些第一次招待的高通量筛选。此外，一些优秀的评论关于优化利用高通量筛选基因的发现，以及如何基于RNAi筛查结果可以集成其他组学为基础的数据集应该是有帮助的15,16。 “点击”高通量筛选方法的选择是另一个讨论的主题是超出了本报告的范围。关于?...

监测多样化的细胞的生物过程的各种荧光素酶为基础的记者，是市售的。这些DNA构建的大部分编码荧光素酶蛋白质的诱导表达特定细胞刺激或扰动。最强大的以转录为基础的记者将表达荧光素酶的酶合成的增强子元件或建立对他们的可靠性报告生理上相关的转录事件1,2的基因的启动子区的控制之下。也有记者反荧光素酶系统，​​利用GAL4-UAS驱动荧光素酶蛋白的表达。 Gal4的是一种酵母转录激活因子，在UAS增强子的Gal4的特异性结合以激活转录的基因序列放置3向下流。这些类型的荧光素酶系统通常支持了DNA质粒 - 一个编码的GAL4融合蛋白的调控Ry的感兴趣的蛋白部分，和第二编码的控制下的一个或更多的UAS序列下的荧光素酶的蛋白质。因此，细胞的荧光素酶的信号反映感兴趣的蛋白质的活性水平。基于荧光素酶的记者的另一个常见用途是用于监测目的蛋白的融合蛋白的稳定性，即荧光素酶4。不管记者的类型，在这里指出，作为一个可以不承担任何记者的特异性已审问一个买者自负。因此，尽职调查是必需的任何研究战略纳入新记者构造。 读出的荧光素酶的选择可以是一样重要的控制其表达的DNA元件的可靠性。鉴于萤火虫荧光素酶（佛罗里达州）是最常用的酶在市售的结构，出现新的荧光素酶报告系统，不需要ATP（为的情况下佛罗里达州基反应），或者采用更稳定的酶，发出更强的信号，承诺这个研究平台，以提高效率和可靠性。荧光素酶的记者关于选择化学研究中一个重要的注意事项是佛罗里达州的易感性化学抑制，可能会引起虚假的报告。根据我们的经验，其他酶如海肾或荧光素酶Gaussia（RL和GL），分别利用腔肠基板不易抑制小分子5。 复用荧光素酶读出的最常见的格式，主要是因为可获得的易于使用的工具包，顺序地从单一样品中酶活性的测量的能力，需要使用FL和RL。在大多数试剂盒，样品第一次接触到荧光素揭示佛罗里达州的活动，然后通过淬火试剂同时沉积腔肠素产生次生次生RL信号。与另外的其他的荧光素酶可分泌，如GL或，使用另一种基片，如海萤荧光素酶（CL），更大数量的可能性，用于产生高的内容数据，使用荧光素酶已经出现。可以在这里找到6全基因组RNAi筛选使用这种技术的一个例子。这些技术进步反过来刺激特定的机制来解渴，以限制串扰7每种类型的荧光素酶的发展。在我们的手中，我们注意到一个更大的频率的“边缘效应”（莫名的趋势与边缘的高滴度文化板相关的细胞的活性），如GL或CL分泌荧光素酶。另一方面，例如分泌的荧光素酶动力学分析是有用的，因为它们使信号采样不掺细胞活力。 这里介绍我们的研究中，我们将同时监测三个癌症有关的活动细胞过程：P53，KRAS，Wnt信号转导通路。在我们的研究中成立记者pp53-TA-吕克FL记者从Clontech公司（以下简称为P53-FL记者）8，一个的记者Elk1-GAL4/UAS-CL系统（以下简称ELK-1记者安捷伦）， 8X的TCF RL记者，集成了多种合成的增强子元件Wnt信号通路的转录调控因子被称为T细胞因子（或粤港科技合作资助计划）9-11确认。

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

使用RNA干扰（RNAi）的基因的功能基因组规模的审讯化学听话癌细胞漏洞的快速鉴定具有很大的前景。限制这一技术的潜力是无法迅速划定机理的基础表型的结果，从而告知的发展，分子靶向治疗策略。我们在这里概括的方法来解构细胞表型诱导RNAi介导的基因打靶使用多路记者关键癌症细胞相关的流程监控系统，使。高内涵筛选方法是通用的，可以很容易地适用于其他类型的大型分子库的筛选。

尽管绘制各种癌症的突变景观采用大规模的基因组测序工作12-14的进步，我们仍然不到位有一个系统的方法将这些意见纳入干预战略。结直肠癌（CRC），预测的突变，影响组件的三个细胞过程 - Wnt基因/β-catenin蛋白，P53，和KRAS信号 - 被发现在所有肿瘤的99％，这表明他们是在肠道细胞转化的关键驱动因素13。因此，癌症基因组数据集可能丰富支持共同点促癌过程的基因，并可能被?...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

实现系统级的细胞过程的理解，是现代细胞生物学的目标。我们在这里描述的各种细胞功能的复用荧光素酶记者的战略点审问基因的功能基因组规模的RNAi库。

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Research

JoVE Journal

Medicine

11.0K 次观看.  UT Southwestern Medical Center. 实现系统级的细胞过程的理解，是现代细胞生物学的目标。我们在这里描述的各种细胞功能的复用荧光素酶记者的战略点审问基因的功能基因组规模的RNAi库。

视频: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

如何从MR图像测量皮质折叠：一个循序渐进的教程，以计算本地Gyrification指数

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

科研

医学

High Content Screening in Neurodegenerative Diseases

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

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

在神经退行性疾病的高内涵筛选

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

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠：A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

筛选生物活性纳米颗粒在噬菌体免疫细胞中的Toll样受体信号传导抑制剂

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

一种计算机辅助心血管分析诊断平台的研究

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

体外培养肺泡上皮细胞中卤代气体浓度的控制方法

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...