我们诚挚地感谢ITS2的组，Biocenter，维尔茨堡大学，为丰富和宝贵的意见。我们也感谢德意志研究联合会（DFG补助Mu-2831/1-1）提供资金。

1。 ITS2序列的正确诠释<ol><li>进入数据库系统发育的ITS2的工作台位置: <a href="http://its2.bioapps.biozentrum.uni-wuerzburg.de" target="_blank">http://its2.bioapps.biozentrum.uni-wuerzburg.de</a> </li><li>开始你的分析，通过点击"注释"一节中的图标"工具"。然后，键入或粘贴到序列编辑器，在网站上方的序列。序列编辑器会自动检查，是否您的ITS2序列是有效的。 </li><li>选择适合您的序列（如植物Viridiplantae）HMM模型。 </li><li>按一下启动过程中的"注释。" </li><li>徘徊在"杂交"的图标，你可以查看5.8S和28S rRNA的混合的形象，作为一个确认的HMM标注的准确性。 </li><li>点击绿色加号所产生的ITS2序列的符号，选择你的二级结构预测方法:为了预测没有已知templat的结构E，点击"预测结构。"如果你想使用自己的模板，同源建模，点击"模型结构。" </li></ol> 2。二级结构预测<ol><li>预测<ol><li>序列编辑器会自动粘贴到注释的ITS2序列。 </li><li>要开始使用默认设置的二级结构预测，点击"预测结构"按钮。 </li><li>保存包括二级结构模型到数据池ITS2序列，并通过点击绿色加号，然后"添加到池。"另外，你可以把它添加到你的数据池，通过拖放（图1）。 </li><li>如果该序列不能直接折叠，同源建模最好的结果。保存最合适的序列结构通过拖放拖放到数据池。另外，用右键，然后在点击保存序列结构"添加到池中。"到的数据池</li></ol></li><li>自定义建模<ol><li>键入或粘贴到一个或多个模板（与已知结构）上的序列编辑器。 </li><li>一个或多个目标序列输入或粘贴到较低的序列编辑器（无结构）。 </li><li>点击"预测最佳模板（S）"，开始使用默认设置的同源建模。 </li><li>显示在结果列表模板目标的最佳组合。 </li><li>通过拖放到数据池或由右键上点击保存您所选择的模型序列结构（S）"添加到池。" </li></ol></li></ol> 3。 MOTIF搜寻<ol><li>在网站顶部的序列编辑器中键入或粘贴到您的查询序列（S）。 </li><li>选择正确的HMM模型（如植物Viridiplantae）。 3.3。 "母题搜索"，点击启动进程。 </li><li> ITS2的序列与强调主题的插图特德在网站的底部。 </li><li>点击图标旁边的序列头显示二级结构中突出的图案。 </li></ol> 4。搜索和浏览<ol><li>搜索<ol><li>键入一个类群名称或序列标识符（GI），将在网站顶部的搜索栏。 </li><li>由现场搜索框出现一个类群名称搜索支持。 </li><li>您可以执行一个由逗号分隔查询多个搜索。 </li><li>点击"搜索"按钮，执行搜索。 </li><li>你的结果会出现一个新的选项卡中列出。 </li><li>点击列名，根据特定的列进行排序的结果。您还可以添加或删除您所选择的列与列菜单。列名之内出现的箭头图标上点击列菜单可以进入。 </li><li>点击"显示细节"查看序列结构的细节。 &lt;/ LI&gt; <li>通过拖放到数据池或由右键上点击保存您所选择的序列结构（S）"添加到池。" </li><li>保存结果到一个外部文件，点击"保存选择"或"全部保存"。 </li></ol></li><li>浏览<ol><li>通过在网站左侧的树状结构浏览浏览ITS2序列数据库。 </li><li>一个加号，点击查看类群低一个级别。 </li><li>一个类群的名称，点击打开一个新标签包含每个类群的序列结构。 </li><li>点击"显示细节"查看序列结构对细节。 </li><li>通过拖放到数据池或由右键上点击保存您所选择的序列结构（S）"添加到池。" </li><li>保存结果到一个外部文件，点击"保存选择"或"全部保存"。 </li></ol></li></ol> 5。 ITS2的高炉<ol><li>键入或粘贴到一个或多个查询序列，序列编辑器。您的序列可以是纯核苷酸序列或序列结构对。你也可以键入一个序列以下几个二级结构。通过检查框"序列化XXFASTA序列"作为单独的查询，随后使用这些结构。 </li><li>到高炉开始使用默认设置，单击"爆炸"。根据您的查询的性质，无论是共同BLASTN程序或ITS2序列结构高炉进行。 </li><li>打开一个子标签内出现标签为每个查询序列"BLAST结果"，以及执行搜索概述。 </li><li>点击"显示对齐"，以查看计算的高炉路线。 </li><li>通过拖放到数据池或由右键上点击保存您所选择的高炉点击"添加到池中。" </li><li>保存结果到一个外部文件，点击"保存选择"或"保存。" </li></ol> 6。多序列结构调整<ol><li>点击"管理数据集"，然后旁边的放大镜符号的游泳池中的序列，在看看你的数据池。另外，您可以点击网站左下方的数据池的迹象。 </li><li>在您的数据池的序列结构对点击查看其详细信息。 </li><li>在游泳池中的所有序列结构对创建多序列结构对齐，单击"分析数据集"，然后"序列和结构。" </li><li>现在，你被要求选择你对齐的图形模式。如果您的对齐只包含几个序列，拒绝按一下苗条模式"号"否则，按一下选择纤细的图形模式"是的。" </li><li>在几分钟，你的对齐显示在新标签页（图2）。此外，它会自动保存到数据池。 </li><li>为了节省您的对齐到一个外部文件，点击"保存对齐"。 </li></ol> 7。进化树<ol><li>计算序列结构的邻接树的多个对齐，单击"分析数据集"，然后"邻接。" </li><li>结果树在新标签（图3）所示。 </li><li>规模与卷轴自由树"缩放树。" </li><li> reroot你的树，树的一个节点或叶上，然后点击"在这个节点Reroot。" </li><li>如果你想删除您的数据池的类群，叶上单击并选择"从池中删除此节点。"现在，您可以重新计算路线和树的样本数量减少。 </li><li>点击"保存树"作为分析的最终结果外部NEWICK文件保存您的系统发育树。 </li></ol> 8。附加软件<ol><li>点击"关于本网站" - "工具"，找到额外的通知关于独立的的工具4SALE和ProfDistS ATION。 </li><li>旁边的路线和邻接ITS2的数据库网络接口提供的功能，你现在可以访问了几个新功能，如物种的划分，根据补偿碱基变化（CBCS）。 </li></ol> 9。代表结果如上所述的工作流程已成功应用在几个开放式访问调查3,4。例子可以通过以下链接查看: <ul><li> <a href="http://www.plosone.org/article/info%3Adoi/10.1371/journal.pone.0016931" target="_blank">http://www.plosone.org/article/info％3Adoi％2F10.1371％2Fjournal.pone.0016931</a> </li><li> <a href="http://www.biomedcentral.com/1756-0500/3/320" target="_blank">http://www.biomedcentral.com/1756-0500/3/320</a> </li></ul>在这些大规模的研究，我们能够解决绿藻的亲缘关系以及Hypnales（苔藓）W第i个高的分辨率。在这两种情况下，收集了详尽的类群取样ITS2的数据库9 4SALE 15,16自动对齐，最后由ProfDistS 17到进化树处理。在所有这些步骤，序列和结构信息，同时使用。引导进化骨干支持实现使用简介邻接（PNJ）19，这是在单机版的ProfDistS的。 一个序列结构对小集，数字1到3直接上新ITS2序列数据库工作台描述这种自动化的工作流程中的关键步骤5:样本数量，多序列结构对齐，并最终进化树计算。 <img src="/files/ftp_upload/3806/3806fig1.jpg" alt="图1" /> 图1。拖放每类群抽样。在任何时间序列或序列结构<html> ê对可以添加数据池，通过拖放的实例。这里使用拖放和二级结构预测的下降后增加的序列结构。蓝色椭圆标志的序列结构面积下降到数据池。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3806/3806fig1large.jpg" target="_blank">点击此处查看全尺寸版本的这个形象。</a> <img src="/files/ftp_upload/3806/3806fig2.jpg" alt="图2" /> 图2。在全图形模式下的多序列结构对齐。对于少数序列的数据池，被选为全图形模式。基地是彩色的;碱基可以通过点击一个碱基或碱基对的支架上用红色圆圈突出。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3806/3806fig2large.jpg" target="_blank">点击这里查看这个图片全尺寸的版本。</a> 3.JPG"ALT ="图3"/&gt; 图3。序列结构的邻居加入树。七类群多序列结构的对齐计算，可自由扩展树保存在NEWICK格式。

