Name: analysis of &#946;-amyloid-induced abnormalities on fibrin clot structure by spectroscopy and scanning electron microscopy
Uploaded: 2018-11-30T14:30:00
Description: Watch this Scientific Journal Video about Analysis of ÃŽÂ²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy at JoVE.com

作者感谢纽约三机构治疗发现研究所 (tdi) 的川崎正森、麻岛一郎和迈克尔·福利合成了 acam 纤维蛋白原相互作用抑制剂及其宝贵的建议。作者还感谢斯特里克兰德实验室的成员进行了有益的讨论。这项工作得到了国家卫生研究院批准 ns104386, 阿尔茨海默氏药物发现基金会和罗伯逊治疗发展基金 h. a., nih 授予 ns50537, 三机构治疗发现研究所, 阿尔茨海默氏症药物发现基金会,rudin 家庭基金会和 john a. herrmann 为 s. s。

1. 用于分析的 aβ42和纤维蛋白原的制备
<ol><li>从冻干粉制备单分子 aβ42<ol>
<li>将 aβ42粉末加热至室温, 并在 1500 x 克的温度下旋转30秒。</li>
		<li>每0.5 毫克 aβ42粉末加入100μl 冰凉六氟异丙醇 (hfip), 并在冰上孵育30分钟。 注意: 在处理 hfip 时要小心, 并在化学罩中执行所有步骤。</li>
		<li>准备20μl 的等价物, 让薄膜在化学罩中干燥 2-3小时, 薄膜可以储存在-20°c。</li>
		<li>在10μl 新鲜二甲基亚硫酸 (d...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminopyrimidine+Class+Aggregation+Inhibitor+Effectively+Blocks+Abeta-Fibrinogen+Interaction+and+Abeta-Induced+Contact+System+Activation.">Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Abeta-Fibrinogen Interaction and Abeta-Induced Contact System Activation.</a> Biochemistry. 57 (8), 1399-1409 (2018).</li><li>Pretorius, E., Mbotwe, S., Bester, J., Robinson, C. J., Kell, D. 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W., Nagaswami, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+fibrin+polymerization+kinetics+correlated+with+electron+microscope+and+turbidity+observations:+clot+structure+and+assembly+are+kinetically+controlled.">Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.</a> Biophysical Journal. 63 (1), 111-128 (1992).</li><li>Chernysh, I. N., Nagaswami, C., Purohit, P. K., Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+clots+are+equilibrium+polymers+that+can+be+remodeled+without+proteolytic+digestion.">Fibrin clots are equilibrium polymers that can be remodeled without proteolytic digestion.</a> Scientific Reports. 2, 879 (2012).</li><li>Klunk, W. E., Jacob, R. F., Mason, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+amyloid+beta-peptide+(Abeta)+aggregation+using+the+Congo+red-Abeta+(CR-abeta)+spectrophotometric+assay.">Quantifying amyloid beta-peptide (Abeta) aggregation using the Congo red-Abeta (CR-abeta) spectrophotometric assay.</a> Analytical Biochemistry. 266 (1), 66-76 (1999).</li><li>Zhao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+amyloid+formation+by+turbidity+assay-seeing+through+the+cloud.">Measurement of amyloid formation by turbidity assay-seeing through the cloud.</a> Biophysical Reviews. 8 (4), 445-471 (2016).</li><li>Bester, J., Soma, P., Kell, D. B., Pretorius, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+and+ultrastructural+characteristics+of+whole+blood+and+plasma+in+Alzheimer-type+dementia,+and+the+possible+role+of+bacterial+lipopolysaccharides+(LPS).">Viscoelastic and ultrastructural characteristics of whole blood and plasma in Alzheimer-type dementia, and the possible role of bacterial lipopolysaccharides (LPS).</a> Oncotarget. 6 (34), 35284-35303 (2015).</li><li>Pretorius, E., Page, M. J., Mbotwe, S., Kell, D. 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这里描述的方法为纤维蛋白凝块体外形成的评估提供了一种可重复和快速的方法。此外, 该系统的简单性使得对 aβ如何影响纤维蛋白凝块形成和结构的解释相对直接。在本实验室的前一份出版物中, 证明了这些检测方法可用于测试化合物抑制 ab-纤维蛋白原相互作用的能力 13,14。利用这两种方法, 分析了一系列合成化合物抑制β纤维蛋白原相互作用?...

