我们感谢Choi和Jung实验室的所有成员进行的方法论讨论。我们感谢 C. Jang 和 D. Guertin 提供的建议和反馈。我们感谢 M.S. Choi 对手稿的批判性阅读。这项工作由 NRF-2022R1C1C1012034 资助给 S.M.J.;NRF-2022R1C1C1007023 至 D.W.C;NRF-2022R1A4A3024551 至 S.M.J. 和 D.W.C.这项工作得到了忠南国立大学对WTK的支持，图1和图2是使用BioRender（http://biorender.com/）创建的。

所有实验均在成均馆大学机构动物护理和使用委员会（IACUC）的批准下进行。将小鼠饲养在IACUC批准的动物设施中，该设施位于22°C和45%湿度的洁净室中，每天进行12小时的光/暗循环。他们被放在通风的架子上，可以随意获得标准的食物饮食（包括 60% 的碳水化合物、16% 的蛋白质和 3% 的脂肪）。垫料和筑巢材料每周更换一次。在这项研究中，使用了12周龄且体重在25g至30g之间的雄性C57BL / 6J小鼠。这些动物来自商业供应商（见 材料表）。
1.通过温度驯化和药理刺激调节棕色脂肪组织的代谢活性
注意:数天至数周的温度适应或使用β-肾上腺素能受体激动剂的药理学刺激是调节 BAT 活性1 的常用方法。因此，下面提供了该方法的简明概述，以便读者能够根据需要选择适当的方法。为了获得代谢不活跃（产热性较低）的BAT，为C57BL / 6J小鼠选择称为热中性的基线温暖温度（28-30°C）。这个范围确保小鼠不需要消耗额外的能量来维持恒定的体温。为了获得代谢适度或高活性（产热）BAT，可以分别选择温和的寒冷（20-22°C）或严寒（6°C）温度。出于该实验的目的，在22°C的标准饲养条件下饲养小鼠，尽管对小鼠来说温和寒冷，但不涉及任何药理学刺激。
<ol><li>温度适应<ol><li>在开始温度适应之前至少1周，将小鼠分开，每个笼子容纳1或2只小鼠。准备配备空气通风、温度和湿度控制的啮齿动物培养箱，并满足所需条件。</li>
		<li>在适当的日子将笼子移至各自的啮齿动物培养箱中，以适应所选的温度。</li>
		<li>确保小鼠数量在所有组中的分布均匀，每个笼子有 1 或 2 只小鼠。单体壳是首选，因为与组壳体相比，单体壳体对温度引起的生理变化更敏感43.以下是每个群体的具体住房条件:<ol><li>热中性（30°C）组:将该组中的小鼠在30°C的温度下连续保存长达四个星期。</li>
			<li>严寒组:最初，将该组中的小鼠饲养在18°C，没有任何筑巢材料。它们将经历每周逐渐下降的温度，到第四周达到 6 °C。温度变化如下:18°C→14°C→10°C→6°C。</li>
			<li>轻度寒冷组（20-22°C）:在与前面提到的标准饲养条件相同的条件下，将该组中的小鼠饲养在啮齿动物培养箱中。</li>
			<li>急性冷挑战:对于急性冷挑战，在没有任何筑巢材料的情况下，每个笼子放置1-2只小鼠，并将它们暴露在6°C的啮齿动物培养箱中长达8小时。 注意:这些住房条件和温度变化对于研究不同温度环境对 BAT 活性和新陈代谢的影响至关重要。</li>
		</ol></li>
		<li>每周更换笼子并补充食物和水。在补充前至少24小时，在各自的温度（啮齿动物培养箱）下预适应笼子。 注意:为了防止适当温度刺激的干扰，重要的是不要提供可能导致筑巢的小鼠富集。为了应对严寒，小鼠会消耗更多的能量来维持体温，从而增加食物摄入量和排泄率。因此，每周至少检查笼子两到三次（遵循当地机构指南）至关重要，以确保小鼠有足够的食物和水供应，并且笼子不会过度潮湿。这种监测对于在研究期间维持小鼠的福祉和健康至关重要。</li>
	</ol></li>
