我们感谢查尔斯·皮诺、梅兰妮·拉加里格和雷吉斯·拉维涅在植物样本的MALDI成像样品准备方面的技巧和技巧。

1. 样品准备
<ol><li>获取生物样本，要么保持新鲜和完好无损（例如，不要强迫它进入管子），要么将其冻结。拟议的协议适用于任何类型的固体生物样本（即植物、动物或人体组织），以本地化特定组织中的化合物。</li>
	<li>冷却低温微分到-20°C。 将样品支架和刀片保持在相同的温度下。</li>
	<li>如有必要，将对象嵌入 M1 嵌入介质中，以便在切割过程中保存它。<ol>
<li>将一些基质倒入放置在低温微分室的塑料模具中。快速添加样品，并倒入一些更多的嵌入介质，以覆盖它。当基质凝固并冷却时，将样品保持在模具中心。 注：并非所有生物物体都需要嵌入。同质物体，如老鼠的大脑不需要嵌入介质，可以切割冻结。</li>
	</ol>
</li>
	<li>将嵌入式或冷冻样品放在低温微分架上，用锋利的刀片切割。5-30 μm 的厚度对植物样本有好处，由于植物样本的异质性，这些样品很难切割。将切割厚度和温度调整到样品中，进行多次切割以找到最佳条件。在提供的示例中，样本被切割为 -20 °C。 考虑将样品保持在 0 °C 以下，以避免样品退化。 注意：使用放置在低温显微镜旁边的双目显微镜可以帮助确定切片的质量。组织必须完好无损。</li>
	<li>使用钳子或小画笔小心地将切片移到 ITO 涂层的滑梯上，然后将手指放在滑梯下，以加热和干燥样品。将幻灯片保存在冷冻微分室中，直到所有样品都准备好。慢慢将滑梯从房间中滑出，以避免热冲击。 注意：要检查幻灯片的哪一侧涂有 ITO 涂层，请在室温 （RT） 下在幻灯片上放置一滴水。掉落将停留在 ITO 涂层侧或未涂层侧的扁平上。</li>
	<li>使用薄记号笔或校正液在幻灯片上绘制标记，并在矩阵沉积前使用高分辨率扫描仪扫描。当样品在质谱仪中时，这些标记将用于确定样品在幻灯片上的确切位置。获得精确参照点的最佳方法是绘制十字架。</li>
</ol>2. 矩阵沉积
<ol><li>准备MALDI矩阵：重70毫克的α-氰诺-4-羟基辛酸（HCCA）基质，并稀释在10mL的水和甲醇溶液（50：50）与0.2%TFA。在RT将矩阵固化10分钟。声波化后可能有额外的固体矩阵。 注：根据感兴趣的分析，可以使用不同类型的 MALDI 矩阵。</li>
	<li>用100%甲醇清洁基质沉积机器人。</li>
	<li>使用甲醇和精密擦拭，清洁滑梯，保持样品不接触他们，不删除痕迹。在没有样品的区域中，在幻灯片上添加干净的盖片。然后将幻灯片的这一部分放置在光学探测器上以跟踪矩阵沉积。 注意：要光学跟踪矩阵厚度，滑梯和盖片必须非常干净。</li>
	<li>使用机器人进行矩阵沉积：在储层中只放置 6 mL 的基质溶液，并添加 2 mL 的 100% 甲醇，总容量为 8 mL。 注：沿沉积的矩阵厚度记录是自动的，可以使用 USB 棒恢复。喷洒方法的开发在这里没有详细说明。</li>
</ol>3. 数据获取
<ol><li>将矩阵的 1μL 放在 MALDI 板上，以使用矩阵峰值作为参考来校准质谱仪。质谱仪采集方法的开发情况没有详细说明。<ol>
<li>将 MALDI 板插入源中，单击 ftms 控制软件中的"加载目标"，然后等待板被加载。</li>
		<li>单击代表 MALDI 板的图像中的点，选择矩阵点的位置。</li>
		<li>在软件的"示例信息"选项卡中注明示例名称和文件夹。</li>
		<li>通过单击"收购"开始获取。</li>
		<li>在收购过程中，使用"MALDI 视频"选项卡上的鼠标指头稍微移动板，使激光点位于不同的点。</li>
		<li>收购完成后，转到"校准"选项卡，在二次模式下选择HCCA 校准列表，然后单击自动。全球校准结果在"校准图"窗口中表示，对于 SolariX XR 7T，应小于 0.2 ppm。</li>
		<li>如果校准良好，请单击"接受"并保存方法。</li>
	</ol>
</li>
	<li>样品上的基质沉积完成后，将幻灯片放在幻灯片适配器中，并使用塑料盖检索标记的位置。</li>
	<li>在灵活的图像软件中，使用打开软件的第一个窗口设置新的成像运行。<ol>
<li>命名成像运行，选择 结果目录，然后单击 "下一步"。</li>
		<li>指示鼠标大小（即用户定义的测量区域），选择要使用的方法（在第 3.1 节校准），然后单击 "下一步"。</li>
		<li>在扫描幻灯片并单击 "下一步"后，加载在第 1.6 步获得的幻灯片的光学图像。</li>
		<li>图像在更宽的窗口中打开。使用幻灯片上的标记来教导幻灯片的位置：在 ftmsControl中，将 MALDI 视频窗口的目标放在标记的确切位置上，然后返回 felx 图像 并单击光学图像上的完全相同的点。重复三个独立点。带有标记的塑料盖被放置在MALDI板上，以恢复其位置并便于教学。 注意：如果标记是用标记制作的，在幻灯片背面添加白色校正液可以使它们在教学过程中脱颖而出。</li>
	</ol>
</li>
	<li>使用"添加测量区域"工具在柔性图像软件中绘制样品感兴趣的区域。</li>
	<li>如果要分析几个样本，则保存成像运行和"自动序列"。</li>
	<li>如果分析多个样本，则使用"自动执行批次流体"启动序列。 注意：定期将数据同步到安全位置。</li>
</ol>4. 数据处理
<ol><li>将原始数据导入可视化软件 （SCILS 实验室），并创建数据集。此软件中分两个单独的步骤完成此操作。<ol>
<li>使用"批量导入器"工具并选择原始数据、指示目标目录并单击"导入"。</li>
		<li>导入后，可以组合多个数据集进行进一步分析。要组合数据集，请使用"文件|新|数据集"工具。</li>
		<li>选择用于获取的仪器类型，通过单击"+"添加导入数据集，并通过单击和拖动对象来排列图像。</li>
		<li>检查质量范围设置或在需要时通过指示兴趣范围进行修改。单击 "下一步 "查看导入摘要并启动导入。</li>
	</ol>
</li>
	<li>可视化样品和/或不同样品中不同组织中感兴趣的 m/z。 只需单击 光谱 即可选择感兴趣的 m/z 或在 m/z 框中键入值。<ol>
<li>如果需要，执行统计测试，以搜索不同组织和/或不同样本之间的共局部或歧视值，以确定感兴趣的 m/z。 这些工具在"工具"菜单中可用，不会在此协议中描述。</li>
	</ol>
</li>
	<li>从对象选项卡中导出感兴趣的m/z（例如，同地化、歧视性化合物）作为.csv文件。如果数据集不是太大（即最多 60，000 行），则也可以导出整个数据集。单击红色箭头指示的兴趣区域右侧的"出口"图标。</li>
	<li>导入.csv文件，以便在注释软件 （Metaboscape） 中创建新的数据集。点击"项目|导入CSV项目"。请注意，将只考虑确切的 m/z， 并且丢失了同位素配置文件。 注：5.0 软件版本允许从可视化软件直接导入数据，从而能够更准确地注释，因为可以考虑同位素配置文件。</li>
	<li>附有定制分析列表的注释，这些列表可以从公开的数据库中提取。软件会给出一个模板来创建分析列表。</li>
	<li>使用预测软件（代谢物预测）对注释化合物的代谢物进行硅基预测。需要开发利益复合物的公式，要么由软件中的用户绘制，要么导入为 .mol 文件。然后，该方法是一个简单的分步协议。</li>
	<li>恢复代谢物列表，创建分析列表，并在注释软件中使用它用预测代谢物注释原始数据。或者，如果代谢物列表很短，则手动按步骤 4.8 搜索。</li>
	<li>可视化软件中原始数据中代谢物的组织分布。</li>
	<li>通过右键单击预测的兴趣代谢物，恢复预测软件中代谢过程中所涉及的酶的名称。</li>
</ol>