阿尔茨海默病 (ad) 是一种导致老年患者认知能力下降的神经退行性疾病, 主要源于β-淀粉样蛋白 (aβ) 的异常表达、聚集和清除障碍, 导致神经毒性1,2. 尽管 aβ集合体与 ad3之间存在良好的联系, 但疾病病理的确切机制尚不清楚4。越来越多的证据表明, 神经血管缺陷在 ad5的进展和严重程度中起着一定的作用, 因为 aβ与循环系统的组成部分直接相互作用。aβ与纤维蛋白原7,8有高亲和力的相互作用, 在 ad 患者和小鼠模型9,10,11中也定位到 aβ沉积。此外, β-纤维蛋白原相互作用诱导异常的纤维蛋白凝块形成和结构, 以及对纤维蛋白溶解的抵抗力9,12。治疗 ad 的一种治疗方法是通过抑制 aβ和纤维蛋白原13, 14 之间的相互作用来缓解循环缺陷。因此, 我们通过高通量筛选和药物化学方法 13, 14, 发现了几种抑制β纤维蛋白原相互作用的小化合物。为了测试光纤 b 系与原相互作用抑制剂的有效性, 我们优化了两种体外纤维蛋白凝块形成的分析方法: 凝块浊度分析和扫描电镜 (sem)14。
凝乳浊度测定是利用紫外可见光谱监测纤维蛋白凝块形成的一种直接快速的方法。随着纤维蛋白凝块的形成, 光线越来越分散, 溶液的浊度也在增加。相反, 当 aβ存在时, 纤维蛋白凝块的结构会改变, 混合物的浊度会降低 (图 1)。抑制化合物的效果可以评估的可能性, 以恢复凝块浊度从β诱导的异常。虽然浊度分析可以快速分析多种条件, 但它提供的关于凝块形状和结构的信息有限。sem, 其中固体物体的地形是由电子探针揭示, 允许分析的三维结构的凝块15,16, 17,18, 并评估如何存在的 aβ和/或抑制化合物改变结构9,14。光谱法和扫描电镜都是经典的实验室技术, 已被用于各种目的, 例如, 分光光度法用于监测淀粉样蛋白聚集19,20。同样, sem 也被用来分析由阿尔茨海默氏症、帕金森病和血栓栓塞中风 21、22、23 例患者的血浆形成的纤维蛋白凝块。这里介绍的协议是优化评估纤维蛋白凝块形成的重现性和快速的方式。
下面的协议提供了在有和没有 aβ的体外纤维蛋白凝块的制备说明。详细介绍了 aβ对纤维蛋白凝块形成和结构影响的分析方法。这两种方法测量抗β纤维蛋白原相互作用的有效性已证明使用 tdi-2760, 一种小型抑制化合物 14.这些方法, 无论是单独和共同, 允许快速和直接的分析体外纤维蛋白凝块的形成。