	<li>使用 β3-肾上腺素能受体激动剂 CL316,243 进行药理学刺激1<ol><li>为了最大限度地提高刺激效果，在注射前将小鼠预置于热中性（30°C）下2-4周。</li>
		<li>驯化期后，腹膜内注射1mg/kg CL316,243。 注意:对于β3-肾上腺素能受体激动剂的慢性刺激（例如 从3天到第1,44,45周），由于药物稳定性，需要每天注射。稀释的CL316,243应在注射当天制备，因为稳定性。</li>
	</ol></li>
</ol>2. 动静脉采血
注意:最好建议12-14周以上的小鼠进行动静脉采血。年轻的小鼠可能没有足够大小的苏尔寿静脉，这是一种独特的血管，专门从肩胛间 BAT46 排出静脉血。
<ol><li>使用校准的汽化器轻轻麻醉，其中 3% 异氟醚用于诱导（在 205 x 265 x 200 毫米大小的腔室中）和 2% 异氟醚用于维护（使用气体麻醉面罩）。 注意:整个动静脉采样程序应在麻醉后立即进行。麻醉期间可以使用水加热的加热垫来维持核心体温。 注意:异氟醚吸入时具有高度挥发性和毒性。因此，一期麻醉必须在通风橱下进行。</li>
	<li>验证动物的反应，例如爪子缩回，以确认麻醉已达到适当的深度。</li>
	<li>从苏尔寿静脉采集静脉血<ol><li>放置鼠标以暴露背部皮肤，在切口前通过脚趾捏合确认麻醉深度。用充足的 70% 乙醇润湿背部皮肤以防止毛发脱落，并沿着胸部下部一直切开一个切口，一直到颈部。 注意:肩胛间 BAT 位于皮肤正下方，由两个脂肪垫组成，上面覆盖着薄薄的白色脂肪组织。重要的是要确保小鼠通过气体麻醉面罩保持麻醉状态。</li>
		<li>用弯曲的尖端镊子轻轻提起肩胛间 BAT，小心地切割附着的组织，其中大部分是肌肉。将脂肪垫抬向头部，继续切割附着的组织并小心地打开该区域，直到苏尔寿静脉（连接到肩胛间 BAT 的两个脂肪垫的"Y"形深红色大血管）暴露出来。 注意:注意不要切割与肩胛间 BAT 前部和侧面区域相连的肌肉组织的毛细血管，因为这样做会显着降低从苏尔寿静脉排出的血液量和质量。</li>
		<li>小心地切开苏尔寿的静脉，并使用底部切口的 200 μL 移液器吸头和 P100 移液器收集约 40 μL 血液。将血液储存在采血管中，并将管子放在冰上，直到血清采集过程。 注意:重要的是从苏尔寿静脉的 Y 形分割点正下方收集血液，以防止血液污染来自上腔静脉47。收集过多的血液会削弱苏尔寿静脉的特征。因此，请务必从苏尔寿静脉中收集所需的最低量血液进行分析。选择采血管时要深思熟虑，因为血清和血浆会产生不同的代谢物谱 48,49,50。对于血浆采集，有必要使用肝素包被的管。在该实验中，选择凝血活化剂包被的管子来获得血清。在任何情况下都不应使用EDTA涂层管，因为EDTA会显著影响质谱信号51,52。</li>
	</ol></li>
	<li>从左心室采集动脉血<ol><li>翻转鼠标而不失去与鼻锥的接触，露出腹侧皮肤。在切口之前通过脚趾捏确认麻醉深度。用足量的70%乙醇润湿腹侧皮肤，以防止毛发脱落，并用剪刀小心地打开胸腔，在不破坏任何内部结构的情况下露出心脏。</li>
		<li>使用 1 mL 注射器和 29 G（1/2"）针头准确刺穿左心室的顶点区域。将三分之二的1/2英寸针插入心尖右侧水平3-5mm处（图1B），然后将注射器向后拉以从左心室收集血液（50-100μL）。左心室的血液是含氧动脉血，呈鲜红色。将血液储存在采血管中，并将管子放在冰上，直到血清采集过程。 注意:应在胸腔内做一个最小的切口，以便进入心脏。切口过多可能导致局部出血，进而导致低血压。这可能会影响从左心室收集的动脉血的质量。避免旋转或翻转心脏，因为这会使确定左心室的精确位置变得困难。</li>