<ol>
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作者没有什么可透露的。

此协议的关键部分是样品准备：样品必须柔软且完好无损。切割是最困难的部分，因为样品的温度和厚度可能因所研究的样品类型而异。动物组织通常是同质的，更容易切割。植物样本通常包含不同的结构，因此由于刀片遇到软、硬或空血管组织，因此更难保持完好无损。强烈建议在与植物样本一起工作时使用新鲜组织，以避免在亲水组织中形成冰并破坏冰。切片存放在 ITO 涂层滑梯上时，必须?...

由于人类活动，异种生物在世界各地广泛分布。其中一些化合物是水溶性，并被土壤1吸收，并进入食物链时，他们积累在植物组织2，3，4。这些植物被昆虫和食草动物吃掉，它们是其他生物的猎物。一些异种生物的摄入及其对植物健康的影响被描述为5，6，7，8，但最近才在组织水平9。因此，目前还不清楚异种生物代谢发生于何处或如何发生，或者特定植物代谢物是否与特定组织中的异生物积累相关。此外，大多数研究忽视了植物中异种生物及其代谢物的新陈代谢，因此对植物组织中的这些反应知之甚少。
这里建议的是一种研究生物样本中的酶反应的方法，该反应可以与基质和反应产品的组织定位相关。该方法可以在一个实验中绘制生物样本的完整代谢剖面，因为分析是无针对性的，并且可以使用感兴趣的定制分析列表进行调查。提供的是在原始数据集中跟踪的候选人列表。如果在样本中注意到一个或几个感兴趣的分析，特定的组织定位可以提供有关相关生物过程的重要信息。然后，利用相关的生物定律在硅中修改感兴趣的分析，以寻找可能的产品/代谢物。然后，通过识别所涉及的酶并本地化组织中的反应来分析原始数据，从而帮助了解发生的代谢过程。没有其他方法提供生物样本中发生的反应类型、感兴趣化合物的本地化及其相关代谢物的信息。一旦有新鲜和完整的组织，并且感兴趣的化合物可以电解，这种方法可用于任何类型的生物材料。拟议的议定书发表在《维莱特》等12 期中，详见此处供科学界使用。