<table>
	<tbody>
		<tr>
			<td>Fibriogen, Plasminogen-Depleted, Plasma</td>
			<td>EMD Millipore</td>
			<td>341578</td>
			<td>keep lid parafilm wrapped to avoid exposure to moisture</td>
		</tr>
		<tr>
			<td>Beta-Amyloid (1-42), Human</td>
			<td>Anaspec</td>
			<td>AS-20276</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol, Greater Than or Equal to 99%</td>
			<td>Sigma-Aldrich</td>
			<td>105228</td>
			<td></td>
		</tr>
		<tr>
			<td>DIMETHYL SULFOXIDE (DMSO), STERILE-FILTERED</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fischer Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fischer Scientific</td>
			<td>BP310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fischer Scientific</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fischer Scientific</td>
			<td>C70</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Syringe, 0.2&#181;M, 25mm</td>
			<td>Pall</td>
			<td>4612</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex Sterile Syringe Filters, 0.1 um, PVDF, 33 mm dia.</td>
			<td>Millipore</td>
			<td>SLVV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid 96 Well Plates High Binding Certified Flat Bottom</td>
			<td>Fischer Scientific</td>
			<td>21377203</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax Plus384</td>
			<td>Molecular Devices</td>
			<td>89212-396</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, 5417R</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 200 Ultrasonic Cleaner</td>
			<td>Fischer Scientific</td>
			<td>15-337-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Rotator</td>
			<td>Thermo Scientific</td>
			<td>2314-1CEQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Spare washers for cover slip holder</td>
			<td>Tousimis</td>
			<td>8766-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Stem Pipets - Sedi-Pet</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>70967-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample holder for CPD (Cover slip holder)</td>
			<td>Tousimis</td>
			<td>8766</td>
			<td></td>
		</tr>
		<tr>
			<td>Mount Holder Box, Pin Type</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>76610</td>
			<td></td>
		</tr>
		<tr>
			<td>Round glass cover slides (12 mm)</td>
			<td>Hampton Research</td>
			<td>HR3-277</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Glutaraldehyde</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>16120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Falcon</td>
			<td>3047</td>
			<td></td>
		</tr>
		<tr>
			<td>Na Cacodylate</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM Stubs, Tapered end pin.</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>75192</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Tabs, Carbon Conductive Tabs, 12mm OD</td>
			<td>Ted Pella</td>
			<td>16084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosamdri-815 Critical Point Dryer with Gold/Palladium target</td>
			<td>Tousimis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Denton Desk IV Coater</td>
			<td>Denton Vacuum</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leo 1550 FE-SEM</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Smart SEM Software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
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analysis of &#946;-amyloid-induced abnormalities on fibrin clot structure by spectroscopy and scanning electron microscopy

本文介绍了用光谱法和扫描电镜 (sem) 法生成β-淀粉样蛋白 (aβ) 蛋白对凝块形成和结构的影响的体外纤维蛋白凝块的制备方法。aβ在阿尔茨海默氏症 (ad) 中形成神经毒性淀粉样聚集体, 已被证明与纤维蛋白原相互作用。这种 aβ-纤维蛋白原相互作用使纤维蛋白凝块结构异常, 对纤溶具有抵抗力。β诱导的纤维蛋白凝血异常也可能导致脑血管方面的 ad 病理, 如微梗塞, 炎症, 以及脑淀粉样血管病变 (caa)。鉴于神经血管缺陷在 ad 病理中的潜在关键作用, 开发能够抑制或减少 aca-纤维蛋白原相互作用的化合物具有很好的治疗价值。在体外的方法,其中纤维蛋白凝块的形成可以很容易和系统地评估是潜在的有用的工具, 开发治疗化合物。本文介绍了一种纤维蛋白凝块体外生成的优化方案, 以及 aβ和β-纤维蛋白原相互作用抑制剂的影响分析。凝块浊度检测快速、重现性高, 可用于同时检测多种条件, 从而可以筛选出大量的β纤维蛋白原抑制剂。可以进一步评估这种筛选产生的命中化合物是否能够使用 sem 改善光纤纤维蛋白凝块结构异常。使用 tdi-2760 (一种最近发现的抗逆转录酶原相互作用抑制剂) 在这里演示了这些优化方案的有效性。

在体外凝血 (浊度) 检测中, 酶凝血酶裂解纤维蛋白原, 导致纤维蛋白网络24的形成。这种纤维蛋白凝块的形成会导致通过溶液的光的散射, 导致浊度增加 (图 1), 在读数周期结束前形成 (图 2, 绿色)。当纤维蛋白原在 aβ42存在下孵育时, 溶液的浊度下降, 曲线的最大高度约为纤维蛋白原的一半 (<strong class="xfig"...

Watch this Scientific Journal Video about Analysis of ÃŽÂ²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy at JoVE.com

Analysis of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

本文介绍了两种方法, 可以单独使用, 也可以结合使用, 分析β-淀粉样蛋白对纤维蛋白凝块结构的影响。其中包括一种用于创建体外纤维蛋白凝块的协议, 其次是血栓浊度和扫描电子显微镜方法。