		<li>执行安乐死，并按照当地机构的指导方针使用适当的方法确保安乐死。例如，安乐死可以通过颈椎脱位进行，并通过心跳停止来确认。</li>
	</ol></li>
	<li>在4°C下以10,000× g 离心血液样品10分钟。 使用移液管小心收集上清液。上清液应含有血清或血浆，具体取决于所用采血管的类型。可以在这里停下来，将样品储存在-20°C下，直到血清处理以进行GC-MS分析。 注意:如果观察到红色上清液，则可能发生溶血。为避免溶血，请在凝血完成前涡旋样品。一些富含红细胞的代谢物（包括谷氨酰胺和乳酸）可能导致数据误解，尽管大多数代谢物可能不会受到溶血的显着影响。建议在一周内分析血清/血浆。</li>
</ol>3. 从血清中提取代谢物和化学衍生化
<ol><li>提取准备 注:包括代谢物提取、衍生化和数据分析在内的所有方法都是先前描述的方法53,54 的略微修改版本。<ol><li>通过向MS级甲醇中加入100μM的DL-正缬氨酸内标溶液（参见 材料表）来制备提取缓冲液。</li>
		<li>确保所有实验程序都在冰上进行。</li>
	</ol></li>
	<li>将从苏尔寿静脉或左心室提取的 10 μL 小鼠血清转移到含有 40 μL 提取缓冲液的 1.5 mL 微量离心管中。</li>
	<li>为了除去细胞碎片和蛋白质，短暂涡旋血清样品，然后在4°C下以最大速度（18,000× g）离心30分钟。</li>
	<li>离心后，小心地将40μL上清液转移到玻璃小瓶中，然后在4°C的真空离心机中干燥3小时。 注意:建议使用玻璃插件（参见 材料表）代替塑料管，因为在随后的衍生化步骤中使用了塑料反应性化学品。</li>
	<li>将干燥的样品进行两个连续的衍生化步骤，以对血清代谢物进行GC-MS分析。 注意: 由于溶剂的刺激风险，应在通风橱下执行以下步骤。<ol><li>将干燥的血清提取物重悬于溶解在吡啶中的30μL10mg / mL甲氧胺盐酸盐（参见 材料表）中，并在37°C下孵育30分钟。</li>
		<li>用70μLN-甲基-N-叔丁基二甲基硅基四氟乙酰胺（MTBSTFA，参见 材料表）在70°C下衍生用于代谢物硅烷化1小时的样品。 注意:由于衍生化溶剂与塑料发生反应，因此建议在以下步骤中使用玻璃注射器而不是塑料尖端。</li>
	</ol></li>
</ol>4. 使用GC-MS进行代谢组学分析
注:采用单联 GC-MS（见 材料表）测量苏尔寿静脉和左心室衍生样品中的各种血清代谢物，包括碳水化合物、氨基酸和 TCA 循环中间体。也可以使用其他色谱柱，尽管包括温度程序在内的实验设置可能会因所用色谱柱的类型而异。
<ol><li>在280°C（入口温度）下，使用氦气作为流速为1.500mL / min（设定点）的载气，以不分流模式将1μL衍生化样品注入GC中。</li>
	<li>将四极杆设置为200°C，GC-MS接口设置为300°C。 注意:所有代谢物分析的烘箱程序从60°C开始，保持1分钟，然后以10°C / min的速率增加，直到温度达到320°C。</li>
	<li>通过设置为 70 eV 的电子电离 （EI） 收集数据，并在扫描模式 （50-550 m/z） 53 下获取样品数据。本研究中使用的所有代谢物均已通过标准品验证，以确认质谱和保留时间。</li>
	<li>使用市售分析软件进行峰面积积分（参见 材料表）。</li>
	<li>将化合物与每个TBMDS衍生物的子离子相匹配。然后，通过将碎片离子的 m/z 值积分到相应峰面积，得到提取离子色谱图（EIC），然后导出。碎片离子如 表1所示。 注:每个样品中每种化合物的EIC均由DL-正缬氨酸归一化。数据由对数2 苏尔寿左心室静脉 （Log2（SV/LV）） 表示，使用每个归一化 EIC 值。</li>
</ol>

