这项工作是支持的国家卫生RO1 - GM085130研究所和国家科学基金会MCB0929394的资助。我们感谢她的盛情款待，让我们在她的实验室电影马里昂施密特博士。

1。足迹试剂的制备<ol><li>准备了10倍的反应缓冲液含有钠cacodylate 100毫米，1毫米EDTA和1个M氯化钾。调整pH值至7.4。使用0.2μm的醋酸纤维过滤设备（NALGENE）过滤器的缓冲区。注：不吸管的RNA直接到10倍缓冲区。 </li><li>准备如表1所示，每个反应的滴定反应混合。滴定混合量（1X缓冲和镁（二）所需的浓度）应为90μL，然后加入1X缓冲的RNA加入10μl。 </li><li>准备一个核糖核酸酶T1消化缓冲液6.63M尿素，柠檬酸钠20MM，为1mM EDTA，0.25微克/μL的tRNA，0.025％二甲苯蓝，0.025％溴酚蓝。这个缓冲区可以存储在4℃至6个月。 </li><li> Fenton反应需要250毫米的铁（NH 4）2（SO 4）2，250毫米EDTA，和500毫米L -抗坏血酸钠新鲜配制的水溶液。浓度在第1章指出.5是平衡的实验，如钠cacodylate缓冲区，不清除自由基和不同的二价离子浓度的最佳选择。 </li><li>过氧化（1.5.1）和氧化（1.5.2）的足迹反应试剂。 <ol><li>过氧化Fenton反应需要100毫米的铁EDTA的解决方案，和50毫米钠L -抗坏血酸和1.5％H 2 O 2，新鲜配制的水溶液。准备用EDTA铁EDTA溶液混合铁（NH 4）2（SO 4）2，有超逾铁EDTA的1.1倍。 </li><li>前发起的氧化裂解反应（3.4.2），创建相结合，2μL的Fe（NH 4）2（SO 4）2（250毫米），EDTA的2.2μL（250毫米），62.5μLFenton反应组合L -抗坏血酸钠（500毫米），22.5μL10X反应缓冲液和135.5μLH 2 O的终浓度达到0（3.4.2）的足迹反应混合物。，分别为10毫米，0.11毫米和6.61毫米。 </li></ol></li></ol> 2。足迹实验RNA的制备<ol><li> RNA可以在体外转录DNA模板6标准生产，如果少于50个核苷酸，购买（例如，在集成DNA技术，www.idtdna.com ）。实验室按照建议的清洁程序，以保持在足迹实验样品RNA的完整性。 </li><li> 8脱磷，在体外转录RNA是必要的前 32 P标签的RNA的5&#39;端。 10 pmol kinasing末端标记的RNA 9解决方案应该是成功的磷酸灭活后的阴天。 </li><li>净化放射性标记的RNA变性凝胶电泳。随后的两个凝胶0.3 M醋酸钠（pH 5.5）中提取的RNA恢复。 RNA沉淀混合水溶液0.5毫升，1毫升乙醇和随后孵育1分钟干冰。在4 ° C，12500 RPM 0.5小时旋转样品。弃上清，用70％乙醇洗涤RNA沉淀。额外的离心去除上清液后，在真空干燥颗粒。 </li><li> P标记的 32 RNA样品溶解在330 UL 1X Buffer.Pipet 30μL的反应管中的RNA溶液，沉淀用乙醇和真空干燥RNA生成参考阶梯（步骤3.5），保持此管。变性加热的缓冲的RNA溶液在95 ° C为2分钟。样品冷却到室温为15分钟，快速旋转，使管放回解决方案的盖子上的冷凝液。 </li></ol> 3。足迹实验<ol><li>电泳梳，确定每分析凝胶的数据点的最大数量。我们使用30梳。参考阶梯占地2井和uncleaved控制之一。准备27个反应管。最后MG（二）concentrati项应均匀分布的对数遍及各地的滴定中点量级的规模。 </li><li>另外，在50 RNA和镁（二）解决方案° C孵育5分钟。 90（二）镁相应数额在1x缓冲液10μLRNA溶液混合，达到100微升的最终体积。孵育30分钟的解决方案，在50 ° C。 </li><li>让我们的解决方案，在25 ° C为1小时的平衡而发生折叠。 </li><li>过氧化（3.4.1）（3.4.2）或氧化羟基自由基足迹反应。 <ol><li>开始放置铁EDTA（100毫米）2μL液滴的过氧化反应，反应的顶部内彼此分开2μLL -抗坏血酸钠（50毫米）和2μlH 2 O 2（1.5％ ）管内含有RNA的解决方案，并开始严厉混合的足迹反应。停止15秒后加入300μL冷无水乙醇的反应。转动管的3-5倍。沉淀，洗涤和在步骤2.4干燥沉淀。 </li><li>加入5μL新鲜配制的Fenton反应混合物（1.6）开始氧化羟自由基footprintingreaction。孵育30分钟，在25 ° C。添加300μL冷无水乙醇淬火反应和组合转向管的3-5倍。沉淀，清洗和干燥的颗粒为在步骤2.4中表示。 </li></ol></li><li>根据标准程序10使用在步骤1.3编写的解决方案生成一个核糖核酸酶T1消化参考样本。 </li><li>溶解于8μL凝胶上样染料II（Ambion公司），并确认RNA的RNA颗粒悬浮使用盖革计数器。 </li><li> 11。负载样本，包括两个引用和控制的标准协议，准备变性，8％聚丙烯酰胺测序胶。独立的RNA片段在60瓦 - 75 W为2.5小时。干凝胶置于存储荧光粉屏幕一夜之间。扫描成像系统胶片放射自显影，如荧光屏。风暴865（GE Healthcare公司）或台风（GE Healthcare公司）。凝胶图像文件传输到您的计算机。 </li></ol> 4。数据分析<ol><li>每个核苷酸的羟基自由基反应，是由每个频带的量化分析凝胶强度。下载，安装和开放SAFA，一个易于使用的开源软件为单波段的装修和量化12，13按照用户手册的说明。简而言之，负载的RNA序列。txt文件的后面。凝胶文件的凝胶图片。调整波段的强度，定义车道，选择一个锚车道，并执行凝胶对齐。频段分配到核苷酸发生在核糖核酸酶T1消化阶梯参考。乐队量化积分和使用的正常化/ colorplot功能正常化和分配潜在不变的残留物。另存为。txt文件的输出。 </li><li> SAFA输出电子表格，其中包含代表胶道和行代表在列tegrated波段相应的RNA片段的单个波段的密度。