<ol>
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没有利益冲突的声明。

ITS2的数据库是一个完整的内部转录间隔区序列结构为基础的系统发育和功能齐全的工作台。网站可以非常快速，直观地操作。而其他基于网络的系统发育工作台像ARB的20或Mobyle 21只能够工作序列和/或共识的结构信息，ITS2的数据库同时认为每个类群和个人的二级结构序列。然而，由于在Web服务器的计算能力的限制，强烈建议使用多个对齐和邻居，4SALE 15,16和17 ProfDistS加入18计算，分别为大型数据集，独立的工具。 ITS2序列的基本结构亲缘关系的工作流程5旁，这些工具具有一些额外的功能，如计算引导复制，资料邻接（PNJ）19或实物的划的基础上补偿碱基变化（CBCS）8。他们可通过"关于本网站" - "工具"下载和详细信息部分。 ，使用4SALE和ProfDistS，要始终把正确的格式的文件转换成。采样要由4SALE处理必须有一个类群的结局。FASTA或TXT，而作为输入ProfDistS序列结构的对齐必须结束。xfasta。 目前，我们正在实施在ITS2的数据库，以及相关工具的系统发育树重建的替代方法。因此，像22序列结构的最大简约和/或最大似然23的方法，将在未来的访问。

<html><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 评论 </td></tr><tr><td>互联网接入</td><td></td><td>最好是高速</td></tr><tr><td> ITS2的数据库9 </td><td>维尔茨堡大学生物信息学系， </td><td>网址: <a href="http://its2.bioapps.biozentrum.uni-wuerzburg.de" target="_blank">http://its2.bioapps.biozentrum.uni-wuerzburg.de</a> </td></tr><tr><td>软件:4SALE 15,16 </td><td>维尔茨堡大学生物信息学系， </td><td>下载: <a href="http://4sale.bioapps.biozentrum.uni-wuerzburg.de/" target="_blank">http://4sale.bioapps.biozentrum.uni-wuerzburg.de/</a> </td></tr><tr><td>软件:ProfDistS 17 </td><td>维尔茨堡大学生物信息学系， </td><td>下载:NI-wuerzburg.de的/"目标="_blank"&gt; http://profdist.bioapps.biozentrum.uni-wuerzburg.de/ </td></tr></tbody></table>

the its2 database

作为进化标记内转录间隔2（ITS2序列）已使用超过二十年。正如ITS2的研究主要集中ITS2序列非常变量，它低级别的系统发育只局限于此标记。然而，ITS2序列和它的高度保守的二级结构的组合，提高进化第1号决议，并允许在多个分类的行列，包括物种划定2-8进化推理。 ITS2序列数据库 ，提出了详尽的内部转录间隔区序列从NCBI GenBank数据库11，准确reannotated 10集。资料隐马尔可夫模型（HMMs）的注释，每个序列的二级结构进行了预测。首先，它是测试是否正确，四螺旋构象的最低能量的结果基于12倍（直接倍）。如果是这种情况并非如此，结构预测同源建模13。在已知的二级结构同源建模，被转移到另一个ITS2序列的二级结构是不能够正确地折叠直接倍。 ITS2的数据库不仅是一个数据库，用于存储和检索ITS2序列结构。它还提供了多种工具，来处理自己的ITS2序列，包括注释，结构预测，主题检测和BLAST 14相结合的序列结构信息的搜索。此外，它集成了4SALE 15,16修剪版本，ProfDistS 17多序列结构比对的计算和邻居加入18树重建。他们一起形成了一个从初始的序列集的连贯序列和二级结构为基础的系统发育分析管道。 简而言之，这个工作台简化首次系统发育分析只几下鼠标点击，而额外提供的综合性大型分析工具和数据。

Watch this Scientific Journal Video about The ITS2 Database at JoVE.com

The ITS2 Database

ITS2的数据库是一个进化的推论，同时考虑序列和二级结构的内部转录间隔2工作台。这包括数据收集，注释准确，结构预测，多序列结构的对齐和快速的计算。概括地说，这个工作台简化点几下首次系统发育分析。

ITS2序列数据库

The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades.  As ITS2 research mainly focused on the very ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

30.5K 次观看.  University of Würzburg. ITS2的数据库是一个进化的推论，同时考虑序列和二级结构的内部转录间隔2工作台。这包括数据收集，注释准确，结构预测，多序列结构的对齐和快速的计算。概括地说，这个工作台简化点几下首次系统发育分析。

视频: The ITS2 Database

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

科研

生物学

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

使用 MALDI 质谱成像进行代谢分析的示例准备

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...