探索棕色脂肪组织中的代谢物交换

作者声明他们没有利益冲突需要报告。

了解 BAT 在全身能量平衡中的代谢潜力的一个关键步骤是确定它消耗了哪些营养物质，它们是如何代谢处理的，以及哪些代谢物被释放到循环中。该协议引入了一种专门的动静脉取样技术，该技术能够进入 C57BL/6J 小鼠肩胛间 BAT 和全身动脉脉管系统的静脉脉管系统，该技术最近由 Park 等人42 开发和验证。以下是您在遵循协议时应认真考虑的关键点。
对于生热...

棕色脂肪组织 （BAT） 具有一种独特的能量消耗特性，称为非颤抖产热 （NST），它涉及线粒体解偶联蛋白 1 （UCP1） 依赖性和 UCP1 非依赖性机制1、2、3、4、5。这些显着特征使 BAT 参与全身代谢的调节和代谢疾病的发病机制，包括肥胖、2 型糖尿病、心血管疾病和癌症恶病质 6,7,8。最近的回顾性研究表明，BAT 质量和/或其代谢活性与人类肥胖、高血糖和心脏代谢健康呈负相关 9,10,11。
最近，BAT被提议作为负责维持NST的代谢汇，因为它需要大量的循环营养物质作为产热燃料6,7。此外，BAT可以产生和释放生物活性因子，称为棕色脂肪因子或BATokines，其充当内分泌和/或旁分泌信号，表明其积极参与系统水平的代谢稳态12,13,14,15。因此，了解BAT的营养代谢应该增强我们对它在人类中的病理生理学意义的理解，超越其作为体温调节器官的传统作用。
使用稳定同位素示踪剂的代谢组学研究，结合使用非代谢放射性示踪剂的经典营养素摄取研究，显著提高了我们对哪些营养素优先被 BAT 吸收以及如何利用的理解 16,17,18,19,20,21,22,23,24,25，26,27.例如，放射性示踪剂研究表明，冷活化的 BAT 会吸收葡萄糖、脂蛋白结合的脂肪酸和支链氨基酸16、17、18、19、20、21、22、23、27.最近的同位素示踪与代谢组学研究相结合，使我们能够测量这些营养物质在组织和培养细胞内的代谢命运和通量24,25,26,28,29,30。然而，这些分析主要集中在营养物质的个体利用上，使我们对BAT在器官代谢物交换中的系统级作用的了解有限。关于英美烟草消耗的特定循环营养物质系列及其在碳和氮方面的定量贡献的问题仍然难以捉摸。此外，对 BAT 是否可以使用营养物质产生和释放代谢物衍生的 BATokines（例如脂因子）的探索才刚刚开始 12,13,14,15,31,32。
动静脉血液分析是一种经典的生理学方法，用于评估器官/组织中循环分子的特异性摄取或释放。该技术以前已应用于大鼠的肩胛间 BAT 以测量氧气和几种代谢物，从而将 BAT 确立为适应性产热的主要位点，具有分解代谢潜力33、34、35、36、37。最近，一项使用大鼠肩胛间 BAT 的动静脉研究与跨组学方法相结合，从而鉴定了由生热刺激的 BAT38 释放的未发现的 BATokines。
基于高灵敏度气相色谱和液相色谱质谱（GC-MS 和 LC-MS）的代谢组学的最新进展重新点燃了人们对动静脉研究的兴趣，该研究用于器官特异性代谢物交换的定量分析 39,40,41。这些技术具有高分辨能力和质量精度，能够使用少量样品对各种代谢物进行全面分析。
与这些进展相一致，最近的一项研究成功地将动静脉代谢组学应用于小鼠水平的 BAT 研究，从而能够定量分析不同条件下 BAT 中的代谢物交换活动42。本文介绍了在 C57BL/6J 小鼠模型中使用 GC-MS 的 BAT 靶向动静脉代谢组学方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-20 &#181;L Filter Tips</td>
			<td>Axygen</td>
			<td>AX.TF-20-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe with attached needle - 26 G 5/8&#34;</td>
			<td>BD Biosciences</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 5977B GC/MSD (mass selective detector)</td>
			<td>Agilent</td>
			<td>G7077B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 7693A Autosampler</td>
			<td>Agilent</td>
			<td>G4513A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 8890 GC System</td>
			<td>Agilent</td>
			<td>G3542A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent J&#38;W GC column (Capilary column) HP-5MS UI</td>
			<td>Agilent</td>
			<td>19091S-433UI</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent MassHunter Workstation software_MS Quantitative analysis(Quant-My-way)</td>
			<td>Agilent</td>
			<td>G3335-90240</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mouse</td>
			<td>DBL</td>
			<td>C57BL/6JBomTac</td>
			<td></td>
		</tr>
		<tr>
			<td>CentriVap -50 &#176;C Cold Trap (with Stainless steel Lid)</td>
			<td>LABCONCO&#160;</td>
			<td>7811041</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Norvaline</td>
			<td>Sigma-Aldrich</td>
			<td>N7502-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5430R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass insert 250 &#956;L&#160;</td>
			<td>Agilent</td>
			<td>5181-1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (LC-MS grade)</td>
			<td>Sigma-Aldrich</td>
			<td>Q34966-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxyamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>226904-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette&#160;200 Serum, 200 &#181;L, cap red, flat base</td>
			<td>Sarstedt</td>
			<td>20.1290.100</td>
			<td></td>
		</tr>
		<tr>
			<td>MTBSTFA</td>
			<td>Sigma-Aldrich</td>
			<td>394882-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine(anhydrous, 99.8%)</td>
			<td>Sigma-Aldrich</td>
			<td>270970-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated CentriVap Complete Vaccum Concentrators</td>
			<td>LABCONCO&#160;</td>
			<td>7310041</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent diet</td>
			<td>SAFE</td>
			<td>SAFE R+40-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent incubator</td>
			<td>Power scientific</td>
			<td>RIT33SD</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Fine Pen Needles - 29 G 1/2&#34;</td>
			<td>BD Biosciences</td>
			<td>328203</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial Cap 9 mm</td>
			<td>Agilent</td>
			<td>5190-9067</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial, ambr scrw wrtn 2 mL</td>
			<td>Agilent</td>
			<td>5190-9063</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial, ambr scrw wrtn 2 mL+A2:C40</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