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

这种基于板的浊度测定的主要优点是，它速度快，允许一次筛选几种淀粉样蛋白-β纤维蛋白原相互作用抑制剂，而无需复杂的设置或要求。可进一步评估纤维蛋白浊度测定中的头部化合物，以恢复通过扫描电子显微镜分析的纤维蛋白血块中的 A-β 诱导结构异常的能力。扫描电子显微镜分析的血块制备的视觉演示至关重要，因为制备中的任何变化都可能导致实验结果的变化。本演示的重点是 A-beta 纤维蛋白原相互作用。该协议可以很容易地修改，以分析与其他蛋白质和化合物与纤维蛋白凝块的其他相互作用。首先，在200微升凝块形成缓冲液中加入1.5微摩尔新鲜准备的纤维蛋白原，每个含有和控制井的 A-beta 阴性42，并在室温下在旋转平台上孵育板。30分钟后，将30微升新鲜准备好的血栓溶液直接加入含纤维蛋白原的井中心，开始形成血块，并立即读取体外血块在350纳米的吸收度，在10分钟内每30至60秒重复一次测量。为了评估 A-β 纤维蛋白原相互作用抑制剂对纤维蛋白凝块结构的影响，首先使用钳子将清洁的 12 毫米硅化玻璃圆盖滑到 12 孔板的单个孔中，并在每个盖滑上添加 80 微升纤维蛋白原，轻轻摊铺溶液，以便均匀分布。向每井添加20微升血栓溶液，以启动血栓形成。在室温下盖上板30至60分钟，然后轻轻将每个血块淹没在两毫升冰冷的卡罗迪酸钠缓冲液中两分钟，在室温下盖上两次，每次洗涤后用1毫升移液器小心地取出缓冲液。上次洗涤后，检查盖滑上的血块形成状态，将血块固定在两到三毫升的冰冷2%谷胱甘肽中，在冰上30分钟。在孵化结束时，轻轻地从每一个井中去除谷胱甘肽，用新鲜的卡多迪酸钠缓冲液清洗血块，如冰上证明的。在分级的五分钟冰冷乙醇中对固定凝块进行水合，在不完全去除洗涤之间的乙醇的情况下，防止血块暴露在空气中。在上次 100% 乙醇清洗过程中，将盖滑移入临界点干燥器样品架中，在每个盖滑之间放置至少 1 个垫圈，并将盖放入充满乙醇的临界点干燥器室中。经过 30 分钟的干燥周期后，使用碳胶带将盖板安装在单个扫描电子显微镜存根上，然后将样品传输到真空溅射涂布机的溅射涂层室。然后溅射涂层小于20纳米的金铂或其他导电材料到样品上25秒，以4个正负的秒，并在扫描电子显微镜上成像样品，该显微镜配备有四千伏的二级电子探测器。纤维素血块的形成导致通过溶液的光散射，导致在阅读期结束时出现稳定度增加。当纤维蛋白原在 A-beta 42 存在的情况下孵育时，溶液的浊度会随着曲线达到仅纤维蛋白原的大约一半的最大高度而减小。在存在 A-beta 42 相互作用阻滞剂的情况下，β-淀粉样蛋白的效果得到改善，浊度高于仅使用 A-beta 的浊度。阻滞剂的效果不会由于背景浊度而出现，因为当 A-beta 不存在时，化合物不会改变纤维蛋白血块浊度。此外，GPRP（一种已知干扰纤维蛋白聚合的肽）存在凝块的形成表明，与没有抑制剂的纤维蛋白凝块形成相比，浊度显著降低。在血栓和氯化钙存在的情况下产生的纤维素原只形成纤维蛋白网，与纤维蛋白的拉长和相互间螺纹以及更大的束。当存在 A-beta 时，纤维素螺纹变薄，在聚合中出现几个粘性团块，表明 A-beta 引起的结构异常。与浊度测定结果一致，在存在 A-β 纤维蛋白酶相互作用阻滞剂的情况下形成血块会部分恢复纤维蛋白血块的结构，从 A-β 诱导的变化中恢复，因为通过这种治疗观察到的团块更少。血块浊度测定和扫描电子显微镜分析方案经过优化，以快速可重复的方式评估几种 A-β纤维蛋白原相互作用抑制剂。不久，数据将提供有关纤维蛋白血块形成的有价值的信息，可用于体外和体内的进一步研究。

Research

JoVE Journal

Biochemistry

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

科研

生物化学

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

线粒体和内质视网膜成像由相关光和体积电子显微镜

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

单颗粒冷冻电子显微镜：从样品到结构

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

微图案透射电子显微镜网格，用于在全细胞冷冻电子断层扫描工作流程中指导细胞定位

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...