作者要感谢让-米歇尔·ANE博士农艺学系威斯康星大学麦迪逊分校提供蒺藜苜蓿样品。这项工作是由美国国家科学基金会（NSF）提供资金支持部分补助CHE-0957784，威斯康星大学的研究生院和威斯康星校友研究基金会（WARF）和Romnes学院研究奖学金计划（以当地雇员）。 EG承认了美国国家科学基金会研究生研究奖学金（DGE-1256259）。

1。仪器仪表<ol><li> MALDI-TOF/TOF微星。使用质谱仪配备了MALDI源小分子的分析（见表材料/设备）。在正或负离子模式取决于目标分析物进行收购。指定所关注的质量范围内，并在50微米的间隔在两个跨样本生成离子图像的表面的x和y维度收集500次激光发射/斑点。激光照射的光栅宽度和数量可以调节，分别得到更高的空间分辨率和最大信号强度。使用DHB基质峰或施加于滑动或直接向组织校准质谱内部标准。 </li><li>高分辨率LC-MS。 （见表材料/设备）运行样品提取物使用任一反相（RP）的LC与C-18柱，或正相（NP）与HILIC柱根据相关分析物的LC-MS分析。用流动相和使之适用于特定样品类型梯度。在正或负的离子模式取决于样品类型进行采集。 </li></ol> 2。组织准备<ol><li>从修剪植物的根瘤，留下3-4毫米根部附着于结节。 </li><li>立即解剖后，使用镊子将组织在低温恒温器杯和盖在明胶（100毫克/毫升的去离子水）。至关重要的是，所述组织被粘在杯的无气泡的底部。 </li><li>闪光通过将杯子在干冰/乙醇浴中，直到明胶硬化并变得不透明冻结的组织。店内样品于-80℃直至使用。 </li><li>从取出样品-80℃冰箱，剪去塑料低温恒温杯和修剪掉多余的明胶。安装包埋组织的低温恒温器与卡盘的最佳切割温度（OCT），媒体一角钱大小的量，而不是让华侨城触碰组织。放置在低温恒温器框，直到华侨城凝固。 </li><li>允许所述卡盘和明胶在低温恒温器箱（设定为-20℃或-25℃）中平衡约15分钟。 </li><li>使用低温恒温器（见表材料/设备），以部分组织1细胞的约的厚度（8-20微米取决于组织类型）和解冻温热的背面（非ITO装载每个片上ITO膜的玻璃涂在手上的背面涂有ITO的玻璃载片的一侧）。将冷冻组织切片靠近温热滑动的带ITO膜的一侧，并允许片粘到滑动。配售部分并拢的幻灯片将微星过程中提供更好的定位。 </li></ol> 3。矩阵的应用<ol><li> MALDI基质的喷笔应用 <ol><li>所有的喷枪程序应在通风橱中进行。 </li><li>彻底清洁喷枪液的容器和喷嘴（见表材料/设备），以甲醇和填充溶液的容器与DHB基质溶液（150毫克/毫升50％甲醇％TFA体积/体积）。 </li><li>从样品约35厘米握住喷枪和应用10-15涂层基体的滑动表面上以10秒的喷射持续时间和在各涂层之间30秒的干燥时间。 </li><li>彻底完成时，避免基质溶液堵塞用甲醇再次清洁喷枪。 </li></ol></li><li> MALDI基质自动喷雾机的应用 <ol><li>遵循自动喷雾器系统的制造商提供的启动指令。 </li><li>在使用40毫克/毫升的根瘤代谢物的MSI（在50％甲醇％TFA体积/体积）DHB作为基质，设定温度为〜80℃，速度到1250毫米/分钟，流速为50微升/分钟，并通到24的数。为了获得最佳覆盖，建议以旋转喷嘴90°和/或偏移每遍之间的喷嘴1.5毫米。启动喷雾器方法。 作为SI去说明，这里使用的特定的喷雾器系统，加热喷嘴的溶剂的蒸发速度更快。作为溶剂蒸发时，该矩阵的浓度迅速增加。施加到样品的喷枪和自动喷雾器中的矩阵具有可比的浓度。 </li><li>遵循由自动喷雾器系统的制造商提供的关机指令。 </li></ol></li><li> MALDI基质的升华应用 <ol><li>称出300毫克DHB进入升华室的底部（见表的材料/设备）。 </li><li>粘在载玻片上的指形冷冻器（在升华室的顶部部分）与组织切片朝下用双面导电胶带。盖在所述双面胶带的滑动，即使导电性的整个背面，产生甚至基质沉积。 </li><li>夹紧升华室的顶部和底部两半连同C形夹。连接真空和广告ð冰和冷水顶端水库。 </li><li>放置升华室中的加热套是在室温下。 </li><li>打开真空泵。等待15分钟，然后打开加热套。加热套应达到120℃以上10分钟的过程。 </li><li> 10分钟后，关闭加热，关闭阀门，以真空（这样所述腔室的内部保持在真空条件下），然后将真空泵关闭。 </li><li>允许该腔室，以恢复到室温，打开阀门释放真空压力，取出样品。在升华室的尺寸将确定矩阵的升华到载玻片上的量。较大的升华室（400毫升烧杯中的尺寸）将使用大约300毫克DHB的，而较小的腔室（150毫升烧杯中的尺寸）将使用大约100毫克DHB的，并且需要切割的玻璃载玻片，以便它适合在该腔室。 </li></ol></li></ol> 4。图像采集<ol> &lt;LI&gt;标记样品的每一个角落+模式与WiteOut涂改液笔被用来作为“教点”。将载玻片放入MALDI滑接板，并用扫描仪取的样品的光学图像。 </li><li>使用仪器公司为50μm的光栅的步长和激光直径等于或小于光栅的步长越小提供的软件设置的图像获取的文件。在这个特殊的仪器，最小激光设置给出的约10微米的和小的激光设置一个激光直径具有40-50微米的直径。 </li><li>加载光学图像进入软件并与光学图像对准板。 </li><li>使用常见的矩阵簇离子，内部标准，或校准组合开始收购前对仪器进行校准。 </li><li>组织指定的区域与MSI进行分析，包括纯矩阵的投影片上的斑点被用来作为一个“空白”。 </li><li>交流开始quisition。 </li></ol> 5。图像生成<ol><li>在由供应商提供的商业软件打开成像文件并提取离子图像。其他开源软件是供微星数据处理19。 </li></ol> 6。代谢物鉴定<ol><li>选择特定米/利息，自质谱使用供应商特定的软件Z（见表材料/设备）。分析物可以从一个矩阵离子进行区分时被分析物峰的质谱选择和产生离子的图像特异性定位于组织部分。 </li><li>生成感兴趣的分析物的列表，并执行MS / MS实验。 见表1和表2中的分析物的样品清单代表性的成果。 </li><li>进行有针对性的LC-MS分析在高分辨率质谱仪（或高分辨率MALDI-MS如果有的话），以获得准确感兴趣的分析物的质量测量，并进行目标分析物的LC-MS/MS得到的特征裂解方式。 </li><li>搜索以确定假定的标识为目标分析物进行数据库。代谢物数据库的例子包括：METLIN，ChemSpider，PubChem数据库，KEGG和HMDB。 </li><li>以确认从精确质量数据库检索推定的标识，匹配的MS / MS的目标分析物的标准文献，和/或碎裂预测软件MS / MS谱图。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