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slips</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">267942</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cryomicrotome</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Excel</td>
			<td style="width:163px;">Microsoft corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">flexImaging</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ftmsControl</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">GTX primescan</td>
			<td style="width:163px;">GX Microscopes</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HCCA MALDI matrix</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8201344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ITO-coated slides</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">237001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">M1-embedding matrix</td>
			<td style="width:163px;">ThermoScientific</td>
			<td align="right" style="width:119px;">1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metabolite Predict</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metaboscape</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Methanol</td>
			<td style="width:163px;">Fisher Chemicals</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MX 35 Ultra blades</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">15835682</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plastic molds</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SCiLS Lab</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SolariX XR 7Tesla</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td>The method proposed is not limited to this instrument</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Spray sheets for ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8261614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">TFA</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
	</tbody>
</table>

investigation of xenobiotics metabolism in salix alba leaves via mass spectrometry imaging

所介绍的方法使用质谱成像（MSI）来建立 S.阿尔巴 叶在接触异种生物时的代谢轮廓。使用非靶向方法，在植物组织中识别和本地化感兴趣的植物代谢物和异种生物，以发现特定的分布模式。然后，在从已识别的异种生物中预测潜在的代谢物（即代谢物和结合物）。当异生物代谢物位于组织中时，记录植物改变其所涉及的酶类型。这些结果用于描述在 S.alba 叶中发生的不同类型的生物反应，以回应叶子中的异生物积累。代谢物在两代人中预测，允许记录连续的生物反应，以改变叶组织中的异种生物。

此协议适用于从暴露于环境中的异种生物的S. alba树中采样的新鲜叶子。这个过程在图1中描述。第一步是准备兴趣样本的薄片。植物样本通常比动物样本更难切割，因为组织是异质的，可以含有水和/或空气。此困难使用嵌入介质处理，在样品周围形成均匀块。矩阵沉积通过使用机器人，避免手部操纵和确保可重复的结果而得到促进。MALDI基质层厚度在整个过程中遵循?...

Watch this Scientific Journal Video about Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging at JoVE.com

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

这种方法使用质谱成像（MSI）来了解在接触异种生物时 ，S.阿尔巴 叶的代谢过程。该方法允许在特定、完整的组织内对感兴趣的化合物及其预测的代谢物进行空间定位。

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

investigation-xenobiotics-metabolism-salix-alba-leaves-via-mass

Research

JoVE Journal

Environment

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

3.0K 次观看.  Université de Strasbourg. 这种方法使用质谱成像（MSI）来了解在接触异种生物时 ，S.阿尔巴 叶的代谢过程。该方法允许在特定、完整的组织内对感兴趣的化合物及其预测的代谢物进行空间定位。

视频: Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

巴西田间放生转基因 伊 蚊的大规模生产

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

科研

环境科学

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

优化烟气设置通过电脑模拟来促进微藻生长的光生物反应器中

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

水生微生物Metaproteomics工作流程：从细胞对胰蛋白酶肽适合串联质谱为基础的分析

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

用氢化物发生-原子吸收光谱法测定多种食品基质中的无机砷。

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

树叶颗粒物去除能力的评价

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

利用目标筛选和非靶向筛选高分辨率质谱相结合的工作流程识别跨氟和多氟化学物种

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

旋转芯在常见菌根网络中植物相互作用的研究

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural losses. To understand the biology of such pest moths, fundamental techniques have been in high demand. Here, methods for mass-rearing, observations, and molecular studies are developed using two tea tortrix, Homona magnanima and Adoxophyes honmai (Lepidoptera: Tortricidae). Insects were mass-reared with sliced artificial diet and maintained by inbreeding for over 100 generations by considering their biological characteristics. Insects have various sex dimorphisms; hence it is difficult to distinguish the sex during the developing stages, which have prevented subsequent assays. The present work highlighted that the sex of tortricids larvae could be determined by observing testes or lactic-acetic orcein staining to visualize the female-specific W chromosome. Moreover, using the sex determination methods, the present study enabled nucleic acid extractions from sex determined embryos and application toward high throughput sequencing. These tips are applicable for other pest insects and will facilitate further morphological and genetic studies.

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

龟科害虫的大规模饲养与分子研究

Tortricidae (Lepidoptera), commonly known as tortrix or leafroller moths, comprises many agricultural and forestry pests, which cause serious agricultural ...

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

通过质谱成像对萨利克斯阿尔巴叶中异种生物代谢的调查