首先，确定保护的网站显示在溶剂可显着的变化，通过比较没有镁（II）与终点毫克（二）浓度在我们的例子（50毫米）的配置文件样本所得的个人资料的保护。该值越低，受保护的碱基对羟基自由基的攻击，反之亦然。 </li><li>创建折叠等温线从个人或群组的核苷酸与镁（Ⅱ）浓度的归波段积分。等温线是单独缩放FI = L +（U - L）的分数饱和度• <img src="files/ftp_upload/3244/3244y.jpg" alt="Ÿ" />其中，f表示乐队的集成密度（S）分析，L和U代表的上限和下限过渡<img src="/files/ftp_upload/3244/3244y.jpg" alt="Ÿ" /> 14的数据都适合使用非线性升是小数饱和。东广场分析程序（我们使用或者原产地（OriginLab）或GraphPad（GraphPad软件公司）。）向山式（1） <img alt="公式1" src="/files/ftp_upload/3244/3244eq1.jpg" /> 其中K，D是平衡解离常数，[M]，对应的变量，可调节的折叠反应，镁（二）在这个例子中的浓度，N H Hill系数。此过程尺度过渡到小数饱和，决定了它的中点，并提供了一​​个过渡是否是S形的现象学测试。来自测序凝胶（图3A）SAFA（图3B）进行了分析和适合如上所述的等温线如图3C所示。如图3所示的分数饱和等温线所产生的电子表格中的缩放SAFA产生对最适合使用的方程分数的上限和下限（U和 L）值=值 /（U - L） - L /（ U - L）。 </li></ol> 5。代表性的成果： 图3显示了代表小四至小六的RNA•OH足迹实验的结果。凝胶图像（左）表示，一）背景乳沟是最小的（右线），B）单核苷酸分配是可能的，因为在T1巷（核糖核酸酶T1劈开每个G）定义的乐队，和c）的羟基自由基诱导的RNA碎片以及上述背景。从低到高镁（二）的过渡是隶属于下降表明RNA三级结构形成的乐队的个人和组的集成条带密度。单次或连续核苷酸的裂解变化随之而来被称为一个“保护”的群体。 •OH保护镁（二）特点的小四至小六RNA介导的折叠密切对应溶剂人迹罕至的地区，其晶体结构中观察到的分子。 折叠的RNA分子的彗星<html>一个有非常明显的结构性或动态的现象，但没有与相关的大专接触保护的不同程度（即，如何接近背景带密度下降）。因此，一些RNA分子将显示定义良好的结构转换，如在图3所示，而有些则不会。乐队强度SAFA 12分析（图3B）量化和规范化。输出是一个“热”的，可视情节对•OH的相对程度的保护。颜色过渡介绍后，镁（二）除无障碍变化。白色红色或蓝色显示更容易或多个受保护的核苷酸，分别。每个阴影的程度，是隶属于一个数值，可以被绘制成一个保护曲线（图3C），如山式（1）通过一项具有约束力的模型分析。解离常数与保护153-155附属的平衡是两倍左右，相应的价值，保护163-164。 ontent“&gt; <img alt="图1" src="/files/ftp_upload/3244/3244fig1.jpg" /> 图1。羟自由基的生产和P4 - P6 RNA折叠。 A）铁（II）催化羟自由基的过氧化氢的生成。抗坏血酸亚铁降低铁（Ⅲ）回。 B）在镁（二）的情况下，没有小四至小六的三级结构形成，使羟基自由基渗透和切割所有可访问的骨干岗位。增加镁（二）启动折叠小四至小六，只允许溶剂可及骨干羟基自由基裂解。 <img alt="图2" src="/files/ftp_upload/3244/3244fig2.jpg" /> 图2。大纲为羟基自由基足迹实验。一）Dephophorylation和32个P 5&#39; -端的RNA标记。二）净化32 P标记在变性聚丙烯酰胺凝胶中的RNA。三）RNA的频段切除，随后的RNA提取，乙醇沉淀。 ð）Prefolding和折叠的RNA。五）新鲜准备的Fenton反应混合物除了生成羟基自由基。 F）的RNA片段，变性聚丙烯酰胺凝胶电泳分离。 G）的定量RNA片段SAFA软件。 <img alt="图3" src="/files/ftp_upload/3244/3244fig3.jpg" /> 图3过氧化芬顿足迹反应后的RNA片段分析。 （一）的RNA被暴露的羟基自由基和卵裂产品使用变性聚丙烯酰胺凝胶电泳（PAGE）得到解决。图片显示，增加镁的浓度（二）过氧化裂解产物。参考和控制车道标记。此图像文件的SAFA的方案，其中定量每个频段的音量输入。 （二）“热”的情节所产生的SAFA。 （三）有关集成条带密度SAFA核苷酸的数量输出值的电子表格是如山equati绑定模型分析（1）。保护区被选为乐队Mg（II）浓度的功能不可或缺的密度增加。在K D是在被监控的网站，其中一半的RNA折叠的Mg（II）的浓度。 Hill系数，N H，是衡量斜坡曲线上具有约束力的协同信息，并提供了一个在这个特定的站点折叠的镁离子的数量的较低的估计。 <img alt="表1" src="/files/ftp_upload/3244/3244table1.jpg" /> 表1卷生成的RNA样品含有不同的Mg（II）浓度的代表。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+SHAPE+and+hydroxyl+radical+analysis+of+RNA+structure+and+ribonucleoprotein+assembly.">High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.</a> Methods. Enzym. 468, 67-89 (2009).</li><li>Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+large+RNA+molecules+with+knowledge-based+potentials+and+structural+filters.">Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters.</a> RNA. 15, 189-199 (2009).</li></ol>