如上所述，样品的准备是在MSI的工作流程中最关键的步骤。不均匀地嵌入组织将导致切片是在某些情况下难于或不可能的。截面尺寸和足够的平衡时间是至关重要的维持组织的完整性，避免折叠和泪水。选择矩阵和应用技术将在确定待检测的分析物的种类的作用，空间分辨率，其结果的再现性。使用矩阵或应用技术的组合可以提供互补的结果。 这个方法是专门为内源性代谢?...

代谢组学领域的发展具有许多重要的生物学应用，包括生物标志物的发现，破译代谢途径在植物和其他生物系统，和毒理学分析4,6-10。研究生物系统时，一个主要的技术挑战是研究代谢途径，而不破坏它们11。 MALDI-MSI允许完整的组织的直接分析，使敏感的检测分析物的单一器官12,13，甚至单个细胞14,15。 样品制备是生产重现性好，质量可靠的光谱图像的关键一步。的图像的质量极大地依赖于因素，例如组织包埋剂，切片厚度，MALDI基质和基质的应用技术。用于成像应用中，理想的部分的厚度是1小区（8-20微米取决于样品类型）的宽度。 MALDI需要有机，晶体沉积Ë基复合材料，通常是弱酸，对样本，以协助分析消融和电离，16个不同的矩阵提供不同的信号强度，干扰离子，以及不同类别的化合物的离子化效率。 矩阵应用技术也起着质谱图像和不同的技术质量的作用适用于不同类的分析物。三个矩阵应用方法载于本协议：喷枪，自动喷雾器，和升华。喷枪矩阵应用程序已被广泛应用于MALDI成像。喷枪矩阵应用的优点在于它是相对快速和容易的。然而，喷枪矩阵应用的质量在很大程度上取决于用户的技能，而且往往是较少重现性，并导致被分析物的扩散，特别是小分子17。自动喷雾系统也有类似的机制，以喷枪矩阵的应用，但甲肝Ë已经发展到去除看到与手动喷枪应用程序的多变性，使得喷雾更具有可重复性。这种方法有时会耗费更多的时间比传统喷枪矩阵的应用。手动喷枪，自动喷雾系统是溶剂型矩阵的应用方法。升华是，正成为越来越流行的代谢物和小分子的质谱成像，因为它减少分析物扩散的干基质的应用技术，但它缺乏必要的溶剂提取，并观察更高质量的化合物18。 自信鉴定代谢物通常需要精确质量测量，以获得公认的鉴定其次是串联质谱（MS / MS）的实验进行验证，与MS / MS谱图相比，目前的标准，文学，或理论光谱。在这个协议中高分辨率（质量分辨60,000 m / z为400的电源），液相色谱（LC）-MS连接到MALDI-MSI同时获得空间信息和内源性代谢物鉴定信心，用苜蓿根系和根瘤的生物系统。 MS / MS实验，可直接执行的组织与MALDI-MSI或组织提取物的LC-MS和用于代谢物的鉴定的验证。 该协议提供了一种简单的方法来映射内源性代谢物M。苜蓿 ，这可适应和应用于在各种组织类型和生物系统的小分子的MSI。