arteriovenous metabolomics to measure in vivo metabolite exchange in brown adipose tissue

棕色脂肪组织 （BAT） 通过一种称为非颤抖产热的独特能量消耗过程在调节代谢稳态中起着至关重要的作用。为了实现这一目标，英美烟草利用多样化的循环营养素菜单来支持其高代谢需求。此外，BAT 分泌代谢物衍生的生物活性因子，可作为代谢燃料或信号分子，促进 BAT 介导的组织内和/或组织间通讯。这表明BAT积极参与全身代谢物交换，这是一个有趣的特征，正在开始探索。在这里，我们介绍了一种 体内小鼠水平 优化的BAT动静脉代谢组学的方案。该方案侧重于产热刺激的相关方法和使用苏尔寿静脉的动静脉采血技术，该技术选择性地引流肩胛间 BAT 来源的静脉血和全身动脉血。接下来，演示了使用这些血液样本的基于气相色谱的代谢组学方案。该技术的使用应通过测量 BAT 对代谢物的净摄取和释放，在器官间水平上扩展对 BAT 调节的代谢物交换的理解。

图 1 显示了 BAT 优化房室代谢组学的实验方案。如协议部分所述，为了获得差异刺激的棕色脂肪组织，小鼠使用啮齿动物培养箱进行温度驯化或接受药理学给药，例如β-肾上腺素能受体激动剂。随后，麻醉小鼠，并收集血液样本进行代谢组学分析（图1A）。对于血液采样， 通过 苏尔寿静脉收集从肩胛间 BAT 特异性引流的静脉血，而直接从心脏左?...

Watch this Scientific Journal Video about Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention at JoVE.com

作者聚焦:阐明脂肪组织代谢和疾病预防的新途径 

在该方案中，概述了与在小鼠模型中使用GC-MS进行BAT优化的动静脉代谢组学相关的方法。这些方法允许在生物体水平上获得对BAT介导的代谢物交换的宝贵见解。

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

我们的实验室有兴趣确定信号通路和代谢通路协同调节脂肪组织代谢和生理学的机制。通过这种方法，我们的目标是找到一种方法来预防或针对由能量失衡引起的代谢疾病，如肥胖和糖尿病。我们方案的主要优点是我们已经从传统的大鼠模型转向使用小鼠模型进行动脉静脉血液采样和代谢组学分析。我们相信，这种转变将使我们能够更快地使用金标准遗传模型来验证我们的发现，以进行棕色脂肪研究。

arteriovenous-metabolomics-to-measure-vivo-metabolite-exchange-brown

Research

JoVE Journal

Biochemistry

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

1.2K 次观看.  Sungkyunkwan University (SKKU). 我们的实验室有兴趣确定信号通路和代谢通路协同调节脂肪组织代谢和生理学的机制。通过这种方法，我们的目标是找到一种方法来预防或针对由能量失衡引起的代谢疾病，如肥胖和糖尿病。我们方案的主要优点是我们已经从传统的大鼠模型转向使用小鼠模型进行动脉静脉血液采样和代谢组学分析。我们相信，这种转变将使我们能够更快地使用金标准遗传模型来验证我?...

视频: 作者聚焦:阐明脂肪组织代谢和疾病预防的新途径 

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

小鼠脂肪组织的采集和 RNA 分析处理

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

大鼠胰腺组织快速、经济高效的RNA提取

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

细胞外葡萄糖消耗作为细胞和组织离体葡萄糖摄取的间接测量

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

动静脉代谢组学测量棕色脂肪组织 中的体内 代谢物交换

动静脉代谢组学测量棕色脂肪组织中的体内代谢物交换