没有利益冲突的声明。

羟基自由基足迹是一个有价值的工具，以评估核酸的溶剂可及表面面积。定性和定量的形成三级结构14，可以遵循作为一个功能参数，如离子种类和浓度，pH值，温度，结合蛋白或折叠的共同因素。引人注目的一条直线前进和廉价的协议，以及由此产生的溶剂可及单核苷酸水平上的折叠信息的结合，使得这种方法非常有吸引力的。传统的•OH足迹反应是增加H 2 O 2，我们称为“过氧?...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA分子在生物学中发挥的重要作用。除了传递遗传信息，RNA折叠成独特的三级结构，实现特定的生物学作用，作为调节剂，粘结剂或催化剂。有关大专院校的联系形成的信息是必须了解的RNA分子的功能。羟基自由基（•OH）是独特的结构由于其反应活性高，体积小的核酸探针。，作为一个足迹探头使用时，羟基自由基地图的溶剂可及表面的DNA和RNA的磷酸骨干如单核苷酸分辨率罚款。羟基自由基足可以用来识别内分子间的接触面，例如在DNA -蛋白质和RNA -蛋白质复合物的核苷酸。 3平衡和动力学4转换可以由羟基自由基足迹作为一个soluti功能进行变量或时间，分别。的足迹的一个主要特点是有限的接触探头（例如，“单次击中动力学”）中的每个核苷酸的聚合物均匀采样的结果。5 在这个视频文章中，我们使用的四膜虫核酶的小四至小六的域名，说明RNA样品制备和一个镁（II）介导的折叠等温线的决心。我们描述了使用众所周知的羟基自由基足迹协议，要求H 2 O 2（我们称之为“过氧化”协议）和一个有价值的，但并不广为人知，替代使用自然溶解O 2的（我们称之为“氧化“协议）。提交的数据压缩，转换和分析程序概述。

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

本协议描述了如何量化的镁（二）依赖RNA的三级结构的形成由两个羟基自由基足迹方法。

监测RNA结构平衡的变动情况“过氧化”和“氧化”羟基自由基足

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K 次观看.  Hunter College. 本协议描述了如何量化的镁（二）依赖RNA的三级结构的形成由两个羟基自由基足迹方法。

视频: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

科研

生物学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

追踪药物诱导Colocalizational变化分析受体后内部贩运

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...