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gelatin</td>
			<td style="width:181px;">Difco</td>
			<td style="width:163px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=214340">214340</a></td>
			<td style="width:611px;">heat to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat- HM 550</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:163px;">956564A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">indium tin oxide (ITO)-coated glass slides&#160;</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25-75-0.8 mm (width-length-thickness)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-Dihydroxybenzoic acid (DHB) matrix&#160;</td>
			<td>ICN Biomedicals</td>
			<td><a href="http://www.labsource.com/Catalog/Item.aspx?ItemID=1787195">PI90033</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Airbrush</td>
			<td>Paasche Airbrush Company</td>
			<td>TG-100D</td>
			<td>coupled with 75 ml steel container</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Automatic matrix sprayer system- TM-Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td style="width:163px;">HTX.TMSP.H021-U</td>
			<td>Specific start-up and shut-down instructions will be given when the instrument is installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sublimation apparatus&#160;</td>
			<td>Chemglass Life Science</td>
			<td style="width:163px;"><a href="http://www.chemglass.com/product_view.asp?pnr=CG-3038">CG-3038-01</a></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>Ultimate Pressure = 1x10-4 Torr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrafleXtreme MALDI-TOF/TOF</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td style="width:163px;">276601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">FlexImaging</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td>269841</td>
			<td>One example of &#34;vender specific software&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MALDI LTQ Orbitrap</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMASZ</td>
			<td>High resolution MALDI instrument for accurate mass measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Q Exactive</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>High resolution LC-MS instrument for accurate mass measurements</td>
		</tr>
	</tbody>
</table>

maldi-mass spectrometric imaging for the investigation of metabolites in medicago truncatula root nodules

用于研究小分子，如药物的药物或内源性代谢物，采用组织提取物需要所关注的组织的均匀化，可能潜在地会改变正在研究1中的代谢途径多数技术。质谱成像（MSI）是一种可以生物组织样品1-5的完整片内提供分析物的空间信息的强大的分析工具。这种技术已被广泛用于研究各种类型的化合物，包括蛋白，肽，脂质和小分子如内源性代谢物。用基质辅助激光解吸/电离（MALDI）的MSI，多个代谢物的空间分布，可以同时检测到。在此，特别为豆科植物的根和根瘤中进行非靶向代谢物组的MSI实验开发了一种方法，提出这可能揭示见解发生的生物过程。该方法，这里介绍显示了一个典型的MSI的工作流程，从样品制备到图像采集，并集中于矩阵的应用步骤，表明是用于检测小分子的有用的几种基质应用技术。一旦生成了多光谱图像，感兴趣代谢物的分析鉴定，讨论和论证。标准工作流程这里介绍可以很容易地修改为不同的组织类型，分子种类，和仪器仪表。

MSI的实验概述示于图1。在实验的开始，样品制备是关键的一步。结节是从植物根修剪和嵌入在明胶。该组织必须被压平的低温恒温器茶杯中，用无气泡，而它被冻结，这将确保组织更容易和正确对齐，而它被切开。当组织已经被切片，重要的是要切断的组织在适当的厚度;太薄部分撕裂，搞坏了组织的完整性，而过厚的部分会降低分析物提取的，并从组织检测到的数量。选择矩阵和?...

Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

质谱成像（MSI）是一种强大的工具，可用于在完整组织中发现和鉴定各化学物种，保护化合物中的其天然环境中，它可以提供新的见解的生物过程。用于本发明的小分子的分析开发出一种MSI方法进行说明。

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

maldi-mass-spectrometric-imaging-for-investigation-metabolites

Research

JoVE Journal

Biology

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

18.8K 次观看.  University of Wisconsin- Madison. 质谱成像（MSI）是一种强大的工具，可用于在完整组织中发现和鉴定各化学物种，保护化合物中的其天然环境中，它可以提供新的见解的生物过程。用于本发明的小分子的分析开发出一种MSI方法进行说明。 

视频: MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

科研

生物学

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

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

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

MRM 微可可性能校准和使用在 22 T 的 Medicago 特伦卡图拉根上演示

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

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 ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...